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Unicorn 4 User Manual - GE Healthcare Life Sciences

1. IV Display tooltip for extended scouting scheme cells Define Clear All Delete Insert Add Senes Help Fig 2 4 Scouting scheme for testing flow rate while holding pH constant 2 6 System Control 2 6 1 Control facilities The system control module allows independent control of up to four chromatography systems from one computer with continuous real time monitoring of the separation process The run status can be displayed as e numerical display of run data from selected monitors e graphical display of curves from monitors e a flow scheme showing the current open flow path in the system e a logbook recording the control events in the run Systems can be controlled either manually with interactive commands or through pre programmed methods UNICORN concepts ez System Control 1 Explorer100 Method c Default Id23 m01 Result c Default Id23001 1es EI E File View Manual System Help Run Hold Pause Continue End fE m OAAS Cone SamplePres Cond pi Pressure Auxint mAU SampleFlow Buffer A2 Univer PumpA 3 AL RA g j Pumpa U6 Inet A WosteF f 0 00 min Run started Logbook 1 60 min Pause 3 92 min End For Help press F1 Controlled By default 4 Fig 2 5 The system control screen
2. T Display tooltip for extended scouting scheme cells Define Clear All Delete Insert Add Fig 5 32 The Scouting tab in Run Set up showing three different column types in three runs 1 3 In Run 1 the default values for the column were accepted In Run 2 the default values for the column were also accepted so the variable values were updated for that run In Run 3 the default values for the column were not accepted and thus the variable values are the same as those in the previous run Inserting scouting runs To insert a new scouting run before an existing run click on a run column on the Scouting tab and then click on Insert The new run will inherit the variable values from the preceding run or from the default values in the method if the run is inserted at the beginning of the scouting series Adding scouting runs To add a new scouting run click on Add The new run inherits the variable values from the highest numbered run or from the default values in the method if the run is added at the beginning of the scouting series Excluding runs In certain situations it may be desirable to skip certain runs within the scouting scheme To do so click on the Run header in the Scouting tab This will change the header from Run to Excluded e p 5d 5 Editing methods Note Note an 18 1138 73 e p76 Deleting scouting runs To
3. 11 4 3 Measuring recovery 0 ee 11 5 Automated quantitation nananana nnn 11 5 1 Creating a quantitation table from standards 11 5 2 Automated Quantitation 22 20000 11 5 3 Automated Update 0 0 cee 11 5 4 Automated update and quantitation in scouting runs 11 5 5 The evaluation procedure instructions for the Analysis Modulens errs ied had oie Rie edand eh 11 6 Measuring molecular size 0 000 eee eee 11 6 1 Overview of molecular size determination 11 6 2 Determining molecular size the process in detail A Evaluation functions and instructions A 1 Smoothing algorithms 0 000 cece eee eee ALT Moving Average riore eee hid wea i Be Pa F A 1 2 Autoregressive 2 0 0 eee Avl 3 Medians 2 ccna hg Gok op oe Vee Ra Bee op ee E a o Ad Savitzky Golay tc a2 wastes he 8 Aovce 2 wed at Kae We a A 2 Baseline calculation theory 00cee eu ee eae 371 A 2 1 Defining baseline segments 2 0 0 0 ce eee 371 A 2 2 Selecting baseline points for Classic algorithm 373 A 2 3 Drawing the baseline 1 ee 373 A 2 4 Estimating the baseline parameters from the source curve for Classic algorithm 374 A 2 5 Measuring the Slope limit using Differentiate and curve co ordinates for Classic algorithm 374 A 3 Peak table column components 000000 ee 375
4. 406 C 3 The statistics available 0 20 eee eee eee 409 C31 Correlation coe tec tet foe A este nae telah tee ay 409 C 3 2 Explained variance 0 00020 eee 409 Introduction E 1 Introduction UNICORN is a control system developed and marketed by Amersham Biosciences AB for real time control of chromatographic separation systems from a personal computer The package operates together with systems in the KTAdesign platform referred to as AKTAdesign systems BioPilot System and BioProcess System from Amersham Biosciences Standard configurations are provided for AKTAdesign systems and BioPilot System while configurations for process scale separation with BioProcess System may be customised for a particular installation UNICORN runs under the operating systems Windows NT workstation 4 0 and Windows 2000 Functional features of UNICORN 4 0 include e One PC may control up to 4 separation systems directly e Network support allows up to 99 systems to be accessed from one PC e A Method Wizard taking the user to step by step through the method development process e Method templates providing method frameworks for most common applications eliminating the need to program methods from scratch e Scouting facilities allowing most parameters to be varied automatically in repeated runs to facilitate process development and optimisation e Dynamic graphical overview of active runs e Comparison
5. Width time or volume base Difference in retention between the peak end and peak start G A in Figure A 3 Area time or volume base Calculated as the area between the curve and baseline between the peak start and peak end shaded in Figure A 3 Height Maximum amplitude above the baseline C F in Figure A 3 Peak endpoint time or volume base Retention value at peak retention start and peak end A G in Figure A 3 e p375 AY Evaluation functions and instructions an 18 1138 73 p376 Width at half height time or volume base Calculated by taking the maximum height of the peak above the base line then determining the peak width at half this value above baseline D B in Figure A 3 where BD bisects CF Percent of total area time or volume base Peak area as a percent of the total area under the curve above the base line Note that this value may differ in time and volume base if the flow rate is not constant throughout the method Percent of total time or volume base Peak area as a percent of peak area the sum of all integrated peaks Note that this value may differ in time and volume base if the flow rate is not constant throughout the method Type of peak limits Identifies the criteria for peak start and peak end as either the baseline intersection or drop line to the baseline Peak endpoint heights Amplitude above the baseline at left A i
6. an 18 1138 73 e p110 Sample_injection 0 0 00 Base SameAsMain 00 Message Load the sample loop with sample then press Continue Screen 00 Hold Sy 10 InjectionValve Inject oO 00 InjectionValve Load 5 00 End_block Pause suspends execution of the method and stops the pumps so that the system comes to a standstill In AKTAdesign systems valves remain in the position they were in before the pause The pause may be defined as indefinite or for a given number of minutes This instruction is most useful for stopping the system in the event of an unexpected condition In both cases the method may be resumed by pressing the Continue button in the System Control toolbar A third way for pausing a method Hold_until is described on page 113 5 10 6 Linear flow rates Linear flow rates cm h can be specified for Flow instructions if a column is selected on the Variables tab or for the Base instruction in the block see Base instruction on page 103 The volume flow rate is calculated from the specified linear flow rate and the column diameter as given in the column definition To use a linear flow rate check the Linear Flow option in the instruction dialogue for the Flow instruction The calculated volumetric flow rate is shown during runs Breakpoint 7 Instructions Parameters FlowRate 0 3055 A Pump ae ve po Hw Gradient Var fooo H emh al Cireman BufferPrep_pH van ere
7. Concentration Values calculated by the Analysis module only available if the Quantitation module is installed Amount As above Molecular size IAs above Sigma E C 4 s50 n l A peak Sigma where n is the number of data points x is the volume or time value y is the amplitude value Xymax 18 the volume or time value at the maximum amplitude value Apeak is the area of the peak The peak width for a Gaussian peak is 4 x Sigma e p377 AY Evaluation functions and instructions Note an 18 1138 73 p378 Resolution Vpo Vay x 1 177 Whi W R h2 where Vga retention volume for peak 1 Vp retention volume for peak 2 Wh1 peak width at half height for peak 1 for Gaussian peaks Wp2 peak width at half height for peak 2 for Gaussian peaks The peak resolution is calculated with one of the following three algorithms 1 Vr2 Vr1 Wbp2 Wp1 2 2 Vr2 Vri Sigma Sigma x 2 3 Vr2 Vaq 2 x Who Wy1 2 354 where Vp 1 Wp1 Sigma and Wy are the retention width sigma and width at half height of the previous peak and VR2 Wp2 Sigma and Wh are the retention width sigma and width at half height of the current peak respectively The UNICORN INI variable EVAL ResolutionAlg determines which of the three algorithms is actually used If this variable has the value 1 2 or 3 then the algorithm used corresponds to the numbered list above If
8. Fig 5 19 Illustration of the different effects of Change and Replace on instructions within and after gradients Moving instructions within a breakpoint To change the order of instructions within the same breakpoint in a block mark the instruction to move with the left mouse button and drag the instruction to its new location holding the left mouse button down You can only move instructions in this way within a group of instructions at the same breakpoint e p63 Editing methods an 18 1138 73 e p64 Gradient Move Gradient 0 00 Base SameAsMain 0 00 Base SameAsMain 0 00 FractionCollector 5 ml 0 00 Gradient 100 00 20 00 Base 0 00 Gradient 100 00 20 00 Base 0 00 FractionCollector 5 ml 20 00 Message END OF GRADIENT Noscreen 20 00 Message END OF GRADIENT Noscreen 20 00 End_block 20 00 End_block Fig 5 20 Instructions can be moved within the same breakpoint by dragging with the shortcut menu Moving instructions between breakpoints To move an instruction to another breakpoint 1 Using the shortcut menu select the instruction to be moved Select Cut from the shortcut menu Edit Cut or lt Ct r1 X gt 2 Select the instruction line just above the insertion point where you want the cut instruction to be pasted 3 Select Paste from the shortcut menu Edit Paste or lt Ct rl V gt The instruction is now removed from its original breakpoint and pasted at the new breakpoint Th
9. Fig 9 50 Setup Chromatogram dialogue Select which chromatogram s to insert from the Selected chromatogram s drop down list Active chromatogram All chromatograms 1 2 etc This inserts the chromatogram that is currently active in the Evaluation window This inserts all chromatograms contained within the Evaluation window This inserts the specific chromatogram corresponding to the selected number name Check the appropriate Settings for the inserted chromatogram s You can select to view and print the chromatograms with Thick lines view the chromatogram s in Landscape orientation insert the chromatogram s so that they Start on new page and or show a chromatogram on a Full page The Original file name can also be inserted on the graph e p237 ED Presenting results Note1 Note2 an 18 1138 73 e p238 3 To define the layout for the chromatogram s click on the Define layout button The Report Chromatogram Layout dialogue is displayed Make the appropriate selections in the various tabs for the report If your chromatogram contains curves with different Y axis units click on the Y Axis tab and select the appropriate curves within each of the axis drop down list boxes Selections made in this layout only affect the report and not the view of the chromatograms in the Evaluation window Scouting variables and other appropriate variables can be selected for the chromatogram header for exam
10. Fig 5 44 The Columns tab in Run Set up 5 5 9 BufferPrep BufferPrep is only available for some KTAdesign systems BufferPrep allows a buffer of any pH and salt concentration to be prepared on line from 4 stock solutions This removes the need to manually prepare new buffers every time the pH needs to be changed Linear and step salt gradients can be run and pH can be used as a variable scouting parameter BufferPrep is optimised for cation and anion exchange chromatography It can be used for gel filtration but not for other chromatographic techniques For a complete description of BufferPrep see the User Manual for KTAdesign systems If a suitable template is not available you can create a BufferPrep method by selecting the BufferPrep tab clicking on the BufferPrep ON radio button The instruction BufferPrep_pH must be available at breakpoint zero at the beginning of the method The method must not contain the instructions PumpAlnlet or PumpBinlet Editing methods 5 Evaluation Procedures Method Information Result Name Start Protocol Frac 950 Variables Scouting Notes Questions Gradient BufferPrep Columns Reference Curves Status C OFF Recipe Stock solutions Piperazine pH 9 4 10 5 Global x Inlet A1 A11 A18 Buffer 100 B 1M 0 1 M Piperazine pH Range 9 4 10 5 Notes BUFFER SOLUTION 2000m Piperazine x 6 H20 38 84g Mw 194 2 ACID SOLUTION 1000m Inlet A2 Acid B
11. Area corresponding to 100 the added amount o M od 15 0 16 0 170 18 0 min Fig 11 8 Peak areas from the sample and the sample with standard addition Analysing results 11 3 Using the evaluation module a Peak integration is performed on the two curves to produce a peak table for both the spiked and unspiked sample 4 The difference in peak area between the spiked and unspiked sample represents the peak area from the added amount 5 Assuming linear proportionality between peak area and amount and knowing the added amount the software calculates the amount of the component of interest in the sample Peak area from unspiked sample Amount in unspiked sample Amount added x Peak area from added amount Reliability of the standard addition technique Standard addition is the least precise of the quantitation techniques since it is restricted to a single concentration level and the amount in the sample is calculated by extrapolation However it is a useful technique when a rapid result is required and high precision is not needed and when you have only a few sample runs to perform e Quantitation requires that the component s of interest are completely resolved from all other components in the chromatogram Overlapping peaks will give unreliable results e The peak integration parameters Baseline settings must be correctly selected The default settings will be satisfactory in many cases but the inte
12. Changing the scale of Y axis The Y axis is automatically scaled for each of the curves To fix the scale of individual curves 1 Select View Properties or select Properties from the shortcut menu The Properties dialogue is displayed 2 Select the Y Axis tab SEAL BULL Fig 6 11 Properties dialogue Y Axis tab Note 4 5 Performing a run GH Select the appropriate curve and click on the Fixed button Enter a minimum and maximum range in the fields within the specified limits Repeat steps 2 to 3 for other curves Click on OK Values on the Y axis apply to the curve with the same colour as the axis markings Click on the legend to get the correct Y axis Changing the scale of X axis Click on the X axis to switch the display between time and volume units The run is controlled according to the time volume base defined in the current block regardless of the base in the curves display Alternatively select the X units in the X Axis tab of the Curve Properties dialogue You can also set the viewed portion of the total run 1 2 3 Select View Curve Properties or select Properties from the shortcut menu The Properties dialogue is displayed Select the X Axis tab Select the appropriate base Time or Volume Curves are collected in time and recalculated for display in volume Thus the resolution of the two bases may appear slightly different Y Axis Curve Style and Colour Fl
13. 06 ATJMFOOB 1_Cone 07 ATJMFOO6 1_pH 08 ATJMF006 1_Pressure 09 ATJMF006 1_Flow 10 ATJMF006 1_Temp gt 11 ATJMFOOB 1 Fractions Curve name oviar Snm 01 COPY Close Help Fig 9 38 Copy curve dialogue 9 4 5 Stacking and stretching curves Several tools are available to stack and stretch curves from different runs to better visualise the differences These tools are normalising curves shifting curves and stretching curves These allow you to manually reproduce the Stack and Mirror functions associated with the Open to compare Curves operation see Alternative A Import chromatograms using Open to compare on page 216 and more besides If the curves have been stacked with the Open to compare curves operation and you want to change the stack offset the easiest way is to repeat the operation with another offset The curves can also be stacked and stretched individually as described below The operations presented below all require the curves to be present in one chromatogram see Comparing curves on page 220 kad Aah Aah M Fig 9 39 Stacking curves Nm 3 0 Presenting results 9 Alternative A Stacking and stretching curves using the normalise function The simplest method to align curves with respect to the X axis or the Y axis for easier visualisation is to use the normalise function To select the curve to be move
14. Any selected as a variable called column 2 To define columns for a scouting run click on the Define button to display the Scouting Variables dialogue Select Column and then OK 3 Click on the Column drop down menu within the desired run A menu is displayed in which you can select the appropriate column Evaluation Procedures Method Information Variables Scouting Notes Questions Result Name Start Protocol Gradient BufferPrep Columns Frac 950 Reference Curves i Superdex_200_PC_3 2 30 Global i Superdex 200_prep_gtade_35 600 Global Initial Elue ButferPrep_ pH S perdex200_prep grade 60 600 Global Superdex_75_HR_10 30 Global Superdex_75_PC_3 2 30 Global Superdex 75_prep_grade_ 35 600 Global Run4 I Display tooltip for extended scouting scheme cells Define Clear All Delete Insert Add Sere Help Fig 5 30 The Scouting tab in Run Set up showing the Column drop down menu 4 When you have selected a column the Column value update dialogue is displayed e p73 Editing methods an 18 1138 73 e p74 Column value update x Update these instructions with column default values Initial_Eluent_Conditions_BP Flow FlowRate Flow_rate 5 Column Value 5 00 ml min Start_Conditions_BP Alarm_Pressure High larm Column_PressureLimit 2 50 Column Value 3 00 MPa Start_Condition
15. IS and Table settings Define components Print Fig 11 18 Quantitation table dialogue Calibration curves will now be produced If internal standard quantitation is used the internal standard must first be selected within the Table settings dialogue This dialogue also allows you to set the basis of the calibration curve on peak area the default or peak height If internal standard is not used you normally do not need to make any changes in the Table settings dialogue in which case you can move on to Entering the data for the standards on page 329 IS and settings To select the internal standard click IS and Table settings in the Quantitation table dialogue IS and Table Settings xi Analysing results CH General Multiply amount with 1 000 0 001 1000 Multiply conc with ji 000 0 001 1000 Amount unit label ma Injection volume 0 050 ml m Internal standard Internal standard peak Internal standard IS conc 0 500 ma ml Quantitation peaks Area Height Cancel Help Fig 11 19 IS and Table settings dialogue Use this dialogue to enter The amount and concentration multipliers you wish to apply When the calibration curve is applied to a sample the amount and concentration of the sample are multiplied by the values The multipliers should normally be set to 1 Change them if you want to determine the amount or concentration in the startin
16. There are two stages to the process e peak data from a standard are used to produce a molecular size curve e from the molecular size curve the molecular size of the components in the sample are determined The process is described in detail in the next Section The following provides an outline of the technique Producing the molecular size curve This process determines the relationship between molecular sizes and corresponding retention values 1 A run is performed with one or more standards Please refer to Chapter 6 The standards should contain a number of components of known molecular size and these should extend beyond the limits of size expected in the test sample 2 Using the Evaluation module peak integrate the standard curve to produce a peak table This process is described in Chapter 10 3 The peak table from the standard is used to produce a molecular size table Each peak is represented by a retention value Relevant peaks are selected in turn and data on the corresponding molecular sizes are entered e p357 ED Analysing results an 18 1138 73 p358 mAU i Standard 400 200 0 0 5 0 10 0 15 0 20 0 25 0ml maAU 600 600 400 200 o 0 5 0 10 0 15 0 20 0 250m Fig 11 42 Chromatographic curves of the standard and the sample The software plots these values as a molecular size curve This shows an inverse relationship between the molecular size and re
17. 0000005 10 3 6 Batch TunSs c0 ose ce Weak ea ira wees eked Alas 10 3 7 Evaluation procedures and reports 00 10 3 8 Placing a procedure on the menu and running 10 3 9 Exporting data or CUIVES 1 eee 10 3 10 Copying results to the clipboard 0 10 3 11 Importing results and curves 0000000005 viii 11 Analysing results 11 1 Introduction 0 0 ccc ee PTs ll OQA tes amp x A Ae a hee oh a de fa TL2 Molecular Sized 3 5 0 ses oe oe A See A O Wee Wadd dee ae LETS SDOTIMITONS tse asd a sce one AP pect a n ghee rA Installations sccei d Poi 4 Behe Sedat Lentek dedente 11 2 Quantitation overview 0 ccc cee ee ee eee 11 2 1 About quantitation 0 0 0 0 0 cee eee 11 2 2 External standard quantitation 11 2 3 Internal standard quantitation 005 11 2 4 Standard addition quantitation 005 11 2 5 Recovery calculation n nannaa aaa anaana 11 2 6 General factors affecting reliability 11 3 Producing calibration curves 00 0 nannan 11 3 1 Preparations before using Quantitate 11 3 2 Creating a quantitation table 0 0 00 005 11 3 3 Editing and updating a quantitation table 11 4 Quantitating the sample 200 e ee eee eee 11 4 1 Quantitation by external and internal standard 11 4 2 Quantitating by standard addition
18. 1 Use the same method as was used for the standards when the quantitation table was created 2 On the Scouting tab click on Clear All to clear the scouting scheme then enter the new values Select the correct concentration level for the standards in the variable Quantitation_Type 3 From the Evaluation Procedures tab select the procedure Update_Quantitation This procedure will automatically integrate the first UV curve using default baseline settings update the selected quantitation table with the new standard and print an update report 4 Click on the Quantify button on the Evaluation Procedures tab and select the quantitation table to be updated This quantitation table will now be copied into the Update_Quantitation procedure 5 By default the quantitation table will be updated by Replace so that all points at the selected level will be replaced with the new point You can only perform one run at each level since the quantitation table will be updated by replacement of the old points 6 If you want to update by Average so that a new average value is calculated from the old points together with the new point you must edit the procedure To do this click Edit in the Evaluation Procedures tab and the Procedure Editor dialogue opens Scroll down the list of instructions in the procedure and select the existing Update instruction Analysing results CH Under Parameters use the scroll bar to locate the Average or replac
19. 9 2 2 Shortcut menu When viewing curves in the Evaluation module the shortcut menu provides a quick alternative to menu commands The following functions are available from the shortcut menu dd Text Edit Text Mode Base Type gt Peak Integrate Copy to Clipboard Copy to Metafile Shift Offset Hatch v Marker Set Marker Ref Point Reset Marker Ref Point Properties Fig 9 3 An example of a shortcut menu within the Evaluation module 9 2 3 Opening the Chromatogram Layout dialogue Most of the changes that you are likely to make regarding chromatogram presentation are made in the Chromatogram Layout dialogue This is opened in one of two ways e Place the mouse cursor in the chromatogram window and select Properties from the shortcut menu options Note that the view from which you activate the Properties command determines the tab that is displayed in the Chromatogram Layout dialogue e Select Edit Chromatogram Layout You can apply any changes made in the Chromatogram Layout dialogue to all open chromatograms by checking the Apply to all chromatograms option Presenting results 9 Curve Style and Colour Edit Texts Layout Library Header Curve Names Y Axis Axis Curve Peak Table Select curves to display 02 HIPREPO2 1_U 2_406nm 03 HIPREPO2 1_U 3_Onm 04 HIPREPO2 1_Cond 105 HIPREPO2 1_Cond 06 HIPREPO2 1_Cone 07 HIPREPO2 1_pH 08 HIPREPO2 1_Pressu
20. By using MethodQueue facilities several methods may be run in a pre defined automatic sequence involving one or more chromatography systems With suitable chromatography system equipment this allows unattended operation of quite complex multi step separation processes 2 6 2 System connections For controlling a separation process the operator establishes a connection between the computer and the chromatography system in one of the system control windows on UNICORN desktop Two kind of connections may be established e Control mode connections which permit full control of the connected system e View mode connections from which the progress of the separation can be monitored but the system cannot be controlled A system can be started from a computer in for example the laboratory Control of the system can be released without affecting the run and the control of the system can be later taken from another computer station for example in the office epll 2 UNICORN concepts an 18 1138 73 e p12 Each chromatography system can have only one control mode connection at any one time but it can have several view mode connections In a network installation the same or different users may establish simultaneous view mode connections to one system on different computers This allows a running process to be monitored from several locations at the same time 2 7 Evaluation The Evaluation module chapter 9 and 10 provides e
21. ED Evaluating results an 18 1138 73 p290 Select help curve s r Y axis scale Minimum jo Maximum jo 03 JAMILO3 1_UV3_254nm 04 JAMILO3 1_Cond 05 JAMILO3 1_Cond 06 JAMILO3 1_Cone O07 JAMILO3 1_pH 08 JAMILO3 1_Pressure 09 JAMILO3 1_Flow 10 JAMILO3 1_Temp 11 JAMILO3 1_Fractions 12 JAMILO3 1_Inject 17 JAMILO3 1_UV1_215nm 01 BASEM 118 JAMILO3 1_UV 1_215nm 01 BASEC Cancel Help Fig 10 36 Create Curve dialogue If you want to create new unit click on the New unit button and enter the new unit name and number of decimal places Create New Unit xi Name Number of decimals jo Cancel Help Fig 10 37 Create New Unit dialogue Click on OK to return to the Create Curve dialogue and again on OK when you have made your selections there With Point Mode selected you can use the left button to insert new curve points on the chromatogram The co ordinates of each new point are automatically entered into the Point list Evaluating results 10 The co ordinates of the mouse cursor are displayed beneath the curves thus allowing you to precisely position a new data point The co ordinates for the new curve are always displayed Selecting Zoom mode allows you to use the mouse to select an area of the view and zoom in The shortcut menu allows you to undo
22. Eluent_B_inlet 01 Flow 1 00 11 End_block End_block 01 Gradient 0 00 B 0 00 base Flow_rate ml min Watch example 5 Collecting three absorbance peaks Collecting three absorbance peaks through outlets F3 F5 and F7 with Waste fractions through outlets F4 F6 and F8 is described below The waste fractions between the peaks are collected through the outlet valve positions F4 F6 and F8 so ensure that the tubing from these positions is lead to a suitably large container It is not recommended to use outlet fractionation for peaks with small volumes i e less than 10 ml The fraction collector should be used for such peaks Editing methods 5 The maximum number of peaks collected in this example is three due to the limited number of positions on the outlet valve When the UV reaches 5 mAU or more the outlet valve is switched to the position for collecting the first peak When the UV reading goes down to 4 75 mAU the outlet valve switches to the next position to separate the waste fraction from the collected peak fraction This process is repeated twice for the next two peaks so that when the UV reading rises above 5 mAU the position switches to collect the peak fraction and the position switches again to collect the waste fraction when the UV reading falls again Note The UV threshold for collecting the waste fraction must be below the threshold for collecting the peak fraction so tha
23. Evaluating results 10 Edit Peak Table 1 OL x id186Level 1 to 4002 1_UV1_280nm id186Level 1 to 4002 1_UV1_280nm 01 BASEM1 ob 5b 100 150 20 0 No Peak name Retention min Peak start min Peak end min rea mAU min pif rf Delete peak Split peak Join left Join right OK Cancel Help Fig 10 25 Edit Peak Table chromatogram window Deleting a peak in the peak table To delete a peak from the table click on the peak in the chromatogram or in the peak table and click on the Delete peak command button Note that the remaining peaks will be renumbered after the deletion Splitting a peak A peak is defined within delimiting drop lines to the left and right of the peak It is possible to split the peak into two new peaks by inserting a drop line The drop line is always inserted at the middle point between two existing drop lines The area under each new peak will not be the same if the symmetry of the original peak was not perfect To make a split select the desired peak in the list or mark in the curve and press the Split Peak button The peaks will be renumbered according to the split Refer to the description below about adjustment of the drop lines Joining a peak It is possible to join the areas of adjacent peaks if separated by a drop line 1 2 Select a peak either on the chromatogram or in the peak table Click on Join left or Join right if you want the peak to be jo
24. External standard quantitation A calibration curve is produced by running one or several concentration levels of the standard containing the component s of interest The amount and concentration of the component in the sample is then determined from the calibration curve This technique is fairly simple and usually gives good precision Internal standard quantitation Peak areas of the components of interest are related to the peak area of an internal standard added in a fixed amount to each concentration level of the standard and to the sample This technique reduces errors that are caused by changes occurring between runs and is therefore the technique that may give the highest precision if a suitable internal standard can be selected e p307 ED Analysing results an 18 1138 73 p308 Quantitation by standard addition The sample is spiked with a known amount of the component of interest The areas of the spiked and unspiked sample are then compared and the amount in the unspiked sample is determined No calibration curves from standards are used Only one component can be quantitated Compared to other techniques standard addition enables a result to be obtained more quickly when you are performing a small number of sample runs However the precision is limited Recovery calculation Recovery is used to determine the losses that may occur during the sample preparation process The sample is spiked with a known amount o
25. If you have an internal standard select it by double clicking on it and give it a new name If you want to exclude a selected peak mark it and press Exclude or double click on the peak again Excluding a peak in one level excludes it from all levels The Peak table within the Define component s dialogue shows three columns the absolute Retention value of the component in level 1 1 the width of each components window If you change the width of a window by adjusting the cursor lines it will be reflected in the Window column the list of components with the currently selected component highlighted Peaks in level 1 1 Black peak markers Retention Window Ce onent name Possible to change 2 min min Maen g 3 59 0 47 Ribonuclease A 10 48 0 47 Cytochrome C 11 69 1 01 Lysozyme Fig 11 14 Peak table columns Peak identification When a component is selected vertical cursor lines show the current identification window The software uses this window to search for peaks in other levels and in the sample run s A peak found in the window is assumed to be the component of interest You can change the limits by dragging a limit cursor line Both cursor lines move symmetrically so that the limits centre on the component peak 1 Set the window to a suitable width The window should be set wide enough to include peaks in the other levels despite minor variations in retention volumes However the window shou
26. Signing View Signatures m Sign as user User Password SS es Full name deta Position deta r Signature Meaning Password IV Lock Cancel Help Fig 4 7 Sign the Method dialogue 3 Type your signature password in the Password field and if desired check the Lock box to permanently lock the method from further changes If the method is locked and you want to make additional modifications select File Save as and give the method a new name e p39 gt Creating methods Note an 18 1138 73 e p40 4 The View Signatures tab provides a list of all signatures associated with the current method 5 Within either the Signing or View Signatures tabs click on OK to complete the signature process 4 5 Saving the method A new method created from a method template or Wizard is untitled and must be saved under a method name before it can be run Click on the Save Method toolbar button or choose File Save to save the method c Default For System Prev Folder Explorer10 474KB Method File Explorer10 312KB Method File Explorer100 310KB Method File 2 Untitled Explorer10 475KB Method File Anion_Exchange Cation_Exchange OK Cancel Help Fig 4 8 Dialogue for saving a method 1 If required select another folder than the default home folder in which to save the method 2 Enter a Method name for the method Method names may be up to
27. amount tmon 11 Ribonuclease A 9 59 12 9160 49 212 0 405 0 203 12 cytochrome C 10 43 28 1310 101 444 0 416 0 208 13 Lysozyme 11 71 139 5769 324 616 0 612 0 306 Fig 11 29 Part of the peak table showing amount and concentration values for quantitated peaks If the amount can not be calculated one of the following signs is shown in the Amount column gt means that the value is higher than the highest value in the calibration curve i e it is outside the valid range of the calibration curve lt means that the value is lower than the lowest value in the calibration curve i e it is outside the valid range of the calibration curve means that the value can not be calculated For example this sign might indicate that the peak could not be identified When the result file is saved it includes the quantitation table that was used for the quantitation You can later show the table that was used by selecting Quantitate Edit Quantitation Table View Current e p341 ED Analysing results an 18 1138 73 p342 If you want to print the table that was used select File Report and select the Quantitate and Mol Size option see Printing reports on page 232 11 4 2 Quantitating by standard addition For an explanation of the standard addition technique see Standard addition quantitation on page 314 There are four stages to the process 1 performing the two runs and then copying the curves into one
28. m Print format Lo Es H B E Pa N Ea Chromatograms in each column fi Chromatograms in each row fi Use thick lines Preview Landscape Cancel Help Fig 9 45 Print Chromatograms dialogue If the Documentation is open click on the Print icon to print any components in the documentation The Preview button opens the Customise Report window allowing for custom set up of the print out Chromatograms can also be printed from the File Report dialogue see Printing reports on page 232 9 8 Printing reports The Evaluation module provides you with extensive tools with which to create detailed reports You can create and save report formats based on either Standard layouts or on Customised layouts To open the report generator interface select File Report or click on the Report toolbar button and the Generate Report dialogue is displayed Note Presenting results 9 Format Contents Global Chromatogram 3U New Global Chromatogram_Peaks Run user Global Full Report Run date amp ti un date amp time Delete Report title Result file name Edit Method file name Page number Chromatogram Current chromatogram Preview Close Help i Fig 9 46 Generate Report dialogue In the dialogue you can see the available formats in the Format field You can select a format from the list and directly apply it Alternatively
29. tn where t current processed point t 1 previous processed point S current source point p smoothing parameter value Increasing the parameter value increases the smoothing effect The filter algorithm accepts integer parameter values between 1 and 25 A 1 3 Median For each data point in the source curve the processed curve is calculated as the median of the data points within a window centred on the source data point The width of the window is determined by the parameter value expressed as number of data points When the source point is less than half the window size from the beginning or the end of the curve the median is calculated symmetrically round the source point over as many data points as possible Increasing the window width increases the smoothing effect To completely remove a noise spike the window width should in principle be slightly more than twice the width of the spike The filter algorithm only accepts odd integer parameter values between 1 and 151 If an even number has been given it is incremented by one A 1 4 Savitzky Golay The Savitzky Golay algorithm is based on performing a least squares linear regression fit of a polynomial of degree k over at least k 1 data points around each point in the spectrum to smooth the data The derivative is then the derivative of the fitted polynomial at each point The calculation uses a matrix formalism to calculate 1st through 9th derivatives The cal
30. 100 i 150 Lettimt E39 Bight limit 14 77632 Cancel Hep Fig 10 6 Peak Window chromatogram window an 18 1138 73 p264 Evaluating results 10 Peak skimming The area under a peak can be calculated either using drop lines or peak skimming Drop lines are vertical marks that split two peaks at the valley This is most commonly used for peaks of relatively similar size In some circumstances for example when a peak has a shoulder use of a drop line will cause too much area of the first peak to be lost to the peak that forms the shoulder Thus the skim peak function can be used when the smaller peak is skimmed off with a straight line starting at the valley between the peaks and ending at the point on the other side of the smaller peak where the slope of the skim line is equal to the slope of the curve In doing so this skims the area under the second peak A B Drop line Fig 10 7 Illustration of how a drop line A and a skimmed peak B affects the area under the main peak and the peak shoulder The Skim peak option can be checked in the Integrate dialogue You can also set the Ratio value to determine when peak skimming should be applied to a peak instead of drop lines the default value for this ratio is 10 The ratio is based on the relationship Biho skim ratio hp2 7 hy Fig 10 8 Skim peak ratio calculation e p265 ED Evaluating results an 18 1138 73 p266 Including neg
31. A 4 Evaluation procedure 000 cece 380 A 4 1 Curve operations s4 vaea setae x naun bee he Eee ae ook 380 AAZ INTC SFATON gos acca eure de yest hee OFA eae adhd eS 383 A43 Fil Qperatlons 2c a o0e c g a oalas a Se ve a uthow adele elk 385 AAA EX POR 5s dae oe awe Pia ee eS Geek Celt a Se 386 A 4 5 Chromatogram functions 0 0 cee 395 AAG One led ncente a ores wii be ddah ok th oe dae ae 396 B Troubleshooting B 1 Logon problems 0 cee eee eee eens 399 B 1 1 Unable to log on to UNICORN 0040 399 B 1 2 Error message Strategy file error n nannaa aaan aana 399 B 2 UNICORN access problems 00 cee eeeeeeees 399 B 2 1 Unable to access certain UNICORN functions 399 B 2 2 Connections are not available 400 B 2 3 Run data Connection in System Control displays a No x 400 B 3 Method and run problems 00000eeeeeeaee 401 B 4 Evaluation problems 0 000 e eee ee ees 403 B 4 1 Incorrect date and time 0002 e eee eee 403 B 4 2 Evaluation procedure aborts 00000 e eee 403 B 5 AKTAdesign system specific problems 403 B 5 1 Connected to a system but no system contact 403 B 5 2 Howseheme sie aea tet Gone te E a a 403 C Mathematical models and statistics C 1 The curve fit models used by the Analysis Module 405 C 2 How the curve fit models are determined
32. CLEAR_ Clears the peak table in Peak table Peak table PEAKTABLE bource from the working memory source of the computer e p383 AY Evaluation functions and i an 18 1138 73 p384 nstructions COPY_ Copies a peak table from Peak Peak table IPEAKTABLE table source to Resulting peak source table Resulting peak table NEGATIVE_ Controls the baseline behaviour in OnOff PEAKS subsequent baseline calculations If OnOff is ON then the baseline Imay be drawn above the curve and Inegative peaks may be detected by IPPEAK_INTEGRATE If OnOff is OFF then the baseline is never drawn above the curve PEAK_ Performs a peak integration on the Source curve INTEGRATE bource curve and stores the result Resulting peak ing peak table in Resulting peak table table It is assumed that the base line is subtracted PEAK_ Specifies which part of the source Source curve WINDOW curve that will be integrated Peaks Left limit between retention Left limit and Right limit Right limit will be detected if the OnOff OnOff parameter is set to On If OnOff is set to Off the whole curve will be used for integration IREJECT_ Any combination of conditions is Height less than PEAKS allowed If all parameters are OFF Width less than then every detected peak are Width more included in the peak table than Area less than Peak must be lone of xx largest SET_ Sets the column height for the Column Height COLUMN_ peak i
33. File name OPEN tthe Result file defined in Filename Peak table name and stores it in the Resulting peak Resulting peak table If is entered as File table Iname the current Result file will be lused The File name parameter Imay include a path from the cur rent users root folder e p385 an 18 1138 73 p386 AY Evaluation functions and instructions Instruction Description Parameters EXPORT_ Exports curve in AIA format Source curve CURVE_AIA Export to file EXPORT_ Exports the Source curve to the Source curve CURVE_ file defined in Export to File in Left limit ASCH ASCII format In the part of Right limit source curve limited by Left limit Every lt n gt sam and Right limit Every lt n gt sam _ ple ples are exported Export to file EXPORT_ Exports the source curve to the _ Source curve CURVE_WKS file defined in Export to File in Left limit Right WKS format In the part of source limit Every lt n gt curve limited by Left limit and sample Export Right limit Every lt n gt samples are to file exported EXPORT_ Exports an evaluation log in Export to file EVAL_LOG_ __ ASCII format to the file defined in ASCII Export to file EXPORT_ Exports an evaluation login WKS Export to file EVAL_LOG_ format to the file defined in WKS Export to file EXPORT_ Exports an evaluation log as XLS Export to file EVAL_LOG_ format to the file defined in XLS Export to fi
34. Gradient Segment 2 zl Block name Column_E quilibration Call Erom Fig 5 12 The Import Block dialogue Note Editing methods 5 2 Choose the method from which you wish to import and select the block to import The name of the selected block is suggested in the Block name field 3 Select a block from the Call drop down list into which the imported block will be placed and enter a breakpoint value 4 Click on Import to import the block The dialogue remains open until you click on Close so that you can import more than one block without closing the dialogue If you use the Import function to copy blocks within a method the blocks are copied from the saved version of the method on disk Any changes you have made in the method but not yet saved will not be copied When importing a block that contains one or more sub blocks you will be asked if the sub blocks are to be imported as well The imported block may not have the same name as an existing block in the method If the default name is not allowed for this reason the Import button will be grey and locked Change the name of the imported block so that the Import button becomes available The block is imported exactly as it appears in the source method If the base of the imported block is defined as SameAsMain the block will inherit the main base in the new method regardless of the base in the source method Also the imported block is inserted with
35. Internal standard quantitation on page 310 for more information Opening the result file 1 From the Main Menu highlight the result file which contains the peak table data from the standard level with the highest concentration 2 Select File Open or double click on the result file name 3 The Evaluation window opens together with the curves window showing the curves and peak tables 4 Select Properties for the chromatogram window and use the Chromatogram Layout dialogue to select the curves and peak table parameters for display Entering the standard data 1 From the Evaluation window menu bar select Quantitate Edit Quantitation Table New Analysing results CH New Quantitation Table x r Standards expressed in Unit C Amount Amount unit label mg Concentration Inj volume fO ml Concentration unit mg ml r Select peak table s Source chromatogram d Default fi xl Level Peak tablefs Nome Ske Peak tablels ae Prev FigT 6Level 1 to 4001 1_UV1_280nm 01 F E Baseline example 1 1216KB Resi ui Baseline example 2 978KB Resi Li Baseline example 3 973KB Resi WJ idts tevel 1to4001 1071KB Resi uy idi6Level 1 to 4002 1158KB Res W idts6Level 1to 4003 1081KB Resi 4 idi86Level 1 to 4004 1083KB Rest h Peak skim example 1153KB Resi 4 i E 4 gt j Current Gelect gt OK Cancel Help Fig 11 11 New Quantitation Table dialogue If the standards are
36. ONOFF Method Info ONOFF Method Notes ONOFF Start Notes ONOFF Run Notes ONOFF Eval Notes ONOFF Sys Set tings ONOFF Cali bration ONOFF Log Book ONOFF Result Name ONOFF Col umn Parame ters NAME Export to file Evaluation functions and instructions A A 4 5 Chromatogram functions Instruction Description Parameters COPY_ Creates a copy of the specified From chromato ICHROM chromatogram If is usedas gram name To source then the current default chromatogram chromatogram is used If is mame used as destination then a default name will be created for the copy CREATE_ Creates a new chromatogram with Name INEW_ the given name If is used for CHROM the chromatogram name a default Iname will be generated and used IDELETE_ Deletes the named chromatogram Chromatogram CHROM If trying to delete the current name default chromatogram a run time error will be caused OPEN_ Opens the specified chromatogram File name Chro CHROM from the specified file Imatogram name RENAME_ _Renames the specified chromato From chromato ICHROM gram If is used as From then gram name To the current default chromato chromatogram gram is used Iname IRESTORE_ Resets the destination for the sub DESTINA sequent curve and peak table oper TION ations to the default CHROM chromatogram Used in pair with the SET_DESTINATION_CHROM instruction SET_DESTI
37. The System Control workspace with Run Data curves flow scheme and logbook displayed 6 1 Starting a method Methods can be started when the system is connected and no other method is running You must have Run methods authorisation to start a method Before starting a method make sure that e The correct system is connected in control mode e The system monitors are correctly calibrated 6 1 1 Starting from the Main Menu You start a method from the Main Menu by highlighting a method in the Methods window and selecting File Run Alternatively highlight the method and select Run from the shortcut menu e p141 ED Performing a run Note Ee Note an 18 1138 73 e p142 Double clicking the method opens the Method Editor with the method loaded 6 1 2 Starting from System Control From System Control select File Run or click on the Run button on the toolbar then double click on the method icon in the displayed dialogue ok Default Test20 0010 Fig 6 2 Starting a method from the System Control module For methods which are used frequently e g column cleaning methods or routine separations it may be convenient to define the methods as commands in the File menu To do this choose File Menu and select the required method The method name will appear as a command in the File menu and choosing the command will start the method 6 1 3 Starting an Instant Run You can start a method templa
38. The box is not allowed to move up above the Max baseline level Evaluation functions and instructions A Max baseline level i Slope limit Fig A 2 Slope limit and Max baseline level When looking for baseline segments the box is virtually moved along the source curve in steps of 1 3 of the Shortest baseline segment A baseline segment is found whenever the currently examined part of the source curve fits completely within the box The found baseline segments are joined by connecting adjacent segments provided that the slope of the joining lines does not exceed the Slope limit A 2 2 Selecting baseline points for Classic algorithm In the second step the baseline segments are replaced by a large number of baseline points A baseline point is placed at the start and end of each segment The line between these will also be filled with points The baseline points are shown as pale blue crosses in the Integrate Edit baseline function see Manually editing a baseline on page 276 A 2 3 Drawing the baseline The baseline points are used to create the baseline curve using a spline interpolation The spline function ensures that the baseline curve is guided by the baseline points but the curve does not necessarily pass through them The baseline will thus be a smoothly curved function passing close to or through the points To reduce the effect of noise on the peak integration the created baseline is adj
39. a Set the desired breakpoint in the Breakpoint field b Choose the instruction type from the various options depending on the strategy and select the desired instruction from the displayed list For short help on the purpose of each instruction click on the instruction and press lt F 1 gt c Enter values for instruction parameters in the Parameters field If a scroll bar appears on the right hand side of the Parameters field additional parameters are required Breakpoint Instructions Parameters Command panel panel panel buttons n C AlaimstMon Mach Fesue ean Eanes ane 4 aE ee Watch Other Timeout 0 0 999 9 Bo 4 f Nees 5 00 ps Ww INFINITE Delete Fig 5 16 The Method Editor Instruction box Click on Insert The new instruction will be inserted in the block either a at the position of the breakpoint of the new instruction if there are no other instructions at that breakpoint immediately after the currently highlighted instruction if the highlight is at the same breakpoint as the new instruction as the last instruction at the breakpoint if there are several instructions at the same breakpoint as the new instruction and none of these is highlighted Note 1 Note 2 Note Caution Editing methods 5 Instructions that are placed at the same breakpoint are executed simultaneously with the exception of Block instructions which are executed in th
40. choose Filter and click on all of the available system check boxes Show method files for the following systems Explorer1 00 Oligopilot Basic10 Show files and folders filtered on name Wildcard can be used Ex Phgrad pee on een Pepe Default Cancel Help Fig 3 7 The Filter dialogue 3 3 5 Logon and file handling es Finding files To find a file e 1 2 ee Choose Find from the shortcut menu or select File Find In the displayed Find files dialogue enter a file name specification in the Search for files filtered on name field You can use standard Windows wildcard characters in the file name specification see above under Filter You can restrict the search further if required Enter a name in the Named field Choose a file type from the pull down menu for Type All Folders Method files or MethodQueue files for the Methods window All Folders Result files or Scouting files for the Results window e Use the Question and Answer fields to enter text strings to identify any desired Question and Answer text identifiers e Click in the Date box and set the date limits for the search e Check Include subfolders to search through all the folders to which you have access If Include subfolders is not checked the search will not access subfolders Named Found folders and files Name Path cA Default r File specification Type Result fil
41. injection volume adding editing 341 installation as stand alone or in network 12 stant Run icon 19 142 instructions adding 59 at the same breakpoint 105 changing 61 conditional 50 112 deleting 61 inserting 60 manual 160 moving between breakpoints 64 moving within a breakpoint 63 viewing 57 watch 50 instructions in methods 7 Integrate_and_Print 83 integrating peaks 259 integration 383 common problems 270 including negative peaks 266 optimising 262 performing 260 selecting part of a curve for integration 264 integration problem noise detected as peaks 269 273 peak limits too high up on the peaks 271 peaks missing 274 internal standard entering the injected amount 329 selecting 328 selecting internal standard peak 324 selecting the component 329 suitable choice 311 internal standard quantitation calibration curve 312 description 307 detailed description 310 reliability of technique 313 IS amount 329 IS and settings 328 IS concentration 329 e 425 um 18 1138 73 426 L last tube illustration 95 setting default position 95 layout saving and applying 195 selecting 147 length entering for a block 53 level definition 305 selecting the standard concentration levels 319 limit value 337 linear flow rates 110 locked by lt user gt 159 log format viewing 106 logbook autoscroll 157 logbook for a run 156 logging off 31 logging on 17 logoff problem logging off 401 logon problem logging on 399 Log
42. result file 2 integrating the curves to produce the peak tables 3 selecting the component to be used 4 evaluating the amount of a component in the sample Preparing for quantitation Perform a run with both the spiked and unspiked sample The result file you use must contain the curves for both the spiked and unspiked samples Open one of the two result files Use File Open Curves to copy the second curve to the opened result file Peak integrate the curves to produce the peak tables for the unspiked and the spiked sample Please refer to Chapter 10 for details on peak integration During integration the sample curves must use the same X axis base unit Time is the recommended unit for highest reliability Check that the integrations are correct If not consult Optimisin 8 P 8 peak integration on page 262 which describes how to optimise integrations Use File Save to save the peak tables before continuing with the quantitation Selecting the component to be used 1 Select Quantitate Standard Addition This opens the Standard Addition dialogue Analysing results Ca Standard Addition x r Select peak table Source chromatogram Addition chromatogram Added amount zji E Peak table s Peak table s Unit label id186Level 1 to 4004 1_UV1_280nm 01 PEAK id186Level 1 to 4004 1_UV1_280nm 01 PEAK mg id186Level 1 to 4004 1_UV1_280nm 01 PEAK1 id186Level 1 to 4004 1_UV1_280nm 01 PEAK1 Fig 11 30
43. 0 Base SameAsMain End_collect 0 Base SameAsMain Here the second Watch instruction will annul the first since a signal can only be watched for one condition at a time In the correct construction above the watch to end collection is not set until Peak_2 is called Note Editing methods 5 Watch example 7 Collecting peaks above a threshold value The example below describes how to use the Watch instruction to collect three peaks above a certain threshold value such as UV instead of using slope for the Peak_Fractionation instruction The number of peaks to be collected can be increased by adding several Start_Threshold and Ending_Threshold blocks When the UV reading goes up to 5 mAU and beyond the fraction collector is started The desired fraction volume must be entered for the FractionCollector instruction When the UV reading goes down to 4 75 mAU the fractionation is ended and the flow directed to Waste This is repeated for the next two peaks The UV threshold for ending fraction collection must be below the threshold for starting fraction collection so that end fractionation conditions will not be fulfilled simultaneously or immediately after start fractionation Main 0 00 Base CV 0 98 ml Mono_S_HR_5 5 0 00 Block Gradient Gradient 0 00 Base SameAsMain 0 00 Gradient 100 Target_ConcB B 20 Length_of_gradient base 0 00 Block Eluate Fractionation Eluate_Fractionation 0 00 Bas
44. 000CV 0 00 min BufferPrep_pH 0 000 For Help press F1 Run Block Scouting 2 Fig 6 15 The logbook panel for a running method The logbook window can autoscroll to display the latest entries if you select the Autoscroll function from the shortcut menu or else check the Autoscroll option in the Properties dialogue 6 3 Manual control 6 3 1 The toolbar The toolbar at the top of the System Control workspace contains a set of buttons for starting and stopping the run Manual Direct Commands Toolbar accessing documentation Windows toolbar and locking the system System toolbar The toolbars can be shown and hidden by clicking on View Toolbars and checking the relevant boxes Run Hold Continue End m a EA Fig 6 16 The toolbar in the System Control workspace Manual direct commands toolbar Run Starts a run when the system is in End state and a method is loaded Hold Suspends execution of a method but continues to pump liquid at the current flow rate and eluent concentration settings Accumulated time and volume continue to be incremented Any method instructions which are set to the time volume when Hold is pressed are executed Later method instructions are not executed until Continue is pressed e p157 ED Performing a run an 18 1138 73 e p158 Pause Behaviour on Pause is strategy dependent Pause suspends execution of a method and stops all pumps so that the system comes to a standstil
45. 1 UNICORN Local fil Niklas result Unicorn4001 1 id186Level 1 to 4001 id186Level 1 to 4002 res BEE id186Level 1 to 4002 1_UV1_280nm id185Level 1 to 4002 1_Inject id186Level 1 to 4002 1_UV1_280nm 01 BASEM id186Level 1 to 4002 1_UV1_280nm 01 BASEM1 id186Level 1 to 4002 1_UV1_280nm 01 1dt 9 47 min 1 595 mAU 8 27 Fig 10 35 Measurement of the Slope limit after differentiation 4 Use the zoom function to magnify the curve over an appropriate area Place the vertical cursor bar at the beginning of a peak where you want the Watch conditions to be fulfilled i e where the slope becomes higher Read the actual slope value in the active Marker box When using the PeakFractionation instruction for AKT Adesign systems measure the slope at the beginning and end of the smallest flattest peak to collect and use these values 5 Inthe Method Editor enter the slope value as a parameter for the Watch instruction or enter the StartSlope and EndSlope values in the Peak_FracParameters instruction 10 2 4 Creating a curve It is possible to create a curve based on any external measurements 1 Select Operations Create Curve In the dialogue that is displayed select one or several help curve s 2 Select the minimum and maximum values of the Y axis Also choose the appropriate units from the Unit list The help curve determines the min and max values for the X axis e p289
46. 256 characters long The method name must be unique for the chosen system within the folder see steps 2 and 3 3 Ifyou have more than one system connected to the computer choose the System for which the method is intended The method can be run on any system that uses the same strategy Remember that different systems may have different configurations and control capabilities Each method is written for a specific strategy The function of the method cannot be guaranteed on systems having other strategies 4 Choose the Technique for which the method was written Creating methods H es 5 Click on OK Note Ifyou receive a syntax error message when the method is saved one or more instructions in the method are invalid These may be calls to blocks which are not defined or instructions which are invalid in your strategy this can also arise if a method is written for one system and saved for another Invalid instructions are marked in red in text instruction mode in the Method Editor see Method instructions on page 57 and must be deleted or replaced before the method can be run The method remains open in the Method Editor when it has been saved so that you can continue editing if you wish Once the method has been saved choosing File Save saves the current state of the method under the same name If you want to save the method under a new name choose File Save As and enter the details as described above 4 6 Star
47. 4 00 End_block 0 00 Block Sample_injection E 0 00 Block Wash_afterinjectiol Sample_injection 0 00 Block Gradient 0 00 Base Volume 0 00 Injection alve Inject 4 00 InjectionVYalve Load 4 00 End_block 0 0 00 Block Clean_after_Gradient 0 00 End_method Fig 2 2 Relationship between blocks and instructions The method left is written as a series of blocks each of which consists of instructions for performing one or more specified tasks right ep7 2 UNICORN concepts an 18 1138 73 p3 Breakpoints Each instruction in a method block is issued at a specified breakpoint according to the method base The first instruction in a block is always at breakpoint 0 and all other breakpoints are counted from this point For example the instruction 5 00 Flow 8 0 will set the flow rate to 8 0 ml min at 5 units minutes ml or column volumes according to the method base after the start of the block Method variables Breakpoint values and instruction parameters may be defined as variables This is a powerful facility for e constructing a framework method with default parameter values which may easily be changed either to create variants of the same method or to adjust the parameter values at the start of a run see Section 4 1 e constructing a method for use in automatic method scouting where one or a few parameters are varied systematically see below and Chapter 7 Using variables make
48. 96 ml Resource_Q 1 ml 0 00 Block EQUILIBRATE Equilibrate 0 00 base SameAsMain 0 00 Watch_UV Less_Than 100 mAU End_block 5 00 Watch_Off UV 5 00 Message The condition was never reached 5 00 End_block Watch example 3 Equilibration with extra safeguard The column is equilibrated until the conductivity is below 5 mS cm If this value is not reached within 6 column volumes the method is paused together with a message Equilibration of the column is continued until the conductivity value is stable allowed to vary by max 2 mS cm over a period of at least 5 minutes If this condition is not met within 10 column volumes the method is again paused At each pause the operator can decide whether to continue or abort the run If you are not using AKTA instruments a delay of 0 1 ml min should be added after the Hold Pause instruction so that the following instruction will not be executed simultaneously with the Hold Pause instruction Main 0 00 Base CV 0 96 ml Resource_Q 1 ml Editing methods 5 0 00 Block EQUILIBRATE Equilibrate 0 00 Base SameAsMain 0 00 Block COND_LESS_THAN 0 00 Block COND_STABLE 0 00 End_block Cond_less_than 0 00 Base SameAsMain 0 00 Watch_Cond Less_than 5 mS cm End_ block 6 00 Message Low conductivity not reached Screen 6 00 Pause 1 00 minutes 6 00 End_block Cond_stable 0 00 Base SameAsMain 0 00 WatchPar_Cond 0 50 m
49. 999999 000 Fractionation_Step Eluate_Frac_Size ml 0 000 0 000 100000 000 Start_Eluate_Frac_at NextTube Ra Peak_Frac_Size ml 0 000 0 000 100000 000 Start_Peak_Frac_at NextTube X Peak_Start_Slope m4U min 100 000 0 010 10000 000 I Show unused variables I Display tooltip for extended variable cells Help Fig 4 5 Variables tab in Run Set up Work through the variable list adjusting the Values to suit your separation To change a variable value simply type the required value in the field Remember that the values you enter here will be default values suggested each time the method is run For some variables pre set values are available within drop down menu boxes For variables with values shown in blue the Value input can be toggled between OFF and a variable range by clicking on the Value cell with the right mouse button Checking the Show details checkbox will show variables that were created with the Visible in details only option This option is used for example to hide less important variables Similarly the Show unused variables checkbox will display variables that are present in blocks but that are not used in the method e p37 gt Creating methods Note 1 an 18 1138 73 e p38 For blocks where the length of the block is defined as a variable e g sample loading column wash setting the length to zero will effectively skip the block Instructions in the block will sti
50. A id186Level 1 to 4002 1 9 2001 8 27 6 9187 28 702 id186Level 1 to 4004 1 9 2001 8 27 6 8896 Mean BER ERB Std Dev ARE R amp I Rel Std Dev AAR AIR Peak B id186Level 1 to 4002 1 9 2001 10 46 3 7804 16 371 2 61 id186Level 1 to 4004 1 9 2001 10 48 2 64 Mean ABD Pal Std Dev ads 7 aa Rel Std Dev are CFR ER abe I Include Wizard Settings 20 Plot 30 Plot Print Save Spreadsheet Save Wizard Settings Close Help Fig 9 24 Data View dialogue The Data View dialogue presents a comparison of the chosen data for the designated peak comparisons Use the Print button to print out the spreadsheet The Save Spreadsheet button allows you to save the data in Excel xls tabbed text txt or FarPoint spread ss3 formats To view the data in 2 dimensional plot click on the 2D Plot button 2D Data View x Peak A 931711996 6712 1998 6 12 1998 6 12 1998 Run Date Select peak Select x axis Select y axis E Fig 9 25 2D Data View dialogue e p213 ED Presenting results Use the Select peak Select y axis and Select x axis lists to select different peaks and different axes A wide array of plot presentation options can be found under the shortcut menu including a Customization Dialogue allowing further customization of the graph owing Style Font Size P aie Style
51. A 4 1 A 4 Evaluation procedure Curve operations Instruction Description Parameters ADD Adds two curves to gain a third curve which is the sum of the two curves The two source curves must have the same Y axis unit and not be fraction or injection curves or else a run time error will occur First source curve Second source curve Target curve position AMP_MUL Multiplies the amplitude of the source curve by the multiplication factor and stores the result in the target curve position Source curve Target curve position Multiplication factor AMP_SHIFT Shifts the amplitude of the source curve by the shift factor and stores the result in the target curve posi tion Source curve Target curve position Shift factor CLEAR Clears specified curve from the working memory of the computer Source curve COPY Copies the source curve to target curve position Source curve Target curve position CUT Cuts out the part of the source curve between Left and Right Limits and stores the result in the target curve position Source curve Target curve position Left limit Right limit DERIVATE Differentiates the source curve first or second order and stores the result in target curve position The Y axis of the target curve position will be a normalised scale without unit Source curve Target curve position First Order or Second Order a
52. CD ROM shipped with your Compaq computer Insert the CD ROM and follow the directions on the screen Note Be sure to always install the latest NT4 service pack after installing something from the Windows NT 4 0 CD ROM e p401 B Troubleshooting an 18 1138 73 p402 There are red instructions in a method Red instructions i e instructions with a red dot in a method are syntax errors and may be due to the following e The method was connected to a wrong system i e the strategy of the system is incompatible with the method e The method instructions do not correspond to the components you have chosen for your system Check your system components under Administration System Setup Components in Main Menu e The Copy function was used instead of Copy from external when importing a method from a diskette e The wrong system may have been selected in the Save As dialogue in Method Editor e You may also have templates not intended for your system which often happens for custom designed systems There are several actions that you can take e Check that the method has been connected to the correct system either in the System Method Connection dialogue when using the Copy from external dialogue or in the Save As dialogue in Method Editor e For custom designed systems go to the Method Editor select the red instruction and either delete it or replace it with a corresponding instruction if available from the Instructi
53. Chromatogram and importing more curves into it or copying curves into the Temporary chromatogram see Temporary chromatogram on page 186 You can perform evaluations in the Temporary chromatogram and transfer the final curves to other destination chromatograms The unwanted contents remaining in the Temporary chromatogram can then be removed using Edit Clear Temporary Chromatogram copy curves Select Edit Copy Curves Select the source chromatogram and the curve of interest Select the target chromatogram Click on the Copy button to effect the copy Stay within the same dialogue to repeat this step for as many other curves you want from the same or different chromatograms When you have copied all desired curves click on Close Open the destination chromatogram and access the Chromatogram Layout dialogue Select in the Chromatogram Layout dialogue the curves that you want to view Curves can be scaled individually or all with the same scale using the All with this unit function see Changing and fixing the axes on page 192 e p225 ED Presenting results an 18 1138 73 p226 Copy Curve xi m Source Chromatogram m Larget Chromatogram 1 05 ATJMF006 1_Cond 06 ATJMFOO6 1_Cone 07 ATJMFOOE 1_pH 08 ATJMFO06 1_Pressure 09 ATJMF006 1_Flow 10 ATJMF006 1_Temp 11 ATJMFOO6 1_Fractions 12 ATJMFO06 1_Iniect Ma 04 ATJMFO06 1_Cond g 05 ATJMF006 1_Cond
54. Click on OK Evaluating results 10 Removing a procedure 1 Select Procedures Edit Delete 2 Select a procedure in the Delete Procedure dialogue 3 Click on OK 10 3 4 Points to watch In recording and editing evaluation procedures for automatic evaluation beware of the following potential pitfalls e Make sure that the procedure addresses the right curves Curves are identified by storage position alone thus the instruction ADD 01 02 03 will try to add curve 01 to curve 02 and store the result in 03 regardless of the contents of 01 and 02 If 03 contains a curve which is not a raw data curve the existing curve in 03 will be overwritten If 03 contains a raw data curve the procedure will stop with an error message The raw data curves will always occupy the same positions for a given strategy e g UV in position 01 If the operation is not valid when the procedure is run the procedure will stop at the instruction with an error message Any subsequent instructions in the procedure will not be executed e In calculating a baseline using the classic algorithm UNICORN suggests default values for the four control parameters Optimising the baseline parameters using a classic algorithm based on the appearance of the curve To instruct UNICORN to use default values appropriate for the curve every time the procedure is run choose the Default setting in the appropriate fields for the parameters For example CALCULATE_BASE
55. Display tooltip for extended scouting scheme cells Define Clear All Delete Insert Add SPIE Help Fig 5 29 Scouting scheme with varying BufferPrep_pH and Column Position in this example You can define up to 99 runs in a scouting scheme Each run can be set to Run or Excluded by clicking on the button at the top of a run column and clicking with the shortcut menu Values can also be changed during the run in System Control The Excluded option simply means that the run is skipped and the next run is taken in sequence The Series button is useful when you want to set up a series of runs with differing integer inputs Select a cell with an integer value and click on the Series button In the resulting Insert Series dialogue type in the selected series of integer values within the specified range Editing methods 5 limits separated by commas Click on OK A new set of runs is inserted into the Scouting tab with the values provided Any changes that you make to variable values when a scouting scheme is run are saved in the result file Results from a scouting run are saved in a scouting folder see Chapter 7 Column scouting It is possible to define different columns for use in the various scouting runs However in selecting a different column other variables may also be changed between runs 1 Choose a method with a column not Any Alternatively you can have a method with CV as the main base and a column not
56. Equilibration 0 00 Base SameAsMain 4 00 End_block During a run the overall time or volume is determined by the sum of the block lengths The length of the main block does not indicate the overall length of the method the main block often consists only of calls to other blocks and has zero length The method length can be checked in the Gradient window of System Control Log format The cumulative method time or volume can be viewed in Method Editor by viewing the Log Format To view the cumulative time or volume for a method select View Log Format or click on the Log Format icon The Log Format dialogue is displayed Editing methods 5 The Log Format dialogue displays the cumulative time or volume for the current method If the method is a scouting run click on the Run button to move between runs Note The cumulative time volume is an approximation and does not take into account time or volume for Watch blocks Wash commands or programmed Hold Log Format x 0 00 Base CY 0 98 ml Alkyl_Superose_HR_5 5 Column W 0 00 Block Start_Conditions_BP H 0 0 00 Block BufferPrep_pH_Flush 0 00 Block Column_Equilibration E 4 12 Block Flowthrough_Collection E 4 12 Block Wash_Out_Unbound_Sample W 4 52 Block Eluate_Fractionation_V H 0 4 52 Block Gradient_Elution H 0 8 51 Block Clean_After_Gradient_BP_ W 12 10 Block Reequilibration_BP 12 10 End_method 0 00 Base CY 0 98 ml Alkyl_Super
57. Evaluating results Note an 18 1138 73 p292 8 Click on OK and save the curve You can change the curve name from the default CreatedCurve and also the curve destination 10 2 5 Measuring salt concentrations in the fractions If you need to know the approximate conductivity or concentration of salt in your fractions it is possible to calculate these from the conductivity curve The conductivity signal is not linear above 0 3 M but you will still gain a relatively good idea of the salt concentrations above 0 3 M A conductivity curve usually given the name Cond is stored in a chromatogram within a result file This curve represents the real conductivity data in mS cm and should be used for calculations Another curve Cond is also present and is the same as Cond but re scaled to display percentage values To make the calculations 1 Select Operations Fraction histogram Select the Cond curve in the left list and the fractions curve should already be selected in the middle list If you have earlier pooled fractions it is possible to select the desired fraction curve Click on OK 2 Select the appropriate fraction curve in the Chromatogram Layout dialogue In the active chromatogram you will see the fraction marks the fraction histogram of the conductivity curve and any other selected curves Select Marker from the shortcut menu to display the vertical cursor line Click on the desired curve legend and the corres
58. Fig 9 68 Documentation Evaluation Log tab an 18 1138 73 e p254 Presenting results 9 Method information The Method Information tab displays information about the method such as method name target system for creation and date of last change Strategy information includes strategy name date and size There is also a tab for Signatures associated with the method Documentation Fig 9 69 Documentation Method information tab Frac 950 The Frac 950 tab displays the setup parameters for the fraction collector You can print out the selected settings by pressing the Print button or return to the main Evaluation window by pressing OK or Cancel e p255 ED Presenting results an 18 1138 73 p256 Documentation x Variables Scouting TextMethod Notes Questions Columns ReferenceCurves Evaluation Procedures Method Information Result Information ResultName Start Protocol Settings Calibration Logbook EvaluationLog Frac 950 r Last tube Tube type Sa Number of tubes 120 120 8 8 Help Fig 9 70 Documentation Frac 950 tab Result information The Result information tab displays information about the method such as method name target system for creation and date of last change Strategy information includes strategy name date and size The Run Summary tab summarises the run in terms of volume or time per block There is also a tab for Signatures associa
59. Fractionation i Fig 9 10 Chromatogram with header information displayed 9 2 10 Saving and applying a layout All configurations that you make in the Chromatogram Layout dialogue can be saved as a layout It is possible to apply saved layouts to other chromatograms All saved layouts are user specific To save a layout 1 Open the Chromatogram Layout dialogue and make the appropriate layout configuration within the various tabs Note that you can return to the chromatogram window by clicking on e p195 ED Presenting results an 18 1138 73 e p196 OK to see the applied affects of a given configuration and return again to the Chromatogram Layout dialogue to perform further changes 2 Select the Layout Library tab and click on Save current layout as 3 Enter a name for the layout in the displayed dialogue If you want the current layout to be the new default layout check the Save as default option 4 Click on OK to save the layout The new name is added to the Saved layouts list To apply a layout 1 Select the Layout Library tab 2 Select a layout from the Saved layouts list and click on the Apply selected layout button The layout is automatically applied to the active chromatogram window If the same layout is to be applied to all chromatograms on the Evaluation workspace select check the Apply to all chromatograms option 9 2 11 Displaying a hatched background in the chromatogram window
60. Global or Personal as required to access the table you wish to rename Select the table you wish to rename Click in the Quantitation table name field and type the new name Click Rename to rename the table or Exit to abort the rename procedure 11 3 3 Editing and updating a quantitation table Quantitate has an editing facility that allows you to return to a quantitation table and alter any of the parameters Note the difference between this function and Update which allows you to incorporate new data into a pre existing quantitation table Update is described in below To edit a quantitation table 1 Select Quantitate Edit Quantitation Table Open from the Evaluation window Use the Open quantitation table dialogue to select the quantitation table you wish to edit Analysing results Ce Selection of the table opens the Quantitation table dialogue The editing facility gives you full access to all the quantitation table parameters If you have used the Update function see below it is not possible to make changes in the Define Component s dialogue 3 Once editing is complete click Save or Save as depending on whether you wish to overwrite the existing table or create a new table under a new name Updating a quantitation table Quantitate includes an update function which allows you to add new peak size data to an existing quantitation table This enables precision to be improved through the use of data from a num
61. Method Editor interface operates in two modes e Text instruction editor e Run Set up editor Switch modes using the icons or the commands in the View menu It is possible to show or hide the Toolbar and or Status bar by clicking View Toolbar or View Status bar Q Method Editor UNTITLED Fie Edit Block View Help 4 tid SAN Fig 5 1 Method Editor toolbar e p45 Editing methods an 18 1138 73 e p46 Icon Menu command Function View Text instructions Open the Text Instructions editor View Run Set up Open the Run Set up editor View Log Format Open the Log Format dialogue E i i P o File Method Wizard Open the Method Wizard dialogue p s 5 1 2 Text Instructions editor The text instructions editor is used for entering and editing method instructions Click on the Text Instructions icon or choose View Text Instructions to display the text instructions editor Method Editor Test20 0010 Bile Edit Block View Help Deas Gelhsoan LELI Main __ 0 00 min Start Condition Column Eauilibr il Wash Qut Unt Fractionation F Affinity Elution il initial Eluent C System Volume 500 min 0 00 min 50 00 ml Complete Same Complete S 2 00 min 1 00 mi Elution step 1 5 00 min 50 0 00 min 8 00 ml ints Ebene C2000 nd a 0 00 min SS eel E Main 0 00 Base Time 20 11 ml HiLoad_16 10_Phenyl_Sepharose_HP Column 0 00 Block Start Conditions AC 0 00 Block C
62. OD Now do a batch run Batch runs on all the desired result files to get the printed reports The procedure can also be saved with a method to get automatic printouts at the end of a run If the selected report format is changed in File Report the new format will be applied when the procedure is run except in cases where the procedure has been imported to a method in these cases the procedure is saved in the method file and cannot be changed If the format is subsequently deleted the procedure cannot be run 10 3 8 Placing a procedure on the menu and running It is possible to choose a maximum of 15 created evaluation procedures to be placed onto the Procedures menu Select Procedures Menu and select the evaluation procedure to be added to the menu Click on OK Activate a chromatogram and select the Procedures menu You will see the procedure that you added to the menu Select this procedure and it is automatically run for the active chromatogram The menu addition is remembered in UNICORN even if the results of the current evaluation session are not saved To take away a procedure from the menu deselect it from the Procedures Menu list 10 3 9 Exporting data or curves Data and curves can be exported to other file formats Select File Export and then the appropriate cascade menu choice Alternatively the Edit Copy to clipboard function can be used which is the quickest and easiest way to copy a chromatogram into for example
63. Other WachPar Presure z Delete Ready I Fig 5 24 Parameters with variable definition are identified by an active VAR button In this example UV1 and UV2 are defined as variables and the UV3 position is fixed All variables are also listed on the Variables tab of the Run Set up see Variables on page 70 grouped according to the block in which they appear 5 4 2 Defining variables To define a new variable i e convert an existing fixed value to a variable 1 In the Text instructions window select the instruction where you want to define the variable The parameters for the instruction are shown in the Instruction box 2 Locate the breakpoint or the required parameter in the Instruction box Click on the Var button 3 Enter a name for the variable in the dialogue and click on OK e p67 Editing methods Note Note an 18 1138 73 e p68 ariable Name Definition Variable name I Visible in details only Cancel Clear Help Fig 5 25 The Variable Name Definition dialogue Variable names may be up to 20 characters long and may contain letters A Z digits 0 9 and the underscore character Use underscore characters instead of spaces if you want to separate words in a name e g Flow_rate Names must be unique within the method The case of letters is retained but is not significant The names Flow_Rate and FLOW_RATE are treated as identical When you define a variable th
64. Peak table s list Highlight the table required 5 Click OK to calculate the molecular sizes Displaying the results The results of the molecular size determination are shown in the Mol size column of the peak table 4 90 No Retention min Mol size kDa Ee 6 42 gt 42 299 43 atl Fig 11 51 Display of results If the molecular size cannot be calculated one of the following signs is shown in the Mol size column gt means that the molecular size is larger than the largest size in the molecular size curve i e it is outside of the valid range lt means that the molecular size is smaller than the smallest size in the molecular size curve i e it is outside of the valid range means that the retention value is negative When the result file is saved it includes the molecular size table that was used for the molecular size determination You can later view the table that was used by selecting Mol Size Edit Mol Size Table View Current If you want to print the table that was used select File Report and select the Quantify and Mol Size option see Modifying an existing report format on page 250 e p367 ED Analysing results an 18 1138 73 p368 Evaluation functions and instructions A A Evaluation functions and instructions This appendix describes the functions implemented in the evaluation module There are four sections in the appendix A 1 describes how the
65. PumpwashE xplorer Va PumpwashPurifier T 3 CoE C AlaimstMon PumpwashBasic a Systemwash C Frac Replace C Watch C Other Delete Fig 5 62 Setting linear flow rate in a method block The linear flow rate option is only available if the base is set in terms of a named column definition If a column is not selected in the Base instruction linear flow will not be available Editing methods 5 5 10 7 Eluent concentrations and gradients Both gradients and immediate changes in eluent concentration are defined in UNICORN methods with the single Gradient instruction The Gradient instruction takes two parameters Target which is the final eluent composition expressed in eluent B Length which is the duration of the gradient not the breakpoint at the end of the gradient The instruction may be read as form a Gradient to reach Target after Length The starting point for the gradient is always the current eluent composition For example 10 00 Gradient 50 B 20 base forms a gradient to 50 B starting at breakpoint 10 with duration 20 method base units i e finishing at breakpoint 30 If the current eluent concentration is greater than 50 B the gradient will be negative Forming a step gradient A step gradient is an immediate change in eluent composition To form a step gradient in UNICORN set the Length parameter to 0 in the Gradient instruction For example 10 00 Gradient 50 B O base forms a step fr
66. Set up Editing methods es Default variable values can be changed either by editing the instruction in the Instruction box or by changing the value in the Variables tab of Run Set up Changes made in the text instruction are automatically updated in the Variables tab and vice versa see figure above Note1 Ifa breakpoint or gradient length is defined as a variable changing the variable value in the Start protocol when the method run is started will shift other instruction breakpoints accordingly This functionality is equivalent to using Change to alter a breakpoint or gradient length see page 63 for how Change affects instructions within gradients Note2 If the same block is called several times in a method the variables in that block will only be displayed once in the Variables tab of Run Set up 5 4 3 Removing a variable To convert a variable to a fixed value 1 In the Text instruction window select the instruction where you want to remove the variable The parameters for the instruction will be shown in the Instruction box 2 Locate the required parameter in the Instruction box Press the VAR button 3 Click on Clear to delete the variable name and click on OK Note Do not remove column variables flow pressure UV averaging time if you want these parameters to be updated if you change column 5 4 4 Renaming a variable To change the name of an existing variable 1 In the Text instruction window sel
67. Standard Addition dialogue Open the Source chromatogram drop down list and select the chromatogram which contains the peak table for the unspiked sample Highlight in the Peak table s list the peak table for the sample Follow the same procedure in the Addition chromatogram section to select the addition peak table for the spiked sample Enter in the Added amount field the amount of the component which was added as the spike Edit if necessary the default unit mg in the Unit label field Click OK to open the Identify Peak dialogue e p343 ED Analysing results Identify Peak x Addition table Retention Fig 11 31 Identify Peak dialogue 8 Locate and select the peak of the unspiked sample You can select the peak either by clicking its black triangle marker or by highlighting its reference in the Source table 9 Locate the peak for the spiked sample Select it either by clicking its blue triangle marker or by highlighting its reference in the Addition table 10 Click OK to confirm the selection Evaluating the amount of the component in the sample The amount of the component of interest is shown in the peak table of the sample chromatogram an 18 1138 73 p344 Analysing results CH we Retention win Area witrwin mount tma Mio TEET 12 9160 26 1310 139 5769 Fig 11 32 The result shown in the Amount column 11 4 3 Measuring recovery For a full explanat
68. The Copy from External dialogue is displayed e p27 E Logon and file handling es 4 Select the desired file s from the relevant source drive and folder Click on the Save button 5 If result file s were selected these will be copied into the previously designated folder in the Results window Copy from External 21 x Copy From s 0 x Sl Temp Unicorn Ewin Fig 3 10 Copy from External dialogue in this example used to copy method files 6 If method file s were selected the Method System Connection dialogue is displayed Method files Select a method file Double click on a system Fig 3 11 Method System Connection dialogue Each copied method listed in the Method files field must in turn be connected to the same type of system same strategy for which the method was originally created listed in the Systems field Highlight a method and double click on a system Click on the OK button an 18 1138 73 e p28 Note Note 1 Note 2 Note 3 Logon and file handling ea The Method System Connection dialogue is displayed again listing the remaining methods to be connected Repeat the process until all methods have been connected Method syntax errors may arise if a method created on one system is connected to a different type of system using the Copy from External facility If at any time you press on the Cancel button the Method System connection dialogue is closed Howe
69. To display a hatched background in the chromatogram window 1 From the shortcut menu select Hatch The hatching can be removed by selecting the Hatch option again When working with more than one chromatogram in the Evaluation window it is easier to apply hatching by 1 In the Chromatogram Layout dialogue select the Curve Style and Colour tab 2 Check the Hatch option then the Apply to all chromatograms option then click OK Hatching is now applied to all open chromatograms To remove the hatching uncheck the Hatch option 9 3 Other presentation possibilities The Evaluation module allows you to perform operations on the curves to optimise the presentation Note Presenting results 9 9 3 1 Changing the size of fraction marks The following steps outline the procedure for changing the size of the Fraction marks 1 Shutdown the UNICORN software 2 Open the file in Notepad and locate the following line EVAL FracSize 10 0 3 Ifthe line is not present add it under the line EVAL Print Margin Bottom 5 4 Find a suitable value for the fraction size by arbitrarily increasing or decreasing the value 10 0 Negative values set size in of window height while positive values set size in character heights If the line EVAL FracSize is not present the default size will be 50 of window height 5 Save the file Do not make any changes in the UNICORN INI file between the lines BEGIN and
70. Wizards and standard templates For example e Changing the method base column volume volume or time e Changing valve specifications for inlet and outlet if the method does not suit your system configuration Creating methods H e Adding or removing variables e Adding conditional events such as equilibrating a column based on a stable conductivity signal e Adding or removing instructions to change the method functionality e Adding or removing blocks to change the method structure To gain an understanding of how method templates are built up and can be modified work through Chapter 5 which gives a full description of method editing facilities e p43 gt Creating methods an 18 1138 73 e p44 Editing methods 5 5 Editing methods This chapter describes the complete facilities for editing methods in UNICORN For many applications suitable methods can be created by changing the default variable values in one of the wizard generated methods supplied with UNICORN Use the more advanced editing facilities described here for e changing selected instructions in the method e g changing the outlet valve position e adding blocks and instructions e g Watch instructions e changing method instructions to adapt to non standard system configurations e creating new methods for applications which are not covered by the templates supplied 5 1 Method Editor interface 5 1 1 Method Editor modes The
71. Yes 5 12 3 Deleting a column To delete a column from the list mark the column and click on the Delete button Be restrictive in deleting global columns since these are available to all users 5 12 4 Selecting or changing the column in a method Any column may be selected together with the appropriate template in the New Method dialogue see Creating a new method on page 33 The column can later be changed in the Instruction box It is also possible to adapt a method to different columns if the column definition name is defined as a variable in the Base instruction as it is in the method templates or Wizard generated methods Changing the column for a method is then done in the Variables tab 5 13 Creating and editing BufferPrep recipes The BufferPrep function is available on some KTAdesign systems Recipes can be edited or new ones created by selecting Edit BufferPrep Recipes in the Method Editor The list of recipes is not linked to a specific method although editing is performed in the Method Editor Recipes are either global i e available to all users or personal i e not available to other users It is best not to edit the global available recipes unless you save the changes under a new recipe name since other users may not appreciate the changes It is recommended that restricted access be given to editing global recipes Note The recipe to use in a certain method is selected in the BufferPrep tab in the Run Set
72. and Delta_Base are entered with the WatchPar instruction e g WatchPar_UV WatchPar_Cond under System Settings in the System Control menu see the UNICORN 4 0 Administration and Technical manual Temporary settings that apply only for the duration of a given run can be entered with the WatchPar in the Alarms amp Mon group in the Method Editor Watch actions The following actions can be taken when a watch condition is met Block name Call the named block Pause Hold Pause or hold the method Continue Continue the method if paused or held End_block End the current block and return to the point from which the block was called End_method End the method Note Editing methods 5 Ready Indicates that the next step in a MethodQueue may start 5 11 3 Watch examples Below follows several examples of how Watch instructions may be inserted into an ordinary template method Some of these methods are also on the UNICORN installation CD ROM Watch example 1 Simple equilibration In the following example The Watch is started on the conductivity signal and the method is then put on Hold When the Watch_cond condition is fulfilled Continue is issued and Watch_cond is turned off automatically Method execution now continues issuing a Watch_UV command Again the method is put into Hold until the Watch condition is fulfilled Note that even though the line Watch_Cond Less_than 5 mS cm Continue is in the method placed before t
73. baseline close to zero AU This can be achieved with baseline subtraction 2 Create a baseline for each UV curve according to the procedures detailed in Sections 10 1 1 and 10 1 2 Subtract the baselines from their respective UV curves using Operations Subtract Alternatively you can subtract the corresponding blank runs from the UV curves if such exist 3 When you have achieved two curves with a zero baseline select Operations Divide and select the two curves for division You have the option to set Threshold values by checking the option and setting the threshold value for each curve This sets the quotient to 1 0 if either of the sample values is closer to zero than the threshold value This is to prevent very high quotient values being created if division is performed with values close to zero Very low quotient values are also prevented Threshold values are suggested by UNICORN although these can be changed Evaluating results 10 Divide x Chromatogram Chromatogram Chromatogram 01 322401 1_ UV1 280mm EN 09 322401 1_ Flow 17 322401 1 4V1 aoci 4 gt I Threshold Result curve name je fe Uv1_280nme ot O1 DI cancel He Fig 10 29 Divide dialogue 4 The resulting curve can then be filtered by Operations Smooth Reducing noise and removing ghost peaks It is suggested that you smooth using the median filter to remove noise that appears as spikes or occurs in a small area of the cur
74. been stored in another result file To access the blank run curve 1 Ensure that the destination chromatogram has been opened and is the active window on the workspace 2 Select File Open and then select Curves from the menu cascade The Open Curves dialogue is displayed Note Presenting results 9 3 Locate and double click on the result file containing the blank run curve The curves in the first chromatogram are displayed Open Curves xi c Default i id2301 4 Baseline example 3 4 Baseline example 2 bU id1 6Level 1 to 4004 bUJid186Level 1 to 4003 e Peak skim example buJid186Level 1 to 4002 idt Level 1 to 4001 Li Baseline example 1 r Select curves Chromatogram Selected curves fi 7 ATJMFOO6 1_U 1_215nm 5 JATJMFOO6 1_Cond Available MATJMFOO6 1_U 1_215nm CIATJMFO06 1_UV2_254nm CIATJMFO06 1_UV3_280nm WIATJMFOO6 1_Cond CIATJMFO06 1_Cond CIATJMFO06 1_Cone JATJMFOO6 1_pH xl Remove Store in chromatogram 1 OK Cancel Help Fig 9 16 Open Curves dialogue with a result file selected 4 Check the curve corresponding to the blank run curve and click on the Select button The selected curve will now be displayed in the Selected curves list To remove a curve from the list check it and click on Remove If there is more than one chromatogram in the result file and the blank run curve resides in another chromatogram select th
75. compensation for losses of sample during the sample preparation process prior to analysis However this technique normally gives good precision and is fairly simple 11 2 3 Internal standard quantitation Internal standard quantitation reduces errors which are caused by changes in the system between successive runs with the sample and the standard concentration levels For example there may be unpredictable losses during the sample preparation procedure or unintentional changes in the amounts injected Internal standard quantitation uses peak tables prepared from the standard as with external standard quantitation However a fixed quantity of an additional component is added to every run including the sample The peak sizes of the standards and the sample are then Analysing results 11 related to the peak size of the internal standard to compensate for any changes that may have occurred between runs The internal standard technique relies on the assumption that any changes in the amount injected of the component s of interest e g due to sample preparation losses correspond to equal changes in the amount injected of the internal standard component A suitable internal standard e must be well separated from all components in the sample not just from the components of interest e must not be present naturally in the sample s To be able to compensate for losses during sample preparation all the standard concentration le
76. components 2 Highlight each component in turn and check that the new point falls within acceptable limits 3 Click Statistics after update to open the Statistics after update dialogue e p337 ED Analysing results an 18 1138 73 p338 Statistics after update x Model T ar Model function values 4 4 08925E 000 Correlati Number of p Fig 11 26 Statistics after update dialogue Use the statistical data to check the curve model after the update Note that the old non updated calibration curve is still shown but the statistics apply to the data after the update If the new point is red the statistics shown will be those for the old curve Click OK to close this dialogue Repeat this procedure for each component Click OK in the Update calibration curve dialogue to progress the update and open the Update report dialogue Update report p General User default Internal standard peak Date 3 31 1999 Internal standard amount Last updated 3 31 1999 Reference peak Table Ext_Std_Chym Nominal reference retention Injection volume Not used Update reference retention Absolute retention and Highest peak maximum ae 5 Retention Checked area Deviation Limit Updated area Averaged Srpna min mAU min mAUmin mAU min mAU min replicates Chymotrypsinogen 8 78 17 2310 1 1063 1 7390 15 0183 3 Bint seve Saveas Hep Fig 11 27 Update repor
77. curve name corresponds to the curve type e g UV1 for UV detection of an eluted component If two or more curves of the same type were created within a result file they will be numbered accordingly e g UV1 UV2 etc For systems using a variable wavelength detector the wavelength for the UV curve is also given e g 280 If you do not want to display the entire names of the curves in both the dialogues and chromatogram windows 1 Click on the Curve Names tab in the Chromatogram Layout dialogue 2 Check the appropriate option boxes for the Curve name appearance 3 Select the appropriate Curve legend position option It is usually sufficient to select the Curve Name option if only one chromatogram is being evaluated However confusion may arise when more than one chromatogram is shown so more complete names may be necessary Curve Style and Colour Edit Texts Layout Library Header Curve Names YArs Axis Curve PeakTable Curve name appearance Curve legend position Left Alignment IV ChromatogramN ame Bight Alignment Vv Curve Name Top Alignment IT Apply to all chromatograms Cancel Help Fig 9 5 Curve Names dialogue an 18 1138 73 e p190 Presenting results 9 9 2 6 Changing the colour and style of curves All curves within a chromatogram are represented by a default colour and line style Curves imported into the chromatogram or newly created curves are a
78. curves To rename a reference curve in a method 1 Select the Reference Curves tab and click on Rename 2 Select a curve from the list and change the name in the Rename item to field 3 Click on Rename The dialogue remains open until you click on Close so that you can rename more than one curve without closing the dialogue e p87 Editing methods Note an 18 1138 73 e p88 5 5 8 Columns You can look at the parameters of the column selected for your method by clicking on the Column tab The column parameters will be displayed in the Column Data field If you are performing scouting runs with different columns all of these will be listed Select the appropriate column to display the parameters Evaluation Procedures Method Information Result Name Start Protocol Frac 950 Variables Scouting Notes Questions Gradient BufferPrep Columns Reference Curves Columns used in method Column Data jAlkyLSuperose_HR_5 5 Parameter Value Unit Height mand 5 0 cm Diameter mand 0 5 cm Column volume 0 982 ml Column volume unit mand ml Technique mand HIC vt 0 69 ml Vo 0 0 ml Max pressure mand 25 MPa Default flowrate mand 0 5 ml min Max flowrate 05 ml min Typ peak width at base 3 ml pH High value longterm 13 pH Low value longterm 2 pH High value shortterm 14 pH Low value shortterm 2 Average particle diam 13 um Code no 17 0586 01 Typical loading range 3 30 mg Scan rate Spectra sec
79. curves X Fom 0 mi d HIPREPOZ i Cone M07 HIPREP02 1_pH 01 HIPREPO2 1_UV1_280nm 02 HIPREP02 1_U Y 2_406nm 03 HIPREPO2 1_UV3_Onm 04 HIPREPO2 1_Cond 05 HIPREP02 1_Cond 06 HIPREP02 1_Cone J07 HIPREPO2 1_pH 08 HIPREP02 1_Pressure 09 HIPREPO2 1_Flow J10 HIPREP02 1_Temp To 220 ml Cut graphically m Reduce number of samples Reduce by factor 15 Max no samples 208 po Select I Normalise retention Remove Cancel Help Fig 10 43 Export Curve dialogue Pressing the Export button will then prompt a dialogue where you can choose the new file name and destination The default name is the same as the name of the current file The extension of the exported file will be asc cdf wks or xls depending on the file format chosen 10 3 10 Copying results to the clipboard A related activity is Edit Copy to Clipboard where the contents of the active window are copied to the Windows clipboard and can be later easily pasted into other programs e g Microsoft Word Curves and Documentation are copied as Windows enhanced metafiles emf while Peak tables are copied as text In the latter case only the columns selected in the spreadsheet will be copied e p301 ED Evaluating results Note an 18 1138 73 p302 10 3 11 Importing results and curves You may import curves in AIA cdf ASCII
80. difference of the two curves The two source curves must have the same Y axis unit and not be fraction or injec tion curves curve Second source curve Target curve position Evaluation functions and instructions A TDIV Divides two curves to gain a third First source curve which is the quotient of the curve two curves The two source curves Second source can have any Y axis unit The curve threshold values are used to avoid Target curve division of numbers close to zero position At those points where source Threshold1 curve 1 has amplitude less than Threshold2 Threshold1 or source curve 2 has amplitude less than Threshold2 the result of the division is defined to be 1 0 The Y axis of the curve will be the same as that of the first source curve A 4 2 Integration Instruction Description Parameters CALCULATE Calculates a baseline from the Source curve BASELINE _ source curve The baseline is Target curve stored in the target curve position position DEFAULT can be selected inthe Noise Window Baseline parameters which will Shortest baseline then calculate default baseline segment parameters for each new curve Slope limit Max baseline level CALCULATE Calculates a baseline from the Source curve L curve crvSrc using a morphologi Destination BASELINE_ kal method The baseline is stored kurve MORPH lin curve crvDst INoise Window Width Distance Between Points
81. factor Fig 9 27 3D Data View dialogue an 18 1138 73 e p214 Presenting results 9 The 3D Data View dialogue is similar to the 2D dialogue with the addition of a list for altering the variable on the Z axis and a different shortcut menu that allows the figure to be viewed by animated rotation viewing Style gt Peak A Customization Font Size General More Font Color Style Numeric Precision Main Tite ne Include Data Labels Peka z Grid Lines gt 7 p Grid Lines Sub Title A Show Bounding Box e QED DE ns Rotation Animation SNE m Numeric Precision j 2 Ce Rotation Increment Color 0 1 2 3 Rotation Detail gt Monochrome Plotting Method gt Monochrome Symbols J Rotation Animation Eont Size Maximize C Lage Med C Small es a 7 CN Customization Dialog de Hotes taps Never Export Dialog Cancel Apply Help Original Export Maximize Help Fig 9 28 Shortcut menu and Customization dialogue within 3D view When viewing results in 2D or 3D views click on Copy to Clipboard to copy and store the figure for transfer to external programs To save the current Wizard settings for future use click on the Save Wizard Settings button Give the Wizard settings a name and if the settings are to be used by all users on the system check the Global wizard settings check box Click on OK The Global wizard setti
82. fo min Cancel Help Fig 11 15 Identification settings dialogue e p325 ED Analysing results Peak identification by absolute retention Use this option when there has been little or no drift in retention between successive runs of the standard Quantitate will find corresponding peaks in these successive runs providing any drift in retention does not move a peak outside the peak window Peak identification by relative retention If peak identification becomes difficult because of drifting retention between runs which moves peaks out of the peak window you can choose to identify peaks according to their position relative to a reference peak This is achieved as follows 1 Open the Define Component s dialogue again and identify a component for use as the reference Choose a peak that is well separated from any other peaks This enables the window to be set relatively wide and the system can accommodate a larger drift in retention value 2 Click Identification settings and set Identify peak on to Relative retention 3 Open the Component menu and select the component to be used as the reference peak and set the window width for that peak an absolute value The width should be set fairly wide to be able to accommodate a larger drift Make sure there are no other large peaks within the window 4 Click OK to return to the Define Component s dialogue The Peak table now includes another column Ret Ref whic
83. from that required for editing and running methods 8 1 Setting up a MethodQueue 8 1 1 Defining a MethodQueue To create a new MethodQueue 1 In Main Menu click on the MethodQueue icon or select either File New MethodQueue or Tools MethodQueue New The MethodQueue Editor dialogue is displayed f System2 System3 System4 MethodQueue Start Immediate Start End of Method Queue Setup Save As Close Help Fig 8 1 MethodQueue Editor dialogue 2 To adda method to the MethodQueue list select the End of MethodQueue instruction in the list 3 Click on the appropriate System button The Load MethodQueue dialogue is displayed Note The number of available System buttons is dependent on the number of systems that were selected during installation of UNICORN De epl77 og MethodQueues an 18 1138 73 e p178 Load MethodQueue xi Name System Size Type Prev Folder fz anion Wizard Method File Dave Wizard Method File DNApay Diigo Method File 1d23 Explorer100 Method File March1 Explorer1 00 Method File E March 2 Explorer100 Method File Fig 8 2 Load MethodQueue dialogue Use the dialogue to locate and select the required method Click on OK or double click on the method file The system type for the selected method e g Explorer 100 is assigned to the selected System button and the System name on the button is replaced with the
84. in the Manual amount is used as short for concentration or amount Each of them can be used to produce the calibration curve When analysing the sample both amount and concentration are calculated The addition of a known quantity of the component of interest to the sample prior to the sample preparation for the run Used throughout the manual as a common term for peak area or peak height The result of a peak integration presented in tabular form The peak table can include for example height area and retention volume After the analysis the peak table contains the values for concentration amount and molecular size The relationship between amount and peak size of a component The relationship can be shown as a curve and as a mathematical expression Analysing results 11 Quantitation table All necessary data required to quantitate one or several components in a sample The Quantitation table contains calibration curve s and peak identification settings Level A known amount or concentration of a standard The Levels are numbered 1 20 in decreasing order of concentration Molecular size The relationship between molecular size and curve retention volume for a number of components The relationship can be shown as a curve and as a mathematical expression Molecular size All necessary data required to determine the table molecular size of one or several components in a sample The Molecular size
85. included as an operation in an evaluation procedure to automate process evaluation and documentation 2 8 Network considerations UNICORN can be installed on a stand alone PC workstation or PC workstations in a network UNICORN concepts ez 2 8 1 Stand alone installation In a stand alone installation up to four chromatography systems may be physically connected to and controlled from the workstation where UNICORN is installed CERREN Elali Chromatography aan i PC with UNICORN HEF KE KE He Fig 2 6 Stand alone installation of UNICORN on a workstation which can control up to four separate chromatography systems 2 8 2 Network control from a remote workstation In a network installation each chromatography system is physically connected to a workstation but may be controlled from any workstation in the network on which the UNICORN software is installed A workstation to which a system is physically connected is referred to as a local station Other workstations in a network installation are called remote stations During installation of UNICORN for the first time on a workstation in a network configuration certain files are copied to the network server These files include UNICORN user files and strategy files and are the global settings for all UNICORN users in the network see Chapter 3 in the UNICORN 4 0 Administration and Technical manual UNICORN program files and templates are locall
86. is called the instructions in the block are executed in the order that they are written until the block is finished or the End_Block instruction is executed Any settings made in a block are valid throughout the method until the settings are changed Editing methods 5 Main 0 00 0 00 Base CV 0 97 ml Resource_S_1_ml Start_condition Block Start_condition 0 00 Base SameAsMain 0 00 BufferValveAl A11 0 00 Buffer valveB B1 0 00 OutletValve 0 00 ColumnPosition Position2 0 00 Gradient 0 00 Concentration_B 0 00 Base 0 00 Flow 5 00 Flow_rate mlin 0 00 End_block Block Equilibration Equilibration 0 00 Base SameAsMain 4 00 Equilibrate_with End_block Block Sample_injection Gosrigleriestion 0 00 Base Volume 0 00 Injection valve Inject 4 00 Empty_loop_with InjectionValve Load 4 00 End_block Block Wash _after_injection Fig 5 7 Illustration of the flow of process control through method blocks Calls may be of two types Unconditional calls are made with the Block instruction Conditional calls are made with a Watch instruction which makes it possible to call a specified block or an instruction when a particular monitor signal meets a given condition As long as the condition is not met the block is not activated Watch instructions can use various conditions to respond to absolute signal values or to rate of signal change Note that the breakpoint when the Watch instruction is issued determines
87. may be volume ml or depending on the scale defined in the system strategy time min or column volume CV defined as a numerical value or taken from the column definition For all blocks other than the main block the base may also be defined as SameAsMain which means that the block will inherit the base defined in the main block Different blocks may use different bases Use the base which most closely suits the purpose of the block Column volume is recommended as the base for most steps in a run In some situations it may be more suitable to use time or volume base for individual blocks Be careful when changing the base for an existing method Changing between time and volume base can affect the relative duration of steps in the method if different steps use different flow rates e p103 Editing methods Note an 18 1138 73 e p104 For method blocks which use a volume or column volume base make sure that the flow rate is not zero Volume breakpoints are calculated from the flow rate of the pump and the method will not progress if the flow rate is zero The parameters for the Base instruction differ slightly according to whether a named column definition is used Column parameter Any If the Column parameter in the Base instruction is set to Any and the Base parameter is set to CV the column volume is set numerically by the Volume parameter The column volume may be defined as a variable allowing the scale
88. messages at critical points in the method e g when Watch instructions are used for conditional events The example block below starts the fraction collector and informs the operator Start_frac 00 Base SameAsMain 00 Message Fraction collection started Screen 00 FractionCollector 5 m1 Oo OS O 00 End_block Messages which are set to Screen will be displayed on the screen during a run and will remain there until acknowledged by the operator Messages can also be set to Noscreen these will be recorded in the run log but not displayed on the screen Editing methods 5 es Set_mark Other text messages can be inserted into the chromatogram at set points using the Set_mark instruction The Set_mark instruction is also a convenient way of inserting a note into both the logbook and the chromatogram during a run This contrasts from Message which is displayed on the screen and entered into the logbook only Set_mark can be used to insert manual notes for example when a problem occurs in the run The instruction can also be incorporated into a method as shown in the following example where Set_mark is used to highlight the start and end of peak fractionation in a method E Main 0 00 Block Start_Conditions_GR W 0 00 Block Column_Equilibration E 0 00 Block Rinse_Sample_Loop W 0 00 Block Load_Sample_Loop E 0 00 Block Flowthrough_Fractionation E 0 00 Block Sample_Injection E 0 00 Block
89. method folder or result file to external Note 1 Select the file to be copied in either the Methods window or Results window 2 Select Copy to External from the shortcut menu or select File Copy to External The Copy to External dialogue is displayed 3 Select the destination drive and folder and click on the Save button Copy to External 24 x Copy to A or B will compress the file zip Select destination drive and folder Cancel Fig 3 9 The Copy to External dialogue Help If you select the 314 Floppy Drive a as the destination drive the files will be automatically compressed into a zip file thus allowing approximately 5 10 times the storage capacity Moreover if the zipped file is greater than the storage capacity of the disk the file saving is automatically spanned across several disks Files are automatically decompressed when using the Copy from External operation see below The zip function does not work if you select the Copy function Copy files from external Method and result files can be copied from external If the selected files have been compressed using the Copy to External function then these will be automatically decompressed To copy a method or result file from external 1 Select the destination folder in the Methods window or Results window 2 Without selecting a file icon bring up the shortcut menu and select Copy from External or select File Copy from External
90. of the run to be decided when the method is actually run Breakpoint p Instructions Parameters Column Inset po Ho C Pump Block Any zi i CF h Continue s ijiisa End Block Base aa Em End Metred a gt AlamstMon Eyajuate ripe Hold Volume a 0 1 999999 Loop Var 0 38 Him Replace Cine Loop_End va zm _Beplace atc Message New_Chromatogram Other Pause z Delete Fig 5 58 With column volume selected for the Base instruction and Any selected for the Column parameter the column volume is entered numerically and may be defined as a variable Column parameter named column If a named column is selected for the Column parameter in the Base instruction the volume specified in the selected column definition will automatically be used for column volume in the method block The column volume for base CV cannot then be changed in the instruction or defined as a variable The Column parameter can however be defined as a variable recommended Choosing a column definition also enables linear flow rate and column performance calculations T Breakpoint TT Instructions Parameters Column me Pump wish oo H VAR Hov Peace Blet DR z End_Block ase jar 7 Change AlarmstMon Eng Method Dee Hold Volume 0 1 999999 C Frac ps Loop 85 lace Can Loop_End m Bece fate Message New_Chromatogram Other Pause a Delete Fig 5 59 Using a named column definition in the Base inst
91. peaks 0 eee eee ees 10 1 1 Baseline calculation for integration 0 10 1 2 Performing a basic integration 0040 10 1 3 Optimising peak integration 00000005 10 1 4 Optimising the baseline parameters using a morphological algorithm 0 0 0 0 0 10 1 5 Optimising the baseline parameters using a classic algorithm ose vrash lt lt a dates Baha vk de a Aes 10 1 6 Manually editing a baseline 0000005 10 1 7 Adjusting the peak limits 000 10 1 8 Measuring retention time and peak heights 10 1 9 Measuring HETP 0 0c ee 10 1 10 Measuring peak asymmetry 0 0 0 0 0 cee 10 1 11 Measuring resolution 0 0 0 eee 10 2 Other evaluations 0 000 ccc ee 10 2 1 Peak purity and peak identification 10 2 2 Simulate Peak Fractionation 10 2 3 Finding the slope values for Peak Fractionation or Watch instructions 200 0000 cee eee 10 2 4 Creating a CU fe eve wae ieee ace i oe eee a he 10 2 5 Measuring salt concentrations in the fractions 10 3 Automated evaluation procedures 05 10 3 1 Recording a procedure 0 eee 10 3 2 Editing an existing procedure 0 000 10 3 3 Renaming and removing procedures 10 3 4 Points to Wath ees 5 4 seg eae ewe peed Cee aes A 10 3 5 Running evaluation procedures
92. remaining instructions in the method are executed Instructions that share the same breakpoint as the Hold_until instruction but are placed after it in the method will be executed after the Hold_until conditions have been met E Hain 0 00 Base C 0 10 Column ml Any E W 0 00 Block Column_Equilibration Column_E quilibration 0 00 Base SameAsMain 0 00 Hold_until Cond Less_than 10 mS cm 5 Timeout base 0 00 AutozeroU 5 00 End_block 0 00 End_method Fig 5 63 Example of a Hold_until instruction in a method 5 11 2 Standard Watch conditions The system strategy includes Watch instruction for each monitor defined in the system A Watch is active from the point at which it is issued until either e the Watch condition is met e anew watch is set for the same monitor e a Watch_Off instruction is issued for the monitor The conditions for which a Watch can be set are as follows for most monitors Greater_than The signal exceeds a specified value Less _than The signal falls below a specified value Slope_greater_than The rate of change of the signal exceeds a specified value expressed in monitor units minute e g mAU min Slope_less_than The rate of change of the signal falls below a specified value expressed in monitor units minute e g mAU min Less_than_or_valley The signal falls below a specified value or a valley is detected A valley is detected only pl13 5 Editing methods Note a
93. remove one run from the Scouting tab click on any variable in the run and then click on Delete Confirm that you want to delete the run To clear all runs click on Clear All and confirm that you want to clear them all Start protocol for scouting runs On the Start Protocol tab see Start Protocol on page 96 you may choose whether to display the Start Protocol at the beginning of the first run only in a scouting series or at the beginning of every run Displaying the Start Protocol at the beginning of the first run only allows unattended operation of the entire scouting series but does not permit variable values and other settings to be adjusted at the beginning of each run Displaying the start protocol at the beginning of every run requires that you go through the start protocol and then press the Start button to start each run You must select Scouting in the Start Protocol tab in Run Set up if you want to change the values for scouting variables before a run Converting a scouting run into a non scouting run To convert a scouting run into a non scouting run i e to have only the original method and variable values click on the Clear All button and confirm the deletion of entries Factorial design The contents of the Scouting tab can be copied lt Ctrl C gt and pasted into a third party factorial design program Processed values can then be pasted lt Ctrl V gt back into the Scouting tab 5 5 3 Questions Ques
94. replaced in the list 7 Ifthe current curve does not prove useful for your comparison use the Delete curve button to delete it from the comparison Also at any point you can use the Back button to navigate back to the Data Selection dialogue and add any new curves to your comparison 8 In the Peak identification settings field each row identifies a peak parameter to be compared among all peaks Use the Delete row button to remove the selected row from the field if you select Delete row without first highlighting a row the first row A is deleted by default 9 When all peak selections and identification settings are complete click on the Next button to proceed to the Peak Data selection dialogue Peak Data Selection x Select Peak Data Peak endpoint retentions width at half height Fercent of total area PFercent of total peak area Type of peak limits Peak endpoint heights Fraction tube id lt Back Cancel Help Fig 9 23 Peak Data Selection dialogue Use the Peak Data Selection dialogue to identify any peak characteristics to be included in your comparisons This is the last point from which you can use the Back button to navigate backwards in the Wizard Click on the Finish button to prompt the Data View dialogue or the 2 D Data View dialogue if Media life time study was chosen Presenting results 9 File Run time Peak name Retention Area Height Resolution min mAU min mAU Peak
95. selecting the addition peak table 343 selecting the components 342 selecting the peak of the spiked and unspiked samples 344 selecting the sample peak table 343 selecting the source chromatogram 343 selecting the unit 343 spiked and unspiked samples 315 the stages 342 standard data entering 320 standard run definition 304 standard definition 304 standards entering standards data 329 entering the amounts 330 start protocol 96 142 start UNICORN 17 e 439 um 18 1138 73 440 starting a run 41 statistics after update 337 correlation 331 details of statistics available 409 explained variance 331 for molecular size curve 363 statistics dialogue 331 363 status bar text messages 159 step gradient 111 store in new chromatogram option 221 strategies what are strategies 3 strategy file error message 399 structure width 266 subtracting a blank run curve 201 204 system connections 11 System button 177 System Control 10 buttons 158 icon 19 monitor signals not appearing 401 overview 4 screen 11 T techniques available using chromatography 35 template creating methods from method templates 33 deleting 100 saving method as template 99 temporary chromatogram 186 temporary chromatogram clearing contents 186 text adjusting free text object for report 235 defing and positioning text for curve 191 entry in chromatogram 204 instructions editor 46 text alignment selecting 155 text instructions editing 42
96. so that you can rename more than one block without closing the dialogue If the block you renamed is used in a Block or Watch instruction the block name in these instructions will be changed automatically 5 2 6 Copying moving and importing blocks By using the shortcut menu or Edit options in the Method Editor you can find move copy cut and paste blocks within a method or import blocks from another method Finding text in the text window This function searches for specific text strings in the method text 1 Choose Edit Find or use the shortcut menu and select Find Find what JT Match whole word only Beara a T Match case 2 Up I Search from top of document Down Fig 5 11 Find dialogue within the Text window 2 Enter the text that you want to search for the search direction and any case matching criteria 3 Click on OK You can also search for the next occurrence of your selected text string by choosing Find Next lt F 3 gt from the shortcut menu Similarly choose Find Previous lt Shift F3 gt to move back to the previous occurrence of the current text string Moving blocks To move a block 1 Using the shortcut menu select the block to be moved and then select Cut from the shortcut menu Alternatively select Edit Cut or lt Ctr1l X gt 2 Select the instruction line just above the insertion point where you want the cut block to be pasted e pod Editing methods a
97. sometimes wavelength For example UV1_280 UV2_280 and UV1_254 are all different curves To search for all UV curves enter UV in the Curve name text field Presenting results 9 Open Curves to Compare xi Chromatogram selection Folder c5 A Defaults Se Browse Result Peak skim example x Browse All I Store in new ch Cond _ Pressure Curve optio UV1_280nm Overlay Stack Miror m Found curves Search Clear Select All Peak skim example HIPREPO2 e aa tit Peak skim example HIPREPO2 1_U 2_406nm Peak skim example HIPREPO2 1_U 3_Onm Peak skim example HIPREPO2 1_U V1_280nm 01 B4SEM Fig 9 34 Open Curves to Compare dialogue Click on Search and a list of found curves will be displayed based on the designated search criteria A new search can be performed with new search criteria without erasing curves located in the previous search Select the curves that you want to be imported Click on Select All if you want to import all of the curves You can import the curves into a new chromatogram by selecting checking the Store in new chromatogram option This is recommended to keep the source chromatogram free of too many additional curves Select how the imported curves will be displayed by clicking on one of the buttons Overlay Stack or Mirror e p221 ED Presenting results 1200 pH 7
98. text and Lotus 1 2 3 wks or xls spreadsheet formats and access results from SMART Manager FPLCdirector and PrimeView A wizard guides the user through the operation if PrimeView is chosen When importing SMART or FPLCdirector files from a diskette it is recommended that you first copy files to the hard disc and then import the files Select File Import and then the appropriate menu choice A new dialogue will then prompt you to choose the source folder and file The imported curve or result file is automatically opened to the screen Analysing results 11 11 Analysing results Note The procedures outlined in this chapter refer to the Analysis module an optional UNICORN feature that must be ordered and installed 11 1 Introduction This chapter describes how to extend the UNICORN Evaluation module with an optional Analysis module to e determine the absolute quantity or concentration of a component e determine the molecular size of a component The Analysis module is accessed in the Evaluation window under two new menus Quantitate and Mol Size The Analysis module uses many of the facilities within Evaluation and it is assumed that the user is familiar with these see Chapters 9 and 10 11 1 1 Quantitate Quantitate extends the facilities in the Evaluation module to provide a wide range of techniques for quantitative analysis e External standard quantitation e Internal standard quantitation e Standard addition e
99. the Edit button Click on the Component button and then check the AutoSampler checkbox This will make the Autosampler A 900 available under the Injection dialogue during the Wizard setup e p347 ED Analysing results an 18 1138 73 p348 11 5 1 Creating a quantitation table from standards 1 Start by creating a wizard utilizing the Autosampler A 900 On the dialogue for Injection settings choose AutoSampler The method and the injection volume must be the same for all the standard runs On the Scouting tab you may select the correct standard concentration levels by double clicking in the field for the variable Quantitation_Type instruction QuantitationData in the text instructions You can also set the level after the run has been completed Note that Each level is an alias for a specific concentration of the standard Level 1 must be selected for the standard with the highest concentration The levels must be set in order of decreasing concentration of the standard All runs with the same concentration must be given the same level Enter data in the scouting scheme for all the standard runs Method Information Result Name Start Protocol Frac 950 Variables Scouting Notes Questions Gradient Columns Reference Curves Evaluation Procedures Block Variable Runi Run2 Run3 Run4_ Excluded Autosampler_Injection_Partial _ VialNumber 1 1 1 1 1 Injection_Volume ul 50 50 50 50 50 Quantit
100. the Run Set up Method templates are supplied with notes describing the system requirements for running the method Read through these notes carefully before using a method e p8l Editing methods an 18 1138 73 e p82 Evaluation Procedures Method Information Result Name Start Protocol Frac 950 Variables Scouting Notes Questions Gradient BufferPrep Columns Reference Curves Start Notes Run Notes Evaluation Notes TEMPLATE Manual Injection Y BufferPrep version 2 00 01 lon Exchange Chromatography prepared for using BufferPrep Injection Manual Loop or Superloop filled before method start Fractionation OutletValve Flowthrough and elution Elution Linear segmented or step gradient RT Connect BufferPrep eluents according to BufferPrep page Prime System Pumps 4 and B with eluents to be used Prime all inlet tubings 411 42 B1 and B2 with correct eluents No air may be left in the tubings HOW TO USE BufferPrep BufferPrep is by default OFF and no recipe is selected Enable BufferPrep on BufferPrep page in Run Setup by setting BufferPrep to ON and then select a BufferPrep recipe Then go to variable page and set BufferPrep pH Default O OFF Equilibration starts with the block BufferPrep_pH_Flushing If the same pH is used in consecutive runs use pH flushing only for the first run Set pH flushing to off by setting Length_of_pH_Flush to 0 ml SAMPLE LOADING Fixed loop or Superl
101. the Text instruction window In the Text instruction window the method is shown as a list of an 18 1138 73 e p48 Unexpanded blocks in a method Expanded block in a method containing text instructions and a sub block Editing methods 5 blocks denoted by the blue square symbols Beside each block is also a symbol which you can click on to expand the view of the instructions within the block Note that a block can also contain sub blocks as denoted by the blue square symbols E Main 0 00 Base CY 0 98 ml Alkyl_Superose_HR_5 5 Column E 0 00 Block Start_Conditions_BP H E 0 00 Block BufferPrep_pH_Flush 0 00 Block Column_Equilibration W 0 00 Block Flowthrough_Collection E 0 00 Block Wash_Out_Unbound_Sample 5 Affinity_Elution 0 00 Base SameAsMain 0 00 Block Elution_step_1_with_B1 0 00 End_block E 0 00 Block Gradient_Elution E 0 00 Block Clean_After_Gradient_BP_ E 0 00 Block Reequilibration_BP 0 00 End_method DEE 0 4 DREK Fig 5 5 Text instructions showing blocks in a method To collapse the view click on the symbol for that block Alternatively you can double click on the block name to view or hide the instructions see Section 5 3 1 In the block window The organisation of blocks in the method is shown graphically in the block window in text instruction mode Each block is represented by a grey bar with the block name and the length of the block The line is shifte
102. the UNICORN PC Control board is configured according to the settings made during the installation of the program i e the same Control unit number Address and IRQ must be set at the Control board e The communication may also fail if the UNICORN PC Control board configurations conflict with other boards in the PC If this is the case select a free Address and a free IRQ during UNICORN installation and at the Control Board If the Run data option Connection says No 3 there is no contact with the OCI e In Main Menu select Administration System Setup In the dialogue select the system with problems and click on Edit Check that the strategy pipe server name and the control number are correct according to installation at the local station physically connected to the system Troubleshooting B e Jf Connecting to a system remotely check that the local station physically connected to the system is turned on and that the network is functioning at both the remote and the local station e Check that the limit of 8 connections to the system has not been exceeded e Try restarting the system B 3 Method and run problems Cannot Quit or Logoff from UNICORN If you are unable to Quit or Logoff from UNICORN for a connection you may be running a scouting method or a MethodQueue These functions require a control mode connection in order to start subsequent cycles correctly Monitor signals do not appear in the system control C
103. the last zoom step with Undo zoom or to reset the default zoom scale with Reset zoom In Zoom mode you can also drag the vertical cursor line and the co ordinates presented reflect where the line bisects the curve of the same colour Create Curve 322401 1_UV1_280nm 322401 1_CreatedCurve 5 34 min 5 773 mAU 2b ob 2b 4b Spline Through f 3 073 min Delete Delete all pamo Draw straight to next point Re EES PSE Fig 10 38 Create Curve chromatogram window To delete a point from the Point list double click on the appropriate choice in the list Alternatively select the co ordinates in the list and click on the Delete button To delete all of the points in the list click on the Delete All button To draw the curve click on either Draw spline or Spline through Draw spline creates a smooth curve from the data but does not necessarily pass through every point that you have entered By contrast Spline through creates a curve that passes through all of the data points In cases where you have created a curve using Draw Spline you may want the curve to pass through a selection of those points currently lying away from the curve You may force a straight line between two points by selecting the first of the two points in the Point list and then clicking on the Draw straight to next point button This may have to be repeated for several consecutive points to achieve the desired curve e p291 ED
104. the run To change settings use the System Settings command in System Control before starting the run see the UNICORN 4 0 Administration and Technical manual Calibration If this box is checked the monitor calibration settings will be displayed at the start of the run If not satisfied with the calibration you can still calibrate the monitors before the run is started by using the System Calibrate command in System Control Result name changeable If this box is checked you can change the result name when the run is started Click on the Browse button to change the result folder If the Result name changeable box is not checked the result name will still be displayed but neither the name nor the folder can be changed Frac 950 If this box is checked you can view and change any of the Frac 950 setup parameters Scouting start protocol Check the appropriate option for the Scouting start protocol display in scouting runs If you check First run only parameters for the scouting runs can be adjusted at the beginning of the first run only and the runs will thereafter be performed automatically without operator intervention If you check All runs the Scouting start protocol will be displayed at the beginning of each run in the scouting scheme Wi Editing methods 5 5 6 Saving the method 5 6 1 Saving a method Click on the Save icon or choose File Save to save the method If you want to save a copy of the method under a n
105. the same breakpoint value as the block selected for point of insertion 5 3 Method instructions Use the Instruction box in text instruction mode to enter edit and delete instructions 5 3 1 Viewing instructions Instructions are displayed in the text Instruction box e p57 Editing methods an 18 1138 73 e p58 G Method Editor 1d23 EI E Main 0 00 Block Start_Conditions_BP W 0 00 Block BufferPrep_pH_Flush BufferPrep_pH_Flush 0 00 Base SameAsMain 0 00 SystemWash Off Length_of_pH_Flush W 0 00 Block Initial_Eluent_Conditions_BP 0 00 End_block 0 00 Block Column_Equilibration W 0 00 Block Flowthrough_Collection 5 W 0 00 Block Wash_Out_Unbound_Sample Wash_Qut_Unbound_Sample 0 00 Base SameAsMain 2 Wash_column_with End_block Ml 0 00 Block Eluate_Fractionation_ W 0 00 Block Gradient_Elution MM 0 00 Block Clean_After_Gradient_BP_ Clean_After_Gradient_BP_ 0 00 Base SameAsMain 0 00 Gradient 100 Conc_of_eluent_B B 0 00 base MM 0 00 Block Gradient_Delay_BP 1 Watch_off Conc 2 Clean_with End_block W 0 00 Block Reequilibration_BP 0 00 End_method Paame a _ Vat foo mimin ch I Linear Flow ee SystemWash Replace Delete Ready I Fig 5 13 The text instruction window top with the Instruction box bottom Instructions are displayed in the text instruction window as follows Blue square beside text Valid call instruct
106. to 5 with Replace Replace Gradient Gradient Base SameAsMain 0 00 Base SameAsMain 0 00 FractionCollector 5 ml pon 0 00 Gradient 100 00 20 00 Base 0 00 Gradient 100 00 20 00 Base 5 00 FractionCollector 5 ml 20 00 Message END OF GRADIENT Noscreen 20 00 Message END OF GRADIENT Noscreen 20 00 End_block 20 00 End_block Fig 5 18 Replace moves only the selected instruction Changing gradient lengths The length of a gradient is changed by altering the Length parameter Note Editing methods 5 in the Gradient instruction If Change is used to alter the length of a gradient the breakpoints for any instructions issued during the course of the gradient will be adjusted proportionately so that they are always placed at the same relative position within the gradient Instructions issued after the end of the gradient will be shifted by the amount of the change Since the gradient functions over time any instruction that you want to insert after a gradient should be placed after the combined breakpoint and gradient length Moving the End_block instruction in a gradient block with Change does not affect the length of the gradient If Replace is used to alter the length of a gradient other instructions are not affected Message Message at 10 at 15 T 10 20 20 30 Gradient_length 20 3 Gradient_length 30 Message at 10 10 20 Gradient_length 20 J Gradient_length 30 Replace
107. up see BufferPrep on page 88 5 13 1 Creating a BufferPrep recipe To create your own recipe do the following 1 Open the BufferPrep Recipes dialogue by selecting Edit BufferPrep Recipes in the Method Editor e p133 Editing methods an 18 1138 73 e p134 r Recipe m Stock solutions ay ob Inlet A1 A11 A18 Buffer 100 B 1M O1M Acetate pH Range 4 57 Notes BUFFER SOLUTION 2000mi Na acetate x 3 H20 27 22g Mw 136 08 ACID SOLUTION 1000mk Inlet A2 Acid Base Use ampoule 0 1M HCI 01M HCI SALT SOLUTION 2000ml NaCl 233 89 Mw 58 44 Inlet B1 Water Default correction factors 0 0 pH at 0 B and 0 1 pH at 100 B Inlet B2 Salt zi 2M Nali Fig 5 69 BufferPrep Recipes dialogue Click on New to open the New Recipe dialogue and select a buffer from the Buffer substances drop down list If the required buffer is not listed see Defining a new buffer substance below Notes Butfer substances M Hydrogen phosphate pH range Correction factors M From f3 To frs M M M m Acid Base r Salt Component definition Buffer substance 0 1 M HCI X vo M NaCl X Bulle substance Salt Save as Cancel Help Fig 5 70 The New Recipe dialogue Insert the intended stock concentration for the selected buffer Use a buffer concentration of 2 4 times higher than the co
108. values graphically 01215 Cone SamplePres Cond pH Pressure Auxint Cond Flow mAU Cond Curves Temp SampleFlow Fig 6 7 The Curves window To select the curves to be monitored on the screen 1 Select View Properties or select Properties from the shortcut menu The Properties dialogue is displayed 2 Select the Curves tab e p149 ED Performing a run Note an 18 1138 73 e p150 Y Axis Curve Style and Colour Flow Scheme Logbook Run Data Groups Run Data Colour Curves Axis Display curves Select All Clear All Fig 6 8 Properties dialogue Curves tab 3 Select the curves to be displayed from the list or click on Select All if you want to view all of the available curves Note that curves will only be shown for components present in the chromatography system To clear the selection click on Clear All The curves in this list are those for which Store is set to On in the system settings see the UNICORN 4 0 Administration and Technical manual together with any reference curves defined in the method Fraction marks injection marks and set marks will always be shown and are not curves in the list 4 Click on OK Vertical cursor line To display a vertical cursor line select Marker from the short cut menu Drag the cursor line with the mouse Where the line bisects the curve the X axis and Y axis value
109. will create folders IEXSC 1 IEXSC 2 etc 5 5 12 Frac 950 User defined options for Frac 950 are selected on the Frac 950 tab The Frac 950 tab allows the user to choose rack type and the fractionation order Fig 5 48 The Frac 950 tab Rack type Begin the fractionation set up by choosing the appropriate rack type from the Rack drop down menu e p93 Editing methods Note an 18 1138 73 e p94 Rack fig amp 30 mmT ubes gt 18 amp 30 mm Microplates amp 30 mm Tubes 30mmT ubes 30mmT ubes PrepMode Fig 5 49 The Rack drop down menu within the Frac 950 tab Fraction order Next use the Fraction order radio buttons to choose the appropriate order for fractionation m Fraction order G Serpentine row SS a LLL HILL Fig 5 50 The Fraction order radio buttons Row by C row C Serpentine column C Column by column In System Control for manual runs the Frac 950 tab cannot be used in Documentation Instead use the manual fractionation instructions starting with Man_ Last tube The last tube position can only be selected from within Start Protocol before starting a run Click the Define box within the Last tube panel and then move the cursor over the appropriate tube circle within the tube matrix and click again Note Editing methods 5 E d EnS 000000000000 So 8O680C88068 5 COOGOAOCOOOOO Sper GOSSSSCERTRAE TIITII a
110. you can create a new report format or edit the existing formats A report format saved with the Current chromatogram does not necessarily print the actual chromatogram as it appears on the screen in the Evaluation workspace If you want to print a number of results with the same report format create a procedure to print one result and then perform a batch run for the required results see Batch runs on page 298 9 8 1 Creating a new customised report format You can create your own report format and save it for later use The customised report interface allows you to choose from a variety of objects including chromatograms methods documentation free text and more Moreover you can decide the placing alignment and sizing of the objects according to the various options or else apply free placement and sizing To access the customised report interface click on the New button in the Generate Report dialogue to display the Create New Report Format dialogue Click on the Customised format radio button the default setting and then on OK The Customise Report window is displayed e p233 ED Presenting results an 18 1138 73 p234 Customise Report Modified Bile Edt View Insert Layout Help New Page Prev Paae OnePage Zoomin Zoom Ou AddPage Delete Page Exit Daas Page tof 1 Edit Mode Fig 9 47 Customise Report window Adding and deleting pages To add new pages to the report click
111. you have made in the initial dialogue The default options on the Method Wizard dialogues can be reset by clicking on the Set Default button on the initial Wizard dia logue The instructions available for a given system are determined by the system strategy and components A method developed for one system may not be valid on another 2 4 1 2 Make the desired choices on each dialogue then click on Next For further information on any of the Wizard input fields click on the Help button On the final dialogue click on Finish to exit the Method Wizard The new method will be displayed in the Run Set up editor where you can if necessary make any modifications to the method Click on the Save icon or select File Save to save the new method Once saved the method is displayed in the Methods window in the Main Menu Template based method creation To create a new method based on an existing template 1 In the New Method dialogue select the Template radio button in the Use field For system Use ig af Template Method Editor r Template selection Technique Method notes Any z TEMPLATE Installation test version 2 00 05 Template Installation Test to check the function of the solvent delivery the autosampler and the UY monitoring system of AKTAbasic10 Purge Sample Pump Follow the installation instructions in the User Manual to perform the run and to evaluate the result CHECKLIST B
112. 0 ho 1000 tah oH 6 0 600 JA fi m pH 4 0 200 N 0 i H 3 0 ImAU 0 20 40 60 60 700 120 ml Fig 9 35 Different presentation options for comparison of imported curves a overlaid curves b stacked curves c mirrored curves an 18 1138 73 e p222 Presenting results 9 Overlay This presents the imported curves overlaid one on another Stack This presents the imported curves with a given offset Y axis value so that the curves are stacked and distinct from one another Mirror This should be ideally used to view two imported curves One curve is inverted in the Y axis and thus appears to mirror the other curve When you have made your selection click on OK If you selected the Stack option see step 5 the Shift Curves by Offset dialogue is displayed You can set the Offset value to increase or decrease the offset distance between the curves To exclude a curve from the comparison check on the associated check box to de select it Click on OK Shift Curves by Offset E Offset Unit 0 a m Selected curves will be included 02 UV 1_280nm Select All 05 UV2_406nm Clear 04 UV1_280nm 06 U 3_Onm 07 UV1_280nm 01 BASEM SSNS Fig 9 36 Stack Offset dialogue Imported curves are displayed in either the source chromatogram or in a new chromatogram that you created Select the curves that you want to
113. 00 Base Time 0 00 Peak_FracStop 0 00 End_block 0 00 End_block 0 00 End_block 20 00 End_block 0 00 End_method 5 12 The Column list It is possible to select a column in combination with a template when creating a new method see Creating a new method on page 33 Certain column specific parameters are automatically copied into the method Available columns are found under parameter Column in the New Method box and new columns can be added to this list and then be selected when a method is created The Column list is not linked to a particular method although the columns are edited within the Method Editor Columns are either globally available i e available to all users or personal i e not available to other users It is best not to edit the globally available columns unless you save the changes under anew column name since other users may not appreciate the changes It is recommended that restricted access be given to editing global columns see the UNICORN 4 0 Administration and Technical manual for details on restricting access 5 12 1 Adding a new column To add a new column to the Column list do the following 1 Select Edit Column List to open the Column List dialogue Selection of a column in the list will display its parameters in the field to the right of the box Editing methods 5 Column List Alkyl Superose_ HR_5 5 Global i 100 ala se i ast_Desalting_ i HiLoad 16 10_Pheryl_S
114. 003 1_UV2_Onm id1 86Level 1 to 4003 id186Level 1 to 4003 1_UV3_Onm id186Level 1 to 4003 id186Level 1 to 4003 1_UV1_280nm t id186Level 1 to 4004 id186Level 1 to 4004 1_UV1_280nm id186Level 1 to 4004 id186Level 1 to 4004 1_UV2_Onm id186Level 1 to 4004 id186Level 1 to 4004 1_UV 3_Onm lid 86Level 1 to 4004 id186Level 1 to 4004 1 uyi soez 4 Fig 9 18 Data Selection dialogue 1 Use the Chromatogram selection drop down menus to identify the results files for comparison then click on the Search button in the Found curves field A list of all curves that matched the search criteria is displayed in the Found curves field 2 Use the check boxes or click on the Select All button to identify the desired curves within the Found curves field then click on Next If any of the chosen curves have not been integrated the following dialogue is prompted Curves not Integrated xi There are some curves that are not integrated You can select to integrate them with default parameters to skip the curves that are not integratet or to quit the wizard Peak must be on of a largest Skip uit wizard Help Fig 9 19 Curves not Integrated dialogue Note Presenting results 9 If desired change the default value for peak number selection fil ter then click on Integrate The Peak Selection dialogue is prompted Peak Selection xi r Curve id18
115. 07 quadratic through origin curve fit mathematics 408 quantitation automated 347 general points 306 preparing for quantitation 340 selecting peak tables 321 selecting quantitation table 341 showing which table was used to calculate a result 341 stages for internal external standard quantitation 340 steps in quantitation 306 techniques available 307 quantitation table automated update 350 creating 320 creating for automated quantitation 348 definition 305 deleting 334 editing 334 naming 333 new 320 opening 333 printing 333 renaming 334 saving 332 saving update 339 selecting for internal external standard quantitation 341 updating 335 questions defining for answer type 77 deleting 80 display at start of run 97 display in start protocol 143 editing 80 Input field 77 inserting 79 multiple choice 78 no answer 78 types mandatory authorised chromatogram 76 value 78 Quick View 21 quit problem quitting UNICORN 401 quitting UNICORN 31 e 433 um 18 1138 73 434 R rack type options 93 ratio value in peak skimming 265 ratios between UV curves 284 recording a procedure 293 recovery calculating recovery 345 entering the injected amount 346 identifying the addition component 346 identifying the peak tables 346 opening peak tables 345 preparing for quantitation 345 selecting the quantitation table 346 recovery calculation description 308 detailed description 316 reliability of technique 318 red bullet beside text 5
116. 1 connection problem instrument sticks on scanning 403 connection status 159 connections limit 400 not available 400 control facilities 10 manual 157 software modules 4 control mode connections 11 controlled by lt user gt 159 controlling from a remote workstation 13 copying blocks 55 files and folders 26 files from external 27 files to external 26 to diskette 26 correlated baseline 259 correlation 331 363 detail 409 cursor line vertical 65 cursor lines vertical 324 cursor vertical 80 150 curve adding curves 86 204 adding points 290 calculating a baseline 204 changing and fixing the axes 192 changing colours and styles 150 changing the colour and style 191 changing the size of fraction marks 197 comparing curves 12 220 comparing curves by mirror image 229 copying curves into one chromatogram 225 creating 289 cut 198 defining and positioning curve text 191 deleting 87 deleting a point 291 export 300 importing a blank run curve 202 importing curves 220 importing individual curves 224 manipulation 12 matching protein activity to a curve 206 mirror imported 223 move 228 moving a curve using the Shift function 228 normalise 228 optimising presentation in Evaluation 196 overlay imported 223 presentation options for comparing imported curves 222 reading coordinates using vertical cursor line 281 renaming 87 206 selecting curves displayed on screen 149 selecting part of curve for integration 26
117. 1 Changing the size of fraction marks 00 197 9 3 2 Showing part of acurve 0 0 00 cee 197 9 3 3 Reducing noise and removing ghost peaks 200 9 3 4 Subtracting a blank run curve 20 0 eee 201 97370 AGING CUVE S aes ols Sak nde RG aS le eel Motte 204 9 3 6 Entering text in the chromatogram 204 9 3 7 Pooling fractions 1 ee 205 9 3 8 Matching protein activity toa curve 0008 206 9 3 9 Renaming chromatograms curves and peak tables 206 9 4 Comparing different runs 2 00 e eee eee eee 206 9 4 1 Multifile Peak Compare 0 2 00 00 cee 207 9 4 2 Wizard SETUP ites ek leek ok ee lees a Mackie e aE 207 9 4 3 Comparing chromatograms from different runs 216 9 4 4 Comparing CUIVES 0 eee 220 9 4 5 Stacking and stretching curves 2 200 226 9 4 6 Mirror images of CUVES 2 eee 229 9 5 Electronically signing results 002 eee eee 230 9 6 Saving results ie ii orate ot Sakis S94 kak oe OS 231 9 7 Printing active chromatograms 002ee eee 231 9 8 Printing reports 0 0 ees 9 8 1 Creating a new customised report format 9 8 2 Creating a new standard report format 9 8 3 Modifying an existing report format 9 9 Run documentation 0 00 0 c eee eee 9 10 Exiting Evaluation 0 0 0 cee 10 Evaluating results 10 1 Integrating
118. 10 Fig 10 13 Baseline calculation with 50 lower Shortest baseline segment The baseline is however still unsatisfactory due to the high slope of the short segments in the region between the second and fourth peak which are still not identified as baseline segments If you increase the Slope limit by a factor 2 5 a correct baseline is calculated Fig 10 14 Correct baseline parameters Peak limits too high up on the peaks This peak integration problem is in most cases caused by a too high value for the Slope limit and or a too high value for the Noise window This can be encountered when the chromatogram includes a very large e p271 ED Evaluating results an 18 1138 73 p272 flow through or solvent peak The large peak affects the calculation of the default baseline parameters leading to too high values for Slope limit and Noise window Fig 10 15 Peak start and end points on peaks caused by an excessively high Slope limit Note the difference between the situations in Figures 10 12 and 10 15 In Figure 10 12 no baseline segments were detected between the second and fourth peaks there are no blue crosses is this region when Edit baseline is selected The baseline follows the curve as a best fit and cannot be drawn above the curve unless Accept negative peaks is selected In Figure 10 15 baseline segments are detected on th
119. 138 73 p396 Evaluation functions and instructions A REPORT Prints a report with the specified Report layout named report layout and title If Report title Title is then the title in the report layout is used If Report Layout is then a default lay out is used RUN_ Starts a program as a separate Program name PROGRAM Jprocess The Program name string contains the program name and parameters to start it with UPDATE Updates a Quantitation table with Peak table new data from one standard source concentration level Global or Personal table The default Limit value of Quantitation 12 5 will be used table name Injection volume in ml Concentration level for the standard Average or replace point Save updated table Print updated table e p397 AY Evaluation functions and instructions an 18 1138 73 p398 Troubleshooting B B _ Troubleshooting B 1 Logon problems B 1 1 Unable to log on to UNICORN Choose your username from the list and enter your password If you have forgotten your password ask the system administrator for a new one If you cannot log on using your correct username and password the USERS30 MPM file in the UNICORN SERVER FIL folder may be corrupt Restore the file from the latest back up copy or reinstall the default user see the UNICORN 4 0 Administration and Technical manual If users are not available on a remote station in a network i
120. 2 2 Software modules UNICORN control software consists of four integrated modules e The Main Menu with functions for file handling and administrative routines such as definition of available chromatography systems and maintenance of user profiles e The Method Editor where methods for pre programmed control of chromatography systems are created and edited e The System Control module which permits manual or method based control of chromatography systems and on line monitoring of separation processes There may be up to four independent system control modules on one computer for controlling up to four separate systems Note UNICORN concepts TA e The Evaluation module with extensive facilities for presenting and evaluating stored results from separation processes These modules are present on the Windows taskbar To minimize a module to the taskbar click on the Minimize button at the right hand end of the window title bar To minimize the whole of UNICORN click on the lt Windows M gt keys on the keyboard Minimizing a module window to the taskbar does not close the module Once opened UNICORN modules remain active until you quit the program A minimised System Control module may thus be actively in control of a running process 2 2 3 On line help A comprehensive on line help utility is included in UNICORN software Entry to the general help utility can be accessed from the Help menu Dialogue or window specific hel
121. 2 B x C Model function values A 2 05453E 002 Fig 3 3 Statistics table for the quadratic model 3 at least 6 recommended Measuring range for the calibration curve within the highest and lowest values for the points e p407 an 18 1138 73 p408 Mathematical models and statistics Quadratic through origin Based on the equation y Ax Bx The constants A and B are determined by linear least squares regression Please see a statistics textbook for further information Minimum no of points required Measuring range for the calibration curve Point to point As these are not based on a single equation no statistical data is available The statistics table contains only information on the number of points in the curve Minimum no of points required Statistics xi Model TE Fig 3 4 Statistics table for the quadratic through origin model 2 at least 4 recommended from the point with the highest value down to the origin Statistics x Model Point to point Model function values Number of point s 4 Fig 3 5 Typical statistics table for a point to point model 2 Measuring range for the calibration curve within the highest and lowest values for the points Mathematical models and statistics 3 The statistics available C 3 1 Correlation For linear models the Analysis Module calculates the correlation coef
122. 4 showing part of a curve 197 size 227 smoothing 200 stack imported 223 stacking and stretching curves 226 stacking and stretching curves using the normalise function 227 stretching and shrinking a curve using multiply 229 subtracting a blank run curve 201 subtracting the blank run curve 204 curve model models available in quantitation 331 selecting 330 curve models 405 curve models for molecular size curve 362 curve offset 223 cut curve 198 e 417 um 18 1138 73 e 418 D_Baseline setting 115 data export 300 date and time incorrect 403 default parameters in methods 8 default peak identification 325 definition amount 304 calibration curve 304 level 305 molecular size curve 305 molecular size table 305 peak size 304 peak table 304 quantitation curve 305 sample 304 spiking 304 standard 304 standard run 304 delete column 133 instruction 61 molecular size table 365 quantitation table 334 deleting blocks 53 files or folders 29 text in a chromatogram 205 Delta_Base setting 115 Delta_Peak setting 114 deviation and limit as buttons 337 deviation column 337 dialogue Batch run 298 BufferPrep recipes 134 Calculate recovery factor 346 Calibration page in Documentation 253 choosing windows displayed in System Control workspace 145 Chromatogram Layout X axis tab 194 Chromatogram Layout Curve tab 189 Chromatogram Layout Y axis tab 193 Column List 129 Column value update 74 Condition 179 Contents tab in
123. 41 molecular size calculation 367 recovery calculation 346 standard addition quantitation 344 W watch actions 116 watch conditions 113 Hold_until 113 watch conditions settings 114 watch example collecting absorbance peaks 122 collecting peaks above a threshold value 125 collecting three absorbance peaks 120 equilibration with extra safeguard 118 equilibration with simple safeguard 118 safe sample injection 119 simple equilibration 117 starting and stopping fractionation at a certain concentration of Buffer B 127 watch instructions 50 wildcards in file name specifications 24 wildcards used in file search 217 window Baseline box with Shortest baseline segment and Noise 372 Create Curve chromatogram 291 curves 149 Customise Report 234 Cut 199 Edit Baseline chromatogram 277 Edit Peak Table chromatogram 279 flow scheme 66 general window techniques 145 hide 145 maximising or restoring 145 Method 21 Method Editor block 50 Method Editor flow scheme 66 Method Editor gradient 64 Normalise 227 Peak Window chromatogram 264 run data 146 Selection of Run Data 149 setting a suitable width 324 text instruction 58 window width absolute and relative 327 WKS export format 301 X X axis base unit 319 changing scale 153 xls 301 Y Y axis adjustment 192 changing scale 152 e 443 um 18 1138 73 444 Z zero baseline 259 zoom function 65 154 undo 65 198 zoom function in chromatogram window 197 in gradient p
124. 6Level 1 to 4002 1_UV1_280nm Peak Table id186Level 1 to 4002 1_UV1_280nm 01 PEAK1 Fig 9 20 Peak Selection dialogue The Peak Selection dialogue displays the integrated peak for the first of the chosen curves the associated peak table as well as a Peak identification settings table that is used to identify which of the peak parameters will be used in the comparison Follow the steps below to complete the dialogue 1 In the curve window choose the desired peak either by double clicking directly on the peak or by clicking once and then clicking on the Select peak button The peak is assigned a letter designation A B C and the peak parameters are displayed in the Peak identification settings table If the peaks in the curve window do not appear to be integrated properly e g ghost peaks are labelled quit the Wizard and perform a Peak Integration on the selected curve Check to see that the resulting curve has been properly integrated then repeat the Multifile Peak Compare Wizard operation e p209 ED Presenting results an 18 1138 73 p210 Peak Selection Fig 9 21 Peak Selection dialogue showing ghost peaks in the curve window Set the desired peak identification criterion by clicking on the desired parameter in the Peak identification settings table For example if you have selected the highest peak in the curve and want to compare the highest p
125. 8 Evaluating results 10 10 Evaluating results This chapter will mainly describe how to e integrate peaks e automate evaluation operations e export data and curves 10 1 Integrating peaks Using peak integration UNICORN allows you to identify and measure a number of curve characteristics including peak areas retention times and peak widths 10 1 1 Baseline calculation for integration Integrating peaks is divided into two steps calculating the baseline and calculating peak areas As a correct baseline is crucial for accurate calculation of peak areas several ways of calculating the baseline are available in UNICORN e Using the Calculate baseline instruction for automatic calculation of the baseline which gives in most cases a very accurate measurement Calculate baseline is the most common method for calculating baselines Baseline calculation can be performed using the Morphological algorithm or Classical algorithm see sections below For a description of the principles of baseline creation see Baseline calculation theory on page 371 e A blank run curve with the same chromatographic conditions as the corresponding sample can be used as the baseline for peak integration Another approach that may improve the peak integration if a blank run is available is to first subtract the blank run from the source curve see Subtracting a blank run curve on page 201 and then perform peak integration on the resul
126. 8 reference component identifying 326 330 reference curves 86 display at start of run 97 display in start protocol 143 reject peaks 263 relationship with instructions 7 reliability general factors affecting reliability 318 remote controlling from a remote workstation on the network 13 renaming molecular size table 365 quantitation table 334 renaming files or folders 29 replace breakpoint 62 replace for update 337 report adding and deleting pages 234 adding objects 234 adjusting available viewing options 243 adjusting chromatogram object settings 236 adjusting documentation object 239 adjusting Evaluation log object 240 adjusting free text object 235 adjusting method object 238 adjusting page layout 244 adjusting settings for Frac 950 object 241 adjusting settings for quantitate and molecular size object 240 creating a customised report format 233 creating a new standard report format 247 customising the format 250 defining placement and or sizing of object 242 defining the layout for the chromatogram 238 modifying an existing report format 250 moving and resizing objects 242 overview 12 previewing and printing 249 previewing contents 248 printing 246 printing report 232 saving report format 246 saving the report format 249 selecting a format 233 selecting standard report options 248 standard format 250 resolution 378 measurement 283 result file changing the storage location 92 construction of name 92 contents 5 copyin
127. 999999 000 Flowthrough_Fractionation Flowthrough_FracSize ml 0 000 0 000 100000 000 Start_Flowthrough_at NextTube Sample_Injection Empty_loop_with ml 2 500 0 000 999999 000 Wash_Out_Unbound_Sample Fractionation_Step Wash_column_with CV Eluate_Frac_Size ml 2 000 0 000 999999 000 0 000 0 000 100000 000 Start_Eluate_Frac_at NextTube 7 Peak_Frac_Size ml 0 000 0 000 100000 000 Start_Peak_Frac_at NextTube ad Peak_Start_Slope m4U min 100 000 0 010 10000 000__ v Show unused variables IV Display tooltip for extended variable cells Fig 5 4 Run Set up with the Variables tab displayed To access information in the Run Set up click on the relevant tab Tabs in the Run Set up editor are described later in this chapter 5 2 Method blocks Viewing a method as a long list of individual text instructions may be confusing and inconvenient Text instructions are therefore conveniently grouped into blocks of instructions that define a specific functional use For example one block might contain the instructions necessary for equilibrating a column and another block contains instructions for loading a sample etc By using such blocks it is easier to build up a total method for a run 5 2 1 Viewing blocks It is possible for one block to contain one or several sub blocks This is most evident in the Text instruction window In
128. COT Cvikd CIVE DATIN zI Reject peak settings for peak integration Fig 10 41 Instruction field in the Procedure editor dialogue To edit the parameters of a selected instruction enter the new value in the appropriate place in the Parameters field When you have made the desired changes click on the Replace command button You will now see that the selected instruction in the evaluation procedure is updated in accordance with the new parameters assigned to it You can insert new instructions after the currently selected procedure instruction Select an instruction type and instruction in the Instructions field and enter the appropriate parameter values in the Parameters field Click on the Insert command button The new instruction will now be added to the evaluation procedure To remove an instruction from the evaluation procedure select it in the procedure listing and click on the Delete button To save the edited procedure select File Save or File Save as from the dialogue menu commands Name the procedure Choose File Exit from the menu bar in the dialogue 10 3 3 Renaming and removing procedures Procedures that you have created can be renamed or deleted from the list of available procedures It is recommended that you exercise caution in renaming and removing existing global procedures Renaming a procedure 1 2 3 Select Procedures Edit Rename Select a procedure in the Rename Procedure dialogue
129. Choose Standard format and OK and then use the tabs to select the report components within the displayed Create Standard Report Format dialogue Note that the tabs for the standard report format are similar to the individual dialogues listed for objects in Customised formats see Creating a new customised report format on page 233 Create Standard Report Format xi Evaluation Log Frac 950 Contents Header Method Documentation Chromatogram The selected items will be included in the report m Select Items Current user Run user Current date amp time Select All Run date amp time Eas Report title Result file name Glos Al Method file name Page number Frames Report title K O O K K SI O KI Preview Fig 9 60 The Create Standard Report Format dialogue Header tab You can preview the report contents by clicking on the Contents tab Clicking on the symbols next to the content headings reveals the contents of each section of the report Note Presenting results 9 Create Standard Report Format xi Header Method Documentation Chromatogram Evaluation Log Frac 950 Contents This is the information included in this report format Run user Run date amp time Report title Result file name Page number E Method Main Method Blocks Documentation Chromatogram xl Print Preview Save As Cancel Em __ Preview em
130. Create Standard Report Format 249 Copy curve 226 Copy from external 28 Copy to external 27 Correction factors 138 Create Curve 290 Create new unit 290 Create Standard Report Format 248 Curve Name 190 Curve Style and Colour tab in Properties dialogue 151 Curves tab in Properties dialogue 150 Define buffer substance 136 Define component s 323 Define salt 137 Differentiate 288 Divide 285 Evaluation log page in Documentation 254 Export Documentation 301 Filter 24 Find file 25 First Header tab in Page Setup 245 Footer tab in Page Setup 246 Generate Report 233 Identification settings 325 327 Identify peak 344 Import Block 56 Import procedures 85 Import Reference Curve 87 Instant Run 19 Integrate 260 IS and Table settings 329 Line Style and Colour 191 Load MethodQueue 178 Log Format 107 Logbook page in Documentation 254 Logon 17 Manual instruction 160 MethodQueue Editing 179 MethodQueue Editor 177 MethodQueue Setup 180 Method System connection 28 Molecular size 366 Molecular size table 361 Multiply 229 e419 um 18 1138 73 420 New block 52 new column 129 New molecular size table 360 New quantitation table 321 New Recipe 134 Notes page in Documentation 253 Open chromatogram 219 Open Chromatogram to Compare 218 Open chromatograms to compare 217 Open Curves 203 Open curves 224 Open Curves to Compare 221 Open Quantitation table 333 Page Setup tab in Page Setup 244 Peak Table
131. EFORE METHOD START Eluent A Inlet A distilled water Eluent B Inlet B 0 4 acetone in distilled water Prime the System Pumps with the eluents xl For column Any hind Cancel Help Fig 4 2 New Method dialogue Template radio button Note 1 Note 2 Creating methods H 2 Choose the system for which the method is intended The instructions available for a given system are determined by the system strategy A method developed for one system may not be valid on another 3 Select a chromatographic Technique The templates available for the selected technique will be displayed in the drop down list box Available techniques may include Anion_Exchange chromatography Cation_Exchange chromatography RPC reverse phase chromatography HIC hydrophobic interaction chromatography Size_Exclusion chromatography and Affinity chromatography Selection of Any causes the templates for all techniques to be displayed 4 A list of ready to run method templates is displayed for the selected technique Available templates are determined by the system strategy Select one of these templates to create customised methods either by adjusting variable values see Chapter 5 or changing method instructions Click on a template to display information about the particular template in the Method notes field 5 In the For column drop down list choose a specific column to be used Only columns for the selected techniqu
132. END as this may severely affect the functionality of UNICORN 9 3 2 Showing part of a curve This section deals with the selection of just part of a curve for purposes of closer examination of details and for presentation This can be done in three different ways e magnification using the zoom function e fixing the axes e cutting the curves The zoom function In the active chromatogram window it is possible to zoom in on a designated area of the chromatogram This is the easiest and quickest way to enlarge different parts of a curve 1 Place the mouse pointer in any corner of the intended area to be magnified 2 Press and hold the left mouse button A magnifying glass icon will will be added to the mouse pointer arrow on the screen e p197 ED Presenting results an 18 1138 73 p198 3 Drag out a box from the point of origin to cover the area to be magnified Release the mouse button The selected region is now displayed in the entire chromatogram window together with appropriate scales for the Y and X axes 322404 1_UW1_280nm Zoomed Curves 2b 25 55 min Fig 9 11 Illustration of the chromatogram Zoom function 4 To move around in the chromatogram at the current zoom scale use the cursor arrow keys on the keyboard 5 To undo the last zoom step select Undo zoom from the shortcut menu To reset all zoom steps at once i e no zoom applied select Reset zoom from the sho
133. Enter the charge of the completely de protonated ion This will be a negative value for an acid and zero for a base e g for NaH PO4 enter 3 for Tris enter 0 Click on OK to add the new buffer substance to the list of available buffers Defining a new salt When defining a new salt ensure that the new salt is inert i e a salt with no buffering properties In the New Recipe dialogue click on the Salt button Click on the New button in the Define salt dialogue that appears and enter the name of the new component Click on OK Editing methods 5 Define salt x Name Nacia aa E Delete Pana Vae J nge Charge of Anion Charge of Cation OK Cancel Help Fig 5 72 The Define salt dialogue 2 Enter the charge of the anion e g for Cl enter 1 for SO4 enter 2 3 Enter the charge of the cation e g for Nat enter 1 for Mg enter 2 4 Click on OK to add the new salt to the list of available salts 5 13 2 Editing a BufferPrep recipe It is possible to edit an existing recipe or a recipe that you have created 1 Select Edit BufferPrep Recipes in the Method Editor The BufferPrep Recipes dialogue will be displayed 2 Select a recipe from the pull down list and all information about the recipe will be displayed 3 Click on the Edit button to display the recipe parameters Change the recipe see BufferPrep on page 88 for a description of the functions and see below for a description of corr
134. Imeri Precision General Piot Subsets Points Axis Font Color Plotting Method gt i sets onl XIS onl olor Data Shadows BEAUT I Show Annotations Grid Lines Le p Numeric Precision Grid in Front Sub Title Si D we me Graph and or Table gt What to Table Viewing Style Grid Lines Point Label Orientation gt Color Both CY CX C None Mark Data Points Monochrome J Grid in front of data gt C Monochrome Symbols Display Eont Size Graph Table C Both Maximize C Lage Med Small r Subsets to Table Customization Dialog Graphed All Subsets Export Dialog Cancel Apply Help Original Export Maximize Fig 9 26 Shortcut menu and Customization dialogue within 2D view Help To export the view select Export Dialogue from the shortcut menu or click on the Export button from within the Customization dialogue To close the 2D Data View dialogue click on the Close button This will end your Multifile Peak Compare session Alternatively click on Spread to return to the Data View dialogue or 3D Plot to open the 3D Data View dialogue 3D Data View x Peak C ES Baseline example 2 2000 Baseline example 2 1500 LI 1000 Baseline example 2 600 jaggline example 1 10 15 Retention e 2 Height Sigma Resolution Capacity factor Capacity
135. LINE 01 06 XXX XXX XXX XXX can be changed to CALCULATE_BASELINE 01 06 DEFAULT DEFAULT DEFAULT DEFAULT 10 3 5 Running evaluation procedures To run a procedure for a specific chromatogram first make sure that the desired chromatogram is active Click on Procedures Run and choose the desired evaluation procedure Click on OK and the procedure runs at once You can also open the Procedure Editor dialogue and select the dialogue Control Run menu command or click on the Play button e p297 ED Evaluating results an 18 1138 73 p298 10 3 6 Batch runs It is possible to apply an evaluation procedure to a designated batch of result files even if they are not open on the Evaluation workspace It is especially useful for example to perform integration with the same parameter settings on many results or to print a number of results with the same settings The batch run is done in the background of the Evaluation module and thus the results of the run are not seen You will of course receive any print outs or report documentation if this was one of the steps in the run procedure Run documentation 1 Select Procedures Batch run and the Open Procedure dialogue is displayed Select the evaluation procedure and click on OK The Batch Run dialogue which allows you to search for the result files and or chromatograms on which you wish to perform the batch run Tra Batch Run x m Chromatogram selecti
136. MN_EQUILIBRATION 0 00 Block LOAD_SAMPLE_LOOP 0 00 Block LOAD_SAMPLE_LOOP double 0 00 Block WASH_OUT_UNBOUND_SAMPLE click SAMPLE_INJECTION 0 00 Block FRACTIONATION 0 00 Base Volume 0 00 Block GRADIENT 0 00 MethodBase GradientPump 0 00 Block CLEAN_AFTER_GRADIENT 0 00 InjectionValve Inject 0 00 End_method 2 5 Empty_loop_with InjectionValve Load 2 50 End_block 0 00 Block WASH_OUT_UNBOUND_SAMPLE 0 00 Block FRACTIONATION 0 00 Block GRADIENT 0 00 Block CLEAN_AFTER_GRADIENT 0 00 End_method Fig 5 14 Displaying and hiding block instructions 5 3 2 Adding instructions To add a new instruction 1 2 In the text instruction window select an appropriate block and display the instructions within the block Select an instruction line in the block P r Gradient Newinstructon at GOD Base Same sMain breakpoint 0 wall 0 00 OutletValve F1 0 00 FractionCollector 5 ml be added here gt 0 00 Gradient 100 00 20 00 Base 20 00 Message END OF GRADIENT Noscreen 20 00 End_block Fig 5 15 Instructions added at the same breakpoint as existing instructions are inserted after the highlight e p59 Editing methods Note an 18 1138 73 e p60 Make sure that the selected instruction line is within the block not the call to the block 3 New instructions are added from the Instruction box Open the Instruction box View Windows if it is not already displayed For the new instruction
137. MPa 2 50 0 00 25 00 Initial Eluent_Conditions_BP BufferPrep_pH 7 000 0 000 14 000 Buffer_Inlet Al x Flow_rate ml min 5 00 0 00 10 00 Start_ConcB B 0 00 0 00 100 00 ButferPrep_pH_Flush Length_of_pH_Flush ml 0 10 999 Column_Equilibration Equilibrate_with Cv S ystem_Volume_Compensatior Compensation_Volume CV Wash_Out_Unbound_Sample Wash_column_with CV Eluate_Fractionation_V Start_Frac_at B Start_Valve_Fractionation Number_of_fractions Fraction_volume ml First_fraction_in 0 000 999999 000 0 000 999999 000 0 000 999999 000 0 000 100 000 2 0 7 5 0 5 0 100000 0 F4 had Gradient_Segment_1 Target_ConcB_1 B 100 00 0 00 100 00 I Show unused variables IV Display tooltip for extended variable cells Fig 5 27 The Variables tab in Run Set up To change the default values simply enter new values in the appropriate fields Use the scroll bar to display additional variables if the variables occupy more than one screen The changed values will be displayed for the corresponding instructions in the Text instruction window Remember to save the method if you want to use the new values The Variables box must be checked in the Start Protocol tab if you want to be able to change variable values at the start of a method Detail variables are only shown if the Show details box is checked Editing methods 5 5 5 2 Scouting A scouting schem
138. NOFF Cali bra tion ONOFF Log Book ONOFF Result Name ONOFF Col umn Parame ters NAME Export to file Evaluation functions and instructions A EXPORT_ Exports the documentation in the ONOFF Varia DOC_XLS current result file in XLS format bles to the file defined in Export to ONOFF Scout file If all parameters to this func ling ONOFF tion are OFF then no documenta tart Protocol tion is exported If at least one of ONOFF Ques them is ON then the documenta tions tion will be exported and the cor ONOFF Ref responding parts will be included Curves in the exported file ONOFF Eval Proc ONOFF Method Info ONOFF Method Notes ONOFF Start Notes ONOFF Run Notes ONOFF Eval Notes ONOFF Sys Set tings ONOFF Cali bration ONOFF Log Book ONOFF Result Name ONOFF Col umn Info ONOFF Buffer Prep NAME Export to file e p393 AY Evaluation functions and instructions an 18 1138 73 p394 EXPORT_ DOC_ASCII Exports the documentation in the current result file in ASCII format to the file defined in Export to file If all parameters to this func tion are OFF then no documenta tion is exported If at least one of them is ON then the documenta tion will be exported and the cor responding parts will be included in the exported file ONOFF Varia bles ONOFF Scout ing ONOFF Start Protocol ONOFF Ques tions ONOFF Ref Curves ONOFF Eval Proc
139. ORT_ Exports the documentation in the ONOFF Varia DOC_400_ current result file in XML format bles XML to the file defined in Export to ONOFF Scout file If all parameters to this func ling ONOFF tion are OFF then no documenta tart Protocol tion is exported If at least one of ONOFF Ques them is ON then the documenta tions tion will be exported and the cor ONOFF Ref responding parts will be included Curves in the exported file ONOFF Eval Proc ONOFF Method Info ONOFF Method Notes ONOFF Start Notes ONOFF Run Notes ONOFF Eval Notes ONOFF Sys Set tings ONOFF Cali bra tion ONOFF Log Book ONOFF Result Name ONOFF Col umn Parame ters NAME ONOFF Result Info ONOFF Method Sign ONOFF Result Sign Export to file e p391 Ad Evaluation functions and instructions an 18 1138 73 p392 EXPORT_ DOC_WKS Exports the documentation in the current result file in WKS format to the file defined in Export to file If all parameters to this func tion are OFF then no documenta tion is exported If at least one of them is ON then the documenta tion will be exported and the cor responding parts will be included in the exported file ONOFF Varia bles ONOFF Scout ing ONOFF Start Protocol ONOFF Ques tions ONOFF Ref Curves ONOFF Eval Proc ONOFF Method Info ONOFF Method Notes ONOFF Start Notes ONOFF Run Notes ONOFF Eval Notes ONOFF Sys Set tings O
140. Opens the named chromatogram Chromatogram NATION as destination for the subsequent mame CHROM curve and peak operations Used in pair with the IRESTORE_DESTINATION CHROM instruction e p395 AY Evaluation functions and instructions BASE Sets the X axis base in which the following calculations will be done in If the value of X axis base is DEFAULT then the default base is used usually the base the method was run in This instruc tion should be the first in the eval uation procedure otherwise it will have no effect at all X axis base Comment Inserts a comment below the marked instruction Comment text ENDLOOP Marks the end of a LOOP state ment LOOP The instructions between this statement and the ENDLOOP statement are repeated n times It is possible to have loops within loops as long as the number of LOOP statements matches the number of ENDLOOP statements n Number of loops MOLSIZE Calculates the molecular sizes from a molecular size curve A Mol size column will be added to the peak table Peak table source Global or Personal table Mol size table name QUANTL TATE Calculates the concentration and amounts in the sample from a quantitation table Amount and concentration columns will be added to the peak table Peak table source Global or Personal table Quantitation table name Injection volume in ml an 18 1
141. PEAK id 18501 1 UV1 280nm 01 PEAK Fig 11 33 Calculate Recovery Factor dialogue 2 From the Quantitation table drop down list select the desired quantitation table If the table was not saved for global use it is necessary to click the Personal button before selecting the table required Note Only external standard quantitation tables will be shown 3 The peak table for the unspiked sample must now be identified Open the Source chromatogram drop down list and select the chromatogram which contains the sample peak table Highlight the peak table in the Peak table s list 4 Identify the peak table for the spiked sample Open the Addition chromatogram drop down list select the source addition chromatogram and then the peak table in the Peak table s list 5 From the Addition component drop down list highlight the component which was added prior to sample preparation 6 Enter the injected amount of this component in the Added amount field 7 Click OK Viewing the results Using the chromatogram layout detailed above the recovery factor calculated by the software is located at the bottom of the peak table You need to scroll to the end of the table to see it e p346 Analysing results 11 Recovery Factor 0 989 Component name Chymotrypsinogen A Calculated from id 16501 1_UV1_260nm Baseline id 18501 1_UV1_280nm 01 BASEC Peak rejection on Maximum number of peaks 20 Recovery applied Curre
142. Recovery calculations Quantitate uses peak data from standard runs to produce calibration curves which can then be used to evaluate the amount and concentration of components in a sample Preparation of calibration curves is described in section 11 3 Calculating amount and concentration is described in Section 11 4 11 1 2 Molecular size The Molecular size function is used to determine the molecular size of components in a sample using a molecular size curve prepared from one or more standards Molecular size calculations are described in Section 11 6 e p303 ED Analysing results an 18 1138 73 p304 11 1 3 Definitions Standard Sample Standard run Sample run Amount Spiking Peak size Peak table Calibration curve A defined concentration of one or several components The concentration does not need to be the same for all components in the standard One or several standards are used to produce a calibration curve For molecular size calculations the standard contains components of known molecular size A sample with an unknown concentration of the component s of interest The concentration is determined by Quantitation For molecular size calculations the sample contains components of unknown molecular size A chromatographic standard run of a specific concentration level of a standard A chromatographic sample run of a sample to be analysed This means injected amount In most cases
143. Rename The dialogue remains open until you click on Close so that you can rename more than one procedure without closing the dialogue Editing procedures To edit a procedure in a method 1 Select a procedure on the Evaluation Procedures tab and click on Edit e p85 Editing methods Note an 18 1138 73 e p86 2 Edit the procedure as described on page 294 3 Choose File Exit from the procedure editor menu File Save is not available in the procedure editor window when you edit procedures in a method Changes are saved automatically when you close the procedure editor Report formats in procedures cannot be edited or viewed 5 5 7 Reference curves Reference curves are curves from existing result files which you can display in the curve window of System Control during a run You can include up to 5 reference curves in a method You choose which curves to display during the run with the View Curve contents command in System Control see Curves on page 149 Reference curves are only displayed during the run and the Y axis can be made active for the reference curve by clicking on its legend Reference curves are not saved in the result file Evaluation Procedures Method Information Result Name Start Protocol Frac 950 Variables Scouting Notes Questions Gradient BuffePrep Columns Reference Curves Curves listed below will appear in the System Control curve window during run of this metho
144. S cm 2 mS cm 0 00 Watch_Cond Stable_baseline 5 minutes End_block 10 00 Message Conductivity not stable Screen 10 00 Pause 1 00 minutes 10 00 End_block Watch example 4 Safe sample injection The example below applies to systems that have air sensors Inject a 50 ml sample with the system pump directly on to the column If there is less than 50 ml of sample so that air is detected in the inlet the block will be ended and the method continued The Watch_AirSensor1 instruction sets a watch on the inlet air sensor Main 0 00 Base CV 0 10 Column ml Any 0 00 Block Load_Sample_with_SystemPump Load_Sample_with_SystemPump epll9 5 Editing methods Note 1 Note 2 an 18 1138 73 e p120 00 00 00 00 00 00 00 50 0 D G O O R O L Base Volume Alarm_AirSensorl Disabled BufferValveAl A18 Sample_inlet Gradient 0 00 B 0 00 base InjectionMark Flow 1 00 Injection_Flow_rate ml min Watch_AirSensorl 0 Volume_of_sample Equal 1 END BLOCK End_block 0 00 Block Complete_Sample_Loading_Sys Complete_Sample_Loading_Sys 0 00 Base Volume 0 00 Watch_off AirSensorl 0 00 Block Initial Eluent Conditions_Sys Initial Eluent Conditions_Sys 20 0 0 0 0 0 0 0 0 0 00 Base Time 00 PumpAInlet A 00 BufferValveA 00 PumpBInlet 1 1 A11 Bl Eluent_A_inlet
145. Save updated table Print updated table MOLSIZE Calculates the Peak table source Global or Personal table Mol size table name DEFAULT here means that the value will be taken from the Injection volume reported by the Autosampler A 900 from the method DEFAULT can only be used when the injection is done by the autosampler DEFAULT here means that the value will be taken from the level entered in the QuantitationData instruction in the method Before any of these instructions can be executed you must make sure that e A suitable Quantitation table is selected This can be done by entering its name in the Quantitation table name parameter in the instruction The easiest way to achieve this is to select the appropriate quantitation table in the Method Editor with the Quantitate button when the procedure is selected on the EvalProc tab Analysing results 11 e A peak table is present in the chromatogram This can be done by simply including the peak integration instructions prior to the MOLSIZE QUANTITATE or UPDATE instruction in the procedure 11 6 Measuring molecular size The molecular size of components in a sample can be determined by size exclusion chromatography The column must first be calibrated with components of known molecular size The retention is inversely related to the molecular size The procedure for determining molecular size is described below 11 6 1 Overview of molecular size determination
146. Setup Documentation xi Selected items will be included M ariables une i Select All MScouting MStart Protocol Clear All MQuestions pe Reference Curves Fonts JE valuation Procedures Minto m MMethod Notes Te MStart Notes gt I Start on new page Cancel Help Fig 9 52 Setup Documentation dialogue 1 Check the items to be included from the list box To clear the current selection click on Clear To select everything click on Select all 2 If required select Start on new page e p239 ED Presenting results an 18 1138 73 p240 3 If appropriate change the characteristics of the Fonts by clicking on the buttons Title and or Text 4 When you have made your selections click on OK in the Setup Documentation dialogue Details from the selected documentation menu are now inserted into the report in the documentation object Evaluation log The Setup Evaluation Log dialogue is used to chose the settings for the evaluation log object Setup Evaluation Log Ea Fonts Title Text I Start on new page Cancel Help Fig 9 53 Setup Evaluation Log dialogue 1 If appropriate change the characteristics of the Fonts by clicking on the buttons Title and or Text 2 If required select Start on new page 3 When you have made your selections click on OK The evaluation log is now inserted into the report Quantitate and Molecular Size This opti
147. T T T T 0 0 10 0 20 0 30 0 m Fig 11 9 The apparent amount of the addition is calculated by subtracting the amount of the unspiked sample from the amount of the spiked sample 6 The ratio of this apparent amount to the amount actually added to the sample determines the recovery of the system Recovery factor Apparent amount added Actual amount added Apparent amount added Amount of spiked sample Amount of unspiked sample For example if 2 mg of the component of interest had been added to the sample and quantitation indicated an apparent amount added of 1 6 mg the recovery factor was 0 8 The recovery factor may be used to manually compensate for losses during sample preparation The apparent amount in a sample is divided by the recovery factor to obtain the corrected amount e p317 ED Analysing results an 18 1138 73 p318 Reliability of the recovery factor result Specific factors that affect reliability are 1 For good precision the sample should be spiked with an amount of the component of interest in the same order of magnitude as the sample It is assumed that the recovery is the same both for the sample and the spiked sample However if the recovery varies according to the amount of the component of interest results are unreliable 11 2 6 General factors affecting reliability These factors are valid for all techniques apart from standard addition Quantitation requires that th
148. ULT DEFAULT DEFAULT SUB 01 17 47 PEAK_INTEGRATE 47 A Instruction Parameter jon NEGATIVE_PEAKS 2 PAEO PEAK_INTEGRATE Minimum peak height Let 2 PEAK WINDOW o C File operation OFF lt Ampl gt __Beploce Pere SET_COLUMN_HEIGHT poe SET_COLUMN VO Minimum peak width Delete Pine SET_COLUMN_VT Other SET_SKIM_SIZE_RATIO C Test Reject peak settings for peak integration nal Close Fig 10 40 Editing procedures in the Procedure editor dialogue There are several types of instructions as denoted by the options buttons e g Curve operation Integration File operation etc in the Instructions field The appropriate option button and instruction therein will be automatically selected when you select an instruction in the procedure The specific parameters contained within the selected procedure instruction are displayed in the Parameters field A simple definition of the selected instruction is displayed at the bottom left hand corner of the dialogue You can also select an instruction and then press F1 or select Help Index Instruction For a list of procedure instructions with fuller descriptions and parameters see Evaluation procedure on page 380 e p295 ED Evaluating results Note an 18 1138 73 p296 7 m Instruction BR o i Bl Integration PEAK INTEGRATE File operation PEAK WINDOW C Export C Chrom SET_COLUMN_HEIGHT ome fue C Test
149. Wash_Out_Unbound_Sample W 0 00 Block Eluate_and_Peak_Fractionation Eluate_and_Peak_Fractionation 0 00 Base SameAsMain 0 00 FractionationStop 0 00 Outlet Valve WasteF1 0 00 FractionCollector 0 00 Eluate_FracSize ml 0 00 Watch_Conc Greater_than 0 Start_Peak_Frac_at B Start_Peak_Fractionation Start_Peak_Fractionation 0 00 Base Time 0 00 Set_mark Peak fractionation starts 0 00 Peak_FracParameters 100 00 Peak_Start_slope mAU min 75 00 Peak_End_slope mAU min 0 15 Minimum_peak_width min 0 00 Peak_Fractionation 1 Peak_FracSize iml E W 0 00 Watch_Conc Greater_than 100 End_Peak_Frac_at B End_Peak_Fractionation End_Peak_Fractionation 0 00 Base Time 0 00 Peak_FracStop _ _ _ _ 0 00 Set_mark Peak fractionation ends 0 00 End_block 0 00 End_block 0 00 End_block E 0 00 Block Gradient_Elution E 0 00 Block Clean_After_Gradient_P E 0 00 Block Reequilibration_GR 0 00 End method 2 B B 2 2 8 A A Fig 5 61 A method containing the Set_mark instruction see arrows to show the start and end of peak fractionation 5 10 5 Pausing a method A method can be programmed to pause at critical points There are three instructions for this purpose Hold suspends execution of the method but continues to pump eluent at the current flow rate and concentration settings This instruction is useful e g for giving the operator time to load a sample loop e p109 Editing methods Note
150. WasteF1 0 00 Watch_Conc Greater_than 0 Start_Frac_at B pStart_ alve_Fractionation 0 00 End_block Number_of_fractions 2 Fig 2 3 Relationship between variables in Text instructions and in the Variables tab of run set up 2 4 3 BufferPrep BufferPrep is available for some AKTAdesign systems BufferPrep allows a buffer of defined pH and salt concentration to be prepared on line from four stock solutions BufferPrep should be used for ion exchange chromatography 2 5 Scouting Scouting is a powerful facility for repeating a method run automatically with predetermined changes in values for one or more variables in the method Typically scouting is used for optimising chromatographic processes or monitoring column performance e g by determining the dependence of resolution on flow rate or gradient slope A scouting scheme in UNICORN is defined as part of the method Values for the scouting variables may be set in the method and or adjusted when the method is started The following scheme for example scouts for flow rate while holding pH constant ep9 gt UNICORN concepts an 18 1138 73 e p10 Evaluation Procedures Method Information Result Name Start Protocol Frac 950 Variables Scouting Notes Questions Gradient BufferPrep Columns Reference Curves Block Variable Run Run2 Runs Initial_Eluent_Conditions_BP BufferPrep_pH 5 000 5 000 5 000 Flow_rate ml min 1 00 2 00 3 00
151. You are also able to enter new comments in the Evaluation Notes tab Documentation x Method Information Result Infomation Result Name Start Protocol Settings Calibration Logbook EvaluationLog Frac 950 Variables Scouting Text Method Notes Questions Columns Reference Curves Evaluation Procedures Method Notes start Notes Run Notes Evaluation Notes TEMPLATE Autosampler Injection FP Segmented version 2 00 06 Gradient techniques without BufterPrep Injection Autosampler PartialFill Fractionation Frac 950 Flowthrough elution and peak fractionation Elution Linear segmented or step gradient ELUENTS Eluent A Pump A Inlet tubing A11 A18 default A11 Eluent B Pump B Default inlet B1 Prime the System Pumps 4 and B with eluents to be used Prime the inlet tubings A11 B1 with the correct eluents No air may be left in the tubings BufferPrep is not supported by this method SAMPLE LOADING Fill the samples in vials and place them in the autosampler The maximum injection volume equals the A 900 LoopVol 2 Default A 900 loop allows 50 ul FRACTIONATION Fig 9 65 Documentation Notes tab Calibration This displays what system calibrations were made when and by whom Variables Scouting TextMethod Notes Questions Columns ReferenceCurves Evaluation Procedures Method Information Result Information ResutName Start Protocol Settings Calib
152. _216nm ATJMFODS 1_Cond mau o Fig 9 1 Evaluation module view To open a result file either e double click on a result file icon in the Results window of the Main Menu e pl85 ED Presenting results Wi Temporary Tiel E an 18 1138 73 e p186 e select a result file icon in the Results window of the Main Menu and select File Open e click on the Evaluation icon in the Windows taskbar select File Open or click on the Open toolbar button and select a result file from the Open Result dialogue e click on the Evaluation toolbar button in the Main Menu and select a result file from the Open Result dialogue All contents of the opened result file are transferred to the Evaluation workspace By default the chromatograms in a run are shown as opened windows The chromatogram window on top is the active window There is also a minimised Temporary chromatogram window 9 1 1 Chromatogram A chromatogram includes a number of curves that have been created during a run such as UV conductivity pH fraction marks etc The original raw data curves cannot be deleted or modified although they can be used as the basis for evaluation procedures and subsequent creation of new curves A chromatogram also contains the curves created and saved during an evaluation session The default name for the first chromatogram in a result file is 1 9 1 2 Temporary chromatogram The Tempo
153. a Fig 9 61 Create Standard Report Format dialogue Contents tab Previewing and printing the report Within any of the tabs on the Create Standard Report Format dialogue you can preview the entire report printout by clicking Preview Similarly once you have made all of the desired adjustments to the report format you can click on the Print button from within any of the tabs Saving the report format 1 2 5 Click on the Save As button In the displayed dialogue enter a name for the format If you want the report format to be saved globally check the Save as global format option if you have Edit global lists authorisation If you want the format to be used as the default format check the Save as default report format option Click on OK If you selected the Save as default report format option the format name is changed to DEFAULT e p249 ED Presenting results an 18 1138 73 p250 9 8 3 Modifying an existing report format Another way of creating a new report format is to edit an existing format 1 Select File Report or click on the Report toolbar button and the Generate Report dialogue is displayed 2 Select the report format of interest and click on the Edit button Select either Standard format or Customised format and click OK Customised format Choosing the Customised format button opens the Customised Report window As these options are dealt with for creating a new customised r
154. a 00668060000 OOCCOCCCCCCO os OOOOOOOOOO0O aah ae OOOOOOOGO000 cm 0000 Dra Help lt Back Next gt Cancel Fig 5 51 The Frac 950 tab in Start Protocol illustrating the Last tube function If the fractionation process attempts to proceed beyond this position during a run an alarm will be executed The lower right box within the Last tube panel shows the currently selected last tube If a new last tube has been chosen but the user wishes to return to the default last tube position simply click on the Set default button This will automatically set the last tube to its default position When using different sized tubes racks 18mm tubes or Microplates the last tube can be set for both tube sizes Use the Tube type drop down menu to choose the desired tube size and then follow the procedures outline above to select the last tube Note that the total number of tubes sampled may differ according to which Fraction order has been chosen The Number of tubes equation in the bottom left corner of the Frac 950 tab shows the current number of available tubes chosen for fractionation followed by the total possible number in parentheses For example in the figure above 92 of the total 120 small tubes and all 8 of the large tubes will be available for fractionation e p95 Editing methods Note an 18 1138 73 e p96 5 5 13 Start Protocol The Start Protocol tab determines which items of the Run Set up are disp
155. a new name after any editing operations and so preserve the original table 4 Click Exit to leave Quantitate Printing the quantitation table The Quantitation table dialogue provides a print function which you can use to print the data on all components and their related calibration curves Click on Print to print the calibration curves To print a quantitation table that was prepared previously 1 From the Evaluation window select Quantitate Edit Quantitation Table Open Quantitation table s Global Personal Quantitation table name i t_Std_Ribo Fig 11 23 The Open quantitation table dialogue e p333 ED Analysing results an 18 1138 73 p334 Select Personal if necessary then highlight the quantitation table required and click OK The Quantitation table dialogue opens The Components field lists all the components for which quantitation data are available Click Print to print the data Deleting a quantitation table 1 3 4 From Evaluation select Quantitate Edit Quantitation Table Delete to open the Delete Quantitation table s dialogue Select Global or Personal as required to access the table you wish to delete Highlight the table in the list Click Delete to delete the table or Exit to abort the delete Renaming a quantitation table 1 From Evaluation select Quantitate Edit Quantitation Table Rename to open the Rename Quantitation table s dialogue Select
156. a word processing program Data can be exported to the following formats e ASCII asc e Lotus 1 2 3 wks or xls e XML xml e AIA cdf Select the format that best matches your other application software Peak tables are exported as text strings in the ASCII format but as numerical values in the Lotus 1 2 3 wks or xls format When exporting peak tables all possible columns in the table are exported Evaluating results 10 Curves are exported as series of numerical co ordinates referring to time volume and signal respectively Multiple curves can be exported in each file When exporting a curve you may choose to export only a portion of the curve by inserting the limiting retention values directly into the boxes in the Cut curve field or by visually selecting the part of the curve using the Cut graphically option To optimise the size of exported files you may want to reduce the number of data points that are exported by pressing the spin buttons in the Reduce number of samples box to change the Max no samples and the Reduce by factor function The Reduce by factor function decreases the frequency with which data points are sampled for export For example reducing the number of data points by a factor of five will export every fifth point to the file By checking the Normalise retention button all curves to be exported are normalised to a common X axis Export Curves x Source chromatogram Curves to export isi Cut
157. ab or in the text instructions Values for variables selected for scouting are grey on the Variables tab and cannot be changed there Scheme set up Each run column in the scouting scheme represents one potential run through the method The runs which will actually be performed are defined by the status of the Run Excluded button at the top of each column Runs can be included or excluded by clicking with the right mouse button on the top of the Run columns Start protocol settings If the Scouting box in the Start Protocol tab is not checked the scouting scheme will run automatically as defined in the method We recommend that the box is always checked for a scouting method There is otherwise no indication for the operator at the start of the run that the method actually performs several runs epl7l Scouting Note an 18 1138 73 e p172 7 2 Running scouting To perform a scouting run 1 Start the method see Starting a method on page 141 The start protocol will display the scouting scheme as defined in the method assuming that the Scouting box is checked in the start protocol definition see above 2 Check through the settings for the scouting scheme and if required e change variable values for the runs e using the right mouse button click on the Run column to toggle the run status between Run and Excluded If the start protocol is displayed at the beginning of each run you can adjust the setting
158. age 80 Amersham e KY Biosciences
159. ailing the variables scouting variables questions and or notes Header information cannot be displayed for imported chromatograms 1 In the Chromatogram Layout dialogue click on the Header tab 2 Check the options to be included in the header of the chromatogram window and click on OK In the chromatogram window place the cursor at the top of the curve window just below the toolbar until the window sizing tool appears then drag the cursor down to expose the header window Q Evaluation id186Level 1 to 4001 1 vAUNICORN Local fil Niklas result Unicorn4001 1 fid186Level 1 to 4001 id186Level 1 to 4001 res k File Edt View Integrate Operations Procedures Window Help Usa S olasy Mono_S_HR_5 5 Column position Position Wavelength_1 280 nm Wavelength 2 OFF nm Wavelength 3 OFF nm UV_Averaging Time 256 sec Pressure Limit 500 MPa Etuent_A Inlet All Eluent_B inlet B1 Flow_rate Start ConcB Equilibrate_with 5 CV Sample _ID Levell Vial Number 1 Injection Volume 50 4 ul oll iate8 Level 1 to 4001 1_UV1_280nm 1d1S6Level 1 to 4001 1_Inject Id180Level 1 to 4007 1_UV1_280nm O1 BASEM Id 160Level 1 to 4001 1_UV1_280nm oT SASEMT coo 4188Level 1 to 4001 1_Logbook 17 29 2 Start_Conditions gment Block Eluate_and_Peak Fractionation Block Autosampler_Injection Block Gradient_Se Block Reequilibration Block Flowthrough
160. alculating the molecular size of components in a sample The molecular size curve defines the relationship between retention values and molecular size This curve can now be used to determine the molecular sizes of components in the sample Preparing for molecular size calculations 1 Perform a run with the sample 2 Open the sample result file and peak integrate the curve to produce a peak table When integrating the sample curve must use the same X axis base unit as the standards Volume is recommended for molecular size calculations 3 Use File Save to save the peak table before continuing with the molecular size calculations Selecting the molecular size table 1 Select Mol Size Calculate Mol Size Molecular Size xi m Molecular size table MolSize_3C 7 Global Personal r Select peak table Source chromatogram LLL OEE Peak table s id 19301 1_UV1_280nm 47 PEAK Fig 11 50 Molecular Size dialogue You use the molecular size dialogue to choose the molecular size table you are going to use and the sample peak table to which it is applied Analysing results 11 2 Select Global or Personal according to the location of the molecular size table 3 Open the Molecular size table drop down list and select the table required 4 To select the sample s peak table open the Source chromatogram drop down list and select from the displayed list The related peak tables are then shown in the
161. aluation procedures can be called automatically at the end of a method to evaluate and or print the results Method templates supplied with UNICORN include procedures named Integrate_and_Print which integrates the first UV curve in the chromatogram and prints out the results Print_Chromatogram which prints the chromatogram from the run with the scouting variables printed at the top User defined procedures are created in the evaluation module and may be saved in method files see Automated evaluation procedures on page 292 Procedures saved with one method file can be imported to another Variables Scouting Notes Questions Gradient BufferPrep Columns Reference Curves Evaluation Procedures Method Information l Result Name Start Protocol l Frac 950 Selected Evaluation Procedures will run at the end of the method _JPrint_Chromatogram V Integrate_and_Print BP_Report_Chromatagram integrate BP Report Delete Rename Edit Fig 5 40 The Evaluation Procedures tab in Run Set up Note 1 A procedure in a method will not be updated when a procedure with the same name is changed in Evaluation The same applies to report formats saved in a procedure e p83 5 Editing methods Note 2 Note an 18 1138 73 e p34 If you use an evaluation procedure to print results automatically from a run controlled from a remote station in a network installation the results will be printed on th
162. am make the changes and select check the Apply to all chromatograms option Note Imported chromatograms cannot be shown with column volume as the X axis base e p219 ED Presenting results an 18 1138 73 p220 9 4 4 Comparing curves Curves from different runs can be imported or copied into one chromatogram for comparison Alternative A Importing curves using Open to compare Result files contained in the same folder can be automatically searched to locate all curves of a specified type for example all UV curves This is especially useful for comparison of curves from scouting runs Moreover the imported curves can be automatically overlaid stacked or be presented as mirror images 1 Select File Open to compare Curves or click on the Open curves to compare toolbar button Select the search criteria for the folder result chromatogram and or curve name using the respective Browse command buttons Wildcard characters and can also be used to further specify the search parameters see Alternative A Import chromatograms using Open to compare on page 216 User entered search filters to a maximum of 10 will be saved in the drop down menus for both Result and Chromatogram selections More than one string can be used by inserting a between strings as a search delimiter and search filters are automatically saved and stored within user profiles The UV curves are identified with number and
163. ardware for interfacing the controlling PC to the chromatography liquid handling module The User manual is organised in 11 chapters and 4 appendices Introductory 1 Introduction material 2 UNICORN concepts 3 Logon and file handling Methods and 4 Creating methods from method templates runs 5 Creating and editing methods 6 Performing a run 7 Scouting 8 MethodQueues Evaluation 9 Presenting results 10 Evaluating results 11 Analysing results Appendices A Strategy for AKTAdesign systems B Evaluation functions and instructions C Troubleshooting D Mathematical models and statistics Microsoft Windows NT Windows 2000 and other Microsoft products mentioned in this User Manual are trademarks or registered trademarks of the Microsoft Corporation For the purposes of this user manual examples are displayed for an AKTAexplorer 100 system You should refer to the user instructions specific to the chromatography system that you are using Preface Two broad assumptions are made in this manual 1 You should be familiar with the chromatography systems in your installation Refer to the appropriate System Manuals for details 2 You should be familiar with the general principles of using Microsoft Windows NT version 4 0 and or Windows 2000 on your PC Although UNICORN is a self contained program package and does not require any direct interaction by the user with Windows the user interface principles follow the co
164. as a user you are given the option of changing your default password default Click on the OK button to log on If you cannot remember your password you cannot log on to UNICORN Ask your system administrator or other user with sufficient authorisation to give you a new password Note If UNICORN has been installed so that no password is required for logon you need only select you username and click on OK to proceed Press the Cancel button to abandon the logon attempt e p17 E Logon and file handling an 18 1138 73 e p18 3 2 Toolbar Guide The UNICORN Toolbar Guide dialogue is shown after start up and provides an introduction to the Main Menu toolbar icons The Main Menu toolbar icons allow you to begin using UNICORN quickly for example to create a new method in the Method Editor start an instant run open a result file for evaluation or execute manual instructions in System Control Toolbar Guide xi Begin your work directly by using the toolbar buttons re SE lt a Fig 3 2 UNICORN Toolbar Guide dialogue The toolbar icons are About UNICORN This gives you information about the UNICORN version installed copyright and web address for obtaining more information Logon Logoff This allows you to log on or off UNICORN as appropriate Note Logon and file handling Ep Instant Run This opens the Instant Run dialogue Fig 3 3 in which you can select the system Wizard or template
165. ase Use ampoule 0 1M HCI 01M HCl SALT SOLUTION 2000ml NaCl 233 8g Mw 58 44 Inlet B1 Water Default correction factors 0 1 at 0 B and 0 3 at 100 B Inlet B2 Salt 2M Nali Corr Factors Fig 5 45 The BufferPrep tab in Run Set up 1 On the BufferPrep tab select a Recipe from the drop down list box either AIEX or CIEX which are recipes covering a broad pH range Otherwise select one of the single buffer recipes for more narrow pH ranges All information relevant to the selected recipe will be displayed on the tab 2 Prepare the required stock solutions The solutions and the inlets to which they should be connected are displayed to the right of the page Accuracy of preparation is essential The four stock solutions consist of i A mix of buffering components There can be up to five different buffering components enabling a broad pH range to be covered ii An acid HCI or base NaOH for pH on line titration iii Distilled water iv An inert salt e g NaCl for salt gradient formation 3 Select the Variables tab Set the required pH for the run in the variable BufferPrep_pH e p89 5 Editing methods Note Note an 18 1138 73 e p90 Alternatively if you want to perform pH scouting click on the Scouting tab and select BufferPrep_pH as a scouting variable and enter the pH values for the different runs The recipe saved in the method the one selected on the BufferPre
166. ata window This is indicated by a double arrow in gt gt a display A particular display can have one or more instructions attached to it In cases where there is more than one instruction one of the instructions is the main instruction To view the instruction or main instruction if there is more than one double click on the display with the left mouse button This opens the manual instructions dialogue for the instruction Execute the appropriate manual commands For more details on using manual instructions please see Manual instructions on page 160 To select an instruction with the short cut menu click on the display with the shortcut menu and select Instruction A cascade menu shows the specific manual instruction s Click on an instruction to select it This opens the manual instructions dialogue in which you can execute the appropriate command Performing a run GH Instruments Connection Run Status Scouting No orm Flow a iim SampleFlow a ire Pressure a uv Cond Temp TubeNumber roo x Renee i r a Hide me sn Layout Colour Settings gt SetCondScale0 SetCondScale1 00 Properties Fig 6 6 Selection of Run Data window showing individual displays with the double arrows gt gt indicating linked manual instruction s In this example the specific instruction s are shown in the short cut menu for the Cond display 6 2 3 Curves The curves window displays monitor signal
167. ation performing 319 peak size definition 304 peak table definition 304 identifying for recovery 346 list 322 opening for recovery 345 removing from selected list 322 renaming 206 select for quantitation 341 selecting addition for standard addition 343 selecting for producing molecular size curve 361 selecting for quantitation 321 e 431 a selecting for standard addition 343 pH fine tuning with correction factors 90 point to point curve fit 408 pooling fractions 205 precision external standard quantitation 310 internal standard quantitation 313 recovery calculation 318 standard addition quantitation 315 pressure curve selecting units 154 pressure limit adjustment 35 previewing first UV curve 21 print active chromatograms 231 can not print screen on printer 402 method 101 molecular size table 364 printer setup 30 printing scouting results 173 quantitation table 333 report 246 249 reports 232 update report 339 Print_Chromatogram 83 printer setting the margins 30 procedure instructions editing the parameters 296 inserting new instructions 296 removing instructions 296 saving the edited procedure 296 viewing the parameters 295 procedures defining and viewing 84 deleting 85 editing 85 importing 84 placing on the menu and running 300 recording 293 removing 297 renaming 85 296 selecting to run 84 protein matching protein activity to a curve 206 um 18 1138 73 e 432 Q quadratic curve fit mathematics 4
168. ation_Type Sample Sample Sample Sample Sample Sample_ID Sample_ID Sampled SampleB SampleC SampleD ID IV Display tooltip for extended scouting scheme cells Define Clear All Delete Insert Add Series Help Fig 11 35 Scouting tab used to enter standard data Open the Evaluation Procedures tab and select the procedure Integrate_and_Print This procedure will automatically integrate the first UV curve using default baseline settings Save the method Perform all the standard runs Analysing results CH 6 From the Evaluation window select Quantitate Edit Quantitation Table New and create a quantitation table manually from the standard runs see Creating a quantitation table on page 320 11 5 2 Automated Quantitation 1 You must produce a quantitation table in the method editor for the components of interest before samples can be quantitated automatically see Section above 2 Use the same method for the sample runs as you used for the standard runs 3 On the Scouting tab click on Clear All to clear the scouting scheme then enter the new values Double click each Quantitation_Type field in turn and select Sample for all sample runs 4 Return to the Evaluation Procedures tab and select only the procedure Quantitate_Sample This procedure automatically integrates the first UV curve with default baseline settings and uses the sele
169. ative peaks If you want to include negative peaks in the integration select check Accept negative peaks in the Integrate dialogue or in the Calculate baseline dialogue The negative peaks will be reported as negative areas in the peak table By default negative peaks are not included in the integration Manually editing the baseline In the event that the automatic baseline calculation does not produce a satisfactory baseline it is possible to edit the baseline manually by inserting and deleting baseline points This is done with the Integrate Edit baseline function Manually editing a baseline Manually editing the peak table The Integrate Edit peak table function Adjusting the peak limits allows you to manually adjust the peak start and end points split and join peaks 10 1 4 Optimising the baseline parameters using a morphologi cal algorithm The first choice when trying to optimise the peak integration is to change the baseline parameters For a brief description of the parameters and a suggested way of estimating settings for the Morphological baseline parameters from the source curve see Baseline calculation theory on page 371 The Morphological algorithm can be described in terms of a straight line that strolls along the chromatogram parallel to the X axis Data points for the baseline are created wherever the straight line touches the curve and the points are joined at the end to create a baseline Struct
170. ator from Administration User Setup in the main menu e p399 B Troubleshooting an 18 1138 73 p400 B 2 2 Connections are not available Check the connection between the PC and the chromatography system Check that the power to the chromatography system is turned on If the connection appears to be correct and the power is turned on switch off the chromatography system and quit UNICORN Shut down and switch off the computer then restart the entire system If a system is not available when you attempt to establish a connection check that you have access rights to the system Access rights are not automatically assigned for a newly defined system If you receive the error message Cannot connect to system in a network installation check e thatthe local computer to which the system is connected is turned on and logged on to the network e that the computer from which you are trying to establish a connection is logged on to the network e that the limit of 8 connections to the system has not been exceeded If you can establish a connection but cannot control the system the manual menu commands in system control are grey check that no other user has a control mode connection and that you have sufficient access rights to control the system manually B 2 3 Run data Connection in System Control displays a No x In System Control if the Run data option Connection says No 1 or No 2 e Check that
171. baseline data points Select the Point mode button Click on the left mouse button to place a new baseline point on the chromatogram Each new point is represented by a cross and its co ordinates are automatically entered into the Point list This is useful when for example you want the baseline to go up to a high valley between two peaks To make your task easier you can click on Zoom mode and zoom in on specific regions of the chromatogram and then insert baseline data points The shortcut menu allows you to undo the last zoom step with Undo zoom or to reset the default zoom scale with Reset zoom Before Fig 10 24 Baseline before and after editing e p277 ED Evaluating results an 18 1138 73 p278 Deleting baseline data points If you want to delete a data point from the Point list select the appropriate point in the list and press the Delete button Alternatively double click on the unwanted data point entry in the list to delete it Delete all removes all baseline data points Drawing the new baseline When you are satisfied with your baseline point selection click on the Draw baseline button The new baseline curve will be drawn as a spline function based on the previous and the new points The spline function is guided by the points but does not necessarily pass through them You may also force a straight baseline between two points by selecting the first of the two points in the point list and then clicking o
172. be selected e p363 ED Analysing results an 18 1138 73 p364 by testing different models and comparing the explained variance values but you still need to examine the molecular size curve visually to ensure a good fit Please refer to Appendix C for mathematical and statistical details Saving and printing the molecular size table 1 Click Save as to open the Save molecular size table dialogue which shows a listing of existing molecular size tables Save molecular size table x Molecular size table s Global Personal f Molecular size table name Cancel Help Fig 11 49 Save molecular size table dialogue 2 Enter the new name into the Molecular size table name window You can choose whether the table will be globally accessible to any user or only to someone using your user id The default is Global Select Personal if you need to restrict access 3 Click OK to save the table Once the table is saved under Save as it can be updated using the same file name by use of the Save button Care is needed as this overwrites the original table You might prefer to use Save as and a new name after any editing operations and so preserve the original molecular size table 4 In the Molecular size table dialogue click Print to obtain a print out of the molecular size curve statistics and molecular size table Analysing results 11 Click Exit to conclude the molecular size
173. ber of standard runs and also simplifies the process of renewing the calibration curves before each analysis The injection volume must always be the same for the new run as it was for the previous standard runs Proceed as follows to initiate the update procedure 1 Select Quantitate Edit Quantitation Table Update from the Evaluation window This opens the Update Quantitation Table dialogue Update Quantitation Table xi m Quantitation table Ext_Std_Chrom he Global Personal r Select peak table Result d Default Source chromatogram Level Peak table s M Baseline example 1 m Baseline example 2 m Baseline example 3 uli id186Level 1 to 4001 Li id1g6Level 1 to 4002 i id1 86Level 1 to 4003 vel 1 to 4004 M Peak skim example 1216KB 978KB 973KB 1071KB 1158KB 1081KB 1083KB 1153KB Result File Result File Result File Result File Result File Result File Result File Result File id186Level id186Level 1 to 4004 1_UV1_280nme t 1 to 4004 1_UV1_ 280nm 0 01 1 Fig 11 24 Update Quantitation Table dialogue ED Analysing results 2 Select Personal if the quantitation table is located in your personal folder 3 Open the Quantitation table menu and highlight the table which is to be updated 4 Double click the result file icon to access the new data or click Current if you wish to use the result f
174. but that are not used in the method Make any changes you require in the variable values Use the scroll bar at the right hand side of the tab if the variables do not fit onto one screen Certain variables use the drop down menus in the Variable column for selecting options Variable values in blue can be switched from single position name e g ON OFF to variable range by clicking on the value with the right mouse button Click on the Add button to copy the values from the previous run and change variable values as required Repeat this procedure until you have defined all the runs you require Up to 99 runs can be defined for scouting You can delete and insert run columns using the Delete and Insert buttons respectively Series of runs for vial number are created by clicking on the variable cell in the run you wish to start the series and then by clicking on the Series button Enter ranges vial number in the Insert series dialogue and press OK If you want to exclude runs from the default scouting scheme right click on the Run button at the top of the Variable column to toggle between Run and Excluded Open the Start Protocol tab in Run Set up Check the Scouting box if you want the Scouting page to be displayed at the start of the run recommended This allows the operator to adjust the Run Excluded status for individual runs and the values for scouting variables before the method starts If this box is not checked running the
175. by checking the Save as global box If the column is instead for your personal use only uncheck the Save as global box A personal and global column cannot be given the same name Click on OK to add your column to the column list Saving column globally requires Edit global lists authorisation in the user profile see the UNICORN 4 0 Administration and Technical manual 5 12 2 Editing column parameters Column parameters may be edited by doing the following 1 Select Edit Column List Select a column and then click on the Edit button to display the Edit column dialogue 2 Use the Values column to alter any desired parameter settings 3 To save the changes in place of the original parameters click on Save To save the changed parameters as a new column click on Save as In the Save As dialogue you can change the name of the column Select check the Save as global box if you want the column to be globally available If the column is instead for your personal use only deselect uncheck the Save as global box Click Editing methods 5 a on the OK button and the new column will be added to the column list Note If parameters for a certain column are changed and the same column was earlier selected and saved in a method the column in the method will not automatically be updated with the new parameters When opening the method a question will appear asking if you want to update the parameters It is recommended that you answer
176. cal to those described in Chapter 5 refer to that chapter for further details 4 2 Editing method variables The method templates are constructed from blocks representing the stages in a typical separation Each block has a set of method variables displayed on the Variables tab in the Run Set up You set default values for the variables in the Method Editor and can change these values for a particular run in the start protocol before the run is started Evaluation Procedures Method Information Result Name Start Protocol Frac 950 Variables Scouting Notes Questions Gradient BuffePrep Columns Reference Curves Block Variable Value Range Main Column ml 0 100 0 100 999999 000 Start_Conditions_CF Column_position Position Bypass Wavelength_1 nm 280 190 700 Wavelength_2 nm OFF 190 700 Wavelenath_3 nm OFF 190 700 UV_Averagina_Time sec 2 56 zi Pressure_Limit MPa 4 00 0 00 10 00 Initial_Eluent_Conditions_CF Start_Eluent_Inlet AI bd Flow_rate ml min 1 00 0 00 100 00 Column _E quilibration Equilibrate_with Cv 5 000 0 000 999999 000 System_Volume_Compensation Compensation_Volume ml 8 000 0 000 999999 000 Flowthrough_Fractionation Flowthrough_FracSize ml 0 000 0 000 100000 000 Start_Flowthrough_at NextTube Sample_Injection Empty_loop_with ml 2 500 0 000 999999 000 Wash _Out_Unbound Sample Wash_column_with CV 2 000 0 000
177. chromatogram a 01 HIPREPO2 1_UV 1_280nm 02 HIPREP02 1_UV2_406nm 03 HIPREPO2 1_U 3_Onm 04 HIPREP02 1_Cond 05 HIPREPO2 1_Cond 06 HIPREPO2 1_Cone 07 HIPREPO2 1_pH 08 HIPREPO2 1_Pressure 09 HIPREPO2 1_Flow 17 HIPREPO2 1_UV1_280nm 01 By zl xi Curve name uvi _280nm 01 SMTH r Filter type Moving average C Autoregressive Median C Savitzky Golay m Filter Parameters Light Hard Filter width fg 0 533 ml 1 151 Lik e __ nee Fig 9 13 Smooth dialogue 3 Select the Filter type to be applied in the smoothing operation This selection can be based on the following criteria e Choose Moving average if you have noise along most of the curve Smoothing with this filter affects peak height but not retention There is little effect on peak area Presenting results eH a e Choose Autoregressive if you have periodic noise along the whole curve Smoothing with this filter will affect peak height and retention although has little affect on peak area e Choose Median if there is only one or a few noise spikes e g caused by air bubbles or if the noise is confined to only a small part of the curve Smoothing with this filter may give flattened peaks and affect peak areas slightly but will not affect the retention e Choose Savitzky Golay algorithm to calculate the smoothing and differntiation of data by a least squares technique 4 Sel
178. chromatogram window in the Chromatogram Layout dialogue 322401 1_UV1_280nm 01 MUL 322401 1_UV1_280nM 01 NORM 20 ob 2b 4b 6b ab 10 0 420 mir Fig 9 43 Two curves displayed in mirror image 9 5 Electronically signing results To electronically sign a result file select File Sign Result The Sign the Result dialogue is opened Sign the Result c Default id186Level 1 to 4004 Signing View Signatures r Sign as user Us aT Password E Full name jea Position sta r Signature Meaning Password l MV Lock OK Cancel Help beet He Fig 9 44 Sign the Result dialogue Note Note Presenting results 9 1 The Sign as user field shows the properties of the current user In most instances you will want to use the user shown in the User drop down list box but it is also possible to select another user from those available on the list 2 In the Meaning field provide a short text description explaining the meaning behind the signature e g Peak integration performed 3 Type your user password in the Password field and if desired check the Lock box to permanently lock the result file from further changes Locking the signature should only be done when you are sure that no further modifications to the file will be necessary 4 The View Signatures tab provides a list of all signatures associated with the current method Informa
179. collect resets the Watch condition so that the method will react correctly to subsequent changes in the UV absorbance Main 0 00 Base CV Resource_Q_1 ml Editing methods 5 0 00 Block ELUTION Elution 0 00 Base SameAsMain 0 00 Gradient 100 20 Base 0 00 Watch_UV Greater_than 100 mAU Peak_1 20 00 Message End of gradient Noscreen 20 00 End_block Peak_1 0 00 Base SameAsMain 0 00 OutletValve F3 0 00 Watch_UV1 Less_than_or_valley 100 mAU Waste 0 00 End_block Waste 0 00 Base SameAsMain 0 00 OutletValve F1 0 00 Watch_UV1 Greater_than 100 mAU Peak_2 0 00 End_block Peak_2 0 00 Base SameAsMain 0 00 OutletValve F4 0 00 Watch_UV1 Less_than 100 mAU End_collect 0 00 End_block End_collect 0 00 Base SameAsMain 0 00 OutletValve F1 0 00 End_block e p123 5 Editing methods Wrong an 18 1138 73 e p124 Peak_1 Waste Peak_2 End_collect OutlatF1 QuiletF4 OutletFi Fig 5 66 Peaks collected by the method in example 4 above The design of a method of this kind with several Watch instructions for the same monitor is important The following construction appears simpler but is incorrect 0 00 Watch_UV Greater_than 100 mAU Peak_2 0 00 Watch_UV Less_than 100 mAU End_ collect 0 00 End_block Peak_2 0 0 0 00 OutletValve F4 0 00 End_block 0 0 0 00 OutletValve F1 0 00 End_block
180. con and click on the object of interest To select several objects hold down the lt Ctrl gt key while clicking on the objects 2 For free object placement of an object hold down the left mouse button and drag the object to its new position Similarly to free size the object click on one of the object border anchors either at the corners or in the middle of a border and drag the box to re size it 3 For defined placement and or sizing of object s select from the following options Oe Matches the left alignment of all chosen Oe objects to that of the highlighted object 5 Matches the right alignment of all chosen m objects to that of the highlighted object Matches the top alignment of all chosen objects to that of the highlighted object Be 38 Matches the bottom alignment of all chosen objects to that of the highlighted object oo Adjusts the chosen object s to the left and right margins ul Presenting results 9 margin H Adjusts the chosen object s to the left Adjusts the chosen object s to the right margin Adjusts the chosen object s to the centre of the page Q Adjusts the chosen objects to the same size H as the highlighted object Adjusts the chosen objects to the same O width as the highlighted object Adjusts the chosen objects to the same T height as the highlighted object Viewing options You have several viewing options available in the View menu or on the toolbar Preview Ed
181. ct all the procedures otherwise they will be run twice 11 This new Update_Average procedure should then be selected on the scouting page for the second and further runs at each standard level concentration The Update_Quantitation procedure Update by Replace should still be used for the first run at each level 12 After the standard runs have been set up in the scouting scheme select the samples to be run Select the Quantitate_Sample procedure in the scouting scheme for all the sample runs Select Sample in the variable Quantitation_Type for all the sample runs Method Information Result Name Start Protocol Frac 950 Variables Scouting Notes Questions Gradient Columns Reference Curves Evaluation Procedures Block Variable Run5 Rung Run Rung 4 Main Procedure Quantitate_Sample Quantitate Sample Quantitate Sample Quantitate_S ample Autosampler_Iniection_Pattial Vial Number Injection Volume ul 50 50 50 50 Quantitation Type Sample Sample Sample Sample Sample_ID Sample_ID SampleE SampleF SampleG SampleH Ki ui On IV Display tooltip for extended scouting scheme cells Define Clear All Delete Insert Add Fig 11 41 The last part of the Scouting page used to set up sample runs 13 Save the method and perform the runs Note Analysing results 11 All standards will now be run automa
182. cted quantitation table to quantitate the sample before printing the amounts and concentrations of the components 5 Click on the Quantitate button on the Evaluation Procedures tab and select from the Global or Personal folder the quantitation table to be used This copies the quantitation table into the Quantitate_Sample procedure Documentation x Method Information Result Information ResultName Start Protocol Settings Calibration Logbook EvaluationLog Frac 950 Variables Scouting TextMethod Notes Questions Columns Reference Curves Evaluation Procedures Selected Evaluation Procedures will run at the end of the method JPrint_Chromatogram Integrate_and_Print _JReport_Chromatogram Integrate_Full_Report Ziduantitate Sample Update_Quantitation Fig 11 36 Evaluation Procedures tab used to select the procedure e p349 ED Analysing results Note Note an 18 1138 73 p350 The procedure can not be executed if a quantitation table has not been selected 6 Save the method with a new name Perform the run s The amount and concentration of the components in the samples will be printed automatically after each run 11 5 3 Automated Update The process of updating a Quantitation table is described in Updating a quantitation table on page 335 A quantitation table for the components of interest must be available before a quantitation table can be automatically updated
183. ction Footer which allows you to select the components to be included in the footer e p245 ED Presenting results e L an 18 1138 73 p246 Page Setup Ea Page Setup First Header Footer Header The selected items will be included in the report m Select Items Logo F ignment Current user _Font T Logo Run user Left Current date amp time Select All CEE Middle Run date amp time Ee Bight Report title Result file name Coal Layout Method file name Bs Page number zj z COL A name T Line under Report title Report Title Cancel Fig 9 58 Page Setup dialogue Footer tab Footer options are similar to those for First Header so you can have all information in either the header or footer or split the information between them as required Printing the report To print the report select File Print or click on the Print icon Select the page range in the displayed dialogue and then on OK Saving the report format To save the report format 1 Select File Save or click on the Save toolbar icon If saving for the first time the Save Report Format dialogue is displayed Presenting results 9 Save Report Format xi Global BP_Chromatogr_Report Global BP_Full_Report Global Chromatofocusing Global Chromatogram Global Chromatogram_Peaks Global Chromatogram_Quant Gl
184. culation is performed with the data in low X to high X order If the input trace goes from low to high it is reversed for the calculation and then re reversed afterwards For more information on the Savitzky Golay algorithm see Steiner et al 1972 Analytical Chemistry 44 1906 Evaluation functions and instructions A A 2 Baseline calculation theory The baseline calculation can schematically be described in three steps 1 Defining baseline segments 2 Select baseline points 3 Draw the baseline A 2 1 Defining baseline segments In the first step baseline parameters are used to find the baseline segments The parameters can be seen in the Integrate Calculate baseline function or by pressing the Baseline settings button in the Integrate Peak integrate function The default values for the parameters are determined from the source curve The baseline segments are found by different parameters based on the type of algorithm selected Morphological algorithm The Morphological algorithm searches for all parts of the source curve which 1 come into contact at both extremes of the Structure width This parameter is based on the widest detected peak in the curve 2 fulfils the Minimum distance between data points This parameter reduces the total number of data points created from a curve Classic algorithm The Classic algorithm searches for all parts of the source curve which 1 are longer than the Shortest baseline segme
185. curve source curve by the Multiplication factor and stores the result in the target curve position Target curve position Multiplication factor e p381 AY Evaluation functions and instructions an 18 1138 73 p382 RET_SHIFT Shifts the retention of the source Source curve curve by the Shift factor and stores Target curve the result in the target curve posi position tion Shift factor SIMULATE_ Simulates Peak Fractionation Source curve PEAK FRAC Target curve position Frac size Width Start slope End slope SMOOTH_ Smooths source curve with an Source curve AR autoregressive filter and stores the Target curve result in target curve position The position Filter parameter decides the Filter strength of the filter SMOOTH_ Smooths the source curve witha Source curve MA moving average filter and stores Target curve the result in Resulting Curve The position Filter width parameter decides Filter width how many samples wide the filter is SMOOTH_ Smooths the source curve witha Source curve MEDIAN median filter and stores the result Target curve in target curve position The Filter position width parameter decides how Filter width many samples wide the filter is SMOOTH____ Smooths curve with Savitzky Source curve SG Golay algorithm Target curve position Filter width Polynomial order SUB Subtracts two curves to gain a First source third curve which is the
186. cut menu or e reverse all of the zoom in actions to the default scale setting by selecting Reset Zoom from the shortcut menu Changing the base You can select the base shown on the X axis either time volume or column volumes Make the appropriate selection from the shortcut menu option Base type Changing the base for the display does not affect the base in the method instructions You can therefore check how long a method will take simply by setting the axis scale to time even if the method blocks are written in volume or column volume base Viewing Hatch marks You can display a hatched screen in the Gradient window by selecting Hatch from the shortcut menu e pod Editing methods an 18 1138 73 e p66 5 3 6 The flow scheme window The flow scheme window displays the configuration of system components This window is static and for information only useful for example in identifying valves for flow path instructions Flow Scheme Buffer A2 ALS Valueht PumpA AL j Pump Al U6 Thier B PumpB 1 PumpB Waste Fi CY Vaive pump i FracF2 Outlet F3 alve Value F4 F5 va F w F Fig 5 22 The Method Editor flow scheme window 5 4 Method variables Variables can be assigned to most instruction parameters including breakpoints Variable values can be changed immediately before the start of a run without using the Method Editor allowing one method to be used fo
187. d ATJMFOOS 1 UV 215m Delete Rename Fig 5 42 The Reference Curve tab in Run Set up Adding curves To add a reference curve from a result file 1 Select the Reference Curves tab and click on Import 2 Choose the result file containing the curve to be added and select the curve from the Select list Note Editing methods 5 3 If desired change the curve name in the Import as field 4 Click on Import The dialogue remains open until you click on Close so that you can continue to add curves from the same or different result files Import Reference Curve o Default Select as mapp104 1_U 1_215nm m ul faul all mapp104 1_UV2_230nm mapp104 1_UV3_254nm Baseline Baseline Baseline mapp104 1_Cone example 1 example 2 example 3 mapp104 1_Pressure mapp104 1_Flow mapp104 1_ U1 215nm 01 BASEM mapp104 1 UV 215nm 01 BASEL amp idi86level1 idi66Level1 idi86Level1 id186Level 1 to 4001 to 4002 to 4003 to 4004 EA ful E id2301 Peak skim example Import as mapp104 1_UV1_215nm 01 BASEC Fig 5 43 The Import Reference Curve dialogue Deleting curves To remove one or more reference curves from the method 1 Select the Reference Curves tab and select curve s from the list 2 Click on Delete and confirm the action Deleting curves from the method does not affect the curves in the result file from which they were imported Renaming
188. d Variables Scouting TextMethod Notes Questions Columns Reference Curves Evaluation Procedures Method Information Result Infomation ResuktName Start Protocol Settings Calibration Logbook EvaluationLog Frac 950 Logging started 2001 07 09 13 34 35 User riklas UNICORN 4 00 11 2001 01 09 13 34 45 Chromatogram change 1 2001 01 09 13 34 45 BASE TIME 2001 01 09 13 34 57 REJECT_PEAKS OFF OFF OFF OFF 4 2001 01 09 1 NEGATIVE_PEAKS OFF 2001 01 09 1 SET_COLUMN_HEIGHT DEFAULT 2001 01 09 13 34 57 SET_COLUMN_VT DEFAULT 2001 01 09 13 34 57 SET COLUMN VO DEFAULT 2001 01 09 13 34 57 CALCULATE_BASELINE_MORPH 01 17 0 151 3 033 2 023 2001 01 09 13 34 57 SUB 01 17 47 2001 01 09 13 34 57 SET_SKIM_SIZE_RATIO OFF 2001 01 09 13 34 57 PEAR_INTEGRATE 47 A Logging started 2001 01 15 10 44 26 User niklas UNICORN V4 00 11 2001 01 15 10 47 22 Chromatagram change 1 2001 01 15 10 47 22 BASE VOLUME 2001 01 15 10 49 26 BASE TIME 2001 01 15 10 53 08 BASE VOLUME Logging started 2 5 2001 11 47 17 AM User default UNICORN V4 00 11 2 5 2001 11 47 33 AM Chromatogram change 1 2 5 2001 11 47 33 AM REJECT_PEAKS OFF OFF OFF OFF 20 2 5 2001 11 47 33 AM NEGATIVE_PEAKS OFF 2 5 2001 11 47 33 AM SET_COLUMN_HEIGHT DEFAULT 33M SET_COLUMN_VT DEFAULT 47 2 5 2001 11 SAAM SET_SKIM_SIZE_RATIO OFF
189. d down to indicate calls to other blocks In the following example the blocks are called in sequence from the Main block at breakpoint 0 Blocks to which there is no valid call are not shown in this window e p49 Editing methods an 18 1138 73 e p50 owthrough Fr Sample Iniectio wash action Gradient Elutior Gradient Elutior adien 0 00 CY 0 50 ml adii eg 0 02 min Gradient Segm 0 02 nt Seqme ac q 20 00 CV 0 00 CY Watch Conc Greater_than 0 B Start_Fractionation Loop Fig 5 6 The Method Editor block window Conditional Watch instructions are indicated by a green line showing the start and duration of the watch The example above has a Watch instruction to start the fraction collector which is active throughout the gradient elution block Loop to repeat a group of instructions and Hold_until instructions are also indicated in the block window If you click on the line representing a block in the block window the first instruction in the block will be highlighted in the text window In the gradient window Blocks are represented in the gradient window by marks on the X axis showing the length of each block The name of the block in which the cursor line is currently placed is shown at the top of the window see Using the gradient window on page 64 5 2 2 Calling blocks To execute the instructions contained within a block in a method the block must be called by the program When a block
190. d elution e optimising gradient length and slope e optimising step gradients This chapter describes how to set up and use scouting schemes in UNICORN 7 1 Setting up scouting Scouting schemes are defined as part of the Run Set up in the Method Editor see Scouting on page 71 Any parameters including breakpoints can be scouted provided that they can be defined as variables in the method To set up a scouting scheme 1 Create a method by selecting a method template or Wizard If a method is created without using a method template or Wizard define appropriate variables in the method 2 Open the Scouting tab in Run Set up e p167 Scouting an 18 1138 73 e p168 Fig 7 1 The Scouting tab in Run Set up showing a scheme for testing flow rate The first time the Scouting tab is opened no variables are selected and the Define Variables dialogue is automatically displayed Otherwise click on Define and choose the variables you wish to scout from the list Scouting Variables C Empty_loop_with O Wash_column_with Fig 7 2 The dialogue for selecting scouting variables Scouting By default no variables in the method are selected Select only the variables you wish to scout Checking the Show details checkbox will show variable details on the Variables tab in Run Set up Similarly the Show unused variables checkbox will display variables that are present in blocks
191. d within a chromatogram 1 2 Select Operations Normalise The Normalise dialogue is displayed Select the curve to be normalised and a reference curve to be normalised against For example if you want to stack curves select the curve at the bottom of the stack to be normalised against and the curve to be moved as normalised Click on OK The Normalise window is displayed A box surrounds the curve selected to be normalised Q Normalise olx 322404 1_UW1_280nm 01 NORM Fig 9 40 Normalise window You can now use the following functions Size Allows the selected curve to be stretched along its Y axis or X axis Click on Size and then drag the coloured box either along its Y axis or X axis This is useful for comparison of curves with for example different gradient lengths e p227 ED Presenting results an 18 1138 73 p228 Move Allows the selected curve to be moved to any position on the chromatogram Axes are automatically re scaled to accommodate the new positioning This function is useful for stacking curves Click on Move and then move the curve with the mouse pointer Click on the mouse button when the curve is in the correct position Normalise The curve to be normalised will be adjusted to the help curve Thus the height of the highest peak on both curves will be the same and will occur at the same retention point The curve to be normalised is automatically moved along
192. dQueue folder is represented by a special icon in the Methods window of the main UNICORN menu Double click on the MethodQueue folder icon to open it A MethodQueue folder contains the MethodQueue definition and copies of all methods included in the MethodQueue MethodQueue definition in MethodQueue folder It is important to realise that the MethodQueue works with copies of the original method files If changes are made in the original method these will not affect the method in the MethodQueue To implement changes in a MethodQueue method edit the method in the MethodQueue folder Alternatively edit the original method then use Method Queue folder MethodQueues 8 the MethodQueue editor to update the MethodQueue replacing the old method with the changed version It is a good idea to make sure that MethodQueue definitions always contain updated methods to avoid confusion between different versions of method files 8 2 Editing MethodQueues To edit an existing MethodQueue open the MethodQueue folder icon with the shortcut menu command Edit The MethodQueue Editor dialogue is displayed for the selected MethodQueue Edit MethodQueue authorisation is required to edit a MethodQueue e To change the start condition or method name select the line to be edited and click on the Condition or assigned system button respectively e To insert a new MethodQueue line after the currently selected line click on Insert e To
193. deleting 53 entering length 53 importing 56 inserting 51 naming 52 renaming 54 skipping 38 time versus cumulative time 108 viewing 48 volume versus Cumulative volume 108 window 49 blocks 7 copying 55 e 413 um 18 1138 73 414 importing 55 in method templates 37 in methods 6 blue square beside text 58 bold text 58 breakpoint changing 62 moving instruction between 64 moving instruction within breakpoint 63 replace 62 breakpoints 8 breakpoints during gradients 112 buffer defining a new salt 136 defining a new substance 135 selection 134 stock solutions preparation 89 BufferPrep 9 88 133 creating a recipe 133 display in start protocol 143 display of recipe at start of run 97 editing a recipe 137 fine tuning recipe with correction factors 138 C calculating amount and concentration in the sample 340 calculating a baseline 204 259 calibration curve basing on peak area or height 328 definition 304 producing 319 calibration data display 144 calibration display of calibration settings at start of run 98 calling block 50 53 calls unconditional and conditional 51 capacity factor 379 changing breakpoints 62 instructions 61 chromatogram adding chromatogram object to report 236 comparing chromatograms from different runs 216 contents 186 copying curves into one chromatogram 225 displaying a hatched background 196 entering text 204 importing chromatograms to compare 216 218 optimising presentation 186 opt
194. dialogue Block name Enter a name for the block in the Name field Block names may be up to 30 characters long and may contain letters A Z digits 0 9 and underscore characters Block names must be unique within the method The case of letters is retained but is not significant the names Start_Frac and START_FRAC are treated as identical Note 1 Note 2 Editing methods 5 Base Choose a base for the block If you choose SameAsMain the new block will inherit the base from the Main block in the method The corresponding Base instruction will be inserted in the block at breakpoint 0 If you choose CV enter a value for the column volume If you chose a specific column a column volume is entered automatically Length You can enter a length for the block if required An End_Block instruction will automatically be inserted in the block at the corresponding breakpoint This field may not be left blank Call You can call the new block from an existing block e g the Main block Choose the block from which the newly created block should be called in the From field and enter the breakpoint at which the call is to be made in the At field If you do not want to call the block e g when the block being created is to be activated by a Watch instruction choose the lt Unused gt line in the From drop down list These blocks are placed last in the method in the Unused category If the Block instruction is placed at the same brea
195. dialogue is displayed The Info tab is shown by default and UNICORN takes a few moments to scan the connected chromatography system for the components present Maintenance manager xi Information Warming Info Warning Scanning instrument s PLEASE WAIT Help Fig 6 18 Maintenance manager dialogue Info tab When UNICORN has completed its scan all system components are displayed Maintenance manager x Information Warning Info Warming 3 System pH Cond Pump Sample Pump amp Valveb Valve FF Help Fig 6 19 Maintenance manager dialogue Info tab with system components displayed an 18 1138 73 e p162 Performing a run 6 2 To view the information about a specific component click on it The information can be viewed on the right hand side of the tab Maintenance manager x Information Warning info Warning System B E pH Cond gt General Specific Pump Sample Pump UV Valve 1 Valve 2 Valve 3 Valve 4 Valve 5 Valve 6 Valve7 Cell constant 40 00 1 cm Slope 100 40 Zero offset 65 94 mV Cell 59 40 my Cell resistance 257513 8 Ohm Cond temp comp 2 00 2 ee Fig 6 20 Maintenance manager dialogue Info tab with information displayed for pH Cond cells You can select to view General information which contains information suc
196. e appropriate chromatogram from the drop down list The curves for that chromatogram are displayed from which you should make the appropriate selection 5 To import the curve click on OK For more detailed information about how to import curves chromatograms and other results see Comparing different runs on page 206 e p203 ED Presenting results an 18 1138 73 p204 Alternative B Calculating a baseline If there is no blank run curve you can instead create a baseline with Integrate Calculate baseline see Baseline calculation for integration on page 259 Subtracting the blank run curve Select Operations Subtract to subtract the blank run curve or the baseline away from the sample curve Click on OK All resulting curves from the subtract operation receive the SUB suffix 9 3 5 Adding curves In some runs several sequential chromatograms may have been created for example when the instruction New chromatogram has been used in the method thus creating different chromatograms during the run In order to view and evaluate the resultant curve of all the chromatogram parts the curves must be added together The common situation is when you have a number of chromatograms within the same result file and you want to add the curves In some circumstances curves may need to be imported from other result files To add curves 1 Select and view the first chromatogram in the sequence 2 Select Operatio
197. e Open Chromatograms to Compare dialogue is displayed Presenting results 9 Open Chromatogram to Compare xi Chromatogram selection Folder c Default ss Browse Result i g Browse All Chromatogram f Sa Browse All m Found chromatograms Clear v Baseline example 1 Baseline example 2 Baseline example 3 Cjid186Level 1 to 4001 id186Level 1 to 4002 jid 1 86Level 1 to 4003 lid 86Level 1 to 4004 gt 4 is Select All di r ee ee ee ee et OK Cancel Help Fig 9 31 Open Chromatograms to Compare dialogue The search will take place in the displayed folder only To select another folder click on the Browse button and open the desired folder The search for chromatograms will take place in all result files within the selected folder as denoted by the asterisk You can instead select a specific result file using the Browse function Moreover you can use wildcard characters to search within result files with a specific name profile You can use standard wildcard characters in the file name specification stands for any number of characters and for any single character For example iex will search files named iex iex will search all files with names beginning iex iex will search all files with names ending iex iex will search only 4 character names ending in iex User entered search f
198. e Peak Table tab of the Chromatogram Layout dialogue Every peak will have a HETP value A narrow peak gives a low value corresponding to a well packed column A broad peak gives a high value indicating a column that is not optimally packed an 18 1138 73 p282 Evaluating results 10 ss HETP is calculated as follows HETP L N N 5 54 x Vp wy assuming a Gaussian peak where N no of theoretical plates L bed height in cm Vr peak retention elution volume or time wp peak width at half height expressed in the same units as VR 10 1 10 Measuring peak asymmetry This function can be used in combination with HETP to help assess column performance A perfect peak will have no asymmetry and after peak integration give a value of 1 0 Any value less than 1 0 means that there is a left skew i e the asymmetry falls on the leading side left of the peak The reverse is true for values greater than 1 0 where the asymmetry comes on the tailing side right of the peak To view the asymmetry data select Asymmetry in the list within the Peak Table tab of the Chromatogram Layout dialogue Click on OK and return to the chromatogram window 10 agl Jele height Asymmetry width B width A where A and B are the partial peak widths measured at 10 of the peak height with A representing the leading part of the peak and B the tailing part of the peak 10 1 11 Measuring resolution Resolution is a measure of the re
199. e Volume 0 00 OutletValve WasteF1 0 00 Watch_UV Greater_than 5 Start_Threshold mAU Start_Fractionation Start_Fractionation 0 00 Base SameAsMain 0 00 FractionCollector 1 Eluate_FracSize ml 0 00 Watch_UV Less_than_or_ valley 4 75 Ending_Threshold mAU End_Fractionation End_Fractionation e p125 5 Editing methods an 18 1138 73 e p126 0 00 Base Volume 0 00 FeedTube 0 00 FractionationStop 0 10 Watch_UV Greater_than 5 Start_Threshold1 mAU Start_fractionationl Start_fractionationl1 0 00 Base SameAsMain 0 00 FractionCollector 1 Eluate_FracSizel ml 0 00 Watch_UV Less_than_or_ valley 4 75 Ending_Thresholdl mAU End_fractionationl End_fractionationl1 0 00 Base SameAsMain 0 00 FeedTube 0 00 FractionationStop 0 10 Watch_UV Greater_than 5 Start_Threshold2 mAU Start_fractionation2 Start_ fractionation2 0 00 Base SameAsMain 0 00 FractionCollector 1 Eluate_FracSize2 ml 0 00 Watch_UV Less_than_ or_valley 4 75 Ending threshold2 mAU waste2 waste2 0 00 Base SameAsMain 0 00 FractionationStop 0 00 End_block 0 00 End_block 0 10 End_block 0 00 End_block 0 10 End_block Editing methods 5 0 00 End_block 20 00 End_block 0 00 End method Watch example 8 Starting and stopping fractionation at a certain concentration of Buffer B Sometimes you may wish to start a
200. e are displayed If you do not find your specific column it can be added to the list see The Column list on page 128 The column volume recommended flow rate pressure limit and averaging time for the selected column will be automatically copied into the method thus reducing the need to edit the method You may have to adjust the pressure limit depending on the configuration tubing etc of your system Except for the column volume the column specific values will only be copied into the method if the corresponding instructions are available as variables If Any is selected you can use any column but must enter the column volume in the method on the Variables tab It is recommended that a specific column is selected 6 Click on OK once you have made your selections The method template will now be opened in Run Set up editor as an untitled method e p35 gt Creating methods 4 1 3 Method Editor To create a new method using the Method Editor 1 Inthe New Method dialogue select the Method Editor radio button in the Use field New Method Explorer 00 l izard Fig 4 3 New Method dialogue Method Editor radio button 2 Click on OK This will prompt the Text Editor Method Editor UNTITLED Fig 4 4 Text Editor screen within Method Editor an 18 1138 73 e p36 Creating methods H The techniques used when creating a new method within the Method Editor are identi
201. e component s of interest are completely resolved from all other components in the chromatogram Overlapping peaks will give unreliable results The peak integration parameters Baseline settings must be correctly selected The default settings will be satisfactory in many cases but the integration results have to be checked for all chromatograms for additional information regarding peak integration see Integrating peaks on page 259 All integrations must be performed using the same X axis base unit Time is the recommended unit for highest reliability The concentration levels of the standard have to be accurately prepared Errors in the amount or concentration values will lead to unpredictable results Self imposed limitations such as the use of a small number of concentration levels of the standard also limit precision Appropriate choice of the concentration range of the standard will improve precision This should extend across the presumed amount in the sample Use of the most appropriate curve model will maximise precision The accuracy is improved if several runs are performed at each level Precision is maximised by performing all runs consecutively and thereby reducing systematic errors For detailed information about quantitative analysis see a statistical textbook such as Statistics for Analytical Chemistry 3rd Edition 1993 J C Miller and J N Miller Ellis Horwood PTR Prentice Hall Analysing res
202. e default layout name Run Data You can choose run data items to display without using named groups simply be checking or unchecking items in the list Run data style To change the colour of the text or text background in the displayed readings boxes 1 With the cursor over the Run Data window select Colour Settings Text or Colour Settings Background from the shortcut menu 2 Select colours in the dialogue and click on OK e p147 ED Performing a run Instructions Maximise Run Data Groups gt Hide Colour Settings 7 bar Properties psi an 18 1138 73 p148 Alternatively 1 Select Properties from the shortcut menu The Properties dialogue is displayed 2 Select the Run Data Colour tab 3 Click on the Text button or Background button and select a new colour Click on OK The result of the colour change is displayed in the tab 4 Make further adjustments to the colours as appropriate 5 Click on OK to close the dialogue and apply the changes Run data pressure units If the Pressure data is displayed in the Run Data window you can set the displayed units 1 2 Left click on the Pressure data to display the shortcut menu Select Set Unit and then the appropriate unit either MPa bar or psi from the menu cascade The selected unit is now displayed Run data manual instructions Some strategies directly link specific manual instructions to the displays in the Run D
203. e drop down list Open the drop down list and highlight Average then click the right hand side Replace button to change the instruction In the Procedure editor dialogue select File Exit Procedure Editor Update_Quantitation File Control Help Mode Edit BASE TIME REJECT_PEAKS OFF OFF OFF OFF 20 NEGATIVE_PEAKS OFF CALCULATE_BASELINE 01 17 DEFAULT DEFAULT DEFAULT DEFAULT SUB 01 17 47 PESK_INTEGRATE 47 A REPORT Chromatogram Peaks UPDATE A GLOBAL Ext Std Chym DEFAULT DEFAULT AVERAGE ON ON Instruction Parameter C Curve operation C Integration Level File operation DEFAULT v Export f a RUN PROGRAM Average or replace point zi ieee AVERAGE h C Test Update Exteral Internal quantitation table Fig 11 37 Procedure editor dialogue 7 You can now save the method and perform the runs The Quantitation table will be updated automatically after each run Make sure that the correct concentration level for the standard is selected in the variable Quantitation_Type for each run 11 5 4 Automated update and quantitation in scouting runs It is possible to run both standards and samples in the same scouting run and to continuously update a previously created quantitation table with new values To do this the template and procedures must be slightly modified 1 You must have a quantitation table available for the components of interest 2 Open the
204. e is a series of runs where chosen variable values are varied When a method is run using scouting the method is repeated automatically for each selected run in the scouting scheme Typically scouting will vary one or more variables in a series of runs e g flow rate or elution gradient See Chapter 7 for a full description of scouting facilities All scouting parameters can be set when a run is started provided that Scouting is checked in the Start Protocol tab The parameter values entered in the Method Editor provide the default settings for the scouting method To prepare a method for scouting 1 Select a method with appropriate variables in the method instructions Open the Scouting tab in Run Set up The Scouting Variables dialogue is displayed automatically if there are no scouting variables defined If scouting variables are already defined and you want to change the selection of scouting variables click on the Define button to display the Scouting Variables dialogue Checking the Show details check box will display variables created with the Visible in details only option Checking the Show unused variables checkbox will display variables that are present in blocks that are not used in the method These blocks can however be utilised in the manual instructions box Scouting Variables M Wavelength_1 O Wavelength_2 O Wavelength_3 V BufferPrep_pH V Flow_Rate C Buffer_Inlet O Start_ConcB C Equilibrate_wi
205. e of curve names Make sure that the curves processed when the procedure is executed are compatible with those processed when it was recorded An evaluation procedure aborts if you try to store resulting curves at the position of an original raw data curve B 5 AKTAdesign system specific problems B 5 1 Connected to a system but no system contact In System Control if the Run data option Connection says Yes but the option Instruments says Scanning and there is no contact with the system after a period of waiting check if e The chromatography system is turned on e All cable connections are intact B 5 2 Flowscheme If the flowscheme is not working properly check that you have selected 65536 colours under Windows NT Start Settings Control Panel Display Settings e p403 Bd Troubleshooting an 18 1138 73 p404 Mathematical models and statistics C Mathematical models and statistics The Analysis Module see Chapter 11 uses data from standard runs to produce calibration curves for use over a range of sample concentrations The accuracy of the interpolation that this provides depends on the quality of the curve fit model employed This appendix describes the models that are available It also describes the Analysis Module s statistical tools which you use to measure how well the model fits the data from the standard s C 1 The curve fit models used by the Analysis Module The Analysis Module provides a co
206. e particular run and will be saved in the result file Runs may also be excluded or included during a run using the System Control documentation Note Editing methods 5 Variable values in the scouting scheme will override any values in the Variables tab Text Method Displays the method instructions Double click on a Block instruction as denoted by the blue square and mark to display or hide the instructions in the called block see Viewing instructions on page 57 Method instructions cannot be changed from this display Questions If this box is checked questions defined in the method will be displayed at the start of the run Since the answers to questions can form an important part of the run documentation you are recommended always to check the Questions box Notes If this box is checked the Notes tab will be displayed at the start of the run You can enter notes in the Start notes tab but not in any of the other tabs You can use the scroll bar if necessary to read notes in the Method notes tab Gradient If this box is checked you can view the gradient at the start of the run BufferPrep Only available to some systems If this box is checked the recipe selected in the method will be displayed at the start of the run You cannot change recipe at the start of a run Columns If this box is checked you can view the available column definitions see Columns on page 88 The column definition us
207. e pasted instruction is inserted with the same breakpoint value as the instruction selected for point of insertion 5 3 5 Using the gradient window The gradient window provides a graphical overview of the block structure and eluent gradient in the current method Block name Eluate_Fractionation Gradient 0 00 mi Fig 5 21 The Method Editor gradient window Editing methods 5 Vertical cursor line A vertical cursor line can be dragged from the Y axis with the mouse As you drag the cursor line e The current position is identified at the top of the window in terms of the block name x position in the currently displayed base and eluent concentration in eluent B e If the text instruction window is displayed the instructions are scrolled so that the instruction at the cursor line is at the top of the window e If the Instruction box is displayed the instruction at the cursor line is shown in the dialogue The zoom function To zoom in on a selected region of the Gradient window 1 Press down and hold the left mouse button and drag a rectangle out on the screen to encompass the area to be viewed 2 Release the mouse button The display is now zoomed in on the selected area 3 Repeat the process for further magnification of selected areas You can reduce the scale of the zoom in function in two ways either e reverse each zoom in action a step at a time by selecting Undo Zoom from the short
208. e peaks open the Chromatogram layout box click on Peak Label select Peak name and click OK Calculating the amount and concentration in the sample 1 From Evaluation select Quantitate Calculate amount and conc This opens the Calculate Amount and Concentration dialogue Calculate Amount and Concentration xi m Quantitation table NE Global Persona r Select peak table Source chromatogram 1 bg Peak table s Injection volume id186Level 1 to 4004 1_UV1_280nm 01 PEAK fo ml id186Level 1 to 4004 1_UY1_280nm 01 PEAKI Fig 11 28 Quantitate Amount and Concentration dialogue Analysing results 11 2 From the Quantitation table drop down list box select the quantitation table to be used If the table was saved using the personal option click the Personal button before opening the drop down list 3 Use the Source chromatogram drop down list box to select the source chromatogram which contains the sample curve 4 Select the sample peak table from the Peak table s list 5 Check the Injection volume Enter or edit this if necessary For internal standard quantitation the injection volume must be the same as used for the standard runs 6 Click OK to complete the Quantitate dialogue Viewing the results of quantitation The results of quantitation are shown in the Amount and Concentration peak table columns wo Peak name Retention min area maUtmin Height mau conc mg m1
209. e printer currently set up on the local station not on the remote station If however you execute the procedure interactively from the evaluation module on the remote station the results will be printed on the printer set up on the remote station where you are working Defining and viewing procedures Evaluation procedures are normally defined in the Evaluation module Procedures imported to a method can also be viewed and edited in the Method Editor select the required procedure in the list and click on Edit see Editing an existing procedure on page 294 for a description of how to edit evaluation procedures Evaluation procedures which process chromatogram data rely on consistent identification of curves in the result file for correct operation If you include evaluation procedures with a method make sure that references to curves in the procedure will be valid when the procedure is executed at the end of the run see Automated evaluation procedures on page 292 for more details Selecting procedures to run The Evaluation Procedures tab lists all evaluation procedures associated with the method Check the procedure s that are to be executed at the end of the run The procedures will be executed in the order they appear in the list Importing procedures To import an evaluation procedure 1 Select the Evaluation Procedures tab and click on Import 2 Choose a procedure from the Select list You can also choose to impor
210. e sequence in which they are written If you are using KTA systems the Pause Hold and Hold_until instructions will stop execution at this breakpoint i e instructions following after Pause Hold and Hold_until at the same breakpoint will not be executed until a Continue instruction is issued 5 3 3 Deleting instructions To remove an instruction 1 Select the instruction in the text instruction window 2 Use one of the following three alternatives select Delete from the shortcut menu or e press Delete in the Instruction box or e press on the Delete key on the keyboard An instruction that has been deleted can only be recovered by re inserting the instruction If you want to suspend execution of an instruction temporarily e g during development work you can replace the breakpoint with a value after the End_block or End_method instruction You cannot delete the Base instruction at the beginning of a block If you delete the End_block instruction the block will end at the last instruction in the block If a gradient is currently being formed the gradient will continue into the next block 5 3 4 Changing instructions There are three possibilities for changing an instruction e change the breakpoint e change parameters including variables see Method variables on page 66 e select another instruction To change an instruction e p61 5 Editing methods an 18 1138 73 e p62 1 Select the i
211. e the file Do not make any other changes in the UNICORN INI file between the lines BEGIN and END since this may severely affect the functioning of UNICORN Ka Note Logon and file handling 3 3 5 Logging off To log off from UNICORN click on the Logon Logoff icon or select the Logoff menu command Processes that are running when you log off will continue to run and may be left locked with a locking password or unlocked see Section 6 5 for more details If the Method Editor module was active at the time of logoff it will be re opened when the same user logs on again UNICORN will still be open after a user has logged off and another user may log on We recommend that you always log off when you leave the computer to prevent other users from accidentally changing or deleting your files or disturbing your runs 3 6 Quitting UNICORN To quit UNICORN and close the program select File Quit program in the Main Menu You will be prompted to save any unsaved data in the Method Editor or Evaluation module If a run is proceeding when you quit do not shut down Windows NT 2000 or turn off the computer while the run is in progress You cannot quit the program if you are performing a scouting or MethodQueue run e p31 3 Logon and file handling an 18 1138 73 e p32 Note Creating methods H 4 Creating methods UNICORN is supplied with a set of Method Wizards that can serve as the starting point
212. e three levels 18 0 min Area Standard Area Internal Standard Area Standard Area Internal Standard Fig 11 6 Standard peak area relative to internal standard peak area is used to produce a point on the calibration curve an 18 1138 73 p312 Analysing results 11 4 Data from the sample are prepared in the same way producing peak sizes relative to the internal standard peak size The resulting relative value is then applied to the calibration curve to determine the amount and concentration of the component of interest mAU Internal Standard Sample 250 200 150 100 50 18 0 min Area component of interest Area Internal Standard 5 v Amount component of interest Amount Internal Standard Fig 11 7 Peak area of the component of interest is related to the internal standard peak area and from the calibration curve the amount is calculated from this ratio Reliability of the internal standard technique This is potentially the most reliable of the quantitation techniques However if the internal standard component is not carefully selected reliability will probably be worse than for the external standard technique see Internal standard quantitation on page 310 for internal standard selection guides Specific factors that affect reliability are 1 If the sample contains many peaks there is an increased risk of overlap when the extra com
213. e up and down slopes of the peaks marked by blue crosses in these regions when Edit baseline is selected By considerably decreasing the Slope limit in Figure 10 16 a better baseline can be constructed leading to an improved peak integration Fig 10 16 Peak integration with decreased Slope limit Evaluating results 10 You may also have to decrease the Noise window to get a perfect peak integration Fig 10 17 Correct baseline after decreasing both Slope limit and Noise window An alternative approach is to exclude the large peak from the peak integration as its presence affects the default baseline parameters and the retention and area of the large peak is in most cases not interesting Using the Operations Cut curve function the appropriate region of the chromatogram can be selected The peak integration can then in most cases be performed with default baseline parameters on the cut curve Noise detected as peaks Sometimes you get too many peaks after the peak integration usually because noise on the baseline is erroneously detected as peaks The cause of this problem is that the Noise window parameter is too low Increase the Noise window parameter in the baseline calculation This may in some cases result in peak limits too high up on the peak slopes see example below e p273 ED Evaluating results noisepeak DefaultNoiseWindow 322401 DetaultNo
214. e value in the parameter field applies as the default value for the variable Check the Visible in details only checkbox to set the variable as a details variable Detail variables become visible on the Variables tab if the Show details checkbox is checked This option is useful for hiding less important variables Only one variable which affects block length breakpoint or gradient length may be defined within each block Any number of other parameters may however be defined as variables within a block UV_Averaging_Time sec Colurn_PressureLimit MPa Initial_Eluent_Conditions_BP ButferPrep_pH Buffer_Inlet Flow_rate ml min Start_ConcB B BufferPrep_pH_Flush Lenath_of_pH_Flush ml Column_E quilibration Equilibrate_with CV System_Volume Ce Bensatior Compensation Volume CY Wash Gat Unbound Sample Wash_column_with CV H E 0 00 Block Start_Conditions_BP E W 0 00 Block BufferPrep_pH_Flush BufferPrep_pH_Flush 0 00 Base SameAsMain 0 00 SystemWash Coto pH Hus 0 00 Block Initial_Eluent_Conditions 6P 0 00 End_block W 0 00 Block Column_Equilibration Column_Equilibration 0 00 Base SameAsMain luate_Fractionation_V Start_Frac_at B H 0 00 Block Syste Start_Valve_Fractionation Number_of_fractions Fraction_volume ml 5 00 End_block First_fraction_in Fig 5 26 Relationship between variables in text instructions and in the Variables tab of Run
215. e values as required Note Scouting Repeat this process until you have defined all the runs that you wish to add 7 3 Scouting results 7 3 1 Viewing results Each result file in the scouting result folder represents the results of one scouting run and can be viewed in the same way as result files from ordinary runs see Chapter 9 The Scouting tab in the documentation saved in each scouting result file shows the whole scouting scheme with the values for the current run displayed in a cell with a yellow background Any changes made to the variable values during a run will only be recorded in the result files for runs after the changes were made 7 3 2 Comparing runs To compare chromatograms in results from different runs in a scouting series open a result file and choose File Multifile Peak Compare or click on the Multifile Peak Compare Wizard icon see Multifile Peak Compare on page 207 7 3 3 Printing results Scouting results can be evaluated and reports can be generated in the same way as for other result files see Chapters 9 and 10 The following settings in File Report in the Evaluation module are important for scouting Documentation Scouting includes the complete scouting scheme but does not indicate which run generated the particular result file Documentation Variables prints the variable values used during the particular run Edit Chromatogram Layout Box Header tab Scouting Variables option pri
216. e x or y plane using the multiply function This function is similar to Normalise Size except that each curve is repositioned with precise numbers instead of by eye and the instruction logged in the evaluation log 1 Click on Operations Multiply and select the curve to be multiplied 2 Select check the appropriate axis for multiplication either Multiply retention and or Multiply amplitude 3 Insert the appropriate multiplication factor and click on OK Multiply Eg Source chromatogram Target chromatogram 03 id2301 1_UV3_Onm 15 04 id2301 1_Cond 16 05 id2301 1_Cond 17 O6 id2301 1_Cone 18 07 id2301 1_pH xl 13 xl Curve name UV1_280nm 01 MUL Multiply type J Multiply retention jo J Multiply amplitude jo Cancel Help Fig 9 42 Multiply dialogue 9 4 6 Mirror images of curves A very useful way of comparing the features of two curves is to produce a mirror image of one curve To achieve this 1 Select Operations Multiply 2 Select the desired curve to be mirrored and select Multiply amplitude in the Multiply type field e p229 ED Presenting results an 18 1138 73 e p230 3 Type in a multiplication integer of 1 and click on OK 4 Shift the mirror image curve downwards for an improved presentation see Alternative B above Now the mirror image of the original curve will be displayed in the active window Select deselect for the other curves wanted in the active
217. eak among all curves check the Height check box Presenting results 9 Peak Selection x r Curve id186Level 1 to 4002 1_UY1_280nm Peak Table id186Level 1 to 4002 1_UY1_280nm 01 PEAKI mAU 10 0 0 0 6 0 10 0 15 0 20 0 min Select peak 6 9187 28 Next curve Pre ious Curve Set as peak Delete curve Peak Name a Delete row m 2 aie 1 m 8 270 10 530 m eos Fig 9 22 Peak Selection dialogue showing selection of initial A peak Use the Next curve and Previous curve buttons to navigate forward and backward among your selected curves manually checking the selections made by the software If desired assign a name to a chosen peak by highlighting the appropriate row and then clicking on the Set peak name button This can be useful when comparing multiple peak parameters and you wish to have peak names other than Peak A Peak B etc Follow steps 1 4 for any other desired peaks in the current curve or navigate to other curves and follow steps 1 4 above To change the designation of a peak either e click on the desired peak in the curves window click on the Set as peak button and choose None Note that you must also select the same peak in the Peak identification settings table and click on Delete row or e p211 ED Presenting results an 18 1138 73 e p212 e select a new peak click on the Set as peak button and click on the letter of the old peak the one to be
218. eak table name is colour coded All the available peaks for all curves are listed together in the Retention Mol size table Selecting the peaks and entering molecular size data Select the components of known molecular size by double clicking and enter their molecular sizes in the table The triangles show the peaks that have been selected When a peak is selected the name of its source peak table is shown above the curve window The text is coloured to correspond with the related curve This is useful when you wish to know which of two closely spaced peaks from different peak tables has been selected Using data on the standard enter the molecular size for each of these peaks as follows 1 Select the first valid peak by double clicking it on the curve or in the table 2 Double click in the Mol size column and enter the known molecular size from the standard 3 Repeat steps 1 and 2 for all the components of known molecular size 4 Ifa mistake is made a peak can be excluded by highlighting its entry in the table and clicking Exclude or by double clicking it again Choosing the molecular size curve model The molecular size curve shows the relationship between molecular size and the corresponding retention The curve is plotted from the Retention Mol size data that you have entered in the table Before this can be done a curve model is needed which describes the relationship between molecular size and retention 1 Choo
219. eaks if used Absolute and Relative window width While Peak identification is set to Absolute retention the peak window width can be displayed as Absolute or Relative by clicking the appropriate button in the Identification Settings dialogue Identification Settings xi rm Peak identification Identify peaks on Absolute retention and Highest peak maximum x Window width as Relative Absolute r Reference peak for relative retention Component z Window jo min Cancel Help Fig 11 17 Identification settings dialogue e p327 ED Analysing results an 18 1138 73 p328 Select Absolute to show the window width of each peak in minutes or ml Select Relative to display the width of each component as a percentage of its retention If Peak identification is set to Relative retention Window is set automatically to Relative except for the reference peak When component selection and identification setting is complete click OK to open the Quantitation table dialogue Quantitation table Int_Std_Ribo x Components r Calibration curve Area Sample Area Intemal Standard Cytochrome C Lysozyme Amount Amount Internal Standard Enter amount or concentration for every level r Curve model C Linear Linear through origin Quadratic C Quadratic through origin Point to point r Statistics Correlation 0 9842 More Dre
220. ect an appropriate smoothing parameter value from Light to Hard for the selected filter This can be done using the Filter width slider or you can manually insert a value in the Filter width text field The smoothing effect increases with increasing parameter values Smoothing is always a compromise between noise removal and preservation of peak shape The easiest way to find the optimum smoothing effect is to start with a low parameter value e g the default value and increase it until the best result is achieved A useful strategy is to increment the parameter value by the default value for each try 5 Click on OK The formulae for the filters are described in see Smoothing algorithms on page 369 9 3 4 Subtracting a blank run curve This is a frequently used function in presentations especially if the curves have a drifting baseline or ghost peaks Note Ifthe ghost peaks come from impurities in the eluents all equilibration of the columns should be the same from run to run If for example the equilibration volume with buffer A is larger before a blank run curve than before a separation your ghost peaks might be higher in the blank run curve e p201 ED Presenting results an 18 1138 73 p202 Fig 9 14 UV curve with baseline prior to subtraction of the baseline Fig 9 15 UV curve after subtraction of the baseline Alternative A Importing a blank run curve If a blank run curve was done this may have
221. ect the instruction where you want to rename the variable The parameters for the instruction will be shown in the Instruction box 2 Locate the required parameter in the Instruction box Press the VAR button 3 Enter a new variable name in the dialogue and click on OK e p69 Editing methods Note Note an 18 1138 73 e p70 5 5 Run Set up The Run Set up is a tabbed window for defining the method properties To access the Run Set up tabs press the Run Set up icon at the top of the Method Editor toolbar To access a given tab in the Run Set up click on the respective tab 5 5 1 Variables The Variables tab lists all variables used in the method with their default values organised by method block You can change the default values to create a variant of the method see Chapter 4 Note that in a scouting method values for variables included on the Scouting tab cannot be changed on the Variables tab Evaluation Procedures Variables Scouting Method Information Result Name Notes Questions Gradient Start Protocol ButterPrep Columns Frac 950 Reference Curves Value Range Alkyl Superose_HR_5 5 v Position Bypass hd 280 190 700 0 190 700 0 190 700 Variable Start_Conditions_BP Column_position Wavelenath_1 nm Wavelenath_2 nm Wavelength_3 nm UV_Averaging_Time sec Column_PressureLimit
222. ection factors and save using either Save or Save as The original recipe will now be updated or a new recipe added to the list respectively e p137 Editing methods Note an 18 1138 73 e p138 If parameters for a certain recipe are changed and the same recipe was earlier selected and saved in a method the recipe in the method will not automatically be updated with new parameters When opening the method a question will appear asking if you want to update the parameters in the method recipe It is recommended that you answer Yes The question will not appear if you only change the correction factors The correction factors in the method recipe will not then be updated Fine tuning with correction factors In order to obtain high pH accuracy a recipe can be fine tuned around a specific pH by setting correction factors Run BufferPrep manually at 0 and 100 and change the default correction factors as follows 1 Select the Recipe under group Other in System Control Manual Click on Execute Set the pH in the instruction BufferPrep_pH in group Pump Click on Execute Set the flow rate to be used during the run in instruction Flow Click on Execute 2 Check the pH reading when stable Allow at least 30 ml of eluent to pass through before expecting a steady pH reading 3 Change to 100 B by setting the Gradient instruction in Manual Pump to 100 for Target and 0 for Length Click on Execute 4 Check the pH reading wh
223. ed A H Make the appropriate Baseline selection from the above mentioned possibilities The Calculate baseline option with the default settings for the parameters is the most common choice Note There are two choices of algorithm for baseline calculation Morphological and Classic The default setting is Morphological for more details see Optimising peak integration on page 262 4 Click on OK to perform the peak integration when you are satisfied with your selections Following integration the peaks in the chromatogram will be automatically labelled with their respective retention times The start and end point of each peak will be marked by drop lines The peak table will be displayed underneath the active chromatogram an 18 1138 73 p260 Evaluation id186Level 1 to 4002 1__ v UNICORN Local fil Niklas result Unicorn40011 tid186Level 1 to 4001 id186Level 1 to 4002 res we File Edt View Integrate Operations Procedures Window Help Evaluating results 10 a n La S w E id186Level 1 to 4002 1_UV1_280nm id188Level 1 to 4002 1_Inject id188Level 1 to 4002 4_UV1_280nm 01 BASEM 1d188Level 1 to 4002 1_UV1_260nm 01 BASEM1 AU 827 p50 foo 11 68 10 46 iso Ho 0 fo 956 po No Retention min Area mAU min te 1 0801 3 7804 7 7133 Fig 10 2 The results after peak integration In addition to peak areas several
224. ed in the method run is selected with the Base instruction and may be changed at the beginning of the run on the Variables tab if the columns parameter is defined as a variable see Variables on page 70 Reference curves If this box is checked the reference curves associated with the method will be displayed at the start of the run You can add delete and rename curves at the start of the method All curves in the list can be displayed in System Control during the run Evaluation procedures If this box is checked the evaluation procedures set to execute at the end of the method will be displayed at the start of the run You can change the choice of procedures to execute but you cannot add or e p97 5 Editing methods an 18 1138 73 e p98 remove procedures Procedures are stored as part of the method file which cannot be changed at the start of the run Method information If this box is checked the method information including creator target system strategy information and date and time of creation and latest change will be displayed at the start of the run You cannot edit the method information There are also tabs for Signatures associated with the method as well as Method Duration The Method Information tab is for information only and cannot be edited Settings If this box is checked the settings including alarms monitors and curve configuration will be displayed for information at the start of
225. ed text field Click on Change text or Delete text 3 Toalter the font or text orientation select the specific text that you want to modify and click on Font or Set Orientation Make the desired changes in the resulting dialogues 4 Click on OK to close the dialogue and apply the changes 9 3 7 Pooling fractions During a separation fractions are sequentially collected with each fraction containing a set volume of sample Each fraction is numbered according to its order in the sequence and this information is saved as a curve under the name Fractions Selection of this curve in the Chromatogram Layout dialogue will display the precise contents of each fraction relative to the information displayed on the UV detection curve Since the peak of interest may not always be collected in one fraction it may be desirable to pool some fractions To pool the fractions 1 Select Operations Pool fractions The Frac curve is automatically displayed for the active chromatogram The chromatogram selection can of course be changed 2 In the Fraction no field check the fractions to be pooled 3 Select the destination for the new curve The default name of the new curve has POOL as its suffix 3 Click on OK e p205 ED Presenting results Note an 18 1138 73 p206 The active chromatogram will now display both the original and pooled fraction curves so uncheck the original fraction curve in the Chromatogram Layout dia
226. ed upon criteria you determine Click on the Reject peaks command button in the Integrate dialogue In the dialogue that is displayed select check the criteria and parameters by which peaks will be excluded from the integration You are able to define the minimum height width and area the maximum width as well as a specified number of the largest size peaks The default criterion is to include only the 20 largest peaks e p263 ED Evaluating results Reject Peaks xi ri ooo mau T Width less than fo 000000 min J Area less than 0 000000 min m U J Width more than o 000000 min JV Peak must be one of 20 largest Cancel Help Fig 10 5 Reject Peaks dialogue Selecting part of a curve for integration To select only a part of a curve for integration click on the Peak window button in the Integrate dialogue A chromatogram window will open containing the curve and two vertical cursor lines These lines can be dragged to define a region between them that will be analysed Alternatively X axis values for the Left limit and Right limit may be typed in Click on OK to return to the main dialogue The baseline will be calculated from the whole curve but calculation of the areas beneath the peaks is only performed on the selected section of the curve The default peak window includes the entire curve from the injection point Peak window _ ol x id186Level 1 to 4002 1_UV1_280nm
227. ee da ho eens ssid 4 idee van L deed 71 55 3 QUESTIONS ies chad Sip dees Sed Maryam sante adie eee ada oleh 76 55 4 Gradient sses derar eek ee ae Aba a hak ae be 80 Soon NOLS is me Ste Sed ane eae oy Leda ee ee ee 8l 5 5 6 Evaluation procedures 0 00 0 ee ees 83 5 5 7 Reference Curves eek bee eee de eee ee 86 5 5 8 COIUMNS nee ne eve oe ee a he ead he eG 88 59 9 BUTERPIED crc che ethno eee coat la ce ve nd ne ee BE ee 88 5 5 10 Method Information 000 000 eee 91 5 5 II sRESUIENAMOX seth ch retains thas ewe kate pale Ad 91 Dod Prac 950 nated eat a e ee ee ae gee an 93 Sole Stare Proto CO oe es eRe eg hee Eee re ees 96 5 6 Saving the method 0 ce eee eee eee eee 99 5 6 1 Saving Method 4 8 4 84 24 64 Sak ote hate he 28 99 5 6 2 Saving as a template 0 0 ee 99 5 6 3 Deleting a template 0 0 0 0c eee 100 5 7 Printing the method 0 cece eee ee eee 101 5 8 Exporting Methods 0 0 ce eee eee 102 5 9 Exporting Run Set up 0 000 cece ee eee 102 ill 5 10 How to use selected unconditional method instructions 103 5 10 1 Base instruction n on aaan anaana eee 103 5 10 2 Instructions at the same breakpoint 105 5 10 3 Block and method length 0050 106 5 10 4 Messages and Set_mark 0 0 ccc eee 108 5 10 5 Pausinga method 0 ce eee 109 5 10 6 Linear flow rates ee ees 110 5 10 7 Eluent conce
228. eleting templates 5 7 Printing the method You can print a copy of the method including items from the method documentation Run Set up and the Text instructions window 1 In the Method Editor select File Print or click on the Print icon The Print dialogue is displayed Only items available from the Method Editor can be selected Method Information C Signatures Method Duration Method Variables O Scouting Text Method O Current Expansion O Exclude Unused Blocks O Questions O Evaluation Procedures O Reference Curves O Result File Setup O Columns O BufferPrep Recipe O Instruction Set C Method Notes Cancel Help Fig 5 55 Print dialogue within the Text instruction view If you would like access to the Current Expansion print option you must select the print command from the Text instructions view By checking the Text method option all instructions will be printed including those in the unused blocks If you check the Print current expansion option the method will be printed according to the current expansion in the Text instructions window If you check the Exclude unused blocks option only those blocks used in the method will be printed 2 Check the options that you want to print out e p101 Editing methods an 18 1138 73 e p102 3 Click on OK to print 5 8 Exporting Methods This dialogue should ideally be opened while in Text Instructions mode To ex
229. ems will be included in the report Documentation rm Select Items VManables nn V Scouting V Start Protocol MQuestions Reference Curves Evaluation Procedures Vlinfo VMethod Notes VStart Notes xl Clear All Print Preview Fig 9 62 Create Standard Report Format dialogue Documentation tab 9 9 Run documentation The full documentation of a run is stored within the result file Some of the documentation functions are described below Chromatogram Once the desired formatting choices have been made clicking on the Preview button will display the page layout Press the Print button to print out the report Also you can save the modified format under a new name by clicking on the Save As button To open the Documentation either select View Documentation or click on the View Documentation toolbar icon To print documentation contents click on the Print button in the Documentation dialogue see Printing active chromatograms on page 231 page and clicking on Save as The contents can be saved as a new method by selecting Method e p251 ED Presenting results an 18 1138 73 p252 Variables The Variables tab lists the parameters that were used during the run Method Information Result Information Result Name Start Protocol Settings Calibration Logbook Evaluation Log Frac 950 Variab
230. en stable at 100 B 5 Ifthe readings are acceptable at both 0 and 100 the correction factors do not need to be changed 6 Ifthe readings are not acceptable click on Correction factors in the Edit Recipe dialogue File BufferPrep recipes Edit in the Method Editor Correction Factors x pH deviation at 0 B fo Cancel pH deviation at 100 B Hel fo rl Fig 5 73 The Correction Factors dialogue Editing methods 5 7 Enter the deviation at 0 and 100 Example if the pH is set to 7 0 and the actual pH is 7 1 enter 0 1 if the actual pH is 6 9 enter 0 1 Note If correction factors already exist the measured pH deviation should be added to the old factors 8 Click on OK and then save the recipe e p139 5 Editing methods an 18 1138 73 e p140 Performing a run GH 6 Performing a run This chapter describes how to perform and monitor runs from the System Control module Calibration of system monitors is also described 9 System Control 1 Explorer100 Method c Default ld23 m01 Result c Default Id23001 res PIE Ee Yen Mend Sytem Hep We Run Hold Ranse Continue End ae ee ace a S Conc SamplePres Cond H Pressure Auxint SampleFlow m iasteF1 g 0 00 min Run sared Logbook 1 60 min Pause 3 92 min End For Help press F1 Controlled By defaut Fig 6 1
231. ents before the Quantitation table can be saved Note If relative retention is used for peak identification and the reference an 18 1138 73 p332 peak is not used for quantitation amounts must still be entered for the reference peak and a model must be selected before the quantitation table can be saved Enter dummy values and select any model Saving the quantitation table As this is a new table it can only be saved at this stage by use of the Save as button Save quantitation table xi Quantitation table s Ext_Std_Chym Int_Std_Ribo protein_std_01 Global Personal i Quantitation table name per ooo oo Cancel Hep Fig 11 22 Save quantitation table dialogue The Save quantitation table dialogue provides a listing of existing quantitation tables and enables you to enter the name for the new quantitation table Analysing results CH You can choose whether the table will be globally accessible to any user or only to someone using your user id The default is Global Click Personal if access is to be restricted 2 Enter a name for the table This name may be up to 20 characters long and may contain letters A Z digits 0 9 and the underscore character 3 Click OK to save the table Note Once the table is saved under Save as it can be updated using the same name by use of the Save button Care is needed as this overwrites the original table You might prefer to use Save as and
232. epharase_HP Global Column volume asl Column volume unit ml i HIC 55 0 0 25 Default flowrate 1 0 Max flowrate 1 0 Typ peak width at base 12 pH High value longterm 13 pH Low value longterm 2 pH High value shortterm 14 H Low value shortterm 2 HiLoad_ 26 60 Superdex _ 2005 _prep_grade Global p ania A HiLoad_26 60_Superdex_20_prep_grade Global seed particle diam n at pm Hiload_26 60_Superdex_75_prep_grade Global Typical loadi 25250 HiPrep_16 10_Butyl Global ypicai loading range mg HiPrep_16 10_CM Global Scan rate Spectra sec HiPrep_16 10_DEAE Global HiPrep_16 10_0ctyl Global Fig 5 67 The Column List dialogue 2 Click on New to display the New Column dialogue New Column T Column volume Column volume unit Technique Ve vo Max pressure Default flowrate Max flowrate Typ peak width at base PH High value longterm PH Low value longterm BH High value shortterm BH Low value shortterm Average particle diam Code no Typical loading range Spectra sec Fig 5 68 The New Column dialogue e p129 5 Editing methods an 18 1138 73 e p130 The Parameter column lists the column parameters that can be altered Some of the parameters are mandatory labelled mand and you cannot save the column until these have been filled in Use the Variables column to enter the desired values The column paramet
233. eport see Creating a new customised report format on page 233 they will be treated here only briefly To add formatting to the page layout 1 Add new objects as desired Alternatively you can Cut Copy Paste or Delete objects using the shortcut menu 2 Edit existing and new objects by moving them to new positions resizing them or changing their properties Change properties by clicking on an object with the shortcut menu This generates a shortcut menu that allows you to the highlighted section 3 You can also modify the contents of the highlighted report section by choosing the Properties command From the shortcut menu click on Properties make the desired choices on the resulting dialogue and press OK 4 Once the desired formatting choices have been made clicking on the Preview button will display the page layout Press the Print button to print out the report Also you can save the modified format under a new name by selecting File Save As Standard format Choosing the Standard format button generates a second Edit Standard Report Format dialogue with tabs representing format options for the major sections of the report The options within the various tabs are identical to those available when creating a new standard format see Creating a new standard report format on page 247 Presenting results 9 Create Standard Report Format xi Evaluation Log Frac 950 Contents Header Method The selected it
234. erase the currently selected line from the MethodQueue click on Delete In the dialogue box check the system s for which the method is to be deleted 8 3 Running a MethodQueue Before starting a Method Queue run make sure that all systems used in the MethodQueue are connected with control mode connections and are in End status The system associated with the first System button in the MethodQueue definition must be connected to System Control window 1 the second system to System Control window 2 and so on The MethodQueue will not start unless all required systems are connected in control mode To start a MethodQueue from the Main Menu select the MethodQueue icon and select File Run or select Run from the shortcut menu The MethodQueue will start in accordance with the conditions defined in the MethodQueue setup You cannot start or end a MethodQueue from the System Control window Run MethodQueue authorisation is required to start a MethodQueue 8 3 1 Method execution in MethodQueues The start protocol for the first and each subsequent method step in the MethodQueue is displayed when the corresponding method is run If you require unattended MethodQueue operation after the start of the first method step make sure that subsequent method steps do not include a start protocol epl8l og MethodQueues an 18 1138 73 e p182 The Condition setting for each step in a MethodQueue determines the relative timing of the steps If s
235. ers are used in UNICORN in the following ways Parameter Height Diameter Column vol ume Column vol ume unit Technique Vt Vo Unit cm cm ml or uL or nl or litre ml or ul or nl or litre ml or ul or nl or litre ml or ul or nl or litre Mandatory yes yes yes no yes no no Comments Calculation of N m Calculation of column volume Calculation of volu metric flow from a set linear flow Calculation of column volume Used in method with CV as base Automati cally calculated from Height and Diameter Not possible for user to set directly The column volume is calculated in the set unit The set unit is also used for Vt Vo and flow rates Decides for which tech nique the column should be available when New Method is selected Total liquid volume used to calculate the capacity factor after an integration Void volume used to calculate K after inte gration Max pres sure Default flow rate Max flowrate Typ peak width at base pH High value long term pH Low value long term pH High value short term MPa ml min or litre h or ul min or nl min ml min or litre h or ul min or nl min ml or litre or ul or nl yes yes no no no no no Editing methods 5 Used for setting pres sure limit automati cally in method Warning when method is saved and started if the pressure limit is set
236. erview 167 performing a run 172 pH scouting example 175 preparing a method 71 printing results 173 results 172 sample volume example 174 scheme 71 scheme set up 171 screening different columns example 174 setting contents of report print 173 setting parameters 71 setting up 167 setting variables in the scouting page 171 start protocol 76 start protocol settings 171 using different columns 73 variables 143 viewing results 173 scouting runs using automated update and quantitation 351 scouting scheme setting up for automated update quantitation 353 screen problem with fit of method editor window on screen 401 search filters 218 security access 14 backup 30 connection 14 data 14 features 14 system in UNICORN 2 select peak table for quantitation 341 source chromatogram 341 um 18 1138 73 e 438 series button 72 set mark during run 108 settings display 144 display at start of run 98 shift curves by offset 223 sigma 377 skim peak ratio calculation 265 slope limit 269 adjustment 272 slope values 287 determination 288 smoothing algorithms 369 curve 200 parameter 201 sorting order of files 23 spiked sample standard addition 315 spiking definition 304 splines through curves 291 stack imported curves 223 stacking and stretching curves 226 stand alone installation 13 standard addition description 308 detailed description 314 entering the added amount 343 preparations 342 reliability of technique 315
237. es Question Answer r Advanced IV Include subfolders C Date trom 17 171880 to 1 16 2001 Close Help Fig 3 8 The Find file dialogue When you have entered all parameters click on Search The result of the search is shown in the Found folders and files field Double click on a file in this list to return to the Main Menu with the selected file highlighted in the appropriate window If you click on Close with or without selecting a file you will return to the Main Menu with the window display unchanged e p25 3 Logon and file handling Note 1 Note 2 Note 3 Note 4 Note 5 an 18 1138 73 e p26 3 3 6 Copying and moving files and folders You can copy and move files and folders to another folder that is specific to your user name You can also copy or move files to and from an external drive and folders available on the network If you copy or move a folder all files within the folder will also be copied or moved Copying or moving files and folders 1 Select one or more files or folders in either the Methods or Results window of the Main Menu To select multiple files or folders use the standard Windows function keys lt Ctrl gt or gt Shift gt 2 Click with the right mouse button on any file folder icon and choose the Copy or Move command from the shortcut menu or select File Copy or File Move The Copy or Move dialogue is displayed respectively 3 Select a
238. ess 3 3 1 Creating a new folder To create a new user specific folder 1 Select the appropriate window Methods or Results in which you want to create a new folder 2 Select File New Folder or New Folder from the shortcut menu The Create New Folder dialogue is displayed 3 Enter the name of the new folder and click on OK The new folder is displayed in the appropriate window Any user that has access set to the main folder in which the new folder was created also has access to the folders and files contained therein 3 3 2 Opening and running method files To open and edit a method file in the Method Editor click on a file in the Methods window and select File Open or click on the file with the right mouse button and select Open from the shortcut menu Alternatively double click on a file to open it Logon and file handling Ep Methods Ee o Default Prev Folder fa 1d23 Wizard Method File fz test2o Wizard Method File Explorer100 Method File a Run g n Wizard Method File Rename Copy Move Delete Copy to External Fig 3 4 The Method window with shortcut menu selected Method files can be run directly in the System Control module Alternatively click on a file in the Main Menu Methods window and select File Run or click on Run in the shortcut menu 3 3 3 Opening result files and Quick View Result files located in the Result window can be opened in the same way as method files
239. ess see Integrating peaks on page 259 nAU 250 200 150 100 50 Analysing results CH Standard 3 levels l OJA 15 0 15 5 16 0 16 5 17 0 17 5 ml Fig 11 2 Chromatographic curves from three levels of the standard The peak tables from the series are used to produce a calibration curve for each component This curve shows the relationship between amount and peak size This curve is used to obtain quantity data from the sample Area Amount Fig 11 3 Calibration curve for one of the components based on peak area versus amount prepared from the standard levels A run is then performed with the sample and the curve is peak integrated The components of interest are identified from the sample peak table by the peak identification settings Using the peak size s the concentration and amount are calculated from the calibration curve e p309 ED Analysing results an 18 1138 73 e p310 mAU Amount Fig 11 4 Calibration curve uses sample peak area to determine amount Reliability of the external standard technique For a description of the general factors that affect the reliability see General factors affecting reliability on page 318 Factors specific to the external standard technique are 1 Precision is limited by changes that may take place between runs e g column degradation and mobile phase variations 2 There is no
240. esults 10 1 Select the Source Chromatogram and thereafter the curve for which a simulated peak fractionation is to generated 2 Select the Destination Curve 3 Enter a Curve name if other than the suggested name 4 Enter values for Parameters Fraction size Min width Start slope and or End slope 5 Click on OK The simulated peak fraction curve is displayed on the chromatogram and alignment of the fraction label text can be changed in the Chromatogram Layout box 10 2 3 Finding the slope values for Peak Fractionation or Watch instructions With AKTAdesign systems it is possible to only collect peaks during fractionation Peak fractionation parameters are set in the Method Editor with the instruction Peak_FracParameters StartSlope and EndSlope values are set The procedures for finding suitable slope values for a particular run are described below It is also possible to set up conditional Watch instructions which allow the progress of a run to be determined by the events during the run e g start collecting fractions when the first peak emerges The slope of the curve may be set as a condition used to satisfy a Watch condition in the method during the run It is therefore important to use accurate slope values for the specific Watch instruction parameter sl Fig 10 33 The slope of a curve e p287 ED Evaluating results an 18 1138 73 p288 To determine slope values you must first make a run with the sam
241. ethod Information tab in Run Set up There are also tabs for Signatures see Signing the method on page 39 associated with the method as well as Method Duration The Method Duration presents the estimated total time and buffer volume required for the method If the method includes a scouting scheme click on the Run x button to see estimated values for the times and volumes for the different scouting runs The Method Information tab is for information only and cannot be edited 5 5 11 Result Name Use the Result Name tab to specify how the result files will be named for the results of a run and where the result file will be saved You can also specify the name of the special scouting folder where results from scouting runs will be stored e p91 5 Editing methods an 18 1138 73 e p92 Variables Scouting Notes Questions Gradient BufferPrep Columns Reference Curves Evaluation Procedures Method Information Result Name Start Protocol Frac 950 No result Result name Serial number is added to all resultnames Method name C Date Name r Result directory A ANiklas Browse Scouting subdirectory Fig 5 47 The Result Name tab in Run Set up The result file name is constructed by adding a 3 digit serial number to one of the base options listed below The serial number is incremented automatically each time the method is run e the Method name plus a 3 digit seria
242. eues 8 1 Setting up a MethodQueuve 0 2c eee eee 177 8 1 1 Defining a MethodQueue 2 0000 eee 177 8 1 2 MethodQueue folders and icons 180 8 2 Editing MethodQueves 200 ee eee eee 181 8 3 Running a MethodQueue 00e eee eens 181 8 3 1 Method execution in MethodQueues 181 8 4 Displaying MethodQueuves 0 200 eens 182 vi 9 Presenting results 9 1 Opening aresultfile 0 cee eee eee 185 9 1 1 Ch hromatogram ns 4 2650 os es Ee ee Se a Ss 186 9 1 2 Temporary chromatogram a a saaana aeaaeae 186 9 2 Basic presentation of chromatograms 186 9 2 1 The chromatogram window 0 000 eee eee 187 92 2 SSMOMCUTIMEMNU Fo niran D tele A g te tech rye E test 188 9 2 3 Opening the Chromatogram Layout dialogue 188 9 2 4 Choosing the curve s you want tosee 189 9 2 5 Displaying curve nameS 0 eee 189 9 2 6 Changing the colour and style of curves 191 9 2 7 Defining and positioning curve text 005 191 9 2 8 Changing and fixing the axeS 0 00 00 00 eee 192 9 2 9 Viewing information about the run 195 9 2 10 Saving and applying a layout 00 195 9 2 11 Displaying a hatched background in the chromatogram window 0 00 eee eee eee 196 9 3 Other presentation possibilities 000 196 9 3
243. ew name choose File Save As and enter the details as described below 1 Enter a name for the method Method names may contain letters A Z and digits 0 9 The case of letters is not significant The method name must be unique for the chosen system within the folder see steps 2 and 3 below 2 By default the method will be saved in your home folder To change the folder double click on the appropriate folder icon in the Methods panel c Default For System Name System Size Type cs Prev Folder tz 1d23 Explorer10 474KB Method File est20 Explorer10 312KB Method File l Test20 0010 Explorer100 310KB Method File Untitled Explorer10 475KB Method File Technique Anion_Exchange Cation_Exchange RPC HIC Eoo Size_Exclusion Affinity Method name a23 Chromatofocusing OK Cancel Help Fig 5 53 Save As dialogue for saving a method 3 Ifyou have more than one system available choose the system for which the method is intended The method can only be run on the strategy for which it is saved Remember that different systems may have different configurations and control capabilities 4 Click on OK 5 6 2 Saving as a template You can save the method as a template if you have Edit global lists authorisation see the UNICORN 4 0 Administration and Technical Manual 1 Choose File Save as Template The Save As Template dialogue is displayed e p99 Editing methods Save as Template
244. executes the currently marked instruction if the list is empty Note that although all instructions are executed simultaneously some for example gradient and fraction instructions may take some time to complete in the liquid handling module The Delete button deletes the selected instruction from the current list One instruction can be deleted at a time Performing a run GH If you close the dialogue by clicking on the Close button without choosing Execute commands in the list will not be executed and will be deleted from the command list Manual instructions can also be issued while a method is running A manual setting applies until the next method instruction of the same type is executed e g a manual Flow instruction will set the flow rate until the next Flow instruction in the method is executed Manual instructions that you issue during a method are recorded in the logbook for the method run 6 3 3 Alarms and warnings The system settings determine the acceptable limits of monitor signals during a run The limits can also be set for the current run using an instruction in the method Limits set with a method instruction override the limits set in system settings If these limits are exceeded in arun a warning W or alarm A dialogue with a message is displayed on the screen e The run continues if a warning is issued e An alarm pauses the system Warnings and alarms are displayed regardless of the activity currently in pro
245. f the component of interest The amount in the spiked sample is then determined from a calibration curve and is compared with the amount in an unspiked sample The recovery can only be determined for one component each time 11 2 2 External standard quantitation External standard quantitation is based on the use of a standard prepared in a number of concentration levels A run is performed for each concentration level and calibration curves are produced showing the relationship between amount and peak size for each component The calibration curves are used to quantitate the components in the sample The standard should contain known amounts of all the components that are to be quantitated in the sample The technique can be based on the use of a single standard concentration level but then the calibration curve is limited to a linear through the origin relationship The use of a number of different concentration levels of the standard broadens the range of the calibration curve allows the development of non linear calibration curves and improves the precision Multiple runs at each level improve precision further The following description is based on the use of a standard which contains two components and which is run at three different concentration levels An outline of the technique 1 A run is performed for each standard level 2 The curves are peak integrated to produce a peak table for each run for a description of the proc
246. ficient which shows how well the data are linearly related The correlation is displayed in the Statistics table If you are producing a calibration curve relating peak area or height to amount or concentration you aim to achieve a high positive correlation coefficient A value of 1 indicates a perfect fit of all the data to the straight line Note If you have only two data points for a Linear model or only one point for a Linear through origin model the fitted straight line will inevitably pass exactly through the points By definition this leads to a correlation of exactly 1 but this does not indicate a good fit but instead too few data points In these cases the Statistics table will show a symbol instead of the correlation value A molecular size curve has a negative slope so the aim is towards a correlation coefficient of 1 with the same note regarding too few points Correlation is derived as follows Pie Correlation X x Eo 37 i where X and Y are the averages of the x and y values respectively For the molecular size model Linear log Mw X is the average of the logarithms of the molecular sizes C 3 2 Explained variance Explained variance provides a measure of how much of the variation in the data points xy pairs is explained by the model The remaining variation can be attributed to noise i e random errors or selection of an inappropriate model This makes it possib
247. for creating customised methods These wizards are defined with variables for critical parameters in the separation so that customised methods can be created for most purposes simply by setting appropriate values for the method variables Different wizards are provided for different system strategies This chapter describes how to create methods for advanced method editing facilities see Chapter 5 Wizards are available for KTAdesign systems delivered with standard strategies 4 1 Creating a new method There are three ways to construct a new method e Using the Method Wizard e From an existing method Template e Within the Method Editor 4 1 1 Method Wizard The Method Wizard is designed as a user friendly option for constructing new methods To start the Method Wizard 1 Click on the Method Wizard icon or select File Method Wizard from within the Method Editor screen or select the Wizard radio button in the Use field of the New Method dialogue This will prompt the initial Method Wizard dialogue Method Wizard for System WINCORN LAB9 NT xj p Technique Column and BufferPrep Technique Column Position Position Bypass BufferPrep C ON OFF Back Next gt Cancel Help Set Default Fig 4 1 The Method Wizard start dialogue e p33 gt Creating methods Note an 18 1138 73 e p34 The options available on the subsequent Method Wizard dialogues depend on which choices
248. g from external 27 copying to external 26 location 92 naming 91 opening 21 320 overview 5 selecting an alternative 321 showing which quantitation table was used 341 specifying name 144 results after peak integration 261 changing result name after start of run 98 copying results to the clipboard 301 opening a result file 185 presenting results overview 185 saving results 231 viewing scouting results 173 viewing the results of internal external standard quantitation 341 viewing the results of molecular size calculation 367 viewing the results of recovery calculation 346 viewing the results of standard addition quantitation 344 e 435 direct measurement of retention time and peak heights 281 displaying peak retention 325 measuring retention time and peak heights 281 Mol size table 362 retention multiply 229 run adding scouting settings during a run 172 changing scouting settings during a run 172 comparing different runs 206 comparing scouting runs 173 control mode connection 41 data 145 documentation 251 editing method variable values 41 logbook 156 messages during run 108 method file 21 MethodQueue 181 monitoring 145 performing 141 319 problems 401 set mark during run 108 start protocol pages 42 starting 41 starting an instant run 142 status display options 10 viewing information about a run 195 run data layout 146 manual instructions 148 pressure units 148 window style 147 run documentation calibration 253 evaluatio
249. g the gradient If an End_Block instruction is encountered during a gradient the gradient formation will continue uninterrupted unless a new Gradient instruction is issued in the next block This means for example that a block can be called conditionally during gradient formation without interrupting the gradient Gradients with variable duration For many purposes it can be useful to define the length of the gradient as a variable When this is done breakpoints for instructions issued during or after the gradient in the same block are automatically shifted in proportion to the length of the gradient with the same functionality as Change in the text instruction editor see Changing instructions on page 61 Gradient functions over time and any instruction that you want to insert after a gradient should be placed after the combined breakpoint and gradient length 5 11 How to use selected conditional method instructions Conditional instructions allow the progress of a run to be determined by the events during the run e g start collecting fractions when the first peak elutes or equilibrate the column until the eluent conductivity has reached a given value This is facilitated by the Watch instructions Editing methods 5 5 11 1 Hold_until instruction Hold_until is a special kind of Watch instruction The method is put on hold until a specific condition is met signal test or value or the timeout is reached Thereafter the
250. g the samples 354 selecting update by replace or average 350 setting the procedure 351 the stages 350 variable name definition 352 autoregressive algorithm 369 autoscroll in logbook window 157 average for update 337 averaged replicates column 339 axes fixed scale 194 198 backup security 30 base changing 65 81 choosing 53 instruction 103 type 65 baseline algorithm used for calculation 262 calculation 259 calculation theory 371 classic algorithm 371 default parameters values 269 defining baseline parameters 371 deleting baseline data points 278 drawing the baseline 373 drawing the new baseline 278 estimating the baseline parameters from the source curve 374 insertion of baseline data points 277 manually editing 276 measuring noise level using curve co ordinates 374 measuring shortest baseline segment using curve co ordinates 374 measuring slope limit using differentiate and curve co ordinates 374 morphological algorithm 371 optimising the baseline parameters using a morphological algorithm 266 269 selecting baseline points 373 selecting the baseline calculation algorithm 263 structure width 266 baseline problem baseline on top of peaks 275 baseline slope does not follow the source curve 270 bases for method blocks 7 batch runs 298 BioPilot System methodbase instruction 105 block adding 51 calling 50 calling from an existing block 53 choosing base 53 controlling block and method length 106 copying within a method 56
251. g volume of the sample instead of in the injected volume of the sample Information about any internal standard being used If you are preparing for external standard quantitation or measurement of recovery factor the default option Not selected should be chosen If internal standard quantitation is being used open the Internal standard peak menu and select the component which represents the Internal standard Next enter the injected IS amount or IS concentration in the standard and sample runs Quantitate can base the calibration curve on peak area or peak height Peak area is usually used and this is the default You may wish to change Quantitation peaks to Height if peaks are not completely separated from those of other components Entering the data for the standards Check that the components selected are correct If you wish to edit the list click Define components to return to the Define Component s dialogue e p329 ED Analysing results Note an 18 1138 73 p330 If relative retention has been used the reference component is marked Ref If an internal standard is in use the related component is labelled IS Quantitation table x Components r Calibration curve Internal standard Ribonuclease Cytochrome C Concentration Enter amount or concentration for every level 16 2530 7 7133 3 7606 Quadratic C Quadratic through origin Point to point r Statistics Exp
252. ging the Column Column_position and Flow_rate for each column 7 4 3 Scouting continuous gradients Scouting continuous gradients involves finding the best final concentration and gradient length for elution of the product from a given column These two parameters are set as variables Scouting Block ___ Runt Run Run3 Gradient_Segment_1 arget_ConcB_1 B 100 00 100 00 100 00 Oooo Cf Length_of gradient 1 Cv 5 000 10 000 0 000 OE S y Fig 7 7 Scouting for optimal gradient slope and length 7 4 4 pH scouting With BufferPrep it is possible to vary the pH automatically in the different runs Block Variable Runi Run Run3 Initial_Eluent_Conditions_BP BufferPrep_pH 3 000 4 000 5 000 a LL i Fig 7 8 Scouting for optimal pH epl75 Scouting an 18 1138 73 e p176 MethodQueues 8 8 MethodQueues MethodQueues provide a means for linking several methods together on the same or different systems For example if a system wash procedure is programmed in a separate method it can be linked in a MethodQueue to a series of different process methods ensuring that the same wash procedure is used before every process Alternatively the product of a separation on one system might form the starting material for a separation on the next allowing fully automated multi step processing Specific user authorisation is required to edit and run MethodQueues separate
253. graphy runs are programmed as methods in UNICORN This section gives a brief overview of the concepts and principles of methods See Chapters 4 and 5 for a description of how to program methods and Chapter 6 for how methods are used to control chromatography systems 2 4 1 Method creation Methods can be developed in a number of ways including the Method Wizard see Method Wizard on page 33 by using an existing method template see Template based method creation on page 34 or by using the Method Editor see Method Editor on page 36 The Method Wizard takes the user step by step through the method creation process Method templates are basic methods which provide convenient starting points for developing customised methods Creating methods with the Method Editor is best suited for methods that require many fine scale modifications Method Wizards or templates for most chromatography techniques are supplied with UNICORN installations for AKTAdesign systems New methods are created by selecting a suitable system technique template and column By selecting a column recommended flow rate column volume and pressure limit for the selected column are copied into the method The method can then if necessary be modified on the Variables tab or in the Text instructions Fully adequate customised methods for many applications can be created simply by adjusting the values of method variables in a suitable template 2 4 2 Meth
254. gration results have to be checked for all chromatograms for additional information regarding peak integration see Integrating peaks on page 259 All integrations must be performed using the same X axis base unit Time is the recommended unit for highest reliability e The standard addition technique assumes a linear through the origin relationship between amount of component and peak size This is a good approximation for small quantities under normal conditions e Changes may take place between runs Standard addition has no way of compensating for these changes However if losses during sample preparation are constant between the two runs they may be accounted for by spiking the sample prior to sample preparation e Precision is maximised by using a spike amount which is of the same order of magnitude as the sample e Precision is maximised by performing all runs consecutively and thereby reducing systematic errors e p315 ED Analysing results an 18 1138 73 p316 11 2 5 Recovery calculation Recovery is used to determine losses which may occur during the sample preparation process Recovery can also be used to determine the recovery factor of a preparative purification or chromatographic process The recovery can only be determined for a single component A calibration curve is produced using a concentration series of an external standard The calibration curve range must cover the amount in both the sample a
255. gress in UNICORN you will be notified of an exceeded limit in a running system even if you are developing a method evaluating data or monitoring a run on a different system Warnings and alarms are also recorded in the logbook for the run In a network installation alarms and warnings are displayed on the controlling station and all stations viewing the system An alarm can be acknowledged only from the computer connected in control mode alarms are displayed but cannot be acknowledged on computers connected in view mode 6 4 If communication fails If the results of a run were to be stored on a server or another location and there is network communication failure during a run that has been started from a remote station the run will continue and the results will be saved in the Failed folder on the local station A control mode connection can be established on the local station to control the running system See the Security section of the UNICORN Administration and Technical manual for more details 6 5 Maintenance Some strategies such as for the AKTA family support the possibility to view system information for the components in a chromatography unit and to define warnings on the components for maintenance purposes epl6l ED Performing a run 6 5 1 Viewing system component information To view the maintenance functions for a maintenance supported chromatography system 1 Select System Maintenance The Maintenance manager
256. h elution and peak fractionation Elution Step gradient 1 step CHECKLIST BEFORE METHOD START ELUENTS Start Eluent Pump A Inlet 411 418 default 411 Eluent 1 Pump B Inlet B1 Prime System Pump A and B with eluents to be used Prime all the inlet tubings 411 and B1 with the correct eluents No air may be left in the tubings SAMPLE LOADING Fixed loop or Superloop manually filled prior to method start FRACTIONATION Connect outlet F3 to a reservoir for collection of flowthrough Frac 950 must be connected to outlet F2 Set start tube for flowthrough eluate and peak fractionation For example set first tube for flowthrough and then next tube for elution and peak fractionation When running a scouting run see to that these variables are set as scouting variables in the Method Fig 4 6 The Method Notes tab within the Notes tab in Run Set up Tip Creating methods H 4 4 Signing the method To electronically sign the method choose File Sign Method The Sign the Method dialogue is prompted 1 The Sign as user field shows the current user In most instances you will want to use the user shown in the User drop down list box but it is also possible to select another user from those available on the list 2 In the Meaning field provide a short text description explaining the meaning behind the signature e g Method now fully tested and approved Sign the Method c Default Id23
257. h a password to prevent other users from changing run parameters e Data security Result files can be saved automatically at pre set intervals during a run to minimise data loss in the event of system failure In a network installation results are saved on the local station if network communication fails UNICORN concepts TA e Electronic signatures Method and result files can be electronically signed thereby providing enhanced file security Security features network and administrative aspects are discussed in more detail in the UNICORN 4 0 Administration and Technical manual epld gt UNICORN concepts an 18 1138 73 e p16 Logon and file handling es 3 Logon and file handling 3 1 Logging on When you start the computer you must log on to Windows NT or Windows 2000 before you can log on to UNICORN and begin working A 1 To start UNICORN locate the program in the Windows Start LA button under Programs Unicorn UNICORN4 00 Alternatively rs double click on the UNICORN icon on the desktop if this option was selected during installation If UNICORN is already started and the previous user has logged off click on the Tools Logon menu command or click on Logon Logoff icon in the Main Menu module a UNICORN logon User name M Password pe o Cancel Help Fig 3 1 The Logon dialogue Click on your username in the list and type your password The first time you log on to the system
258. h as serial number version number etc or Specific information which contains information such as how long the component has been used how many hours for example a pump has run etc 6 5 2 Setting up maintenance warnings To set up a maintenance warning 1 Click on the Warning tab All of the system components are displayed 2 Click on a specific component for which you want to set up a warning and select Warning New from the menu Alternatively click with the shortcut menu on the specific component and select New from the displayed menu e pl63 6 Performing a run an 18 1138 73 e p164 Maintenance manager Fig 6 21 Maintenance manager dialogue Warning tab Enter the appropriate values for Periodicity enter the desired Pop up text and enter a Name for the warning type Click on Save to save the warning The new warning is added to the specific component Maintenance manager i a Valve 7 Fig 6 22 Maintenance manager dialogue Warning tab with a defined warning set up for the UV lamp Performing a run GH 5 Repeat the process for setting up warnings for the same component or other components in the list 6 Click on Close when you have made the appropriate selections 6 5 3 Viewing and resetting the warning parameters A counter is set up linked to the Periodicity that you defined for a new warning message see above By entering the edit warning mode you are able to
259. h shows the retention value of each component relative to the retention value of the reference component This reference component is marked Ref in the Window column The Window column shows the window width for each peak expressed as a percentage of its relative retention value Peaks in level 1 1 Black peak markers Ret Ref eee Component name Possible to change 2 38 Internal standard 1 98 Ribonuclease A 1 82 Cytochrome C Ref Lysozyme an 18 1138 73 p326 Fig 11 16 Peak table columns for relative retention Analysing results CH Identifying a peak within a window If any of the windows shown in the Define Component s dialogue includes more than one peak Quantitate must be advised how peaks are to be identified Use the second Peak identification menu in the Identification settings dialogue which offers the following options e Highest peak maximum the default e Closest to retention i e closest to the centre of the window see retention column in the table in the Define component s dialogue Avoid this option in combination with relative retention e Maximum peak area Examine the nature of the peaks enclosed by the window then select the option which differentiates between the wanted and unwanted peaks If there are large peaks from components which are not going to be quantitated use closest to retention The selection applies to all peaks even the internal standard and reference p
260. have shortcut keys on the keyboard which are written within lt gt marks CONTENTS 1 Introduction aisha nnana de os a ee che wee 2 UNICORN concepts 2 1 UNICORN control software 0 000 cee e eee 3 2 SUATEBIES os acres tals atte Sheds ods Sob asta bee tates 3 2 2 UNICORN user interface 0 0 00 cece eee eee 3 2 21 Toolbar GUdE ss tin eyn Se tol es ot Fe a Re ee AD 3 2 22 Software MOGUIeS aie sou xes easiest a bw ee Se an 4 223 ONIN Help erene Re ae Se KA aN 5 2 3 Files and folden iaa cee cae espa a aa aaa aa ase 5 231 Method MES 5 sieea Ae Bete dk A A rS hae ae RIN 5 Besi2 RESUMES 25 3 ctor Saas A EE E ack AANE OE ai agate 5 24 Methods eave sa oc iees sat a a a a aa a ea a oe lee ate 6 2 4 Method ier ations wwe csi ea cate sti oie ea a deren 4 6 ZA 2 Method Structures lt oxi ccek 26nd aR S oa a Bea AF 6 Z4 Buffer ENE pie naed neice Aba ra a O n aah bee ab ats 9 2 5 Scouting 6 203550 i die ei on ES eee 9 2 6 System Control 2 cee ee 10 2 61 COntrOl AGES n sna etd his ee wae ei eles eed 10 2 6 2 System connections 0 0 0 eee 11 2 1 EVAIUARION sexes aaar eis oh BROS ees 12 2 8 Network considerations 0 00 00 cee e eee eee 12 2 8 1 Stand alone installation 0 000000000000 00000 13 2 8 2 Network control from a remote workstation 13 2 9 SECUI gt seicclnc oan batece ie Oley Naas bid ae eaten ee Suh ote 14 3 Logon and file handl
261. he Hold the method is put on hold first and then continued only after the conductivity has reached a level less than 5 ms cm This is because Hold is an instruction which will be executed at its breakpoint while Continue is not an instruction but rather an action for the Watch instruction This method works satisfactorily although one drawback is that it might never end and thus consume all of the buffer if the conditions for some reason are unfulfilled See the Watch example 2 below for equilibration with a safeguard If you are not using AKTA instruments a delay of 0 1 ml min should be added after the Hold Pause instruction so that the following instruction will not be executed simultaneously with the Hold Pause instruction Main 0 00 Base CV 0 96 ml Resource_Q 1 ml 0 00 Block EQUILIBRATE Equilibrate 0 00 Base SameAsMain 0 00 Watch_Cond Less_than 5 mS cm Continue 0 00 Hold epll7 5 Editing methods Note an 18 1138 73 e p118 0 10 Watch_UV Less_than 100 mAU Continue 0 10 Hold 0 10 End_block Watch example 2 Equilibration with simple safeguard This is an example of equilibrating the column until the UV has reached a level below 100 mAU or until the column has been equilibrated with five column volumes of buffer whichever condition is met first In this way it is possible to equilibrate the column without the risk of running out of buffers Main 0 00 Base CV 0
262. he peak tables associated with this chromatogram appear in the Peak table s list Increment the level number for each new standard concentration if not set automatically The levels must be in consecutive order of decreasing concentration of the standard You can incorporate up to 10 peak tables at each level prepared from runs repeated at the same concentration Quantitate will later allocate each with an incrementing suffix e g 1 1 1 2 etc 8 Ifyou wish to remove a selected table from the list highlight it and click Remove 9 Click OK and the New Quantitation Table dialogue is replaced by the Define component s dialogue Examining the components You use the Define Component s dialogue to select the components that will be used to produce the calibration curve s Quantitate must be able to identify these components in all levels Setting the criteria by which peaks are identified is a part of this dialogue Analysing results CH Define Component s x 1 Show curve for level 1 1 fad Double click on a peak to select it ___ A A A A 44 4 44 a Peaks in level 1 1 Black peak markers Retention Window min min 0 20 Internal standard 0 19 Ribonuclease A 0 19 Cytochrome C Component name Possible to change Identification settings Exclude Hp Fig 11 13 Define Component s dialogue Looking at the contents of each concentrati
263. he same X axis base unit Time is the recommended unit for highest reliability Faroe Lever 4 to 4002 1_Uv1_260nm id e0Level 110 2002 1 inject 1d 6Level 1 to 4002 4_UV4_200nm O1 BASEM 1a ToeLevel 1 to a0021 rar Eva 1168 Fig 11 10 Curves window showing peak data from a standard curve e p319 ED Analysing results an 18 1138 73 p320 4 Check that each integration is correct and consistent If not see Optimising peak integration on page 262 for directions on how to optimise the integrations If many small irrelevant peaks are detected it may be an advantage to re integrate after adjusting the Reject peaks criteria The number of largest peaks to detect has a default of 20 and it may be helpful if this is set to a smaller value 5 Use File Save to save all the peak tables That completes preparations prior to the use of Quantitate 11 3 2 Creating a quantitation table The quantitation table contains all the necessary data such as the calibration curves needed to quantitate one or several components in a sample The procedure for creating quantitation tables is the same for both external standard quantitation and for recovery calculations They both use absolute values of standard peak data For quantitation with internal standard the peak sizes relative to the size of the internal standard peak are used to create a calibration curve Please see
264. he sample peak table 367 molecular size curve 358 choosing the molecular size curve model 362 definition 305 entering the molecular size data 362 example 358 linear logMw curve model 362 linear curve model 362 point to point log Mw curve model 362 point to point curve model 362 preparations for producing 360 quadratic logMw curve model 362 quadratic curve model 362 selecting the source chromatogram 361 selecting the standard peak table 361 selecting the standard peak tables 360 selecting the standard peaks 362 statistics 363 the stages in production 357 molecular size determination the process 360 molecular size table definition 305 deleting 365 opening 365 production from standards 357 renaming 365 saving and printing 364 showing which table was used in a determination 367 morphological algorithm 262 moving average algorithm 369 moving files and folders 26 multipliers amount and concentration 329 multiply retention or amplitude 229 name component 324 naming method 40 network considerations 12 e 429 um 18 1138 73 e 430 example 14 installation 13 new column 129 new folder 20 new method creation 33 New Method icon 19 noise window 269 noise window adjustment 273 274 noise reducing 200 nominal reference retention 339 normalise function 227 not found 339 notes 38 81 display at start of run 97 display in start protocol 143 editing 82 entering 82 in run documentation 253 tab in Run setup 38 0
265. hould be set large enough so that signal noise does not activate the conditions and small enough so that the condition is activated close to the valley or peak As a general guideline set the value to 2 3 times the noise level and 5 10 of the smallest expected peak height Setting a value that is too high can prevent a new peak from being detected after a local minimum Delta_Base Delta_Base helps the software to determine when the baseline is considered to be stable i e it defines the permitted variation for the eplld Editing methods an 18 1138 73 e p116 Stable_baseline condition For this condition to be activated the signal may not vary by more than the Delta_Base value up or down over the time interval specified in the Stable_baseline condition in the watch instruction The Delta_Base setting affects only the Stable_baseline condition WATCH WATCH Stable_baseline interval Stable_baseline interval Delta Base condition met condition not met Fig 5 65 Illustration of the Delta_Base setting The condition Watch Stable_baseline is met if the signal does not deviate by more than Delta_Base from baseline during the time interval specified for the watch The baseline value is determined by the signal at the start of the watch If the condition is not met a new interval is started with a new baseline value defined by the signal level at the start of the new interval Permanent settings for Delta _Peak
266. ht of the flat plateau of the peak using the Marker icon on the chromatogram Measuring retention time and peak heights Insert a value somewhat lower than the plateau height as the Max baseline level in the baseline calculation Fig 10 22 Correct baseline after decreasing Max baseline level If there are several rather short segments that erroneously have been incorporated in the baseline an alternative remedy is to increase the Shortest baseline segment setting 10 1 6 Manually editing a baseline Once a baseline has been calculated it is possible to add or remove baseline points on it and then draw a new baseline from the new set of data points The edited baseline curve can then be used in a new peak integration 1 Select Integrate Edit baseline 2 Choose the desired baseline from the displayed dialogue and click on OK A window will appear displaying the baseline and the curve from which it was calculated Additionally blue crosses are displayed the baseline points and their co ordinates in the Point list Evaluating results 10 Edit Baseline olx 322401 1_UV1_280nm 322401 1_UV1_280nm 01 BASE o ETE ease SEPEN 2b ob 2b ab eb sb wo 1420 mi faia H ps3 min Delete Delete all Point mode fp3629 mau Draw straight to next point Zoom mode OK Cancel Help Fig 10 23 Edit Baseline chromatogram window Insertion of
267. ic runs the curves must be peak integrated to produce the peak tables During integration the standard curves must all use the same X axis base unit Volume is the recommended unit for molecular size determination Use File Save to save the peak table before continuing Selecting the standard peak tables 1 From the Evaluation module select Mol Size Edit Mol Size Table New The New Molecular Size Table dialogue opens New Molecular Size Table x m Unit Molecular size unit label kDa r Select peak table s Source chromatogram Peak table s d Default i jid186Level 1 to 4001 1_UV1_280nm 01 PEAK Name Size Peak table s F id186Level 1 to 4001 1 UV1 28 L Baseline example 1 CON m su Baseline example 2 978KB U Baseline example 3 973KB lui id186Level 1 to 4001 1071KB sui id186Level 1 to 4002 1158KB m id186Level 1 to 4003 1081KB uli id1S6Level 1 to 4004 1083KB Ui Peak skim example 1153KB a T omen Fig 11 46 New Molecular Size Table dialogue an 18 1138 73 p360 Analysing results CH Select a result file by double clicking its icon Open the Source chromatogram drop down list and highlight the source chromatogram required Clicking Current at any time returns you to the chromatogram which was active before entering Mol Size Highlight a peak table which was prepared from the standard and click Select or double click the peak table to t
268. ical algorithm settings and B morphological algorithm with increased structure width value e p267 ED Evaluating results an 18 1138 73 p268 Conversely a too large Structure width value means that narrower peaks especially in fluctuating curves may not be properly followed This may arise when an artifact in a curve is identified by the morphological algorithm as the widest peak and hence used to set the default Structure width value Minimum distance between points The Minimum distance between points is a measure of the distance between the data points used to generate a baseline The largest number of data points is produced at the slopes of the curves and so by increasing the Minimum distance between points value fewer points will be collected on the slopes Zoomed Curves Zoomed Curves 0 0 10 0 20 0 30 0 40 0 50 0 min Fig 10 11 A curve in a chromatogram integrated using the morphological algorithm contains many data points when the Minimum distance between points parameter is set to a low value A The number of data points is reduced when the Minimum distance between points parameter is set to a higher value B Evaluating results 10 Noise window Sometimes you get too many peaks after the peak integration usually because noise on the baseline is erroneously detected as peaks The cause of this problem is that the Noise window parameter is too low Increase the Noise window parameter
269. ick on the X Axis tab in the Chromatogram Layout dialogue e p193 ED Presenting results Chromatogram layout 1 xi Curve Style and Colour Edit Texts Layout Library Header CurveNames Y Asis Axis Cuve PeakTable Axis scale C Auto 7 Fixed fo js ml IV Adjust retention zero to injection number 1 x I Apply to all chromatograms Cancel Help Fig 9 9 Chromatogram Layout dialogue X Axis tab 2 Select the appropriate choice from the Base field either Time of retention Volume or Column volume Note Some calculated curves e g baselines exist in only one base and may seem to disappear when the base is changed Curves are collected in time and recalculated for display in volume Thus switching the base between Time and Volume may slightly alter the resolution 3 To manually set the axis limits click on the Axis Scale Fixed option Type in the desired minimum and maximum values for the X axis 4 The Adjust retention zero to injection number checkbox is checked by default This function sets the time volume to zero at the injection mark i e when the sample was injected The time and volume before injection will become negative values The injection mark to be used as zero is denoted by the number an 18 1138 73 e p194 AF Presenting results 9 9 2 9 Viewing information about the run You may wish to display header information at the top of a chromatogram det
270. idth 327 activity histogram 206 added amount 343 adding blocks 51 curves 86 204 new column 128 addition component identifying for recovery 346 adjust retention zero to injection number 194 AIA export format 301 AKTAdesign system problems 403 alarms and warnings 161 algorithm autoregressive 369 median 370 moving average 369 smoothing 369 all with this unit axis function 192 amount definition 304 measuring in sample 340 amount and concentration multipliers 329 amount unit label 321 amplitude multiply 229 Analysis module evaluation procedure instructions 355 introduction 303 licence agreement 305 mathematical models used 405 overview of techniques 303 answers input field 77 multiple choice 78 no answer 78 value 78 ASCII export format 301 assigning curve names 189 e411 um 18 1138 73 e 412 asymmetry 379 measuring in peak 283 authorised questions 77 automated evaluation procedures 292 automated quantitation 347 edit the scouting variables list 353 in scouting runs 351 integrating the curves 348 performing many runs at each level 353 preparing the quantitation table 349 selecting standard concentration levels 348 selecting the quantitation table 349 351 selecting the samples 354 setting the procedure 351 the stages 349 variable name definition 352 automated update 350 edit the scouting variables list 353 in scouting runs 351 performing many runs at each level 353 selecting the quantitation table 351 selectin
271. iewing hatch marks You can display hatching in the curve window by selecting Hatch from the shortcut menu Selecting curve pressure units If the Pressure curve is displayed in the curves window you can set the displayed units 1 Click on Pressure in Run Data to display the shortcut menu 2 Select Set Unit and then the appropriate unit either MPa bar or psi from the menu cascade The selected unit is now displayed on the Y axis Alternatively 1 Select Properties from the shortcut menu Performing a run GH 2 Inthe displayed Properties dialogue select the Y Axis tab 3 Select the Pressure curve and then the appropriate pressure unit radio button Click on OK to implement the change Selecting the text alignment You can select the way that text is aligned for the logbook and fraction curve 1 Select Properties from the shortcut menu 2 In the displayed Properties dialogue select the Curve Style and Colour tab 3 Select the Logbook or Fraction curve as appropriate 4 Select the appropriate Text alignment option from Horizontal Vertical or Flyover Flyover displays the text if you place the mouse pointer over the generated mark Click on OK to implement the change 6 2 4 Flow scheme The flow scheme is a graphical representation of the chromatography system During a run the flow scheme displays open flow path s in colour and monitor signals with numerical displays The flow scheme thus shows the current
272. ile already open in Evaluation 5 Use the Source chromatogram menu to select the source chromatogram required and highlight the peak table which contains the new data 6 If the level has not already been set in the method open the Level menu and select the level which you wish to update 7 Ifthe selected quantitation table is based on concentration check or edit the Inj Volume field 8 Click OK and the Update calibration curve dialogue opens r Level no 3 Calibration curve before update p Update by Area Average Replace Deviation and limit as ae C Relative Amount Absolute Retention c Area Deviation Limit min Tanaan ell mAU min mAU min mAU min 8 78 Chymotrypsinogen A 13 9120 1 1063 1 7390 Statistics after update Cancel Help Fig 11 25 Update calibration curve dialogue Data on the selected components for the curve to be updated are shown in the table When a component is highlighted in this table its calibration curve is displayed in the window above an 18 1138 73 p336 Analysing results 11 The calibration curve to be updated is shown without taking the new point into consideration A new point is shown either in green or red If it is green the area falls within the set Limit value and this point will be used for calculation of the new calibration curve instead of the old point If it is red it falls outside this range and
273. ilot BioProcess UNICORN and kta are trademarks of Amersham Biosciences Limited or its subsidiaries Amersham and Amersham Biosciences are trademarks of Amersham plc Windows Windows NT and Windows 2000 are trademarks of the Microsoft Corporation Amersham Biosciences AB Bj rkgatan 30 SE 751 84 Uppsala Sweden Amersham Biosciences UK Limited Amersham Place Little Chalfont Buckinghamshire HP7 9NA England Amersham Biosciences Inc 800 Centennial Avenue PO Box 1327 Piscataway NJ 08855 USA Amersham Biosciences Europe GmbH Munzinger Strasse 9 D 79111 Freiburg Germany Amersham Biosciences K K Sanken Building 3 25 1 Hyakunincho Shinjuku ku Tokyo 169 0073 Japan All goods and services are sold subject to the terms and conditions of sale of the company within the Amersham Biosciences group that supplies them A copy of these terms and conditions is available on request Amersham Biosciences AB 2001 All rights reserved Preface Preface About this manual This manual provides a reference to User functionality in UNICORN version 4 0 from Amersham Biosciences AB UNICORN is a complete package for control and supervision of chromatography systems suitable for use with Amersham Biosciences systems for the AKTA design platform AKTA design systems BioProcess System and BioPilot System UNICORN consists of software which runs on an IBM compatible PC under Microsoft Windows NT 4 00 or Microsoft Windows 2000 and h
274. ilters to a maximum of 10 will be saved in the drop down menus for both Result and Chromatogram e p217 ED Presenting results an 18 1138 73 e p218 selections More than one string can be used as a search delimiter 6 by inserting a between strings and search filters are automatically saved and stored within user profiles Chromatogram selection Foe e stas Browse Result Baseline example 3 Peak Skim Example x Browse All Chromatogram il z Browse aj Search Clear Select all Fig 9 32 Open Chromatogram to Compare dialogue showing search delimiters To return to the default setting to search in all result files click on All Click on the Search button and a list of chromatograms will be displayed based on the designated search criteria A new search can be performed with new search criteria without erasing the first found chromatograms from the list Check the chromatograms that you want to import If you click on the Select All button all of the displayed chromatograms are selected for importing If you want to clear the list of displayed chromatograms click on Clear Click on OK and all selected chromatograms are shown on the Evaluation workspace Alternative B Importing using Open 1 2 Select File Open Chromatogram Select the desired result file by double clicking on it and all of the chromatograms contained within will be displayed Normally
275. imising the workspace 187 printing active chromatograms 231 renaming 206 temporary 186 viewing the curves 187 window adjustments 187 chromatogram layout choosing curves displayed 189 editing 188 opening the chromatogram layout dialogue 188 chromatogram questions 77 chromatographic techniques available 35 chromatography systems connecting several to one PC 13 classic algorithm 263 closest to retention 327 colour changing colour of curve 191 changing curve colour 150 column adding a new column 128 choosing 35 deleting 133 display selected columns in start protocol 143 editing parameters 132 list 128 parameter any 104 parameter named column 104 selecting or changing in a method 133 viewing parameters 88 columns display of definitions at start of run 97 communication failure consequences 161 comparing chromatograms from different runs 216 comparing curves 220 comparing runs 206 component examining components 322 identifying reference component 326 naming 324 selecting components for quantitation table 323 e415 um 18 1138 73 e 416 selecting the components for standard addition quantitation 342 selecting the internal standard component 329 compression of files 27 concentration entering standard concentrations 321 measuring in sample 340 concentration and gradient eluent 111 conditional Watch instructions 50 calls 51 instructions 112 connection control and view mode connections 11 status 159 system 11 types 1
276. in the baseline calculation This may in some cases result in peak limits too high up on the peak slopes Optimising the baseline parameters using a classic algorithm for a description of Noise window with the classic algorithm Another possibility is to use the Reject peaks function in the Integrate dialogue to reduce the number of peaks based on an appropriate criterion e g the number of peaks or the minimum peak height 10 1 5 Optimising the baseline parameters using a classic algo rithm The first choice when trying to optimise the peak integration is to change the baseline parameters For a brief description of the parameters and a suggested way of estimating settings for the baseline parameters from the source curve see Baseline calculation theory on page 371 The Integrate Edit baseline function may provide additional information about the cause of the unsatisfactory peak integration by revealing where the baseline points have been placed by the automatic baseline calculation algorithm When optimising the baseline calculation a change in the Slope limit will very often give the expected result An example of the effect of a too high Slope limit is that the up slopes of the peaks may be recognised as baseline segments Baselines that deviate substantially from the source curve may arise from a combination of a too long Shortest baseline segment value with a too high Slope limit A recommended strategy is to change the basel
277. ine parameters step by step and to check the resulting baseline after each change The size of the initial change depends on the cause of the peak integration problem but some general guidelines can be given When the desired effect is achieved it is advisable to go back and check a parameter value in between the two last settings to avoid an unnecessarily low or high value The default baseline parameters can be restored by clicking on the Default command button Baseline parameter Recommended initial change Shortest baseline segment 20 50 Noise window 10 30 Slope limit 25 50 Max baseline level Usually not necessary to adjust e p269 ED Evaluating results an 18 1138 73 p270 Below are some examples of common problems with peak integration and baseline calculation as well as suggestions for improvements The following problems will be discussed Baseline does not follow the source curve Peak limits too high up on the peaks Noise detected as peaks Peaks missing Baseline on top of peaks Baseline slope does not follow the source curve J Laem Fig 10 12 Baseline calculation with default parameters The calculated baseline does not follow the source curve because short curve segments between peaks in the middle of the chromatogram are not identified as baseline segments If you decrease the Shortest baseline segment by 50 the following baseline is calculated Evaluating results
278. ined with the peak to its left or right respectively The original intervening drop line is removed and all peaks are renumbered e p279 ED Evaluating results an 18 1138 73 p280 Adjusting peak start and end points The beginning of each peak is marked with a drop line above the curve and the end of each peak is marked with a drop line below the curve Peak start Fig 10 26 A drop line at the start and end of a peak Where there are two peaks beside one another the end of the first peak will be at the same point as the beginning of the next peak Thus there will be a drop line below and above the line at the same point End of first peak start of second peak Fig 10 27 A drop line between two peaks It is possible to move the drop lines for a selected peak and thus affect the area beneath the peak Evaluating results 10 1 Click on the peak of interest on the curve or in the peak table and two vertical cursor bars become superimposed on the left and right drop lines that delimit the selected peak 2 Drag the left and right drop line bars to define the new left and right limits respectively for the selected peak The drop lines can never be moved beyond any other drop line The new left and right limits are now represented by a drop line above and below the curve respectively and the peak areas are automatically recalculated 3 Drop lines on preceding or following peaks can be similarly adju
279. ing 3A LOGGING ON coe aaa a a eek 2 ER ee 17 3 2 Toolbar Guide ss aia iran renna Sadan a a aA 18 3 3 UNICORN Main Menu windows 0eceeeeeeeeee 20 3 3 1 Creating a new folder 0 00 00 ccc ee eee 20 3 3 2 Opening and running method files 000 20 3 3 3 Opening result files and Quick View 21 3 3 4 gt Presenting filGS 2 sc 3 eliotn Beek Wale r ee oe ba 22 3 3 5 Finding files 2 co cc ee ee EE PAE E E a 25 3 3 6 Copying and moving files and folders 4 26 9 3 7 Deleting TIES 88 fe i oa ale hs ith etd ie Mee ole 29 3 3 8 RENAMING Ml S v2 desi oy Sore Mae eae ea ea eke hake 29 3 3 9 Backup security 0 0 cee ee 30 3 4 Printer setup narr aonainm ose cits eos es ati eee 30 3 4 1 Setting the margins ie sie be eee tak oie eet Sea 30 3 0 LOSING Off ccs ves eee eee ee A a ed te we eS 31 3 6 Quitting UNICORN 0 cee es 31 4 Creating methods 4 1 Creating anew method 0 0 0 cee ees 33 4 1 1 Method Wizard 2 0 6 0 4 6 605 eb oe eee ee een 33 4 1 2 Template based method creation 0 00005 34 Alo Method EGitOr oor enet ths Bed oes gery wise de ea ede de dees 36 4 2 Editing method variables 2000 eee eens 37 4 3 Method notes 200 a eee eee 38 4 4 Signing the method 0 0 00 cee eee ees 39 4 5 Saving the method nananana cee eee ee 40 4 6 Starting a run 2 eee 41 4 7 Editing text ins
280. ing a method 141 starting a method from the Main Menu 141 starting a method from the System Control 142 structure of a method 6 text instructions display in start protocol 143 using selected unconditional method instructions 103 112 e 427 um 18 1138 73 428 variables 8 66 viewing cumulative method time or volume 106 viewing notes 81 method editor in text instruction mode 46 interface 45 overview 4 selecting the windows to display 47 method file copying from external 27 copying to external 26 method files overview 5 method information display 144 method templates start protocols 42 method creating 319 Methodbase instruction 105 MethodQueue adding more method steps 178 changing the start condition or method name 181 Condition command 180 condition setting 182 defining 177 displaying 182 editing 181 End 183 erasing a line 181 execution of methods 181 Exit 183 facilities 11 folders and icons 180 Immediate start command 179 inserting a line 181 overview 177 Ready instruction 182 Restart 183 running 181 selecting the method 178 setting a time interval for starting a selected step 179 setting up 177 Start at 183 Start time command 179 minimum distance between points 268 MinPeak setting 114 mirror images of curves 229 mirror imported curves 223 models curve fit 405 molecular size calculating in sample 366 introduction to principles of measurement 357 selecting the molecular size table 366 selecting t
281. inutes fo Cancel Help Fig 8 4 Condition dialogue The timing of MethodQueue steps performed on different systems can also be controlled by the Ready instruction see Method execution in MethodQueues on page 181 in the method Click on Setup to define the starting time for the MethodQueue Immediate Start sets the MethodQueue to start as soon as you request Run from the Main Menu Start time sets the MethodQueue to start at a pre set time of day up to one week after Run is requested If no day is specified the MethodQueue will start as soon as the pre set time is reached i e within the next 24 hours Click on OK to complete the dialogue and return to the MethodQueue Editor dialogue e p179 og MethodQueues Note Note an 18 1138 73 e p180 MethodQueue Setup xi Start Mode Immediate Start Starttime 03 42 AM vy eT Cancel Help Fig 8 5 MethodQueue Setup dialogue The MethodQueue setup time defines the starting time for the MethodQueue as a whole Do not confuse this with Condition which defines the relative starting time for a step within a MethodQueue 8 Click on Save to save the MethodQueue Enter a MethodQueue name in the dialogue Edit MethodQueue authorisation is required to define a new MethodQueue 8 1 2 MethodQueue folders and icons MethodQueues are saved in a separate MethodQueue folder within the folder that you specified during the save The Metho
282. ion of recovery factor measurement see Recovery calculation on page 316 Preparing for quantitation An external standard quantitation table for the component of interest must first have been prepared see Creating a quantitation table on page 320 Internal standard quantitation tables cannot be used 1 Perform a run with the unspiked sample and a run with the spiked sample Integrate the curves to produce peak tables During integration the sample curves must use the same X axis base unit as the standards Time is the recommended unit for highest reliability Check that the integration is correct If not consult Optimising peak integration on page 262 which describes how to optimise integrations The peak tables for the unspiked and the spiked sample must be present in the same result file Open one of the sample result files Use File Open Peak Tables to copy the other peak table to that result file Use File Save to save the peak tables before continuing with the recovery calculations Calculating the recovery 1 Select Quantitate Calculate Recovery to open the Calculate Recovery Factor dialogue e p345 ED Analysing results an 18 1138 73 r Quantitation table Global Personal Addition component Added amount Ext_Std_Chym b Chymotrypsinogen A x 0 3 r Select peak table Source chromatogram Addition chromatogram 1 1 x Peak table s Peak table s id 18501 1 UV1 280nm 47
283. ions i e Block and Watch instructions to other blocks in the method Bold text Valid instructions Red check mark Instructions with invalid syntax These may be a calls to blocks which are not defined in the method or b instructions which apply to a different system strategy these can arise if a method is written for one system and saved for another or c instructions for components that have not been selected in the System Set up All such instructions must be deleted or changed before a method can be run see Changing instructions on page 61 n Editing methods 5 Normal text Instructions which will not be executed because they are either after the end of a block or method or constitute a block to which there is no call Text with a loop symbol When a block is called from within itself this will generate a potentially infinite loop which might exceed the maximum number of calls allowed in a method Double click on a Block or Watch instruction to display or hide the instructions in the called block or click on the symbol for the block respectively Double clicking on the Main keyword at the beginning of the method will show or hide instructions in all blocks in the method Main Main 0 00 Base CV 0 10 Colurnn ml Any 0 00 Base Cy 0 10 Column ml Any 0 00 Block BASIC_START_CONDITIONS_IX 0 00 Block BASIC_START_CONDITIONS_Ix 0 00 Block COLUMN_EQUILIBRATION 0 00 Block COLU
284. ionship can be linear quadratic or point to point Quantitation is usually based on a number of runs using a standard at several concentration levels The amount and concentration of the component s of interest in the sample are then determined from the peak size of the component using the calibration curve The different steps in Quantitation For all quantitation techniques except standard addition you need to perform the following 1 Run the different concentration levels of the standard 2 Integrate the curves to produce peak tables Check the integration 3 Identify the components for which calibration curves will be produced see Producing calibration curves on page 319 4 Enter the known concentrations for the different standards to produce a calibration curve for each selected component see Producing calibration curves on page 319 5 Run the sample and integrate the curve 6 Let the program calculate the concentration and amount of the components of interest in the sample see Quantitating the sample on page 339 Analysing results 11 s NWN sample to be Ne quantitated amount quantitated sample calibration curves standards at different concentration levels Fig 11 1 Quantitation overview The quantitation techniques available The different techniques available are summarised below Each technique is described in greater detail in the following sections
285. is co ordinates using the appropriately selected Y axis scale to calculate the noise range as the difference between the max and min values Add an extra 20 and insert this value as the Noise window value A 2 5 Measuring the Slope limit using Differentiate and curve co ordinates for Classic algorithm To measure the slope at any point on the curve 1 Select Operations Differentiate A dialogue will appear 2 Select the desired source curve check that a First order calculation is selected and click on OK The differentiated curve will appear in the active chromatogram 3 Measure the Y axis values on the differentiated curve using the Marker curve co ordinates function Remember to select the appropriate Y axis scale Any Y axis value is interpreted as the UV curve slope at the selected retention point If the differentiated curve is very noisy it can be filtered using a light Moving average filter in the Operations Smooth function see Smoothing a curve on page 200 4 Determine the highest slope value of the baseline non peak part of the curve Add 10 and insert this value as the Slope limit Evaluation functions and instructions A A 3 Peak table column components C Fig A 3 Diagram illustrating peak parameters See the parameter list below for explanations Column name Description Peak name Name of peak Retention time or volume base Retention at the peak maximum C in Figure A 3
286. iseWindow_UV1_280nm 322401 DetaultNoiseWindow_UV1_280nm 01 BASEM3 Fig 10 18 A Noise detected as peaks B Peak integration after increase of Noise Window Another possibility is to use the Reject peaks function in the Integrate dialogue to reduce the number of peaks based on an appropriate criterion e g the number of peaks or the minimum peak height Peaks missing In cases where obvious peaks are not detected in the peak integration a probable cause is that the Noise window parameter is too high Fig 10 19 Peak integration with too high Noise window an 18 1138 73 p274 Evaluating results 10 Decrease the Noise window parameter until the peaks are detected Tala Fig 10 20 Correct peak integration after decreasing Noise window Another possible cause of missing peaks is that an improper reject criterion has been used Check the criteria used for Reject peaks in the Integrate dialogue as well as Filter peaks in the Chromatogram layout function Baseline on top of peaks In rare cases the top of a broad flat peak will be incorporated as a baseline segment Fig 10 21 Baseline segment on top of peak e p275 ED Evaluating results an 18 1138 73 p276 This is one of the very few situations where it is useful to change the Max baseline level Measure the heig
287. it This toggles between looking at the print preview mode and edit mode One Page Two Pages This toggles between viewing single pages or pairs of pages where there is more than one page Next Page This displays the next page or pair of pages where there is more than one page e p243 ED Presenting results Note an 18 1138 73 p244 Previous Page This displays the previous page or pair of pages where there is more than one page Zoom In This increases the magnification of the view Zoom Out This decreases the magnification of the view Changing the page layout To change the page layout double click on the header region of the page to display the Page Setup dialogue Page Setup First Header Footer m Margins Top cm Bottom 2 54 cm Left cm hin Right em m Settings IV Same header on all pages I Draw a frame around the pages Fig 9 56 Page Setup dialogue Page Setup tab There are three tabs for changing different aspects of the page layout Page Setup which allows you to set the page Margins and the Units cm or inch You can check the option to have the Same header on all pages and also you can check the option to Draw a frame around the pages First Header which allows you to select the components to be included in the header for the first page If you have not selected the setting Same header on all pages on the Page Setup tab then a fourth tab option Header i
288. it is only one chromatogram and is named 1 Presenting results 9 3 Check the chromatogram s of interest and press the Select button Selected chromatograms are added to the Selected chromatograms list Chromatograms can be deselected by using the Remove command button o Default sie Type Modified ES Prev Folder u Baseline example 1 1216KB Result File 4 8 1999 10 36 E Baseline example 2 978KB Result File 3 30 1999 11 2 ai Baseline example 3 973KB Result File 4 8 1999 10 39 ijid1 86Level 1 to 4001 1205KB Result File 2 12 2001 10 5 i id186Level 1 to 4002 1158KB Result File 1 9 2001 1 36 P ilj id1 86Level 1 to 4003 1094KB Result File 2 12 2001 10 5 ai id186Level 1 to 4004 1083KB Result File 1 9 2001 1 37 P ai id2301 779KB Result File 7 28 1999 12 4 FT a PEE saran manh ile Antenne anan r Select Chromatograms Available Selected MBaseline example 1 1 Select gt gt Remove OK Cancel Help Fig 9 33 Open Chromatograms dialogue 4 Repeat steps 2 3 for chromatograms in other result files 5 Click on OK Viewing all chromatograms 1 Simultaneously display the chromatograms by selecting Window Tile or layer them by selecting Window Cascade 2 Chromatogram windows can be individually sized and the presentation of the curves changed 3 If you want to have the same scale on all of the chromatograms open the Chromatogram Layout dialogue for any chromatogr
289. kpoint as the End_Block instruction the Block instruction will be placed immediately before End_Block Do not call a block from within itself You will generate a potentially infinite loop which exceeds the maximum number of calls allowed in a method A loop symbol is displayed at the beginning of the line if this occurs Press OK to add the new block 5 2 4 Deleting blocks You can delete a block instruction by either i clicking the Delete button in the Instruction box ii by using the shortcut menu iii using the Block Delete Block command or iv clicking on the lt Delete gt key on the keyboard Deleting blocks using the shortcut menu 1 Click on the desired block in the method with the right mouse button to display the shortcut menu 2 Select Delete A warning dialogue is displayed requesting if you want to totally delete the block instruction from the method If you choose Yes and the block contains sub blocks another warning dialogue is prompted asking if you want to delete the sub blocks as well e p53 Editing methods an 18 1138 73 e p54 Answer as appropriate Yes The block is totally removed from the method If the block is called several times in the method all the blocks will be deleted Blocks deleted in this fashion can not be called again in the method No The block is deleted from the method and transferred to the Unused line If the block is called several times in the method h
290. l For KTA design systems valves remain in the position they were in before the pause Accumulated time and volume is not incremented during Pause Any method instructions which are set to the time volume when Pause is pressed are executed Later method instructions are not executed until Continue is pressed Continue Resumes execution of a paused or held method End Terminates method execution and puts the system into End state These commands can also be located under the System Control Manual menu The available buttons in System Control are dependent on the control status of the connection Status Available buttons End Run Running Hold Pause End Manual Run Pause End Hold Pause Continue End Method pause Hold Continue End Manual pause Run Continue End Windows toolbar The Windows toolbar has three buttons Opens a dialogue for choosing which window panels to display Clicking on this button is equivalent to choosing the menu command View Window es Opens the documentation pages Run notes may be entered in the Notes tab and settings may be changed Other tabs are displayed for information only Clicking on this button is equivalent to choosing the menu command View Documentation Note Performing a run GH Lu T Opens the properties pages Clicking on EOU this button is equivalent to choosing the menu command View Properties System toolbar The System toolbar has two functions Discon
291. l number e the Date of the run in an 8 digit format determined by the country setting in Windows NT plus a 3 digit serial number e a freely specified Name within the file naming restrictions in the operating system plus a 3 digit serial number If the result file folder already contains files with the same file name base the serial number is incremented automatically For scouting runs the 3 digit serial number will be the number of the executed run column in the scouting scheme By default result files are stored in the home folder of the user who starts the run To change the folder where the result file will be stored press the Browse button double click on the required folder icon and press Close If the run will contain information that is not important you can save file space by checking the No result checkbox thereby storing the result in the Temporary folder named Manual Runs where only the latest 10 result files are saved Editing methods 5 Note The result name may be specified as changeable in the Start protocol see below In that case the specification in the Result tab serves to generate the suggested result name which may be changed at the start of the run Scouting results will be saved in a special folder on the result file path Enter a name for the folder in the Scouting folder field Each time the scouting method is run a new folder will be created with the name and a serial number e g entering IEXSC
292. l software e UNICORN user interface e Files and folders e Methods and method structure e System Control e Scouting e Evaluation e Network considerations e Security and administration 2 1 UNICORN control software UNICORN runs under the Windows NT and Windows 2000 operating systems and provides facilities for method controlled operation of chromatography systems as well as real time monitoring and subsequent evaluation of the separation process 2 1 1 Strategies Part of UNICORN software referred to as the strategy is system specific The strategy defines what is available in method and manual instructions system settings run data curves and method templates Most of this manual describes the user interface in UNICORN independent of the strategy Strategy dependent instructions are listed in Appendix B 2 2 UNICORN user interface 2 2 1 Toolbar Guide The UNICORN Toolbar Guide dialogue is shown after start up and logon and provides an introduction to the Main Menu toolbar buttons ep3 2 UNICORN concepts an 18 1138 73 p4 Toolbar Guide xi Begin your work directly by using the toolbar buttons KEKIT Fig 2 1 UNICORN Toolbar Guide dialogue The Main Menu toolbar icons allow you to begin using UNICORN quickly for example to create a new method in the Method Editor start an instant run open a result file for evaluation or execute manual instructions in System Control see Section 3 2 2
293. lained variance 99 895 Correlation 0 9996 More i Update report IS and Table settings Define components Eime Fig 11 20 Quantitation table dialogue Select each component in turn and enter the amounts at each level 1 From within the Quantitation table dialogue select the first component at the top of the Components menu Do not select an internal standard component if available as the amount for this has already been entered and does not change between the levels 2 Double click in the first row Level 1 in the Amount column and insert the amount of the component in the standard at this level This is the amount corresponding to the injected volume not the total amount used when the standard level was prepared 3 Repeat this operation for this component at the other levels You can use the cursor keys to move rapidly between levels in this column 4 Use the Curve model radio buttons to select the curve model that gives the best fit Analysing results CH e Linear recommended e Linear through origin e Quadratic e Quadratic through origin e Point to point The resulting curve is shown in the Calibration curve window together with points representing each of the levels of the component Any of the points in this window can be selected by highlighting its entry in the table underneath If more than one run has been performed for some level all points in tha
294. large icons View Large Icons or small icons View Small Icons The details list includes a small icon identifying the type of object file name file type the last modified date and time and the date and time of file creation Logon and file handling es Methods Miel E E Methods o cA Default cA Default z J i E Name System Sre Type Modified Created L HE iE iE j Prev 1423 test20 Test200010 Untitled 31423 Wizard 474KB Meth 8 30 1999 9 6 1999 3 19 PM st20 Wizard 312KB Meth 3 24 2000 9 6 1999 5 10 PM is Test200010 Explorer100 310KB Meth 3 24 2000 3 24 2000 9 22 ka Untitled Wizard 475KB Meth 8 30 1999 9 6 1999 4 12 PM New Folder o New Folder A List Find Large Icons if ier Small Icons Ei Find Previous Level Small Icons Copy from External Copy from External Fig 3 6 Large icon left and detail right display modes illustrated for the Methods window Sorting order In the details list viewing mode files can be sorted in the window according to one of the following Name alphabetical order or reverse alphabetical order System alphabetical order or reverse alphabetical order Method window only Size smallest or largest files first Type alphabetical order of file extension type Modified last recently modified files first Created most recent creation dates first To change the sor
295. lative separation between two peaks and can be used to determine if further optimisation of the chromatographic procedure is necessary If the resolution value is 1 0 then 98 purity has been achieved at 98 of peak recovery provided the peaks are Gaussian and approximately equal in size Baseline resolution requires that the resolution value is greater than or equal to 1 5 At this resolution purity of the peak is 100 To view the resolution data select Resolution in the list within the Peak Table tab of the Chromatogram Layout dialogue Click on OK and return to the chromatogram window The resolution value for each peak shows the resolution with respect to the previous peak e p283 ED Evaluating results an 18 1138 73 p284 R Whit Who where Vr2 gt Vri Vga retention elution volume for peak 1 Vp retention elution volume for peak 2 Wh1 peak width at half height for peak 1 for Gaussian peaks Wh peak width at half height for peak 2 for Gaussian peaks See Appendix Section D 3 for alternative calculations of the resolution 10 2 Other evaluations 10 2 1 Peak purity and peak identification Ratios between UV curves measured at different wavelengths give useful information about peak purity or peak identity The Operations Divide function can be used when you have a result file with run detected at more than one wavelength 1 Before dividing the curves you must make sure that both curves have a
296. layed and may in appropriate cases be altered at the start of a run Click on the Start Protocol tab and check the items that are to be displayed Variables p Checked it Scouting Evaluation Procedures Notes Questions Method Information are displayed before method is started T Columns Reference Curves Frac 950 BufferPrep l Columns l Gradient Start Protocol Result Name I Scouting J Reference curves T Text method I Evaluation procedures IV Questions IV Method information IV Notes Tl Settings T Gradient I Calibration IV BufferPrep IV Result name changeable I Frac 950 r Scouting start protocol First run only Allrun Fig 5 52 The Start Protocol tab in Run Set up Variables If this box is checked values for method variables will be displayed and can be changed at the start of the run These values will override the default values for the particular run and will be saved in the result file The default values stored in the method are however not affected Variables defined for scouting cannot be changed in the Variables tab If the Variables box is not checked the run will be executed with default values for all variables as defined in the method unless scouting is used see below Scouting If this box is checked the scouting scheme will be displayed and can be changed at the start of the run Changes will override the default settings and values for th
297. ld also Analysing results CH be narrow enough to exclude unwanted peaks which will interfere with the quantitation 2 Go through the other levels and check that the width is suitable the window width is the same in all levels To display the actual retention for a peak click on its lower green or black triangle 3 Repeat for all selected peaks Overlapping windows are not allowed By default peaks are identified by their absolute retention values and the highest peak maximum within the window In most cases it is not necessary to change these default settings in which case you can click on OK and move directly to the IS and settings section on page 328 If you have drifting retention and or the presence of nearby or slightly overlapping peaks which may cause the wrong peak to be selected you may need to change the identification settings The criteria by which peaks are identified are set using the Identification Settings dialogue The criteria are valid for all the selected peaks in the Define Component s dialogue These settings also affect the information provided in the Peak table within the Define Componeni s dialogue Click on Identification Settings to open the dialogue Identification Settings xi m Peak identification Identify peaks on Absolute retention and Highest peak maximum Window width as Relative ponse m Reference peak for relative retention Component z Window
298. le EXPORT_ Exports a method to the file Main Blocks METHOD_ defined in Export to file in ASCII Export to file ASCH format If all parameters are OFF then no method is exported If Main is ON then the main method is included and if Blocks is ON then all blocks are included in the exported file Evaluation functions and instructions A EXPORT_ Exports a method to the file Main Blocks METHOD_ defined in Export to file in WKS Export to file WKS format If all parameters are OFF then no method is exported If Main is ON then the main method is included and if Blocks is ON then all blocks are included in the exported file EXPORT_ Exports a method to the file Main Blocks METHOD _ defined in Export to file in XLS Export to file XLS format If all parameters are OFF then no method is exported If Main is ON then the main method is included and if Blocks is ON then all blocks are included in the exported file EXPORT_ Exports multiple curves previ Export to file MULTI_ ously defined with CURVES_ EXPORT_SEL_CURVES instruc ASCH tions in ASCII format to the file defined in Export to file EXPORT_ Exports multiple curves previ Export to file MULTI_ ously defined with CURVES_WKS EXPORT_SEL_CURVES instruc tions in WKS format to the file defined in Export to file EXPORT_ Exports multiple curves previ Export to file MULTI_ ously defined with CURVES_XLS EXPORT_SEL_CURVES instruc ti
299. le SampleB SampleC SampleD ID IV Display tooltip for extended scouting scheme cells Define Clear All Delete Insert Add Series Help Fig 11 40 The first part of the Scouting tab used to set up the standard runs If you do not need to perform many runs at each level skip the following instructions and go on to step 12 below If you need to perform many runs at each level the Update_Quantitation procedure must be edited to perform the update by average and given a new name First a copy of the procedure must be created Click Import in the Evaluation Procedures tab and select the current method used Select Update_Quantitation and give it a new name under Import as for e p353 ED Analysing results an 18 1138 73 p354 example Update_Average Click Import then Close This returns you to the Evaluation Procedures tab 9 Highlight the name of the new procedure and click Edit and the Procedure Editor opens Highlight the existing Update instruction Use the Parameters scroll bar to locate the Average or replace point drop down list and use it to select Average Click on the Replace button to change the instruction Then select File Exit 10 Select in the Procedure Editor dialogue the Update_Average procedure click on the Quantitate button and select the quantitation table and click on OK After this is done de sele
300. le to use the explained variance value for model selection e g to decide if a quadratic model fits the data better than a linear one indicated by a higher explained variance value The explained variance is not calculated for curve models drawn through the origin e p409 Mathematical models and Note an 18 1138 73 p410 statistics The explained variance value is equal to R adjusted for degrees of freedom see a statistical textbook for further information SS residuals k 1 Explained variance 100 x 4 P E ST 7m t where n a 2 SS residuals gt y Y Residual Sum of Squares i l n ox D SS rotal 2 y Total Sum of Squares i l y Average of all y values J Function value using the fitted model A 2 For example Y Axi Bx C n Number of points xy pairs k Number of x terms in the model For example 1 for linear and 2 for quadratic You can only obtain a value for explained variance if you have sufficient data points on the curve For instance if you only have two points for a Linear model or only three points for a Quadratic model the fitted curve will pass exactly through the points By definition this leads to an undefined value for explained variance In these cases the Statistics table will show a symbol instead of the explained variance value INDEX A About UNICORN button 18 absolute and relative window w
301. les Scouting TestMethod Notes Questions Columns Reference Curves Evaluation Procedures Block Variable Value S Main Column Mono_S_HR_5 5 Start_Conditions Column_position Position8 Wavelength_1 280 Wavelength 2 OFF Wavelength 3 OFF UV_Averaging_Time 2 56 Pressure_Limit 5 00 Initial_Eluent_Conditions Eluent_A_Inlet A11 Eluent_B_inlet B1 Flow_rate 1 00 Start_ConcB 0 Column _E quilibration Equilibrate_with 5 Autosampler_Injection Sample_ID ID Vial Number 1 Injection Volume 50 Quantitation Type Sample Flowthrough_Fractionation Flowthrough_FracSize ml 0 000 Start_Flowthrough_at NextTube Wwash_Out_Unbound_Sample Wash_column_with 2 Start_Frac_at 0 z Eluate_and_Peak_Fractionation Fig 9 63 Documentation Variables tab Scouting This displays the whole scouting scheme with the values for the current result file displayed in yellow cells Documentation Method Information Resul Information ResultName Start Protocol Settings Calibration Logbook Evaluation Log Frac 950 Variables Scouting Text Method Notes Questions Columns Reference Curves Evaluation Procedures F Display tooltip for extended scoutin Deme Gear All Dee Help Fig 9 64 Documentation Scouting tab Presenting results 9 Notes This displays notes that you have made at various points during the run
302. lick on OK to add the recipe to the BufferPrep list Defining a new buffer substance Before defining a new buffer substance ensure that all pKa values for the substance are available The pKa values entered should be true pKa i e the pKa value at indefinite dilution as opposed to apparent pKa i e measured at a non zero concentration The pKa values should be given at 25 C 1 In the New Recipe dialogue click on the Buffer substance button Click on New button in the Define buffer substance box that appears and enter the name of the new component Click on OK e p135 Editing methods an 18 1138 73 e p136 Define buffer substance x Name Testsalt2 oa 0 0 0 1 0 1 0 0 0 1 0 1 0 0 0 1 0 1 Number of acidic protons 0 0 3 Charge of deprotonated ion 0 3 0 pKa values given at 25 C OK Cancel Help Fig 5 71 The Define buffer substance dialogue Enter the appropriate values in the Value field for each pKa and dpKa dT parameter All values must fall within the stated Range limits Up to three values can be entered for each buffering component When the buffering component has less than three pKa s the other values should be set to zero A pKa value of zero means that the pKa does not change with temperature Enter the number of acidic protons for the buffer substance in the form that it is actually weighed in e g for NaH PO enter 2 for Na HPO enter 1 for Tris enter 0
303. ll C Multiple choice 5 Preview C No Answer ess Fig 5 33 The Questions tab in Run Set up Questions may be defined to accept four types of answers e Input field accepts any alphanumerical input as the answer Input field questions may have a default answer m Question Sample volume and type J Mandatory J Authorized IV Chromatogram Answer type r Default text Input field C Multiple choice C No Answer C Value Fig 5 34 Options for Input field questions ep Editing methods an 18 1138 73 e p78 Multiple choice allows the user to choose one of a defined set of answers To allow a blank answer enter a space in one of the pre defined answers m Question fis the pH monitor calibrated J Mandatory T Authorized V Chromatogram Answer type Alternatives C Input field Add Delete Multiple choice 7 es C No Answer No C Value Fig 5 35 Options for Multiple choice questions No Answer is not possible to answer This kind of question may be to display important information or to split a question over more than one line by setting all but the last line to No answer Text Use pH standards 4 00 and 7 01 T Mandatory P Authorized J Chromatooram Answer type C Input field Multiple choice No Answer Value Fig 5 36 Options for No Answer questions Value accepts only numerical answers Value questi
304. ll be executed but in an immediate sequence of zero duration You can get a graphical view of the gradient profile and the length of the blocks in a method by viewing the Gradient window in the Method Editor View Windows Gradient or in the Gradient tab of Run Set up Click on the X axis button in the graphical display to change a base for the graphical display Changing the display base will not affect the base in the method 4 3 Method notes Click on the Notes tab in the Run Set up to show the Notes tabs Read through the Method Notes and click on the printer icon or choose File Print to print the method notes The method notes provided with each template describe the important information about the template and if relevant how the system should be connected for the method to work correctly If your system does not correspond to the description either rearrange the valves and tubing connections in accordance with the method notes description or edit the method instructions see Chapter 5 in accordance with your system setup Evaluation Procedures Method Information Result Name Start Protocol Frac 950 Variables Scouting Notes Questions Gradient BuffePrep Columns Reference Curves Start Notes Run Notes Evaluation Notes TEMPLATE Chromatofocusing version 2 00 07 a Chromatofocusing Injection Manual Loop or Superloop filled before method start Fractionation Frac 950 Flowthroug
305. logue 9 3 8 Matching protein activity to a curve The Evaluation operations allow you to combine data from the results of protein activity assays for example ELISA with the data contained on the UV curve A presentation comparing the activity curve with the UV curve can then be made 1 Select Operations Activity histogram A dialogue is displayed with the fraction curve for the specific chromatogram already selected Change the source and target chromatogram selection if desired 2 All the component fractions of the fractions curve are listed For each fraction you can type in the corresponding activity value When all values have been entered click on OK 9 3 9 Renaming chromatograms curves and peak tables Sometimes it may be desirable to change the name of a chromatogram curve or peak table To do this close the Chromatogram Layout dialogue and then 1 Select Edit Rename and the relevant menu cascade option Chromatogram Curve or Peak Table 2 Select the appropriate object in the displayed dialogue and type in the new name Click on OK The new name will replace the old one rather than creating a new curve or chromatogram The original raw data curves cannot be renamed and are not therefore given as options 9 4 Comparing different runs The previous sections dealt with the manipulation of single curves within a chromatogram The following sections describe how to make comparisons between and how to present two or mo
306. manual chromatography software UNICORN Version 4 0 User Manual Amersham um 18 1138 73 e UV Biosciences UNICORN 4 0 User Manual 18 1138 73 Edition AA 2001 04 to order e Asia Pacific Tel 852 2811 8693 Fax 852 2811 5251 Australasia Tel 61 2 9894 5152 Fax 61 2 9899 7511 e Austria Tel 49 761 4093 201 Fax 49 761 4903 405 e Belgium Tel 0800 73 888 Fax 03 272 1637 Canada Tel 1 800 463 5800 Fax 1 800 567 1008 e Central East South East Europe Tel 43 1 982 3826 Fax 43 1 985 8327 e Denmark Tel 45 16 2400 Fax 45 16 2424 e Finland amp Baltics Tel 358 09 512 3940 Fax 358 09 512 1710 e France Tel 0169 35 67 00 Fax 0169 41 9677 Germany Tel 0761 4903 201 Fax 0761 4903 405 e Italy Tel 02 27322 1 Fax 02 27302 212 e Japan Tel 81 3 5331 9336 Fax 81 3 5331 9370 e Latin America Tel 55 11 3667 5700 Fax 55 11 3667 87 99 e Middle East and Africa Tel 30 1 96 00 687 Fax 30 1 96 00 693 Netherlands Tel 0165 580 410 Fax 0165 580 401 e Norway Tel 2318 5800 Fax 2318 6800 e Portugal Tel 01 417 7035 Fax 01 417 3184 e Russian amp other C 1 S amp N I S Tel 7 095 232 0250 956 1137 Fax 7 095 230 6377 e South East Asia Tel 60 3 8024 2080 Fax 60 3 8024 2090 e Spain Tel 93 594 49 50 Fax 93 594 49 55 Sweden Tel 018 612 19 00 Fax 018 612 19 10 e Switzerland Tel 01 802 81 50 Fax 01 802 81 51 UK Tel 0800 616 928 Fax 0800 616 927 USA Tel 1 800 526 3593 Fax 1 800 329 3593 BioP
307. method will perform scouting as defined in the method If the Variables box is also checked the start protocol will display the Variables page as well as the Scouting page Values for variables defined for scouting are however grey in the Variables tab and cannot be changed there On the Start Protocol tab in the Scouting start protocol field check the First run only radio button if you want the start protocol to be displayed only before the first run If this button is selected the settings entered in the start protocol for the first run will apply e pl69 Scouting an 18 1138 73 e p170 10 11 throughout the run and the scouting series will be performed automatically without user intervention Select All runs if you want the start protocol to be displayed before each run in the scheme giving the operator an opportunity to change variable values or e g fill the sample loop before each run The operator must then press Start before each run in the scouting scheme Variables Scouting Notes Questions Gradient BuffePrep Columns Reference Curves Evaluation Procedures Method Information Result Name Start Protocol Frac 950 r Checked items are displayed before method is started Variables I Columns T Reference curves Text method Evaluation procedures I Questions J Method information I Notes T Settings I Gradient I Calibration M ButferPrep Result name changeable I Frac 950 eati
308. mprehensive range of curve fit models For the production of calibration curves these are e linear e linear through origin e quadratic e quadratic through origin e point to point Note that it is the average peak size for all points at a specific level that is used for calculating the calibration curve The following curve fit models are available for molecular size curves e linear e linear log Mw e quadratic e quadratic log Mw e point to point e point to point log Mw For all but the point to point models the Analysis Module provides values for the appropriate constants used in each curve equation It also provides statistical data that you can use to assess the quality of fit of the curve to the data e p405 Mathematical models and statistics 2 How the curve fit models are determined In the following descriptions the text to the left describes the model used for the curve fit and the mathematics used by the Analysis Module to evaluate the constants The screen image to the right is typical for the model described and you obtain it by clicking the Statistics button in the Quantitation table Mol size table dialogue boxes You can find further information about explained variance and correlation in Section D3 Linear Based on the equation y Ax B y A x B The constants A and B are determined by linear least squares regression Please see a statistics textbook for further info
309. n 18 1138 73 p380 Evaluation functions and instructions A DIV Divides two curves to gain a third First source curve which is the quotient of the curve two curves The two source curves Second source can have any Y axis unit The Y jcurve axis of the target curve position Target curve will be a normalised scale without position unit HISTO Creates a histogram from any First source GRAM non fraction curve source curve _ curve 1 and a fraction curve source Second source curve 2_frac and stores the result curve in the target curve position If Target curve source curve 2 is not a fraction position curve a run time error will occur The Y axis of the target curve position will be the same as that of the first source curve INTEGRATE Performs a mathematical integra Source curve tion of the source curve and stores Target curve the result in Result curve This position instruction is not the same as Peak integrate which performs a real peak integration POOL_ Pools fractions from the source Source curve FRACTIONS curve and stores the result inthe Target curve target curve position The frac position tions are pooled from the first First fraction to selected fraction to the last pool selected fraction If source curve is Last fraction to not a fraction curve or First or pool Last is not an existing identifica tion a run time error will occur RET_MUL Multiplies the retention of the Source
310. n 18 1138 73 e p114 after a peak_maximum has been detected and the valley is defined by a local minimum followed by an increase to 102 of the local minimum value plus the Delta_Peak value see below Peak_max The signal falls to a specified fraction of the most recent peak maximum minus the Delta_Peak value see below Factor 1 detects peak maximum Stable_baseline The signal is stable within the limits of the Delta_Base value see below for the period specified by the minutes parameter To determine suitable values for watch conditions it is often most convenient to examine data from a test run For slope values use the Differentiate function in the evaluation module to measure the slope of the test chromatogram see Finding the slope values for Peak Fractionation or Watch instructions on page 287 The Simulate Peak Fractionation technique see Finding the slope values for Peak Fractionation or Watch instructions on page 287 can also be used to find the slope values Two conditions apply for air sensors although may be handled differently dependent on the system Equal 0 No air detected Equal 1 Air detected To use the Watch_AirSensor instruction for air sensors the Alarm_AirSensor setting in Alarms amp Mon must be disabled use the Method Editor to disable the alarm locally in a method or the settings to disable the alarm for all methods The Alarm_AirSensor setting overrides any Watch_AirSensor instructi
311. n 18 1138 73 e p56 3 Select Paste from the shortcut menu Edit Paste or lt Ctr1l V gt The block is now removed from its original breakpoint and pasted at the new breakpoint The pasted block is inserted with the same breakpoint value as the block or instruction selected for point of insertion Copying blocks To copy blocks within a method 1 Select the block to be copied using the shortcut menu Select Copy from the shortcut menu Edit Copy or lt Ct r1 C gt 2 Select the instruction line just above the insertion point where you want the cut block to be pasted 3 Select Paste from the shortcut menu Edit Paste or lt Ctrl V gt A dialogue requests if you wish to rename the pasted block Click on Yes to rename the block before insertion or No to directly insert the copied block The pasted block is inserted with the same breakpoint value as the block selected for point of insertion Importing blocks Blocks may be imported from other method files to which you have access including the current method file in which you are working 1 Select Block Import Block As Import Block xi Select block BufferPrep_pH_Flush c Default Name Clean After Gradient BP V 474KB Method File 312KB Method File 310KB Method File 475KB Method File Explorer10 Explorer10 Explorer100 Explorer10 Eluate_Fractionation_V Flowthrough_Collection Gradient_Delay_BP Gradient_Elution Gradient_Segment_1
312. n Fig ure A 3 and right peak limits E G in Figure A 3 Fraction tube id Fraction number at peak start peak maximum and peak end Baseline height Baseline amplitude at peak start peak maxi mum and peak end A F and G in Figure A 3 Sigma Standard deviation for a Gaussian shaped peak For definition see below Resolution Peak resolution For definition see Measuring resolution on page 283 and below Capacity factor For a definition see below The Capacity factor will only be calculated when the chroma togram is in volume base The total liquid vol lume Vt must be entered in the Integrate dialogue for this parameter to be calculated Evaluation functions and instructions A Kav Gel phase distribution constant in gel filtration For definition see below Kav will only be calculated when a gel filtration column was used and when the chromatogram is in volume base The void volume VO must be entered in the Integrate dialogue for this parameter to be calculated Plate height Height equivalent to theoretical plate and HETP plates metre The column height must be entered in the Integrate dialogue for this param eter to be calculated For definition see Meas luring HETP on page 282 and below Asymmetry Peak asymmetry indicator of column packing For definition see Measuring peak asymme try on page 283 and below
313. n and drag a rectangle out on the screen to encompass the area to be viewed Editing methods 5 2 Release the mouse button The display is now zoomed in on the selected area 3 Repeat the process for further magnification of selected areas You can reduce the scale of the zoom in function in two ways either e reverse each zoom in action a step at a time by displaying the shortcut menu and selecting Undo Zoom or e reverse all of the zoom in actions to the default scale setting by displaying the shortcut menu and selecting Reset Zoom Changing the base You can select the base shown on the X axis either time volume or column volumes Make the appropriate selection from the shortcut menu option Base type Changing the base for the display does not affect the base in the method instructions You can therefore check how long a method will take simply by setting the axis scale to time even if the method blocks are written in volume or column volume base Viewing hatch marks You can display a hatched background in the Gradient tab by selecting Hatch from the shortcut menu 5 5 5 Notes Notes are descriptive comments that form part of the method documentation There are four separate notes fields for method editing start up run and evaluation respectively Only the method notes can be edited from the Method Editor the other notes are accessible at the respective stages in a run To view the method notes open the Notes tab in
314. n available folder or the diskette drive to which you want to copy or move the file folder and click on OK Copied files and folders are user specific You cannot copy or move files between the Methods and Results windows of the Main Menu Explicit authorisation is required to copy or move files see Chapter 4 in the UNICORN 4 0 Administration and Technical manual To copy a file within the same folder open the file in the relevant UNICORN module e g a method file in Method Editor or a result file in the Evaluation module and use the File Save As command in the module to save the file with a different name from the original When copying to a diskette a use Copy to External so that the files are automatically compressed If you are moving a method to another system you must always use the Copy to External Copy from External functions This will give you the possibility of connecting the method to the appropriate system The extension for the method file name is used to identify the system for which the method has been created An incorrect extension may result in syntax errors in the method or the method not being visible in the Methods window of Main Menu Copying files to external Copying files to external may be useful when you want to store all results documentation etc in a common project folder on the network or want to back up the files in a special place Logon and file handling es Tocopya method file scouting
315. n log 254 logbook 254 notes 253 scouting 252 variables 252 Run setup 70 Buffer Prep page 89 Columns page 88 Evaluation Procedures page 83 Method Information page 91 um 18 1138 73 436 Notes page 82 pages 48 Questions page 77 Reference Curve page 86 Result Name 171 Result Name page 92 Scouting page 73 75 scouting page 168 Start Protocol page 96 170 Variables page 48 70 Run Excluded toggle 172 S salt concentrations measuring in the fractions 292 same header on all pages 244 sample calculating molecular sizes 366 definition 304 save edited procedure 296 layout 195 method 40 99 method as a template 99 molecular size table 364 peak tables 320 procedures 294 quantitation table 332 report format 246 249 results 231 updated quantitation table 339 save as 332 Savitzky Golay algorithm 201 scale changing scale of X axis 153 changing scale of Y axis 152 scouting adding scouting settings during a run 172 changing scouting settings during a run 172 choosing the variables 168 comparing runs 173 continuous gradients scouting example 174 defining runs in scheme 72 defining variables 71 deleting and inserting run columns 169 e 437 a deleting columns 76 description 9 display of scheme at start of run 96 displaying start protocols in scouting run 98 examples 174 excluding runs 75 169 factorial design 76 in run documentation 252 insert series 72 inserting scouting runs before an existing run 75 ov
316. n the Procedure Editor dialogue Editing an existing procedure and you follow the above procedure new instructions will be added to the currently open procedure i e you will not be creating a new procedure To create a new procedure select File New Procedure or Procedure Edit New to display the Procedure Editor dialogue To begin recording click on the Rec button or select the dialogue Control Record menu command 10 3 2 Editing an existing procedure Evaluation operations are represented by instructions in the Procedure Editor dialogue These may be modified to suit specific evaluation needs and be saved for later use see Evaluation functions and instructions on page 369 You are recommended to be restrictive in editing existing global procedures It is recommended to open an existing procedure and save a copy under a new name and use this copy to perform any editing procedures 1 Click on Procedures Edit Open and select the desired evaluation procedure from the list The Procedure Editor dialogue is displayed for the selected procedure Evaluating results 10 2 To view the parameters contained within a specific instruction in the procedure select it and view the information contained within the Instructions and Parameters fields of the dialogue amp Procedure Editor Integrate_and_Print BE File Control Help REJECT PEAKS OFF OFF OFF OFF 20 NEGATIVE_PEAKS OFF CALCULATE_BASELINE 01 17 DEFAULT DEFA
317. n the Draw straight to next point button Click on OK and the new baseline will be saved with the default name Edited Baseline This may now be used as the baseline in a new peak integration 10 1 7 Adjusting the peak limits Once a peak table has been generated using the appropriate baseline it is then possible to split or join peaks and to manually adjust the peak start and end points The peaks will then be renumbered and the peak areas will be recalculated 1 Itis recommended that you first access Curve Style and Colour tab in the Chromatogram Layout dialogue and select either Number or Retention for labelling the peaks The former option will sequentially number each of the peaks in the chromatogram which is opened during the edit mode of a peak table The latter option will display the retention volume or time for each peak 2 Select Integrate Edit peak table Select the desired peak table from the displayed dialogue Note that name of the baseline on which the selected peak table was based is displayed at the bottom of the panel 3 Double click on the desired peak table in the list or click on OK A chromatogram window is displayed containing the selected peak table with corresponding curve and baseline The various editing features are described below As an aid it is possible to use the zoom function 4 Once you have completed your changes click on OK and verify the destination of the new edited peak table Note
318. name of the system A line is also inserted into the MethodQueue list before the previously selected End of MethodQueue instruction The new line contains the name of the selected method To add more method steps click on the Insert button and repeat steps 3 4 Note that you must use a new System button to add methods written for a different system type For example if you have designated System 1 to methods for the Explorer 100 system then only methods written for Explorer 100 can be added with this button If you want to add to the list methods written for for example Basic 10 then you must use another free System button such as System 2 In this case System 2 will be assigned and renamed Basic 10 and only methods written for this system can be added using this button MethodQueues 8 MethodQueue Editor Untitled x Explorer100 miga Systema Gysteng MethodQueue Start Immediate Start Condition ASAP 11d23 l End of MethodQueue Delete Setup Save As Close Help Fig 8 3 MethodQueue Editing dialogue with a method selected for an Explorer 100 system By default each method step will start as soon as possible ASAP after the completion of the previous method step To set the time interval for starting a selected step click on the Condition button In the Condition dialogue set the time when the step is to start Click on OK As soon as possible Wait Hours ft 4 M
319. ncentration used in the normal preparation When BufferPrep is used the buffer will be diluted between 2 10 times dependent on the amount of acid base that has to be used to reach the desired pH Up to five buffering components can be selected in the Buffer field To prevent too high ionic strength the sum of the concentrations for all buffers selected should be between 0 03 M and 0 2 M typically 0 1 M Note Editing methods 5 3 Select either HCI acid or NaOH base from the Acid Base pull down list and insert the required stock concentration typically 0 1 4 Select a salt from the pull down list To create new salts see Defining a new salt below Set the maximum outlet concentration of the salt for 100 B usually 1 0 M 5 Enter the desired pH range of the buffer To set a useful pH range the pKa must be known Click on the Buffer substance button and select the buffer component The pKa values are shown in the list Typically a pH range 0 5 units around the pKa is useful For exact ranges check buffer tables 6 To add notes about the recipe click on Notes and enter the required text in the displayed box Click on OK 7 When you have created your recipe click on Save as If you have entered values into your recipe that are not feasible a warning message will be displayed that tells you what is wrong Enter a name and save the recipe either as global or personal by checking unchecking the Save as global box C
320. nd copying 26 opening and running 20 presentation options 22 renaming 29 result files overview 5 searching techniques 25 sorting order options 23 file name using wildcards 24 filter files displayed 24 filter type 200 autoregressive 201 median 201 moving average 200 filtering peaks from view 262 finding files 25 first header 244 fixed Y axis 192 flow rate linear 110 flow scheme adjusting display 155 viewing manual instructions 156 flow scheme display 155 flow scheme window 66 folder creating a new folder 20 deleting 29 moving and copying 26 renaming 29 footer selecting contents 245 Frac 950 tab within Documentation 255 tab within Run setup 93 fraction histogram 12 e 423 um 18 1138 73 424 fraction order options 94 fractions pooling 205 framework in method 8 G ghost peaks removal 200 gradient changing lengths 62 display at start of run 97 display in start protocol 143 forming a step gradient 111 window 50 64 window zoom 65 with variable duration 112 gradient page 80 gradient profile graphical view 38 H hatch marks 81 viewing 65 154 hatched chromatogram window 196 help on line 5 HETP 379 calculation 283 measurement 282 highest peak maximum 327 hold instruktion 109 Hold_until Watch instruction 113 identification settings 325 import chromatograms to compare 216 218 procedures 84 importing blank run curve 202 blocks 55 importing blocks 56 injected amount entering for recovery 346
321. nd stop fractionation at a certain concentration of Buffer B The example below shows you how to do this with the Watch instruction Set the desired start fractionating concentration to the first Watch_Conc instruction and insert the desired slope and peak fractionation size to the Peak_Frac_Parameters and Peak_Fractionation instructions The peak fractionation will then start at the desired concentration Set also the desired end concentration of buffer B to the second Watch_Conc instruction to end the peak fractionation Main 0 00 Base CV 0 10 Column ml Any 0 00 Block Gradient Gradient 0 00 Base SameAsMain 0 00 Gradient 100 Target_concB B 29 Length_of_gradient base 0 00 Block eluate_and Peak Fractionation Eluate_and_Peak_Fractionation 00 Base SameAsMain 00 FractionationStop 0 0 0 00 OutletValve WasteF1 0 00 FractionCollector 0 00 fracsize m1 0 00 Watch_Conc Greater_than 0 Start_Peak_Frac_at B Start_Peak_Fractionation Start_Peak_Fractionation 0 00 Base Time 0 00 Peak_FracParameters e p127 5 Editing methods an 18 1138 73 e p128 100 00 Peak_Start_Slope mAU min 75 00 Peak_End_slope mAU min 0 15 Minimum_peak_width min 0 00 Peak_Fractionation 0 5 Peak_FracSize ml 0 00 Watch_Conc Greater_than 100 End_ Peak Frac_at B End_Peak_Fractionation End_Peak_Fractionation 0
322. nd the spiked sample Two runs are performed one with the sample and the second with the sample that was spiked with a known amount of the component of interest prior to sample preparation Quantitation of the data from the two sample runs allows the recovery factor of the sample preparation to be calculated The recovery is measured as the recovery for the sample preparation not for the separation during the chromatographic analysis An outline of the technique 1 A run is performed with each level of the standard 2 The curves are peak integrated to produce a peak table for each level 3 The data from the peak tables are used to produce a calibration curve This is the same process as is used in external standard quantitation 4 A portion of the sample is spiked with a known amount of the component of interest prior to sample preparation Both an unspiked and a spiked sample are then run and peak integrated to produce respectively the sample and sample with addition peak tables 5 The amounts for unspiked and spiked sample are calculated from the calibration curve The difference between these amounts provides the apparent amount of the addition Analysing results CH AU 0 10 4 Sample unspiked 0 05 n 0 00 ae T T T 0 0 10 0 20 0 3500 AU Area corresponding eye 0 10 4 to sample is ui i spike 0 05 Area corresponding to addition 0 00 _ e
323. nect Connect System It is possible to P fd A disconnect left icon the system and leave it in locked or eee state Use the connect icon right to connect the system Leave Take Control of the System It is possible to EA kua leave left icon the system in locked or unlocked state Use the Take Control icon right to take control of the system Status bar The status bar also displays a text message indicating the connection status of the window Controlled by lt user gt The indicated user has a control mode connection to the system Other users may establish a view mode connection Locked by lt user gt The indicated user has left the system in a locked state Users who can supply the required password may unlock the system and establish a connection Note that the password is case sensitive It is possible to unlock with the lock password or with the UNICORN logon password If using the UNICORN logon password the user must have the Unlock systems access rights The lock password is the password entered by the user who locked the system and is case sensitive System is available Any user may establish a connection e p159 ED Performing a run an 18 1138 73 e p160 6 3 2 Manual instructions The chromatography system can be controlled with manual instructions issued from the Manual menu The available instruction options are dependent on the strategy and instructions for the components defined fo
324. new question to the list If the list is empty the Insert operation creates the first question in the list e p79 Editing methods an 18 1138 73 e p80 Editing an existing question To change the definition of an existing question select the question to be changed Change the text status type and answer as required and click on Replace Deleting a question To remove a question select the question and click on Delete To remove all questions click on Delete All 5 5 4 Gradient The Gradient tab provides a graphical overview of the block structure and eluent gradient in the current method For scouting runs click on the Run button to see the gradient for each run Method Information Result Name Start Protocol Frac 950 Questions Variables Scouting Notes Gradient BufferPrep Columns Reference Curves Evaluation Procedures Base Gradient Block name 0 00 0 00 Main Fig 5 38 The Gradient tab in Run Set up Vertical cursor line A vertical cursor line can be dragged from the Y axis with the mouse As you drag the cursor line the current position is identified at the top of the page in terms of the block name x position in the currently displayed base and eluent concentration in eluent B The duration of variable length blocks are based on the default variable values The zoom function To zoom in on a selected region of the Gradient tab 1 Press down and hold the left mouse butto
325. ng start proloco First run only Fig 7 3 Settings on the Start Protocol tab in Run Set up determine whether the Scouting page will be displayed at the beginning of scouting runs Open the Result Name tab in Run Set up and enter a name for the scouting folder Change the path for the result file if required The scouting results will be saved in a folder on the result file path named according to the specified scouting folder name with a three digit serial number Save the method Scouting Variables Scouting Notes Questions Gradient BulferPrep Columns Reference Curves Evaluation Procedures Method Information Result Name Start Protocol Frac 950 m Result name Serial number is added to all resultnames Method name C Date C Name p Result directory fieXANiilas Browse Scouting subdirectory Fig 7 4 Settings under Result Name on the Result tab in Run Set up determine the name of the folder where scouting results will be stored Scouting variables The variables that appear in the scouting scheme are usually a subset of those on the Variables tab of Run Set up However while the default values in the method can be set either on the Variables tab or in the text instructions see Method variables on page 66 values in the scouting scheme can only be set on the Scouting tab Changing variable values in the scouting scheme does not change the values on the Variables t
326. ngs check box can also be used to toggle between lists of stored global and user settings Save Wizard Settings xi Stored user wizard settings I Global wizard settings Delete Aename Wizard settings name Cancel Help Fig 9 29 Save Wizard Settings dialogue e p215 ED Presenting results an 18 1138 73 e p216 From the Data View dialogue click on OK to leave the Multifile Peak Compare Wizard 9 4 3 Comparing chromatograms from different runs To import chromatograms from other result files into an already opened result file two functions can be used namely File Open to compare or File Open The former option is most useful in searching for many chromatograms in a specific folder based on defined selection criteria The latter option is best used to import any individual chromatograms from result files in different folders The imported chromatograms are sequentially numbered 11 12 13 etc for identification purposes Up to 10 chromatograms can be made available at the same time on the evaluation workspace 322401 1_UV1_280nm F 322401 2_0V1_280nm I 2b 3b 322401 3_UV1_280nm Fig 9 30 Windows Tile function to display many chromatograms Alternative A Import chromatograms using Open to compare This method is useful for example when importing chromatograms from all files of a scouting folder 1 Click on File Open to compare Chromatograms Th
327. ns Add Add the first curve in the sequence to the second curve in the sequence from the appropriate chromatograms 3 Add the result of the previous step to the next curve in the sequence 4 Repeat this process until all curves have been added together The final curve should be the cumulative curve for the whole run All curves created using the Add operation receive the ADD suffix 9 3 6 Entering text in the chromatogram Basic annotations can be added to the chromatogram 1 Place the mouse pointer in the curves view of the chromatogram window and select Add text from the shortcut menu Alternatively select the Edit Text Add command The mouse pointer is replaced with an abe pointer 2 Position the pointer where you want to insert text in the chromatogram and click the left mouse button once 3 Type in the desired text and then click outside the text box to set the text Presenting results eH If you want to edit change fonts move re orient or delete an inserted text either e Place the cursor anywhere over the active curve and select Edit Text Mode from the shortcut menu highlight the text then choose the desired function from the shortcut menu or 1 Open the Chromatogram Layout dialogue and select the Edit Texts tab Alternatively select the Edit Text Edit command and the Edit Texts tab is displayed automatically 2 Select the specific text that you want to edit and make the appropriate changes in the Select
328. nsaan anaana nannaa 157 6 3 1 THE tol ba er cece ee a nee ee age ke ee 157 6 3 2 Manual instructions 0 0 0 0 0 00000 eee 160 6 3 3 Alarms and warnings n nananana 0000 e ee ee eee 161 6 4 If communication fails 00ccce cece ee nanna 161 6 5 Maintenance 0c eee eee eee 161 6 5 1 Viewing system component information 162 6 5 2 Setting up maintenance warnings 005 163 6 5 3 Viewing and resetting the warning parameters 165 6 5 4 Getting awarning 0 6 0 eee 165 7 Scouting 7 1 Setting up scouting 00 ce eee eee eee 167 7 1 1 Scouting variables 0 0 0 eee 171 PAZ SCHEME SEEUP si asi e SAN hah Beale Gk ea ee sdewtins 171 7 1 3 Start protocol settingS 0 eee 171 7 2 Running scouting 00 eee eee eee ees 172 7 2 1 Changing scouting settings duringarun 172 7 2 2 Adding scouting runs during arun 0 00000 172 7 3 Scouting results 0 0 eee eee 173 7 3 1 Viewing TESUNES si oi dah bd OA wd DAD Rds ood del Re ean 173 7 3 2 COMPAN TUS Eis oo eee EE aL ehh ean weed 173 7 3 3 Printin ireSultS ic eer r s Bead eae ES 173 7 4 Scouting examples 0 00 cece eee 174 7 4 1 Scouting sample volume 0 0 ee 174 7 4 2 Screening different columns 2 000 174 7 4 3 Scouting continuous gradients 2 1 eee 174 LA ASDA SCOUTIN Gs 4 used a dias cide a a ah dh dinette ale uP ores 175 8 MethodQu
329. nstallation the user list in the Logon dialogue box is empty make sure that the computer is logged on to the network before starting UNICORN A remote station accesses the user list directly from the network server If the user list on a local station in a network installation is not up to date make sure that the computer is logged on to the network before starting UNICORN The user list is stored locally on a local station and is updated automatically from the network server if the computer is logged on to the network B 1 2 Error message Strategy file error If you receive the error message Strategy file error Can t load strategy in a stand alone installation the strategy file is probably corrupt Reinstall the strategy as described in the UNICORN 4 0 Administration and Technical manual In a network installation the error may appear if you try to create a method for a system not physically connected to the computer Make sure that the computer is logged on to the network before UNICORN is started so that the strategy file on the server disk is accessible B 2 UNICORN access problems Note When experiencing UNICORN access problems it will often be necessary to make reference to the UNICORN 4 0 Administration and Technical manual B 2 1 Unable to access certain UNICORN functions UNICORN functions to which you do not have access appear grey in the menu and cannot be used Your user profile is determined by the system administr
330. nstruction in the text instruction window The instruction with its current parameters will appear in the Instruction box In the Instruction box make the required changes to the breakpoint or parameters or choose a new instruction Press Change or Replace These buttons are equivalent unless changes are made to the breakpoint or the length of a gradient Changing breakpoints Change and Replace have different functions if the breakpoint is changed Change shifts all subsequent instructions in the block according to the change in the breakpoint Change does not affect the relative order of instructions in the method You cannot change the breakpoint of an instruction to earlier than the nearest previous breakpoint in block For example changing FractionCollector from breakpoint 0 to 5 with Change Change Gradient Gradient 0 00 Base SameAsMain 0 00 Base SameAsMain 0 00 FractionCollector 5 ml 500 FractionCollector 5 ml 0 00 Gradient 100 00 20 00 Base 500 Gradient 100 00 20 00 Base 20 00 Message END OF GRADIENT Noscreen 25 00 Message END OF GRADIENT Noscreen 20 00 End_block 25 00 End_block Fig 5 17 Change moves the selected instruction and all subsequent instructions Replace moves the selected instruction but does not change the breakpoint of any other instruction Replace can change the relative order of instructions in the method For example changing FractionCollector from breakpoint 0
331. nt This parameter determines the minimum length for a part of the source curve to be considered a possible baseline segment 2 have no point outside the Noise window The noise window is defined as a rectangular corridor parallel to the slope of the curve and centred on the first and last points within the currently inspected segment 3 slope less than the Slope limit e p371 AY Evaluation functions and instructions an 18 1138 73 p372 This limits the maximum slope of the baseline to differentiate baseline segments from peaks 4 are lower than the Max baseline level Determines the highest acceptable signal level for the baseline This parameter is by default set to have no influence on the baseline calculation and is seldom necessary to adjust The parameters can be illustrated as a rectangular box in which the source curve has to fit to be identified as a baseline segment Fig A 1 on page 372 The length of the box corresponds to the Shortest baseline segment and the height of the box corresponds to the maximum level of noise on the baseline segments and is referred to as the Noise window Shortest baseline segment Noise Window Fig A 1 Baseline box with Shortest baseline segment and Noise window The rectangular box is allowed to be tilted with a maximum slope corresponding to the Slope limit see Fig A 2 on page 373
332. nt peak filter settings Maximum number of peaks 20 Fig 11 34 Recovery factor result If the recovery can not be calculated one of the following signs is shown gt means that one of the amounts concentrations is higher than the highest value in the calibration curve i e it is outside the valid range of the calibration curve lt means that one of the amounts concentrations is lower than the lowest value in the calibration curve i e it is outside the valid range of the calibration curve means that the recovery factor can not be calculated For example this sign might indicate that the peak could not be identified in both runs 11 5 Automated quantitation Some method wizards designed for quantitation are available for AKTAdesign systems supplied with Autosampler A 900 These can be used to quantitate a sample automatically or to update a quantitation table These procedures do not work on other systems For a general description of procedures please refer to Other evaluations on page 284 A quantitation table must be produced from standards before samples can be quantitated The same method must be used for all standard and sample runs The Basic10 or Explorer10 strategies contain the Autosampler component option To add the Autosampler to the strategy go to System Control and select Administration System Setup In the resulting System Setup dialogue select either Basic10 or Explorer10 and then click on
333. ntegration calculation of the IHEIGHT IHETP value The Column height lparameter is the height of the col umn in centimetres If Column height is OFF then the HETP value is not calculated for the following integrations Evaluation functions and instructions A SET_ Sets the void volume for peak inte Void volume COLUMN_V gration calculation of Kav 0 SET_ Sets the total liquid volume for Total liquid vol COLUMN_V peak integration calculation of the ume IT capacity factor SET_ Sets the Skim size ratio to be used Ratio SKIM_SIZE_ ln the following peak integra RATIO tion s A 4 3 File Operations CURVE_ Opens the curve specified in the File name OPEN Result file defined in File name Curve name and stores it in target curve posi Target curve tion If is entered as File name position the current result file will be used The File name parameter may include a path from the users root folder IMPORT_ Imports a curve to the current Chromatogram CURVE chromatogram from another chro name Imatogram in the current file and source curve stores it in the target curve posi _ Iarget curve tion position IMPORT_ Imports a peak table to the current Chromatogram PEAKTABLE chromatogram from another chro name Imatogram in the current file and Peak table stores it in the target curve posi _ source tion Resulting peak table PEAKTABLE Opens the specified Peak table in
334. ntial Number or by user defined Peak name The Peak label text can be set to either Vertical or Horizontal Checking the No Peak Label box removes peak labels e p191 ED Presenting results an 18 1138 73 e p192 Both Fraction text and Logbook text can be set to Vertical Horizontal or Fly Over alignment by checking the appropriate box Fly Over alignment sets text labels as hidden text which appear only when the cursor is carefully positioned over a curve line Within the Logbook text alignment field clicking on the Filter button opens the Filter Logbook dialogue Choose the radio button for the logbook information to be filtered set the maximum block depth then click OK To see the mark you must also select the curve position 22 in the curve list Filter Logbook xi Select logbook type to show C Block start Enrors alarms and warnings C All Set maximum block depth to show fi Cancel Help Fig 9 7 Filter Logbook dialogue 9 2 8 Changing and fixing the axes By default the Y axes are automatically scaled for each curve to show the whole curve The X axis automatically scaled to display the run from the point of injection ml or minutes It is possible to fix the minimum and maximum values for the axes of any curve and thereby select a specific part of the curve to be displayed Y Axis 1 Click on the Y Axis tab in the Chromatogram Layout dialogue 2 Select the appropriate cur
335. ntrations and gradients 111 5 11 How to use selected conditional method instructions 112 5 11 1 Hold_until instruction 0 0 000000 eae 113 5 11 2 Standard Watch conditions 0000 113 5 133 Watehvexamples x3 os Ba eek ee wad eee BS Y 117 5 12 The Column list 00 cee eee eee eee 128 5 12 1 Adding anewcolumn 2 0000000 eee 128 5 12 2 Editing column parameters 0 0 00000 132 5 12 3 Deleting a column 0 ce ee 133 5 12 4 Selecting or changing the column in a method 133 5 13 Creating and editing BufferPrep recipes 133 5 13 1 Creating a BufferPrep recipe 000000 133 5 13 2 Editing a BufferPrep recipe 2 eee 137 6 Performing a run 6 1 Starting a method 00 eee 141 6 1 1 Starting from the Main Menu 0 000000 141 6 1 2 Starting from System Control aaa aa aaa 0 0 0 0 000 142 6 1 3 Starting an Instant Run 0 0 0 eee 142 6 1 4 Start protocol eds stien Aye Be eae ee dy Beer wei des 142 6 2 Monitoring a run 0 eee eee eee 145 6 2 1 General window techniques 0 00000 e eee 145 6 2 2 RUN datars sxe 2 ea ewes ba eee ee die I Sed deb 145 6 273 CUINES raean shel oP athe ala boas bie oh teat ees 149 6 2 4 gt FIOWSCHEME fone ti eers ee Row eed a beets 155 O22 BD LOSDOO Kijeen 3 eros 4 Bie dete es eta e tn tee cece Ay ad 156 6 3 Manual control
336. ntrations and gradients 111 eluent setting the initial eluent composition 11 enter molecular size data 362 error cannot connect to system 400 connections not available 400 couldn t create result file 401 Method System Connection dialogue keeps appearing 401 No x 400 strategy file 399 evaluating results 259 e 421 exit 257 icon 19 evaluation instruction mol size 356 procedures necessary before executing 356 quantitate 355 update 356 Evaluation log adding Evaluation log object to report 240 Evaluation module functions and instructions 369 overview of Evaluation facilities 12 problems 403 evaluation procedures 83 abort problem 403 automated evaluation procedures 292 chromatogram functions 395 curve operations 380 display after run 144 display at start of run 97 editing 294 export 386 file operations 385 instructions in Analysis module 355 integration 383 miscellaneous 396 potential problems 297 recording operations 12 reporting 299 running 297 explained variance detail 409 in calibration curve 331 in molecular size table 363 export data or curves 300 external standard quantitation calibration curve 309 description 307 detailed description 308 reliability of technique 310 um 18 1138 73 e 422 file automatic compression to zip 27 changing the sorting order 23 copying from external 27 copying to external 26 deleting 29 display all 24 filtering files displayed 24 finding 25 method files overview 5 moving a
337. nts the scouting variable values for the particular run as part of the diagram header For reports generated from scouting runs always make sure that Scouting Variables is selected in Edit Chromatogram Layout box Header tab so that the scouting variable values for the particular run are included epl73 Scouting an 18 1138 73 e p174 7 4 Scouting examples Below are some typical examples of scouting illustrating the way scouting schemes can be set up for different purposes 7 4 1 Scouting sample volume Sample volume scouting is applicable to both adsorption and gel filtration techniques Superloop can be filled with sample and the sample volume varied with the Empty_loop_with variable Block Variable Runi Run Run3 Run4 Empty_loop_with ml S Fig 7 5 Scouting settings for Sample_Injection 7 4 2 Screening different columns Scouting can be used to screen different column types for an application For screening different columns a suitable scheme may include the variables Column e Column_position switches the ColumnValves to the chosen column e Flow_rate Block Variable Runt Run2 Run3 Run4 Main Column im 10 000 onoo fotoo foio Start_Instructions Column_Position Position1By Position Position3 Position4d Column_PressureLimit 4 00 4 00 4 00 4 00 id Flow_Rate ml min 2 00 4 00 6 00 8 00 Fig 7 6 Screening different columns with facilities for chan
338. nventions set by Windows programs Many of the menu commands in UNICORN can be activated using the toolbar buttons keyboard shortcuts and the shortcut menu The availability of these command options is dependent on the active field or window in which you are currently working The function of a toolbar button is displayed when you place the mouse pointer over a button Shortcut menu commands are quickly found through use of the program Typographical conventions Important Note that UNICORN 4 0 is designed to run under English keyboard settings Menu commands the names of dialogue boxes and windows the contents of dialogue boxes windows and option buttons are written with a bold helvetica typeface Menu commands are written in the order of the menu name and then the command separated by a colon For example Select File Save As to display the Save As dialogue Locate the destination drive and folder and enter a file name Click on Save This directs you to click on the File menu and select the command Save As A dialogue called Save As is displayed in which you must locate the destination folder for the saved file and give the file a name You then click on the button called Save in the dialogue to execute the save command Atypewriter like typeface is used for instructions as they appear in the text editor for methods and evaluation procedures These are normally entered automatically by UNICORN Some menu commands also
339. obal Chromatogram_Report Global Full Report Global Installation_T est Report format name I Save as global format J Save as default report format Cancel Help Fig 9 59 Save Report Format dialogue 2 Enter a name for the format 3 Ifyou want the report format to be saved globally check the Save as global format option if you have Edit global lists authorisation 4 Click on OK Note If you selected the Save as default report format option the format name is changed to DEFAULT To save a copy of the format under another name select File Save As and enter a new name in the Save Report Format dialogue Exiting the Customise Report window To exit the Customise Report window select File Exit or click on the Exit button You will be prompted to save unsaved formats When you have exited the window the newly created format is displayed in the Generate Format dialogue 9 8 2 Creating a new standard report format It you do not want to create a new report layout using a customised format you can instead use the fixed layout in the Standard report e p247 ED Presenting results an 18 1138 73 p248 formats With Standard formats you can still select the objects that are included in the report and save the format for later use Selecting standard report options 1 Select the New button on the Generate Report dialogue This prompts the Create New Report Format dialogue
340. od structure Blocks Methods in UNICORN are usually divided into blocks Blocks typically represent well defined steps in the separation procedure such as e column equilibration e sample application e elution e column wash UNICORN concepts ez Blocks are also used for operations which are to be performed conditionally e g starting fraction collection if a UV absorbance peak is detected The user determines the number of blocks in a method as well as the names and functions of the blocks Method base Method blocks are written in one of three bases which defines the unit for the breakpoints in the block e time min e volume ml or according to the strategy e column volume set by the user Different blocks in the same method may be written on different bases for example a column wash block might be written in terms of column volumes while sample loading might be more appropriate in absolute volume Instructions Each block in a method consists of a series of instructions that request specific operations in the system For example Flow 8 0 ml min sets flow rate for the pump to 8 0 ml min Start_condition 0 00 Base SameAsMain 0 00 BufferValveAl A11 0 00 BufferYalveB B1 0 00 OutletValve F1 0 00 ColumnPosition Position2 0 00 Flow 5 00 0 00 End_block Main 7 0 00 Base CV 097miRes Equilibration 0 00 Block Start_condition 0 00 Base SameAsMain 0 00 Block Equilibration
341. of peak parameters from multiple runs e User definable alarm and warning limits for monitor signals e Programmed sequential operation of multi step separations e Batch operation and process documentation in accordance with the requirements of Good Manufacturing Practice GMP and Good Laboratory Practice GLP e Comprehensive data evaluation software including an Analysis module enabling quantitation and molecular size determination epl E Introduction Jh addition UNICORN offers a comprehensive security system e Password control for all users with access authorisation for other users method and result files e Electronic signature handling providing enhanced security for method and results files e Customised definition of access control levels e Audit trail for system operation Note UNICORN must be correctly installed for stand alone or network operation before the software can be used Network considerations software installation and administration of system and user definitions are described in the UNICORN 4 0 Administrative and Technical Manual an 18 1138 73 e p2 UNICORN concepts TA 2 UNICORN concepts This chapter introduces the basic concepts that are specific to UNICORN For a description of how to work with the Windows NT or Windows 2000 operating system see your Windows system documentation Material in this chapter is divided into 9 sections dealing with e UNICORN contro
342. off or restore the iconised Procedure Editor dialogue and click on the stop button or select the dialogue Control End Record menu command Procedure Editor Untitled ox Eile Control Help m Parameter UICTORDAKM Fig 10 39 Procedure Editor dialogue 6 The recorded procedure can be viewed in the Procedure Editor dialogue Restore the iconised dialogue if necessary e p293 ED Evaluating results Note Note Note an 18 1138 73 p294 7 More steps can be added to the evaluation procedure by repeating steps 2 6 The new steps are added to the previous procedure New lines will be inserted into the procedure after the selected line in the currently listed procedure This can be used to insert new instructions between existing instructions 8 If required edit the evaluation procedure Editing an existing procedure 9 Select File Save or File Save as from the dialogue menu and enter a name for the procedure The evaluation procedure is saved within UNICORN and is specific to your user name If you have Edit global list s access you may also check the Global procedure option to make the procedure available to all users Global procedures are marked with Global before the name Even if the results of an evaluation session are not saved the created evaluation procedure s are saved 10 Choose dialogue File Exit menu command If you already have an existing procedure open for editing i
343. ogram 1 OK Cancel Help Fig 9 37 Open Curves dialogue Select File Open Curves The Open Curves dialogue is displayed Select the folder in which to search for curves Click on the result file of choice and where appropriate the specific chromatogram containing the desired curve The chosen chromatogram and curves contained therein will be listed Presenting results 9 Select the desired curve and click on the Select button The selected curve will now be displayed in the Selected curves list To remove a curve from the Selected curves list check the check box then click on Remove If you want to choose more curves from other chromatograms repeat steps 3 4 When all the desired curves have been selected click on OK Restore the chromatogram window and open the Chromatogram Layout dialogue Select the curves that you want to view Curves can be scaled individually or all with the same scale using the All with this unit function see Changing and fixing the axes on page 192 Alternative C Copying curves into one chromatogram Curves can be copied between chromatograms present in the Evaluation desktop For effective comparison of curves it is suitable to transfer all relevant curves to a single chromatogram This is best achieved by To creating a new chromatogram using File New Chromatogram and copying curves into it from other chromatograms or copying an existing chromatogram using Edit Copy
344. olumn_Equilibration S E 0 00 Block Flowthrough_Fractionation Flowthrough_Fractionation 0 00 Base SameAsMain 0 00 OutletValve F3 0 00 End_block MH 0 00 Block Load_Sample_with_SystemPump 0 00 Block Wash_Out_Unbound_Sample 0 00 Block Fractionation_F_AC 0 00 Block Affinity_Elution Trstructions C Pump C Flowpath AlaimstMon Frac C Watch Other LI Ready Fig 5 2 The Method Editor in text instruction mode showing the Block window top Text instruction window centre and Instruction box bottom Editing methods 5 Up to four windows can be displayed together with the Instruction box Click on the View Windows icon or select View Windows Customise to open a dialogue for choosing which windows to display Individual screens can be selected for viewing through the View Windows drop down menu options or hidden by selecting Hide from the shortcut menu Customise Windows xi Text Flow scheme Instruction box Block LSS Cancel Help Fig 5 3 The Customise Windows dialogue for selecting which windows to display in the Method Editor Method editing operations which can be performed in the various windows are summarised in the table below Window Functions See Section Block window Select block 5 2 Text window Display and hide block instruc 5 3 tions Select current instruc tion Move instructions within a breakpoint Gradien
345. om the current eluent composition to 50 B at breakpoint 10 The method continues with 50 B Setting the initial eluent composition To set the initial eluent composition set a step gradient to the required concentration at breakpoint 0 at the beginning of the method This instruction can suitably be placed in the Main block e g to start at 50 B Main 0 00 Base CV 10 Column_volume 0 00 Gradient 50 B O base eplll 5 Editing methods Note an 18 1138 73 e p112 Breakpoints during gradients The breakpoint for a Gradient instruction defines the time or volume according to method base for the start of the gradient A gradient with a non zero duration occupies time and volume in the method and breakpoints for other instructions may be set to occur before the gradient is completed For most instructions the instruction is simply carried out at the requested breakpoint while the gradient is forming The following instructions affect the gradient however e anew Gradient instruction will start a new gradient at the requested breakpoint Any remaining duration of the previous gradient is ignored e anew Flow instruction will change the eluent flow rate at the requested breakpoint If the current base is volume or column volume the duration of the gradient will be changed If the method base is time the volume of the gradient will be changed e an End_Method instruction will stop the whole method interruptin
346. on Folder c Defaults X Browse Result Baseline x Browse All Chromatogram f hd Browse All r Found chromatograms Ee Search Baseline example 2 1 Baseline example 3 Clear kk Select all Fig 10 42 Batch run dialogue 2 The search will take place in the displayed folder only To select another folder click on the Browse button and open the desired folder 3 By default all chromatograms are searched for within the designated folder and result files as denoted by the asterisk As for result files you can select a specific result file using the Browse function or you can use wildcard characters to search for chromatograms with a specific name profile Evaluating results 10 You can use standard wildcard characters in the file name specification stands for any number of characters and for any single character For example iex will search files named iex iex will search all files with names beginning iex iex will search all files with names ending iex iex will search only 4 character names ending in iex User entered search filters up to a maximum of 10 will be saved in the drop down menus for both Result and Chromatogram selections More than one string can be used as a search delimiter by inserting a between strings Search filters are automatically saved and stored within user profiles To return to the defaul
347. on and if the alarm is enabled the method will pause when air is detected Settings for watch conditions The mode of function of the WatchPar Monitor instruction in AKTA design systems is affected by certain parameters within the instruction see below Delta_Peak The Delta_Peak setting helps the software to detect valleys peaks and Peak maximum and to ignore noise in the chromatogram Editing methods 5 The Delta_Peak setting a sets the threshold for signal increase after a local minimum which will be interpreted as a valley for the Less_than_or_valley condition A valley and a new peak are detected when the signal increases to 102 of the local minimum plus the Delta_Peak value Note that a valley is detected only after a peak_maximum has been detected For example if there is a local minimum at 0 05 AU and a Delta_Peak of 0 01 AU a valley will be detected at 1 02 x 0 05 0 01 0 111 AU b sets the threshold for signal decrease after a local maximum which will activate the Peak_max condition Peak_max is detected when the signal falls to the specified fraction of the most recent peak maximum minus the Delta_Peak value Peak max Delta_Peak detected No valley y detected Valley detected Fig 5 64 Illustration of the Delta_Peak setting Peak max is detected when the signal falls by Delta_Peak from a local maximum if the Peak_max factor is set to 1 The Delta_Peak value s
348. on Logoff icon 18 loop symbol with text 59 M Main Menu for starting a method 141 functions overview 4 mandatory questions 76 manual control 157 manual instructions 160 Manual menu 160 margins adjusting in page layout 244 mathematical models used by Analysis module 405 mathematics for linear curve fit 406 for linear through the origin curve fit 407 for quadratic curve fit 407 for quadratic through the origin curve fit 408 Max baseline level adjustment 276 maximum peak area 327 median algorithm 370 menu manual 160 messages during run 108 method adjusting method object in report 238 base 7 blocks 48 concepts and principles 6 construction of name 99 controlling block and method length 106 creating 33 creating a new method 33 default notes in template 82 display of instructions at start of run 97 display of method information at start of run 98 255 display of notes at start of run 97 editing operations summary 47 editing the variables 37 editor modes 45 entering editing and deleting instructions 57 execution in MethodQueues 181 framework 8 in automatic method scouting 8 information 91 instructions 7 location in filing system 99 moving to another system 26 naming 40 notes 38 notes entry 82 open file 20 overview creating and editing 45 pausing 109 preparing for scouting 71 printing 101 problems 401 red instructions 402 running file 21 saving 40 99 saving method as a template 99 saving with a new name 41 start
349. on box Repeat this for all red instructions before saving the method I ve logged out of Windows NT and then logged in again but I can not get system connection in UNICORN only for local systems not remote If you shut down Windows NT using the command Start Shutdown Close all programs and log in as a different user you will not be able to obtain a system control connection in UNICORN the next time you or another user logs on This is because the aforementioned shutdown procedure automatically shuts down a number of processes including those needed for system connection that are only started when the computer is booted up In other words you must restart the computer in order to obtain a system connection in UNICORN Print screen does not send a copy of the screen to the printer Print screen only makes a copy of the screen to the clipboard and not to the default printer If you wish to make a print out of the view on the screen press the lt Print Scrn gt key and paste the image from the clipboard into an appropriate program such as Microsoft Paint and then print out the image Troubleshooting B B 4 Evaluation problems B 4 1 Incorrect date and time The date and time recorded in the result file are taken from the PC system clock setting If these are not correct check the system clock setting B 4 2 Evaluation procedure aborts Instructions in an evaluation procedure address curves by identification number irrespectiv
350. on is only available if the Analysis module has been purchased and installed The Setup Quantitate dialogue is used to define the settings for the Quantitate and Molecular Size object Presenting results 9 Setup Quantitate x Settings Eont M Starton new page Fig 9 54 Setup Quantitate dialogue 1 If appropriate change the characteristics of the Fonts by clicking on the buttons Title and or Text 2 When you have made your selections click on OK The Quantitate and Molecular Size object is now inserted into the report Frac 950 The Setup Frac 950 dialogue is used to define the settings for the Frac 950 object Settings Font I Start on new page IV Include rack layout Cancel Help Fig 9 55 Setup Frac 950 dialogue 1 Click on the Font button to set the font for the text 2 The Include rack layout check box is checked by default This will display the rack layout used in the run 3 Click on OK to set the Frac 950 object into the report e p241 ED Presenting results Note an 18 1138 73 e p242 Moving and resizing objects Objects can be freely moved or moved and sized according to the various commands in the Layout menu or toolbar Resizing the width of the objects with Make same size or Make same width can only be performed on chromatograms free text and pictures 1 To select an object for moving or sizing click on the Select toolbar i
351. on level Initially the Define Component s window displays the components from level 1 1 the peak table from the highest concentration of the standard Use the Show curve for level menu to examine the curve for each standard run As you select levels down the list there is a progressive reduction in the size of the components reflecting the decreasing concentration of the standard If an internal standard has been incorporated its peak remains about the same size in each level Selecting the components Use level 1 1 to select the components Each peak detected during the peak integration i e present in the peak table is identified by a lower triangle black in level 1 1 green for other levels There may be different peaks detected for different levels Upper triangles will later identify peaks selected for quantitation 1 Click on a peak to highlight its position in the table 2 Double click on a peak or click on Include to select it for quantitation More than one peak can be selected to produce calibration curves for several components of interest e p323 ED Analysing results an 18 1138 73 p324 Selected peaks will be shown with an upper triangle and with the name component no in the table The selected peaks are the same in all levels Change the component name by marking it and enter a new one When clicking on a selected peak vertical cursor lines will show its identification window see below
352. on line help 5 open chromatogram layout dialogue 188 method file 20 molecular size table 365 result file 185 320 result files 21 out of limit 339 overlay imported curves 223 P pages start protocol pages for method templates 42 password entry 17 PC connecting to more than one chromatography system 13 peak adjusting peak start and end points 280 adjusting the limits 324 adjusting the peak limits 278 adjusting the reject peaks criteria 320 changing peak labels 262 deleting a peak in the peak table 279 direct measurement of retention time and peak heights 281 displaying the retention 325 excluding 324 excluding peaks from integration 263 filtering peaks from view 262 identification 284 324 identification names for peaks 281 integrating 259 integration 12 joining a peak 279 measuring peak asymmetry 283 measuring retention time and peak heights 281 optimising peak integration 262 peak table column components 375 purity 284 purity and identification 12 reject peaks 263 selecting area or height for calibration curve 328 selecting for quantitation 323 selecting the peaks of the spiked and unspiked samples for standard addition 344 selection for molecular size curve 363 skimming 265 splitting a peak 279 viewing peak table data 282 peak identification by absolute retention 326 by relative retention 326 closest to retention 327 default settings 325 highest peak maximum 327 maximum peak area 327 settings 309 peak integr
353. on the Add Page button A new page is added after the last page To delete a page while in single page mode click on the Delete Page button Confirm the deletion To delete a page in dual page mode click on an object on the page and then click on the Delete Page button Confirm the deletion Adding objects To add an object to the report 1 Select an object from the Insert menu or click on the appropriate toolbar icon 2 Move the mouse pointer into the page area of the window You will notice that the pointer has an additional symbol according to the object type you selected to insert 3 Press down and hold the left mouse button and drag out a box to the desired size Release the mouse button A dialogue is displayed specific to the type of object inserted Make the appropriate Presenting results 9 selections in the dialogue and then click on OK to view the inserted object The various object types and dialogues are as follows Free text The Setup Free Text dialogue is used to define the desired text and the settings for the free text object Setup Free Text x Settings I Start on new page Font IV Size to content I This position on all pages Cancel Help Fig 9 48 Setup Free Text dialogue In the dialogue you can 1 Enter text in the edit field 2 If appropriate insert the text box on a new page by checking Start on new page 3 Automatically size the free text box by checking Si
354. ons in XLS format to the file defined in Export to file EXPORT_ Normalises retention when None NORMALISE_ exporting multiple curves RETENTION EXPORT_ Exports the peak table in Peak Peak table PEAKTABLE_ table source to the file defined in source Export ASCII Export to file in ASCII format to file e p387 AY Evaluation functions and instructions an 18 1138 73 p388 EXPORT_ Exports the peak table in Peak Peak table PEAKTABLE_ table source to the file defined in source WKS Export to file in WKS format Export to file EXPORT_ Exports the peak table in Peak Peak table PEAKTABLE_ table source to the file defined in source XLS Export to file in XLS format Export to file EXPORT_ Exports the peak table in XML Peak table PEAKTABLE_ format source XML Export to file EXPORT_SEL Selects a curve for subsequent Source curve CURVES export using the Left cut limit IEXPORT_MULTI CURVES_ instruction The curve is cut according to the right and left cut limit and the number of points to be exported may be set by the Export every for example fifth point parameter Right cut limit IExport every Evaluation functions and instructions A EXPORT_ Exports the documentation in the ONOFF Varia DOC_400_ current result file in ASCII format Ibles ASCII to the file defined in Export to ONOFF Scout file If all parameters to this func ling ONOFF tion are OFF then no documenta Start Pr
355. ons may have specified maximum and minimum limits and may be defined to accept only integer values Editing methods 5 r Question Sample volume and type I Mandatory J Authorized r Answer type Value Input field N Min C Multiple choice C No Answer Max Value Integer Fig 5 37 Options for Value questions On the Questions tab press the Preview button to display the questions as they will appear when the method is run Alternative answers to multiple choice questions are not shown in this mode From preview mode press Edit to return to question editing mode Inserting a question To insert a new question after an existing question 1 Click on the existing question in the questions list 2 Enter the text status type and answer for the new question as required The Answer type panel determines what is displayed in the question definition field e Input field questions Enter a default answer if required e Multiple choice questions Click in the text field under Alternatives enter the answer and click on Add Delete Repeat this procedure to add other alternatives New alternatives are always added at the end of the list To remove an alternative mark the alternative in the scroll list and click on Add Delete e Value questions Enter maximum and minimum limits Check the Integer box if the question is to accept only whole numbers as answers 3 Click on Insert to add the
356. oop manually filled prior to method start Fig 5 39 The Notes tab in Run Set up The notes are entered as free text and may be edited using standard Windows editing functions to edit the notes Words wrap automatically at the end of the field Keystroke Action lt Ctrl X gt Cuts the marked text saving it to the clipboard lt Ctrl C gt Copies the marked text to the clipboard lt Ctrl V gt Pastes the contents of the clipboard at the cursor lt Delete gt Clears the marked text without saving it in the clipboard or deletes the character to the right of the cursor if no text is marked lt Backspace gt Clears the marked text without saving it in the clipboard or deletes the character to the left of the cursor if no text is marked In the default method templates supplied with UNICORN the method notes describe the system setup required by the method e g eluent and Editing methods 5 sample inlets outlets column connections and so on We recommend that you use method notes for this purpose in your own methods to provide documentation of the method requirements Bear in mind that method notes are saved with the method and apply to all runs made with the method Use the start or run notes for run specific information The date and time when the method was created and last edited are saved automatically in the method information and need not be entered in the method notes 5 5 6 Evaluation procedures Ev
357. ose_HR_5 5 Column Mi 0 00 Block Start_Conditions_BP 0 00 Block BufferPrep_pH_Flush Mi 0 00 Block Column_Equilibration Ml 0 00 Block Flowthrough_Collection 0 00 Block Wash_Out_Unbound_Sample W 0 00 Block Eluate_Fractionation_ W 0 00 Block Gradient_Elution E 0 00 Block Clean_After_Gradient_BP_ fj 0 00 Block Reequilibration_BP 0 00 End method e Unit Time Volume min Print Close Help Fig 5 60 Log Format dialogue displaying the cumulative time for a method block breakpoints shown in rectangle with arrow e p107 5 Editing methods an 18 1138 73 e p108 The concept of block time or volume versus cumulative time or volume is illustrated in the following table Cumulative Block Instruction time vol time vol Main 0 0 0 00 Block EQUILIBRATE Equilibrate 0 0 0 00 Base SameAsMain 4 4 4 00 End_block 4 0 0 00 Block SAMPLE_INJECT Sample_inject 4 0 0 00 Base SameAsMain 4 0 Seve 9 5 5 00 End block 9 0 0 00 Block GRADIENT Gradient 9 0 0 00 Base SameAsMain 9 0 0 00 Gradient 100 20 29 20 20 00 End_block Depending on how conditional calls are used see How to use selected conditional method instructions on page 112 the overall method time or volume may vary according to watch events during the run 5 10 4 Messages and Set_mark Messages Use messages to inform the operator of the progress of the run It is a good idea to issue
358. other peak characteristics such as retention time and peak width are automatically calculated The characteristics displayed in the peak table may be selected in the Chromatogram Layout dialogue Peak Tables tab Chromatogram layout 1 x Curve Style and Colour l Edit Texts Layout Library Header CurveNames YaAxis XAxis Cuve Peak Table Select peak table to display Select peak table columns width Area MHeight Peak endpoint retentions Width at half height Peaktable H _ Percent of total area CPercent of total peak area xl Filter Peaks I Height less than btw IT Width Jess than J Area less than a I Width more than jf J7 Peak must be one of jo largest T Apply to all chromatograms Cancel Help Fig 10 3 Chromatogram Layout dialogue Peak Table tab e p261 ED Evaluating results an 18 1138 73 p262 Select the options that you want to be displayed from the Select peak table columns list Most characteristics are automatically calculated for each integrated peak when the peak integrate function is used although only the selected items will be displayed in the peak table Changing peak labels As alternatives to using retention times as peak labels the peaks can be sequentially numbered or be marked with specific identification tags The choice of label type is made in the Curve Style and Colour tab in the Chromatogram Layout dialogue see Changing
359. otocol tion is exported If at least one of ONOFF Ques them is ON then the documenta tions tion will be exported and the cor ONOFF Ref responding parts will be included Curves in the exported file ONOFF Eval Proc ONOFF Method Info ONOFF Method Notes ONOFF Start Notes ONOFF Run Notes ONOFF Eval Notes ONOFF Sys Set tings ONOFF Cali bration ONOFF Log Book ONOFF Result Name ONOFF Col umn Parame ters NAME ONOFF Result Info ONOFF Method Sign ONOFF Result Sign Export to file e p389 AY Evaluation functions and instructions an 18 1138 73 p390 EXPORT_ DOC_400_ WKS Exports the documentation in the current result file in WKS format to the file defined in Export to file If all parameters to this func tion are OFF then no documenta tion is exported If at least one of them is ON then the documenta tion will be exported and the cor responding parts will be included in the exported file ONOFF Varia bles ONOFF Scout ing ONOFF Start Protocol ONOFF Ques tions ONOFF Ref Curves ONOFF Eval Proc ONOFF Method Info ONOFF Method Notes ONOFF Start Notes ONOFF Run Notes ONOFF Eval Notes ONOFF Sys Set tings ONOFF Cali bra tion ONOFF Log Book ONOFF Result Name ONOFF Col umn Parame ters NAME ONOFF Result Info ONOFF Method Sign ONOFF Result Sign Export to file Evaluation functions and instructions A EXP
360. ow Scheme Logbook l Run Data Groups Run Data Colour Curves Axis m Base C Volume m Axis scale Total C Window fio Tit I Adjust retention zero to injection Fig 6 12 Properties dialogue X Axis tab e p153 ED Performing a run Maximize Hide Next Layout Colour Settings gt Set Unit iv MPa mae ba Properties a pe an 18 1138 73 e p154 5 Select the appropriate Axis scale either Total or Window The Total option will show the curves as far as they have come in the run The Window option allows you to set the portion of the total window to be displayed either in minutes or ml depending on the selected base The Adjust retention to zero box which influences the curve view and records in the logbook is checked by default Click on OK The zoom function To zoom in on a selected region of the curve window 1 3 Press down and hold the left mouse button and drag a rectangle out on the screen to encompass the area to be viewed Release the mouse button The display is now zoomed in on the selected area Repeat the process for further magnification of selected areas You can reduce the scale of the zoom in function in two ways either reverse each zoom in action a step at a time by displaying the shortcut menu and selecting Undo Zoom or reverse all of the zoom in actions to the default scale setting by displaying the shortcut menu and selecting Reset Zoom V
361. owever only the row with the block currently marked in the Text instruction window will be deleted In this case the block will not be placed in the Unused section since the block is still used in the method Blocks deleted in this fashion can be called again in the method Deleting blocks using the Block Delete Block command To delete a block from the method using the Block Delete command 1 Select the menu command Block Delete Block The Delete Block dialogue is displayed with all blocks listed in alphabetical order 2 Select the block to be deleted and click on Delete 5 2 5 Renaming blocks To rename blocks choose Block Rename Block or select a block in the method using the shortcut menu and select Rename from the shortcut menu In both instances the Rename Block dialogue is displayed Rename Block Select the block to rename Clean_After_Elution_with_B2 Column_E quilibration Direct_Sample_Injection Flowthrough_Fractionation Flush_Sample Fractionation_Step Initial Eluent_Conditions Step Prepare Sample Loading Reequilibration_Step Rinse_Sample_Flow_Path Start_Conditions_Step System_Volume_Compensation Wash_Out_Unbound_Sample Fig 5 10 The Rename Block dialogue Editing methods 5 By default the block that is currently highlighted in the method text instructions is automatically selected in the dialogue Enter the new name in the New Name field and press Rename The dialogue remains open until you press Close
362. p tab cannot be edited although fine tuning is possible see below However the recipes in the list of all BufferPrep recipes Edit BufferPrep Recipes can be edited see Editing a BufferPrep recipe on page 137 New recipes can also be created see Creating a BufferPrep recipe on page 133 Fine tuning with correction factors In order to obtain high pH accuracy the recipe can be fine tuned around a specific pH by setting correction factors Run BufferPrep manually at 0 and 100 and change correction factors in the method as follows 1 Select the Recipe under group Other in System Control Manual Click on Execute Set the pH in the instruction BufferPrep_pH in group Pump Click on Execute Set the flow rate to be used during the run in instruction Flow Click on Execute 2 Check the pH reading when stable Allow at least 30 ml of eluent to pass through before expecting a steady pH reading 3 Change to 100 B by setting the Gradient instruction in Manual Pump to 100 for Target and 0 for Length Click on Execute 4 Check the pH reading when stable at 100 B 5 Ifthe readings are acceptable at both 0 and 100 the correction factors do not need to be changed 6 Ifthe readings are not acceptable click on Corr Factors in the BufferPrep tab in the method 7 Enter the deviation at 0 and 100 E g if the pH is set to 7 0 and the actual pH is 7 1 enter 0 1 Enter 0 1 if the pH is 6 9 If correction facto
363. p topics can be obtained by clicking on the Help button in the dialogue or by pressing lt F1 gt on the keyboard In the dialogues for method instructions procedure instructions and system settings pressing lt F1 gt when an instruction is highlighted will display an information box with short help on the function and use of the selected instruction 2 3 Files and folders UNICORN Main Menu interface divides user files into two categories for methods and results Only folders to which the current user has access are shown in the Main Menu windows Method window and Results window Files may be displayed in several viewing options see Chapter 3 for more details 2 3 1 Method files Method files contain instructions for controlling a run and are shown in the Methods window of the Main Menu The Methods window also displays icons for MethodQueues which allow several methods to be run in an automatic pre programmed sequence on the same or different systems 2 3 2 Result files Result files are created by UNICORN when a method is run and contain e Run data from the monitors in the chromatography system e g UV absorbance flow rate conductivity etc ep5 2 UNICORN concepts an 18 1138 73 e p6 e Saved results from evaluation of the run data see Chapter 10 e Run documentation including information on for example the Logbook calibration settings scouting parameters text method etc 2 4 Methods Chromato
364. ple if you wish to mark the chromatogram with the sample ID when using the autosampler Click on OK when you have made your selections to return to the Setup Chromatogram dialogue 4 If appropriate change the characteristics of the Fonts by clicking on the buttons Chromatogram Peak table and or Header text 5 When you have made your selections for the chromatogram s click on OK in the Setup Chromatogram dialogue The chromatogram s are now inserted into the report Method The Setup Method dialogue is used to define the settings for the method object Setup Method xi Selected items will be included Main Method Blocks Title Text Fonts Settings Start on new page V Expand main Cancel Help Fig 9 51 Setup Method dialogue Presenting results 9 1 Select the items to be included Main Method This is the method on which the run was based Blocks These are the blocks that were used in the method 2 Select as appropriate for the inserted method to Start on new page and or to Expand main to show the expanded view of the method 3 If appropriate change the characteristics of the Fonts by clicking on the buttons Title and or Text 4 When you have made your selections click on OK in the Setup Method dialogue The method is now inserted into the report Documentation The Setup Documentation dialogue is used to define the settings for the documentation object
365. ple you intend to purify Then use this result to find slope values in the Evaluation module 1 Ensure that you have selected Time as the X axis scale for retention in the Chromatogram Layout dialogue X Axis tab 2 Select Operations Differentiate Select the desired UV curve check that a First order calculation is selected and click on OK The differentiated curve will appear in the active chromatogram Differentiate xi Source chromatogram Target chromatogram i 01 ATJMFOO6 1_UV1_ 215mm 02 ATJMFOO6 1_UV 2_254nm 03 ATJMFOOB 1_UV 3_280nm 04 ATJMFOOB 1_Cond 05 ATJMFOO6 1_Cond 06 ATJMFOO6 1_Conc 07 ATJMFOOB 1_pH ond roe gt 08 ATJMFOO6 1_Pressure Cutve name nn AT nRACONe 4 Fie P uv1_21 5rm 01 A dt First order Secondorder C Inverse Cancel Help Fig 10 34 Differentiate dialogue 3 Measure the Y axis values on the differentiated curve by selecting Marker from the shortcut menu choosing the Y axis differentiate scale and reading the curve co ordinates in the active Marker box It may be necessary to smooth the differentiated curve The units for the differentiated curve is mAU min or AU min Co ordinates are based upon the position of the vertical line on the chromatogram in relation to where it bisects the curve Any Y axis value for the differentiated curve is the UV curve slope at the selected retention point Evaluating results 10 LITEZ 1 to 4002
366. ponding Y axis is displayed 3 Use the mouse to drag the vertical bar back and forth along the X axis For a given fraction its conductivity is displayed in the marker box 10 3 Automated evaluation procedures An evaluation procedure is a recorded sequence of interactive operations in the Evaluation module which can be executed for automated data evaluation and report generation It can be used for single chromatograms and for a number of chromatograms in different result files The concept is analogous to the macro facility provided in many word processing and other programs Evaluation procedures can also be called from methods making run execution evaluation and documentation fully automatic Automation is achieved using the Procedures menu Evaluating results 10 A procedure can be recorded and run using the Procedures menu commands or from the commands available in the Procedure Editor dialogue The Procedure Editor dialogue also allows you to view and edit the instructions within a procedure 10 3 1 Recording a procedure 1 Open the appropriate results file in the Evaluation module 2 To begin recording a procedure select Procedures Record on The Procedure Editor dialogue is displayed in Record mode 3 Minimize the Procedure Editor dialogue 4 Perform the evaluation steps that the procedure is to contain These steps are recorded as you perform them 5 To stop the recording either select Procedures Record
367. ponent internal standard is added 2 The addition of the internal standard must be accurate in both standards and samples otherwise the precision of the quantitation will be reduced dramatically e p313 ED Analysing results an 18 1138 73 p314 11 2 4 Standard addition quantitation This is a simple way to obtain measurements of amount in your sample concentration is not calculated It requires only two runs one with the sample and a second with the sample which has been spiked with the component of interest The technique is straight forward and relatively quick when you are running only a few samples Standard addition might be useful when you want to use the internal standard technique but do not have a suitable internal standard The disadvantages of this technique are its limited precision compared to the external and internal standard techniques and its ability to measure only one component An outline of the technique 1 Arun is performed with the sample The procedure for performing a run is described in Chapter 6 2 A second run is performed with a sample that has been spiked prior to sample preparation with a known quantity of the component of interest mAU 250 4 Sample unspiked 200 4 150 l 100 7 50 J vA N 0 D a eee 15 0 16 0 17 0 18 0 min mAU 950 4 7 Sample with P standard addition Area corresponding ae ae to the sample _ spiked St o
368. port a method select File Export Method to access the Export Method dialogue Export Method X Export Method Block list Options I Main only I Current expansion MV Exclude unused blocks h Cancel Help Fig 5 56 The Export Method dialogue Select whether the current method should be exported as a Method or as a Block list Check the appropriate Option boxes to define the level of detail in the information Select Export to prompt the Export Method to file dialogue Enter a file name and select the target drive and folder Click on Save 5 9 Exporting Run Set up This dialogue is used to export information in Run Set up Select File Export Run Set up to access the Export Run Set up dialogue Editing methods 5 Export Run Set up x Select parts to include in the export Method Information ViStart Protocol v Result Name Cancel Help Fig 5 57 The Export Run Set up dialogue Check the boxes to select the parts of the Run Set up to be exported Select Export to prompt the Export dialogue Enter a file name and select the target drive and folder Click on Save 5 10 How to use selected unconditional method instructions This section provides recommendations for how to use some common programming features in UNICORN methods 5 10 1 Base instruction Every method block must start with a Base instruction defining the base for calculating breakpoints The base
369. process To print a molecular size table that was prepared previously 1 Open the molecular size table see Opening Renaming or Deleting a molecular size table on page 365 2 Click Print to print the data Opening Renaming or Deleting a molecular size table To open a previously saved molecular size table 1 Select Mol Size Edit Mol Size Table Open 2 If you wish to access tables available only to your user id click Personal 3 Highlight the name of the table you require in the Molecular size table s list to copy it to the Molecular size table name field 4 Click OK to open the table or Cancel to abort the operation To rename a molecular size table 1 Select Mol Size Edit Mol Size Table Rename 2 If you wish to rename a table available only to your user id click Personal 3 Highlight the name of the table you wish to rename in the Molecular size table s list 4 Type the new name for the table in the Molecular size table name field 5 Click OK to rename the selected table or Exit to abort the rename procedure To delete a molecular size table 1 Select Mol Size Edit Mol Size Table Delete 2 If you wish to delete a table available only to your user id click Personal 3 Using the Molecular size table s list highlight the table to be deleted e p365 ED Analysing results an 18 1138 73 p366 4 Click Delete to delete the table or Exit to abort the delete procedure C
370. r runs under a variety of conditions see Chapter 4 Variables also form the foundation for automatic method scouting see Chapter 7 Variables are defined with names which can be explicit descriptions of the variable function e g Sample_volume Gradient_length Suitable choice of variable names can make the method easier to read and understand and also help the operator in setting variable values at the start of a run Each parameter defined as a variable is also assigned a default value which is used if no changes are made to variable values at the start of a run Up to 500 variables can be defined in a single method 5 4 1 Identifying variables Parameters defined as variables are identified in two ways e in the text instruction window the parameter is given as the default value in parentheses followed by the variable name e g 4 00 Empt y_loop_with Editing methods 5 Sample_injection 0 00 Base Volume 0 00 InjectonValve Inject 0 00 Outletvalve F2 4 00 Empty_loop_with InjectionValve Load 4 00 End_block Fig 5 23 Default values for variables appear in parentheses in Text instructions e when the instruction is shown in the Instruction box the VAR button beside the parameter field is active in capital letters i e VAR not Var p Parameters AutoZeroUY VAR va a Insert AlarmstMon _ var oe Eai I OFF ome De Var e e ues I OFF Replace C Watch C
371. r the system are displayed To save the results of a manual run issue the instruction Record_on in the Other instruction group at the beginning of the run UNICORN will prompt for a result file name at the beginning of the run When you choose to run the system manually as opposed to a Method run the results are stored in a folder called Manual Runs The Manual Runs folder stores the ten most recent results from your manual runs To more permanently save a result from a manual run you need to move or copy it to another location The Manual menu opens a dialogue similar to the text instruction box in the Method Editor Explorer100 Flowpath Instructions Ea r Instructions r Parameters Pump Connect Position Insert olumnPosition OutletValve Load x Delete BufferValveA1 AlarmstMon Pump lnlet f Frac PumpBlnlet SampleValve Close C Other FlowDirection xl Help Fig 6 17 The Manual instruction dialogue The name of the connected system is displayed in the title bar of the dialogue Available instructions are determined by the strategy and selected optional components for the connected system Manual instructions are entered in the same way as method instructions from the dialogue in the Method Editor The Insert button places the current instruction in the list at the bottom left of the dialogue Clicking on Execute executes all instructions in the list at the same time or
372. ransfer it to the right hand side Peak table s list You can select further peak tables providing all the runs are made under the same conditions If you need to de select a table highlight it in the right hand window then click on Remove Peak tables from other chromatograms can be added to the peak tables list To do this select a result file as before highlight the source chromatogram and select the peak table s required Once the Peak table s list contains all the items required click OK to open the Molecular size table dialogue If the unit of size measurement is different from the default kDa enter the appropriate unit in the Molecular size unit label field Molecular size table Peak table id 19301 1_UV1_280nm 47 PEAK Double click on a peak to select it r n Enter molecular size Retention Mol size 4 Retention Fuya model min kDa C Linear 4 44 Linear log Mw 0 80 C Quadratic 0 82 C Quadratic log Mw 1 68 Point to point 6 42 63 300 Point to point log Mw 7 90 42 700 y 8 42 Statistics 9 59 19 500 Explained variance 98 039 10 43 13 700 Conelation 0 9934 WA i a 12 85 z Tog Mol size ae Excude Print Save Save as Help Fig 11 47 Molecular size table dialogue e p361 ED Analysing results an 18 1138 73 p362 You use this dialogue to select the peaks which will be used to produce the molecular size curve Each curve and its p
373. rary chromatogram is essentially an empty chromatogram and is specific to the Evaluation module Thus curves can be copied into Temporary using Edit Copy Curve and comparisons and or evaluations can be performed This is particularly useful if you do not want to clutter up your original chromatograms with a large number of curves It can also be used to keep blank run curves or curves to compare when opening different result files Information contained within the Temporary chromatogram is automatically saved from one evaluation session to the next but is not saved within the result files Click on the window restore button or select Windows Temporary The contents of the temporary chromatogram can be removed by selecting Edit Clear Temporary Chromatogram 9 2 Basic presentation of chromatograms This section gives directions on how to access result files and optimise the presentation of a chromatogram and its curves via the Chromatogram Layout dialogue The last evaluation operation that is performed can be undone using Edit Undo Presenting results 9 9 2 1 The chromatogram window The chromatogram window is divided into three main views for header information run curves and peak tables The displayed areas for the views can be adjusted by dragging the borders with the mouse cursor between the views Viewing the curves The first time a chromatogram window is opened and viewed a default layout is applied to display all of the original c
374. ration Logbook EvaluationLog Frac 350 SamplePump Calibrated 01 01 26 11 21 by Ingrid Reference Value 1 167000 ml Pump Calibrated 01 01 27 12 56 by Ingrid Fig 9 66 Documentation Calibration tab e p253 Presenting results This displays what happened during a run It is possible to view information concerning alarms the method manual changes during the run errors and the system Variables Scouting TextMethod Notes Questions Columns Reference Curves Evaluation Procedures Method Information Resul Information ResutName Start Protocol Settings Calibration Logbook EvaluationLog Frac 950 p Fiter M Alam F Method Manual I Enors M System 5 00MPa O 00MPa Pump lnlet A1 BufferValveA1 A11 Ww mnl min a Gradient 0 00 B 0 000 CV Volume End Block End Block Adjust retention zero gt Block Column_Equiibration ea e Base SameAsMain End Block Block Autosampler_Injection Base SameAsMain l Message Level l Quantitate Info Injection Volume 0 050 ml i Hold 2001 01 09 09 26 30 InjectionPartial 1 50 ul Yes Air ml Samplelnject 0 r Base C Time 223 JAJAHANE S 8 2 33233 2 Fig 9 67 Documentation Logbook tab Evaluation Log This lists all of the evaluation operations that you have performed for the current result file for all sessions including procedures executed at the end of the metho
375. rd or template for AKTAdesign systems select Sample in the variable Quantitation_Type 11 4 1 Quantitation by external and internal standard The processes involved in external or internal standard quantitation of a sample are very similar The procedural differences occur mainly during the creation of the quantitation tables A quantitation table is specific to either internal or external standard quantitation e p339 ED Analysing results an 18 1138 73 p340 For an explanation of the external and internal standard techniques please see External standard quantitation on page 308 and Internal standard quantitation on page 310 respectively Preparing for quantitation A quantitation table for the components of interest must first have been prepared Creating a quantitation table on page 320 1 Perform a run with the sample If internal standard quantitation is being used the internal standard must have been added to the sample prior to the sample preparation procedure The injected amount must be the same as in the standard levels 2 Open the sample result file and peak integrate the sample curve to produce a peak table During integration the sample curve must use the same X axis base unit as the standards Time is the recommended unit for highest reliability 3 Use File Save to save the peak table before continuing with the quantitation If you want to show the names of the components above th
376. re 09 HIPREPO2 1_Flow 1110 HIPREPO2 1_Temp 011 HIPREPO2 1_Fractions 112 HIPREPO2 1_Inject Clear Select all I Apply to all chromatograms Cancel Help Fig 9 4 Chromatogram Layout dialogue Curve tab The layout of the three views for header curves and peak table can be modified in the various tabs that are displayed in the Chromatogram Layout dialogue You can work freely in the Chromatogram Layout dialogue and all of the configurations are applied when you click on the OK button If instead you want to close the dialogue without applying the changes you have made click on the Cancel button The main features of the Chromatogram Layout dialogue regarding chromatograms are described in the sections below Features regarding peak tables are described in chapter 10 9 2 4 Choosing the curve s you want to see In the Curve tab of the Chromatogram Layout dialogue is a list of all curves contained within the chromatogram numbered from 01 onwards Select the curves you want to see in the chromatogram Click on OK to return to the active chromatogram window 9 2 5 Displaying curve names By default names are sequentially built up from three components e result name e chromatogram name e curve name e p189 ED Presenting results For example a curve with the name 9139401 1_UV1_280 is derived from the result named 9139401 The chromatogram name is a number automatically given during a run e g 1 The
377. re curves or chromatograms from different runs It is possible to e compare curve parameters among curves from different runs e view several chromatograms at the same time e overlay curves from different runs in one chromatogram Presenting results 9 o stack curves from different runs in one chromatogram e stretch curves to make comparisons easier e create mirror images 9 4 1 Multifile Peak Compare 9 4 2 Wizard set up To open Multifile Peak Compare choose File Multifile Peak y Compare Start Wizard or click on the Multifile Peak Compare toolbar icon This will prompt the Multifile Peak Compare Wizard entry dialogue Click on the Next button to display the Operation dialogue Operation xi r Select operation Compare scouting runs C Media life time study r Select retention unit Cmn Cl Co Cy Col lt Back Cancel Help Fig 9 17 Operation dialogue within Multifile Peak Compare Select from the available operations choose a retention unit and click on Next This will prompt the Data Selection dialogue e p207 ED Presenting results Note an 18 1138 73 p208 Data Selection xj j Chromatogram selection Folder esetu Browse Result FSCS Browse Al Chromatogram fi amp l Browse af Curve name pm Browse aj j Found curves id1 86Level 1 to 4003 id186Level 1 to 4003 1_UV1_280nm lid186Level 1 to 4003 id186Level 1 to 4
378. rmation A variant of this model is available for use in the production of a molecular size curve This uses the logarithm of the molecular size as the x value in the above expression Model function values Fig 3 1 Statistics table for the linear model Minimum no of points required 2 at least 4 recommended Measuring range for the calibration curve within the highest and lowest values for the points an 18 1138 73 p406 Linear through the origin Based on the equation y Ax The constant A is determined by linear least squares regression Please see a statistics textbook for further information Minimum no of points required Mathematical models and statistics Statistics x Model i x Model function values Corr 3949 Number of point s 4 Fig 3 2 Statistics table for the linear through origin model 1 at least 2 recommended Measuring range for the calibration curve from the point with the Quadratic Based on the equation y Ax Bx C The constants A B and C are determined by linear least squares regression Please see a statistics textbook for further information A variant of this model is available for use in the production of a molecular size curve This uses the logarithm of the molecular size as the x value in the above expressions Minimum no of points required highest value down to the origin Statistics x Model y x
379. roups Curve Style and Colour Run Data Colour Flow Scheme Logbook Curves Axis WhRun Data MVGroup VGroup3 Instruments Connection Run Status Scouting No Acc Yolume Block Volume Acc Time Block Time Flow Pressure OS ampleFlow CS amplePres UV1_215 Rand D K K K K K K K K i J Edit Group Delete Group Fig 6 5 Properties dialogue Run Data Groups tab 3 Select individual run data parameters to view and click on OK an 18 1138 73 e p146 Note Performing a run GH Alternatively select a group either an available group Single or multiple selections of available groups can be made by clicking the check boxes in the list to the left edit an available group Select the group from the list on the left and click on the Edit Group button Modify the included readings in the list to the right Finish the dialogue by clicking on OK create a new group Click on the New group button and select the readings that you want to view from the list then enter a name for the group Finish the dia logue by clicking on OK For systems with optional components parameters are not shown for components that are not included in the system 4 To delete a group click on Delete Group and click on the check boxes in the delete layout dialogue 5 Click on OK to view the selection in the Run Data window The name of the layout selected replaces th
380. rs already exist the measured pH deviation should be added to the old factors 8 Save the method When changing the correction factors for the recipe selected in the method the recipe with the same name in the list of all BufferPrep recipes is not affected The changes will only apply in the specific method Editing methods 5 For changing the default correction factors in a recipe not saved in a method see Editing a BufferPrep recipe on page 137 5 5 10 Method Information The Method Information tab displays information about the method such as method name target system and date of last change Strategy information includes strategy name date and size The Strategy Notes button displays a list for which systems programs and file versions the strategy is designed for Variables Scouting Notes Questions Gradient BufferPrep Columns Reference Curves Evaluation Procedures Method Information Result Name Start Protocol Frac 950 jl Signatures Method Duration Method information Method name d Default Id23 m01 Method created by Niklas Date of creation 7 28 1999 12 26 21 PM Created for system Explorer100 Method last modified by default Date of last modification 3 28 2001 3 39 42 PM Strategy information Strategy name 100 200 Strategy date 1 17 2001 11 43 28AM Strategy size 1965456 bytes Strategy Notes Fig 5 46 The M
381. rtcut menu Alternatively Use the lt Page Down gt and lt Page Up gt keys to zoom in and zoom out respectively on the whole chromatogram Fixed scale axes Another way to display only part of a curve is to choose or fix the minimum and maximum values of the Y and or X axes in the Chromatogram Layout dialogue see Changing and fixing the axes on page 192 Cutting curves The cut curve function allows a region of the curve between two values on the X axis to be cut and stored as a new curve This is done in the following way 1 Select Operations Cut curve Presenting results 9 In the displayed dialogue select the curve s to be operated on Click on OK 3 The selected curves will now be shown in a new window which also contains two vertical cursor lines To facilitate the cutting process it is possible to use the zoom function within the window Q Cut olx id186Level 1 to 4001 1_UV1_280nm mau ob 5b 100 150 200 25 0 Left limit 11 8002 Bight limit 13 38409 Cancel Help Fig 9 12 Cut window To select the region to be cut either e drag the two cursor lines to define the left and right limits of the cut area or e type the desired left and right limit values in the boxes marked Left limit and Right limit Note The areas outside of the Left limit and Right limit will not be saved in the newly created cut curve Thus the X axis of the new saved curve
382. ruction The column volume is taken from the column definition and cannot be changed in the instruction The column name can be defined as a variable Note 1 Note 2 Note 3 Editing methods 5 In cases where a template including Wizard generated methods and column are chosen it is easy to select other columns for that template in the Variables tab To select a column for a method when the method is not selected from a template 1 Define new columns in the Columns list if required see Adding a new column on page 128 2 In Text instruction mode mark the Base instruction in the method the first instruction 3 For the Column parameter in the Instruction box choose the required column from the drop down menu 4 Define the Column parameter as a variable recommended This will make it easy to change the column selection for different runs 5 Click on Change in the Text instruction box A selected column definition applies locally within the block for which it is selected and is not transferred to other blocks We strongly recommend that the column definition is selected for the main block A column definition can be chosen and defined as a variable even if the base for the block is set to volume or time Parameters in the column definition will then be used for linear flow rate and column performance calculations If you want parameters to be updated when you change the column you must define flow press
383. s are displayed at the top right corner of the window Changing the curve colours and styles The curves window displays graphs for the selected curves in different colours with any reference curves included with the method as dashed lines The curve colours and styles can be changed Performing a run GH Select View Properties or select Properties from the shortcut menu menu The Properties dialogue is displayed Select the Curve Style and Colour tab Properties X Run Data Groups Run Data Colour Curves Axis Y Axis Curve Style and Colour FlowScheme Logbook SamplePres xl C sranta Clans m Fraction text alignment Logbook text alignment Horizontal Horizontal C Vertical Vertical C Fly over Fly over Filter Cancel Help Fig 6 9 Properties dialogue Curve Style and Colour tab Select a curve from the list and then select an appropriate colour and style Select the Fraction text alignment as desired Select the Logbook text alignment It is possible to filter the type of information overlaid on the curve window by clicking on the Filter button Select the information by pressing the radio buttons and set the maximum block depth Click on OK to return to the Curve style and Colour tab e p151 6 Performing a run an 18 1138 73 e p152 Filter Logbook Fig 6 10 The Filter logbook dialogue 5 Click on OK to save the Curve style and colour settings
384. s for the current and subsequent runs at the beginning of every run If the start protocol is not displayed at the beginning of each run the sequence of runs will be performed automatically according to the settings entered in the start protocol for the first run 3 Work through the rest of the start protocol then press Start The results of a scouting run are saved in a special scouting folder as defined in the Results tab of the start protocol Within the folder each run is saved in a separate result file named according to the usual naming rules see Result Name on page 91 If the start protocol is displayed for each run in a scouting scheme you may be able to change the result file name during scheme execution 7 2 1 Changing scouting settings during a run At any time during a run you can click on the Documentation icon in System Control and change the scouting settings in the Scouting tab for runs which have not yet been started Settings for the run which is currently in progress cannot be changed You can use this feature to adjust settings for scouting even if the start protocol is not displayed at the beginning of each run 7 2 2 Adding scouting runs during a run As long as the last run has not yet started it is possible to add new scouting runs To do this click in one of the variable fields in the first empty column The values from the previous run will be copied into the new column and you can change the variabl
385. s in the event of hard disk failure backup copies should be taken at regular intervals This can be best achieved by having the UNICORN folders on the server if available and working directly from these folders Alternatively you can use the File Copy to External function to save files onto the network server It is standard practice for backups to be made of the server folders The responsibility for making backup copies rests entirely with the user Amersham Biosciences cannot undertake to replace method programs lost as a result of computer failure or other incident 3 4 Printer setup UNICORN 4 0 uses the default printer and printer settings installed on your computer To change the choice of printer either change the default settings in Windows NT 2000 or set up your choice of destination printer for the current working session by selecting File Printer setup in the Main Menu module and selecting the desired printer 3 4 1 Setting the margins The default margins for the printers can be changed 1 Locate the file UNICORN INI found under C ANUNICORN BIN for example by using Windows NT Explorer 2 Double click on the file to open it and locate the following lines EVAL PrintMarginLeft10 EVAL PrintMarginRight5 Eval PrintMarginTop5 Eval PrintMarginBottom5 The values in the lines set the margins based as a percentage of the full width and height of the paper 3 Change the values as appropriate and sav
386. s it easy to adapt a method to a particular chromatography run For example in the block below the breakpoint for switching the InjectionValve from Inject to Load is defined as a variable with the name Empt y_loop_with The value supplied for this variable when the method is run will set the volume with which the loop is emptied Sample_Injection 0 00 Base Volume 0 00 InjectionValve Inject 4 00 Empty_ loop with InjectionValve Load 4 00 End_Block Variable names in method instructions are always preceded by the character The value for the variable is shown in parentheses in the instruction in the example above 4 00 is the value for the variable Empty_loop_with By using variables a method may be displayed either in detail as Text instructions or in a condensed form as variable values in Run Set up mode This is illustrated in Figure 2 3 The Run Set up mode is displayed when the method is run allowing variable values to be set at the beginning of the run Up to 500 variables can be included in a single method UNICORN concepts ez Ml 0 00 Block Wash_Out_Unbound_Sample Wash_Out_Unbound_Sample 0 00 Base SameAsMai Wash_Out_Unbound_ Sample Wash_column_with CV 2 000 q 2 Wash_column_wit P gt End_block Eluate_Fractionation_V Start_Frac_at B 0 000 SM 0 00 Block Eluate_Fractionation_ Start_Valve_Fractionatio Eluate_Fractionation_ 0 00 Base SameAsMain 0 00 Outlet alve
387. s programmed to start The actual time of start is shown for a currently running MethodQueue The buttons in the MethodQueue display have the following functions Restart Restarts the currently running MethodQueue if a start protocol has been terminated by Cancel End Terminates a running MethodQueue after the current step Any methods currently in operation will continue to run and must be terminated with End in the System Control window if they are not to run to completion Clicking on End for a pending MethodQueue deletes the MethodQueue from the pending list Exit Closes the MethodQueue monitor panel e p183 og MethodQueues an 18 1138 73 e p184 Presenting results 9 9 Presenting results A result file is automatically generated at the end of a run and contains a complete record of the run including method system settings curve data and run log The Evaluation Module offers extensive facilities for presenting and evaluating curve data This chapter describes how to e view the documentation from a run e present the chromatograms and curves of your result file e compare chromatograms and curves e print reports 9 1 Opening a result file Evaluation Peak skim example 1 _ c UNICORN Local fil Default result Peak skim example res 4 Ele Edit yiew Integrate Operations Procedures Window Help 2 8 UsSel sao hal E HIPREPO2 1_UV1_280nm HIPREPO2 1_UV1_280nm 01 BASEM ATJMFOO8 1_UV1
388. s visible in the Page Setup dialogue The Header tab allows you to select items to include in the headers of the individual pages except for the first page Page Setup xi Page Setup First Header Footer Header The selected items will be included in the report m Select Items m Logo Presenting results cE Eurentuser a Font C Loge ea Left Run user Select Al C Middle Current date amp time C Bight Run date amp time lear All gam Lyu Report title I Line over K K K K Result file name Method file name Page number CL aueut mame x I Line under lt Free text Font Report title Report Tite Font Cancel Fig 9 57 Page Setup dialogue First Header tab 1 6 T Check the items to be included in the header Click on Select All or Clear to facilitate your selection as appropriate If appropriate click on the Font button to alter the font characteristics Add Free Text and a Report Title as appropriate You can also change the font style for these options If you have a logo in bitmap format this can be added to the header Check the Logo option and then use the Browse function to locate your bmp logo file Select the alignment of the logo either Left Middle or Right Select if you want a Line under or Line over the header Click on OK to implement the sele
389. s_BP 0 00 AveragingTimeUY AvTimeUVHUY_Averaging_Time 5 12 Column Value 5 12 sec Fig 5 31 Column value update dialogue This dialogue asks if you want to update the method for the particular scouting run with the default values for the column parameters The answer that you give to this dialogue has different consequences in terms of the scouting variables Yes No The method for that scouting run is updated with variable parameter values for the selected column consisting of UV averaging time pressure limit flow rate etc These parameter values are added to the scouting variables present on the Scouting tab Note that the updated parameter values may differ from the values for the same variables in other scouting runs No changes are made The method retains the parameter values corresponding to the column that were either i originally selected during creation of the method or ii included in an earlier version of the method on the Scouting tab where the default values for that column were accepted Editing methods 5 1 2 3 Evaluation Procedures Methqd Information Result Name Start Protocol Frac 950 Variables Scouting Notes Question Gradient BufferPrep Columns Reference Curves Block Variable Main Column Alkyl Supe HiLoad_16 Superdex_i Start_Cond Column_positiof Position By R Positiont By Position By UV_Averaging Column_Press Tritial_Eliel Flow_rate mi7
390. same method as was used to create the quantitation table from the standard runs 3 Click on the Evaluation tab in Run Set up Highlight the Update_Quantitation procedure from the list and click on the Quantitate button Select the quantitation table to be used and click on OK Deselect the Update_Quantitation procedure e p351 ED Analysing results an 18 1138 73 p352 Repeat this for the Quantitate_Sample procedure After this is done make sure both procedures are de selected otherwise they will be run twice Enter the text instruction mode select View Text instructions and select the last instruction in the method End_Method Select Other in the Instruction box Highlight Evaluate in the list open the Procedure drop down list and select a procedure e g Update_Quantitation p Method Editor 1423 LEL C oe et ea FSI sH I Te Daca Sm wOlan H Main 0 00 Base CY 0 98 ml Alkyl_Superose_HR_5 5 Column Ml 0 00 Block Start_Conditions_BP Ml 0 00 Block BufferPrey sh Breakpoint Parameters 2 Insert 0 00 Ho Var Change Replace Delete Fig 11 38 Instruction box used to select Evaluation procedures Click VAR in Parameters to open the Variable name definition dialogue box Type a variable name for example Procedure and click on OK The Evaluate instruction will be automatically inserted in the method H 0 00 Block Reequilibration 0 00 Evaluate Update_Quantitation Proced
391. se the appropriate Curve model by clicking its button from the options available Linear Linear logMw theoretically this is the best choice Quadratic Quadratic logMw Point to point Point to point log Mw Analysing results CH Each of the peaks selected is represented by a point in this curve which is drawn according to the best fit that can be achieved using the model selected Try different models until a satisfactory fit is found See Statistics below 2 You can select a peak by clicking it in the curve clicking its entry in the Retention Mol size table clicking its plotted point in the molecular size curve Whatever method is used the peak or peak reference will be highlighted in all three views Statistics Apart from the two point to point models the molecular size curves can be expressed as a mathematical expression This expression and related items can be viewed by clicking on Statistics Statistics x Model f log x B Model function values Help Fig 11 48 Statistics dialogue The expression shown at the top of the window is followed by the values for the constants it contains Correlation which is only displayed for linear models should be as close as possible to 1 00 The explained variance value should be as close as possible to 100 but is usually rather high even for poor models For instance a value of 90 indicates a very poor model The best model may
392. see Section 3 3 2 A result file is opened in the Evaluation module see Chapters 9 and 10 It is also possible to preview the first UV curve in the first chromatogram for a selected result file without opening it in the Evaluation module This facilitates selection of the correct result file for opening 1 Select the result file s to be opened and select Quick View from the shortcut menu The Quick View dialogue is displayed containing the chromatogram with the first UV curve displayed ep2l 3 Logon and file handling an 18 1138 73 e p22 Quick iew c Default id2301 res 1d2301 1_UV1_280nm Previous Fig 3 5 Quick View dialogue 2 Ifyou choose to view several result files use the Next and Previous buttons to move between them 3 To open a result file in the Evaluation module directly from the Quick View dialogue click on Open 4 Click on Cancel to close the dialogue 3 3 4 Presenting files The way files are presented in the windows can be set from the File menu or from the shortcut menu Presentation options are e view mode e sorting order e filter for displaying only a chosen set of objects e g methods for one system View mode From the View Show menu or from the View options in the shortcut menu you can select to display the contents of the windows in several Windows based alternatives View the files either as a details list View Details a simple list View List
393. smoothing functions are calculated A 2 gives an basic introduction into baseline calculation theory which is an essential part of peak integration A 3 describes the peak table column components A 4 the Procedure Editor instruction types are described which are used to build up an evaluation procedure A 1 Smoothing algorithms A 1 1 Moving Average For each data point in the source curve the processed curve is calculated as the average of the data points within a window centred on the source data point The width of the window is determined by the parameter value expressed as number of data points When the source point is less than half the window size from the beginning or the end of the curve the average is calculated symmetrically round the source point over as many data points as possible Increasing the window width increases the smoothing effect The filter algorithm only accepts odd integer parameter values between 1 and 151 If an even number has been given it is incremented by one A 1 2 Autoregressive The first data point in the source curve is copied to the processed curve For each subsequent data point the previous processed point is multiplied with the parameter value and added to the current source data point The result is then divided by the parameter value plus 1 according to the following formulae e p369 AY Evaluation functions and instructions an 18 1138 73 p370 t S4 pt tna Sn p 1
394. status of the run at a glance Stretching a flow scheme The flow scheme can be stretched to fit the screen by selecting Stretch from the shortcut menu Alternatively select the Properties option and the Flow scheme tab is shown in the Properties dialogue Check the Stretched box to display a stretched view of the window A12 Buffer AZ ALS ValveAt Pump Ald 5 Al RA gt ALS Al ve ump Al B2 PumpB me PumpB Inlet Flow Scheme A Injection CY Wate WasteF1 Outlet Value wt ii Fig 6 13 The flow scheme for a run e p155 ED Performing a run an 18 1138 73 e p156 Flow scheme manual instructions Some strategies directly link specific manual instructions to the components in the flow scheme window The components in the flow scheme that are associated with instructions are indicated with double arrow gt gt A particular component can have one or more instructions attached to it In cases where there is more than one instruction one of the instructions is the main instruction e To view the instruction or main instruction if there is more than one double click on the component with the left mouse button This opens the manual instructions dialogue for the instruction Execute the appropriate manual commands For more details on using manual instructions please see Manual instructions on page 160 e To select an instruction using the shortcut men
395. sted Movement of these drop lines can be up to but never beyond any other drop line A drop line may also not be moved beyond a point where the peak meets the baseline Identification names for peaks Double click on the peak of interest in the peak table spreadsheet cell and edit the name directly in the cell You will see the peak names in the chromatogram and the spreadsheet cell only if you have earlier accessed Curve Style and Colour tab in the Chromatogram Layout dialogue and selected Peak name for labelling the peaks Note also that in order to have Peak name listed within the spreadsheet cell you must choose the Peak name option under the Peak Table tab within the Chromatogram Layout dialogue 10 1 8 Measuring retention time and peak heights It is possible to determine the co ordinates of any point on a curve and thus obtain values for retention and peak height This is a useful tool for many other functions such as for measuring the parameters used in baseline calculations Co ordinates can be obtained in two ways e Direct measurement e Viewing peak table data Direct measurement 1 From within the chromatogram area display the shortcut menu and select Marker A vertical line is displayed on the chromatogram and can be dragged left and right with the mouse pointer The marker box at the top left corner of the window contains coloured text which shows the X axis co ordinate and Y axis co ordinate both with their respec
396. t a procedure from another method Select a method to show the procedures stored in the method If you have chosen a method click on Evaluation Procedures to return to the complete list 3 If desired change the procedure name in the Import as field 4 Click on Import The dialogue remains open until you click on Close so that you can continue to add procedures from the same or different method files Note Editing methods 5 ci Default Select Global BP_Report_Chromatagram HE iE iE Global InstEXP10XT Global Chromatofocusing 1d23 test20 Test20 0010 Global Integrate_and_Print Global Integrate_BP_Report Global Integrate_Full_Report Global Print_Chromatogram Global Report Chromatogram Global Quant Sample Global Update_Quant Untitled Import as Quant_Sample Procedure List Close Help Fig 5 41 The Import procedures dialogue Deleting procedures To remove one or more procedures from the method 1 Select the Evaluation Procedures tab and check the procedure s to be deleted 2 Click on Delete and confirm the deletion Procedures that you delete from the method are immediately removed from the method file Renaming procedures To rename a procedure in a method 1 Select the Evaluation Procedures tab and click on Rename 2 Select a procedure from the list and change the name in the Rename item to field 3 Click on
397. t dialogue Analysing results 11 This report provides a summary of the proposed update so that you can assess its viability Components that will not be updated are shown in the column Updated area or Updated ratio if internal standard is used with the text Out of limit If the text Not found appears no peak was found within the set peak window The column Averaged replicates shows the number of points used to calculate the average area value After each update by Average the number is increased by one After an update by Replace the number will be one Nominal reference retention shows the retention for the reference peak in level 1 1 Update reference retention shows the retention for the reference peak in the new peak table e Use Save or Save as depending on whether you wish to overwrite the existing table or create a new table under a new name e Click Print to obtain a print out of the Update report Click Exit if you do not wish to proceed with the update or to close the dialogue after saving the table 11 4 Quantitating the sample This section describes how samples are quantitated using calibration curves Calibration curves are applicable to external and internal standard quantitation and to recovery factor measurement Standard addition measurements are also described Note that the method used for the sample runs must be the same as for the standard runs If the method was created from a Wiza
398. t level will be shown However the average of these points will be calculated and then this average value will be used for producing the calibration curve For more information on the curve fit click on Statistics to open the Statistics information box For mathematical and statistical details see Mathematical models and statistics on page 405 Statistics Eg Model UE Model function values 4 5 43308E 001 B 2 34518E 002 Explained variance 99 877 Correlation 0 9996 Number of point s 4 Fig 11 21 Statistics dialogue The correlation which is only displayed for linear models should be as close as possible to 1 00 The best model may be selected by testing different models and comparing the explained variance values but you still need to examine the calibration curve visually to ensure a good fit The explained variance value should be as close as possible to 100 but is usually rather high even for poor models For instance a value of 90 e p331 ED Analysing results indicates a very poor model The explained variance will not be shown for curve models drawn through the origin If the point to point model is selected no statistics are available 5 Complete the quantitation table by entering data and the curve model for the remaining components in the Components list You will then have one calibration curve for each component The data must be entered and models selected for all compon
399. t setting to search for all chromatograms click on All Click on the Search button and a list of chromatograms will be displayed based on the designated search criteria Select the chromatograms that you want to import If you click on the Select All button all of the displayed chromatograms are selected If you want to clear the list of displayed chromatograms click on Clear Click on Run to perform the batch run Any created curves and peak tables will be saved in each result file automatically To view the results of a batch run on a specific result chromatogram open this in the Evaluation workspace 10 3 7 Evaluation procedures and reports The creation of evaluation procedures combined with batch runs is a very useful tool to produce printed documentation simultaneously for many result files e g for a number of scouting runs This removes the necessity to open import result files onto the Evaluation workspace Begin recording a new procedure by selecting Procedures Record on 2 Select File Report and choose a report format Run documentation e p299 ED Evaluating results Note rocedures Record On Edit Run Batch Run 0 Global Integrate_and_Print 1 Global Integrate_Full_Report an 18 1138 73 p300 Select Print in the Generate Report dialogue as the final instruction Stop the record function by selecting Procedures Record Off Save the procedure Doo w
400. t the waste condition will not be fulfilled simultaneously or immediately after peak collection Main 0 00 Base CV 0 10 Column ml Any 0 00 Block Gradient Gradient 0 00 Base SameAsMain 0 00 Gradient 100 Target_ConcB B 20 Length_of_gradient base 0 00 Block Eluate Fractionation Eluate_Fractionation 0 00 Base SameAsMain 0 00 FractionationStop 0 00 OutletValve WasteF1 0 00 Watch_UV Greater_than 5 mAU Peak Peak 0 00 Base SameAsMain 0 00 OutletValve Feed 0 00 Watch_UV Less_than_or_valley 4 75 mAU Waste Waste 0 00 Base SameAsMain 0 00 OutletValve Feed e p121 5 Editing methods an 18 1138 73 e p122 0 00 Watch_UV Greater_than 5 mAU Peak1 Peak1 0 00 Base SameAsMain 0 00 OutletValve Feed 0 00 Watch_UV Less_than_or_valley 4 75 mAU wastel wastel 0 00 Base SameAsMain 0 00 OutletValve Feed 0 00 Watch_UV Greater_than 5 mAU Peak2 Peak2 0 00 Base SameAsMain 0 00 OutletValve Feed 0 00 Watch_UV Less_than_or_ valley 4 75 mAU END_BLOCK 0 00 End_block 0 00 End_block 0 00 End_block 0 00 End_block 0 00 End_block 0 00 OutletValve WasteF1 0 00 End_block 20 00 End_block Watch example 6 Collecting absorbance peaks Collect one or two absorbance peaks through outlets F3 and F4 respectively with waste fractions collected through outlet valve F1 waste Each called block except End_
401. t window Display block duration and elu 5 3 5 ent gradient throughout the method Flow scheme window For information only The flow 5 3 6 scheme picture is static and is therefore not updated according to system status or changes in the method Instruction box Specify breakpoints instruc 5 3 2 tions parameters and varia bles Insert change and delete instructions e p47 Editing methods 5 1 3 Run Set up The Run Set up is a series of tabs for defining the method properties To access the Run Set up tabs press the Run Set up icon or select View Run Set up from the menu Evaluation Procedures Variables Scouting Method Information Notes Questio Result Name ns Gradient BufferPrep Start Protocol Columns Frac 950 Reference Curves Block Main Column ml Variable Value 0 100 Range 0 100 999999 000 Start_Conditions_CF Column_position Position Bypass 280 Wavelength_1 nm 190 700 Wavelength_2 nm OFF 190 700 Wavelenath_3 nm OFF 190 700 UV_Averaging Time sec 2 56 Re Pressure_Limit MPa 4 00 0 00 10 00 Initial_Eluent_Conditions_CF Start_Eluent_Inlet Al Flow_rate ml min 1 00 0 00 100 00 Column_E quilibration Equilibrate_with Cv 5 000 0 000 999999 000 System_Volume_Compensation _ Compensation_Volume ml 8 000 0 000
402. tab in Chromatogram Layout 261 Print 101 232 Procedure editor 293 Procedure editor Editing procedures 295 Procedure editor instructions field 296 Quantitate 340 Quantitation table 328 330 Quick View 22 Reject Peaks 264 Rename Blocks 54 Run Data groups tab 146 Save As for saving a method 99 Save quantitation table 332 Save Report Format 247 Saving a method 40 Scouting page in Documentation 252 Scouting Variables 71 168 Select level 322 Settings 263 Setup Chromatogram 237 Setup Documentation 239 Setup Evaluation Log 240 Setup Free Text 235 Setup Method 238 Setup Quantitate 241 Shift 228 Smooth 200 Stack Offset 223 Standard addition 343 Statistics after update 338 Toolbar Guide 4 18 Update calibration curve 336 Update quantitation table 335 Variable name definition 68 Variables page in Documentation 252 Windows 47 X Axis tab in Properties dialogue 153 Y Axis tab in Properties dialogue 152 displaying curve names 189 divide curves 284 documentation for manual runs 94 printing 255 draw frame around the pages 244 spline 291 straight to next point 291 edit BufferPrep recipe 137 chromatogram layout 188 column parameters 132 editing text inserted in a chromatogram 205 existing procedure 294 method instructions 46 method variables 37 MethodQueues 181 methods overview 45 parameters in procedure instruction 296 procedures 85 text instructions 42 46 edit quantitation table 334 eluent conce
403. table cancel the method start re calibrate the monitor s with the System Calibrate command see the UNICORN 4 0 Administration and Technical manual and restart the method The names of the result file and scouting folder are specified here This page is displayed if there are any other pages in the start protocol The names may be changed if this is permitted in the start protocol Displays the set up for the Fraction collector Also allows the user to set the last tube If there are any questions in the start protocol that require authorised confirmation you will be asked for a username and password when you attempt to leave the screen containing the questions Only users with Confirm Sign authorisation may authorise answers to such questions Each question that requires an authorisation must have a separate authorisation If the start protocol for a method in the queue is cancelled the MethodQueue is paused Select MethodQueue Display Running in the Main Menu and Restart or end the run in the displayed dialogue Performing a run GH 6 2 Monitoring a run The System Control workspace displays the status of the current system On the Windows taskbar there may be up to four System Control modules available that can be connected to different systems Separate systems may be controlled and displayed independently of each other Each System Control workspace displays up to four windows for monitoring different aspects of the r
404. table contains the molecular size curve 11 1 4 Installation If you are using a network UNICORN 4 0 must be present on all the computers but this is not the case for the Analysis module which can be installed on just one or on several of the computers See the licence agreement for information on legal aspects of the installation Install the Analysis module as follows 1 Insert the diskette in floppy A 2 From the Desktop close all applications on the computer 3 Open My Computer and double click the 3 floppy A icon 4 Double click Setup exe in the floppy drive file window to run the installation program Follow the instructions on the screen 5 When installation is complete remove the diskette and restart the computer 11 2 Quantitation overview 11 2 1 About quantitation Quantitation is used to determine the amount or concentration of components in a sample This chapter gives an overview over four e p305 ED Analysing results an 18 1138 73 p306 quantitation techniques provided by the Analysis module in UNICORN Quantitation should only be performed on chromatograms that have been saved in the current result file s General points Most quantitation techniques use peak integration data from standards to produce calibration curves These curves show the relationship between the amount of the components of interest and the peak sizes at different concentration levels of the standard The relat
405. te or wizard directly by clicking on the Instant Run icon in the Main Menu toolbar or by selecting File Instant Run in System Control Use of this function requires that templates or wizards are defined Standard systems are supplied with templates or wizards Custom systems require that the user creates templates 6 1 4 Start protocol If the method is defined with a start protocol this will be displayed before the method actually starts Work through the start protocol answering questions as required As each screen is completed click on Next to move to the next screen or Back to return to the previous screen The last screen has a Start button to start the run At any stage click on Cancel to abort the method start Performing a run GH The following start protocol items may be displayed see Run Set up on page 70 Variables Scouting Text Method Questions Notes Gradient BufferPrep Columns Reference Curve All the variables defined in the method instructions organised by block Values for variables which are not defined as scouting variables can be changed here for the current run Variables for scouting see Chapter 7 Values for Scouting variables may be changed for the current run and override the corresponding values on the Variables tab It is also possible to include or exclude runs in the scouting scheme with the Run Excluded buttons Text instructions for the method These are displa
406. ted with the result xi Variables Scouting TextMethod Notes Questions BuffePrep Columns Reference Curves Evaluation Procedures Method Information ResultInfomation ResultName Stat Protocol Settings Calibration Logbook EvaluationLog Information Signatures rel Time C Yolume min Block Start End ength Main nc 111 77 111 77 Start_Conditions IX 0 05 012 0 07 Initial_Eluent_Conditions_X 0 12 0 12 0 00 BufferPrep_pH_E quilibration 0 12 6 26 614 Initial_Eluent_Conditions_IX 6 26 6 26 0 00 Column_E quilibration 6 26 19 92 13 66 System_Volume_Compensation 6 26 7 86 1 60 Flowthrough_Fractionation 19 92 19 99 0 07 Sample_Iniection 19 99 20 10 an wash_Qut_Unbound_Sample 20 10 28 15 8 04 Eluate_Fractionation 28 15 28 42 0 27 Gradient_Elution 28 42 104 84 76 42 Gradient_Segment_1 28 42 88 71 60 30 Statt_Fractionation 28 48 28 48 0 00 Gradient_Segment_2 88 73 96 77 8 03 Gradient_Segment_3 96 73 104 82 8 03 Clean_After_Gradient_IX 104 84 Bal Fig 9 71 Documentation Result Information tab Presenting results 9 9 10 Exiting Evaluation If you want to quit from the Evaluation module of UNICORN select File Exit You will then be asked if you want to save the results of the evaluation session that you have performed If you answer Yes the previous version of that result file will be unconditionally over written e p257 ED Presenting results an 18 1138 73 p25
407. tention Retention 0 20 0 25 0 5 0 75 1 0 1 25 log Mol size Fig 11 43 Molecular size curve Analysing results CH Calculating the molecular sizes in the sample The molecular size table is used to calculate the molecular size of components in the sample 1 The sample peak table is used to obtain retention values for each of the components of interest mAU Sample 400 200 0 0 5 0 16 0 15 0 26 0 25 0 ml Fig 11 44 Chromatographic curve of the sample 2 The molecular size curve is used to obtain the molecular sizes of the components in the sample The molecular sizes are presented in the peak table Retention 3 13 5 T I T 025 0 5 075 10 1 25 Molecular size of ae HA log Mol size Fig 11 45 Molecular size curve e p359 ED Analysing results 11 6 2 Determining molecular size the process in detail This Section describes how to e produce the molecular size curve e calculate the molecular sizes of the components in the sample Producing the molecular size curve Before creating the molecular size curve you need to perform chromatographic runs with an appropriate standard with components of known molecular size The standard should contain components of size which extend over the range expected in the sample If you are using many components it may be better to split them into two or more standard runs After the chromatograph
408. th O Flowthrough_FracSize C Empty_loop_with O Wash_column_with zi E Chai ee I Show details I Show unused variables Cancel Help Fig 5 28 The dialogue for defining scouting variables e p71 Editing methods Note an 18 1138 73 e p72 Check the required variables in the variable list and click on OK 4 Make any changes you require in the variable values For variables with text values e g column position select the required value from the drop down list Values for variables selected for scouting under Define cannot be changed on the Variables tab Changes made on the Scouting tab do not affect the default values in the method instructions 5 To adda new Run column click on Add to copy the values from the last run column and then change variable values as required 6 Repeat this procedure until you have defined all the runs you require The example below shows scouting set for four flow rates Evaluation Procedures Method Information Result Name Start Protocol Variables Scouting Notes Questions Gradient BuffePrep Columns Reference Curves Block Yariable Runt Run2 Run3 Excluded Start_Instrj Column_Positiq Position By Position1By Position2 _ Position Bypas Wavelength 2 130 730 130 9 BufferPrep BufferPrep_pH 6 000 6 000 6 250 D 3 D t A fo I
409. the X axis and stretched along the Y axis 4 When all operations have been performed click on OK to save the new normalised curve Open the Chromatogram Layout dialogue to select the normalised curve for viewing 5 Repeat the procedure for all curves you want to stack or stretch Alternative B Moving a curve using the Shift function If more precise positioning of curves is required then the shift function should be used This function is similar to Normalise Move except that each curve is repositioned by a precise value instead of by eye and the instruction logged in the evaluation log 1 Select Operations Shift The Shift dialogue is displayed Select the curve to be shifted Shift Lx Source chromatogram Target chromatogram am 01 id2301 1_UV1_ 280nm 02 id2301 1_UV2_Onm 14 03 id2301 1_UV3_Onm 15 04 id2301 1_Cond 16 05 id2301 1_Cond 17 06 id2301 1_Cone 18 x 07 id2301 1_pH S 08 id2301 1_Pressure Curve name 09 id2301 1_Flow UV1_280nm O1 SHFT 10 id2301 1 Temo xl UV1_280nm 01 SHFT Shift type I Shift retention 0 ml J Shift amplitude 0 mAU Cancel Help Fig 9 41 Shift dialogue Presenting results 9 2 Select the axis along which the shift is to be made i e along the X axis Shift retention or the Y axis Shift amplitude Enter the shift value and click on OK Alternative C Stretching and shrinking a curve using multiply Curves can be stretched or shrunk in th
410. the colour and style of curves on page 191 If you do not want to view the peak labels e g for presentation purposes select the No peak label option Filtering peaks from view It is possible to temporarily remove peaks from display in a peak table based upon the criteria you determine 1 In the Chromatogram Layout dialogue click on the Peak Table tab 2 Select check the filter criteria in the peak table and specify the values used to filter the peaks i e the minimum height width and area the maximum width as well as a specified number of the largest size peaks Click on OK If you later want to include the peaks again you have to deselect the options The difference between Filter peaks and Reject peaks is that the latter function permanently excludes peaks from the integration and affects the calculation of total peak area etc 10 1 3 Optimising peak integration If the results from the peak integration are unsatisfactory there are several possibilities to improve the results Morphological and Classic baseline calculation You can use one of two baseline calculation algorithms depending on the type of peaks to be integrated e The Morphological algorithm is set as the default and gives the best results in curves with drifting baseline and peak clusters Optimising baseline calculation using the Morphological algorithm is also relatively easy since there are only three baseline parameters namely structure width noise
411. the variable has the value 0 or if the variable is not defined or has a value other than 0 1 2 or 3 then the default 3 algorithm is used To change the peak resolution algorithm edit the UNICORN INI file by 1 Minimize UNICORN and locate the file UNICORN INI within C UNICORNIBIN 2 Open the file and locate the following line EVAL ResolutionAlg If the line does not exist then add it before the Begin line 3 Choose the desired value for the algorithm 4 Save the file Do not make any changes in the UNICORN INI file between the lines Begin and End as this may severely affect the functionality of UNICORN Evaluation functions and instructions A A Capacity factor Va Vi V k where Vp retention volume V total liquid volume RRER K av where Vp retention volume Vo void volume Vc column volume SAsymmetry Asymmetry width B width A where A and B are the partial peak widths measured at 10 of the peak height with A representing the leading part of the peak and B the tailing part of the peak 10 peak height AB HETP HETP L N N 5 54 x Vp wy where N no of theoretical plates L bed height in cm Vp peak retention volume or time Wp peak width at half height expressed in the same units as Vg Plates meter is the number of theoretical plates per meter N x 100 L e p379 AY Evaluation functions and instructions
412. tically and the quantitation table will be updated after each run Then all the samples will be run automatically and the amount and concentration of the components of interest will be printed after each run The result files will include an additional chromatogram labelled 12 containing a small part of the curves collected during the execution of the evaluation procedure 11 5 5 The evaluation procedure instructions for the Analysis module Three new procedure instructions are available in the Analysis module Other procedure instructions available in UNICORN are described in the UNICORN 4 0 Administration and Technical manual Instruction Description Parameters QUANTITATE Calculates the concentration and amounts in the sample from a quantitation table Amount and concentration columns will be added to the peak table Peak table source Global or Personal table Quantitation table name Injection volume in ml e p355 ED Analysing results an 18 1138 73 p356 molecular sizes from a molecular size curve A Mol size column will be added to the peak table UPDATE Updates a Peak table source Quantitation table with new data from Global or Personal one standard table concentration level Quantitation table The default Limit name value of 12 5 will be used Injection volume in ml Concentration level for the standard Average or replace point
413. ting curve using the Calculate baseline option In addition to blank runs it is possible to select any curve present in the current chromatogram e g an edited baseline see Manually editing a baseline on page 276 as baseline e Using a Zero baseline i e no baseline subtraction at all e Reusing an already existing baseline for the selected curve by selecting the Correlated baseline option This is the default alternative whenever possible e p259 ED Evaluating results 10 1 2 Performing a basic integration To perform a basic integration ful 1 Select Integrate Peak integrate or click on the Peak Integrate toolbar icon The Integrate dialogue is displayed Integrate x Chromatogram Baseline Target peak table 1 hd Calculate baseline 7 Peaktable B Peaktable C Peaktable D Baseline settings Peaktable E hzi J Peaktable F Grope Peak table name Peak window JU 1_280nm 01 PEAK M gt Reject peaks Column height fio 00 cm I Peak skim 10 act esting 1708 o 01 HIPREPO2 1_UV1 280nm PN 02 HIPREPO2 1_U 2_406nm 03 HIPREPO2 1_U 3_Onm 04 HIPREPO2 1_Cond 05 HIPREPO2 1_Cond 06 HIPREP02 1_Conc 07 HIPREPO2 1_pH Cancel Help Fig 10 1 Integrate dialogue Choose the source curve to be analysed usually the first UV curve and a peak table destination from the peak table list Any chromatogram can contain up to eight peak tables designat
414. ting a run This section briefly summarises how to start a run with a method The method must be saved before it can be started See Chapter 6 for more details of how to run a method Note If you are editing the method in the Method Editor and have made changes that you have not yet saved these changes will not apply during the run Similarly if you edit the method while it is running the run will not be affected It is the version of the method that is saved on disk at the time when the method is started that controls the run 1 Establish a control mode connection to the system where the method is to be run You cannot start a method without a control mode connection to the appropriate system 2 Choose File Run from System Control for the required connection and select the method to run Alternatively click on the method in the Methods window of the Main Menu and select Run from the shortcut menu Do not double click on the method icon in the Main Menu as this will open the Method Editor 3 Change the method variable values if required The suggested values are those saved in the method Any changes you make will apply only for the current run and will be recorded in the run documentation 4 Go through the rest of the Start Protocol entering information where appropriate Use the Next and Back buttons to move e p41 gt Creating methods an 18 1138 73 e p42 through the Start Protocol Start Protocol tabs for most Wi
415. ting order choose Sort from the shortcut menu or File Sort and choose the appropriate sorting order from the menu cascade Alternatively click on the column headers in the window for Name System Size Type Modified and Created to toggle file sorting accordingly Click a second time on the same sorting option and the files are sorted in reverse alphabetical order increasing file size etc as appropriate to the selection Changing the sorting order affects only the currently active window e p23 3 Logon and file handling an 18 1138 73 e p24 Filter To restrict the files displayed according to file name or the system in the Methods window only with which they are associated choose Filter from the shortcut menu or select View Filter Mark the system s for which you want to display files and enter a file name specification if required Click on OK to activate the filter The filter only affects the display in the Methods window You can use standard Windows wildcard characters in the file name specification stands for any number of characters for any single character For example iex will display only files named iex iex will display all files with names beginning with iex iex will display all files with names ending with iex 1ex will display only 4 character names ending with iex If a filter is active this is indicated in the title bar of the panel e g c Default Filter on system To display all files
416. tion on this tab is for viewing purposes only and may not be changed 5 Within either the Signing or View Signatures tabs click on OK to complete the signature process 9 6 Saving results Any changes to chromatograms including all new created curves and all imported or created chromatograms can be saved in either of two ways using File Save or the Save toolbar icon which saves all changes in the original result file or e File Save as which allows you to create a new result file in the specified target folder All curves created during the manipulations will also be saved This may not always be desirable Before saving remove unwanted curves from a chromatogram using Edit Delete Curve The original curves can never be deleted 9 7 Printing active chromatograms To print out the open chromatogram s select File Print or click on the Print toolbar icon If you want to print out several chromatograms ensure that these are open on the workspace before selecting File Print A dialogue appears allowing you to select the print format and if applicable the number of chromatograms in each column and row in e p231 ED Presenting results 3 an 18 1138 73 e p232 the printed document It is also possible alter line thickness and present landscape views by selecting the checkboxes Click on Preview to view the page layout before printing Click on OK to print out the chromatograms Print Chromatograms y x
417. tions provide a means for entering structured run specific information at the start of a run Method Wizards and templates supplied with UNICORN are defined with a set of questions for sample column and eluent identification To define questions open the Questions tab in Run Set up For questions to be shown in the start protocol the Questions option must be checked in the Run Set up Start Protocol tab Questions may have the following status e Mandatory these questions must be answered before a method is started Editing methods 5 o Authorised answers to these questions must be acknowledged by a user with Confirm Unlock authorisation see the UNICORN 4 0 Administration and Technical manual The user s password must be given to acknowledge the answers e Chromatogram these questions will be printed with the answers on the same page as the chromatogram if Diagram header is chosen in an evaluation report see Printing reports on page 232 Evaluation Procedures Method Information Result Name Start Protocol Frac 950 Variables Scouting Notes Questions Gradient BufferPrep Columns Reference Curves E ilnput field volume and type No2 input field Column No3 Input field Eluent A No4 Input field Eluent B No5 Input field Remarks Question meimei R I Mandatory IT Authorized F Chromatogram Bece F let gt Answer type Default text 2e Input field siz Delete a
418. tive units The colour and units of the Y axis information corresponds to a specific curve of the same colour e p281 ED Evaluating results id186Level 1 to 4002 1__ UNICORN Local fil Niklas result Unicorn4001 1 lid186Level 1 to 4001 id186Level 1 to 4002 res E id186Level 1 to 4002 1_UV1_280nm id186Lewel 1 to 4002 1_Inject id186Level 1 to 4002 1_UV1_280nm 01 BASEM id186Level 1 to 4002 1_UV1_280nm 01 BASEM1 9 56 min 4 060 mAU 827 Fig 10 28 Active bar linked to a marker for determining curve point co ordinates 2 Click on the desired curve legend and the correct Y axis is displayed 3 To close this function de select from the shortcut menu Viewing peak table data The retention time and amplitude of any peak can be directly viewed ina peak table after an integration if selected for in the Chromatogram Layout dialogue 10 1 9 Measuring HETP HETP height equivalent to a theoretical plate calculations allow you to check how well the column has been packed 1 Perform a run with injection of a non interactive substance for example a small volume of acetone Note that the injection must be at zero time 2 In the Integrate dialogue type in the column height cm in the Column height field Perform the peak integration according to your other selected parameters 3 To view the results of the integration select Plate height HETP in the list within th
419. to be expressed in concentration rather than amount click Concentration and check or edit the injection volume in the Inj Volume field Note that the software will always calculate both amount and concentration for the sample The default Amount unit label is mg Edit this if necessary The name of the active chromatogram should appear in the Source chromatogram field If you need to use an alternative result file double click on its icon within the Result field and select the source chromatogram Clicking Current at any time returns you to the chromatogram that was active before entering Quantitate Highlight the standard peak table of level 1 in the Peak table s list This should be the table for the highest concentration of the standard Click Select or double click the peak table to include it in the Level Peak table s list If the level has already been set in the method the level is automatically copied into the Level Peak table s list If not a Select Level dialogue will appear Open the Level menu highlight 1 and click OK It is useful to think of each level as an alias for a specific concentration of the standard e p321 ED Analysing results an 18 1138 73 p322 Select Level Level n a ok Fig 11 12 Select Level dialogue 7 Repeat this procedure with all the standard peak tables Open another result file by clicking on its icon in the Results field and then select the new source chromatogram T
420. to higher value Used to set the flow rate automatically in a method If the method uses linear flow the parameter value is automatically calcu lated Used to give a warning when saving or starting a method if a higher flow rate is chosen Used to set averaging time for UV detector Used to set peak frac tionation parameters Used to give a warning when saving or starting the method if the BufferPrep_pH is higher than the set value Used to give a warning when saving or starting the method if the BufferPrep_pH is lower than the set value Used to give a warning when saving or starting the method if the BufferPrep_pH is higher than the set value e p131 5 Editing methods pH Low no Used to give a warning value short when saving or starting term the method if the BufferPrep_pH is lower than the set value Average par pum no Information only ticle diame ter Code No no Information only Typical load mg no Information only ing range Scan rate spectra sec no Information only Note 1 The values for the parameters Max pressure Default flowrate and Typical peak width at base used to set average time and peak fractionation parameter MinWidth are only copied into the method if the corresponding instructions are available as variables Note an 18 1138 73 e p132 4 Click on Save as and enter the name of your column You can choose to save the column globally i e available to all users
421. to run Press on the Run button to view the Start protocol and to start the run template or view the Wizard For system Use Wizard Template m Template selection Method notes i Fig 3 3 The Instant Run dialogue Use of this function requires that Wizards or templates are defined Standard systems are supplied with Wizards and templates but custom systems require that the user makes templates New Method This immediately starts the Method Editor module and displays the New Method dialogue see Creating a new method on page 33 System Control This activates the first connected System Control and displays the Manual instruction dialogue see Manual instructions on page 160 Evaluation This displays the Open Result dialogue Select a result file and click on OK to launch the Evaluation module see Presenting results on page 185 e p19 3 Logon and file handling an 18 1138 73 e p20 MethodQueue Prompts the MethodQueue Editor dialogue see Editing MethodQueues on page 181 Display Running Prompts the Running MethodQueue dialogue see Displaying MethodQueues on page 182 3 3 UNICORN Main Menu windows The two Main Menu windows display the folders to which you have access within UNICORN and the method and result files within the currently open folder respectively You can only see method files written for systems to which you have acc
422. tructions 000 cee eee 42 5 Editing methods 5 1 Method Editor interface 0 0 cee ee eee es 45 5 1 1 Method Editor modes 0 000 0000 000 ee 45 5 1 2 Text Instructions editor 2 0 0 00 00 000 46 Boe RUN SECU Dp eein ca toh k eters hale We AG Rta Ea see 48 5 2 Method blocks 0 0000 cee e eee eee ees 48 5 2 1 Viewing bIOCKS 0 ee eee 48 52 2 CallinS loCKS a S anha Ke ok Pale ea eae eae Vale 2 eds 50 523 Adding DIOGKS sate whe Ook aA ate Stee We eee etc 51 524A Deleting Bleks ima ie od nk Ee eee eee wh ea 53 5 2 5 Renaming DOCKS sas ipari uaan oa An Daa ee ees 54 5 2 6 Copying moving and importing blocks 55 5 3 Method instructions 2 00 2 eee eee 57 5 3 1 Viewing instructions atelen ceca scale asuna aaneen annaa 57 53 2 Adding INSTTUCTIONS 2 0260 ea d n ei Pe oad ewe 59 5 3 3 Deleting instructions 0 0 0 0 0 eee 61 5 3 4 Changing instructions 0 0 0 0 eee 61 5 3 5 Using the gradient window 0 ee 64 5 3 6 The flow scheme window 00 002 e eee eee 66 5 4 Method variables 00 c eee eee eee eee 66 5 4 1 Identifying variables 0 0 0 ee 66 5 4 2 Defining variables 0 0 0 0 cee ees 67 5 4 3 Removing a variable 6 eee 69 5 4 4 Renaming a variable 0 0 ee ee 69 5 5 RuN S t Up fcok oSai eee A Sa ie eae oi eer s 70 5 5 1 Variables sea cc 0 et Sas eels 2 ee a hati ak ee 70 6 5 2 SCOURING koiri 4 e
423. u click on the component with the right mouse button and select Instruction from the shortcut menu A cascade menu shows the specific manual instruction s Click on an instruction to select it This opens the manual instructions dialogue in which you can execute the appropriate command Flow T Pump lInlet Maximise 3 Pump WashExplorer Hide SystemWash PumpB T Stretch ii Copy to Clipboard Properties Outlet Valve Fig 6 14 Selection of Flow scheme window showing components with manual instruction s In this example the specific instruction s are shown in the shortcut menu for the Injection Valve 6 2 5 Logbook All actions including method start and end base instruction method instructions and manual interventions such as Pause or Hold and unexpected conditions such as warnings and alarms are logged for every run with date time and current username where appropriate The date and time are taken from the system clock in the PC The logbook thus provides complete history of any given run The log is saved in the result file Performing a run GH 0 00 min Run 96 02 14 10 03 17 Method hl01 Result q ingridlhI01 res Manual Logbook 0 00 min Call BASIC_START_CONDITIONS_IEX 0 00 min ColumnPosition Position Bypass 0 00 min Wavelength 280 nm Onm Onm 0 00 min AveragingTime 0 32 sec 0 00 min Alarm_Pressure Enabled 5 00MPa 0 00 MPa 0 00 min BuffervalveAT A11 0 00 min Gradient 0 00 8 O
424. uccessive methods are run on the same system the timing set in Condition applies from the completion of one method to the start of the next If successive methods are run on different systems the Ready instruction in one method can be used to trigger the start of the next method i e with the Ready instruction you will be able to start the next method already before the current method has ended The Condition setting then applies from the Ready instruction to the start of the triggered method This is useful for example in situations where a method on one system prepares the starting material for the next and then continues to wash the system System 1 System 2 Apply sample Elute READY Apply sample Wash Elute 8 4 Displaying MethodQueues Click on the Display running MethodQueue icon or choose MethodQueue Display running to display pending and running MethodQueues A pending MethodQueue is one for which Run has been requested but which has not yet started either because the system is not available or because the Setup time has not been reached Running MethodQueue Eg M01 v Friday 21 30 iex2293 End of Sequence Fig 8 6 Displaying running and pending MethodQueues MethodQueues 8 Use the list box at the top left corner of the dialogue to select a MethodQueue The list shows pending and running MethodQueues For the selected MethodQueue Start at shows the time when the MethodQueue i
425. ults CH 11 3 Producing calibration curves This section describes how peak data from standards are used to prepare calibration curves for use with external standard internal standard and recovery quantitation 11 3 1 Preparations before using Quantitate 1 Create a method to be used for all the standard runs The method and the injection volume must be the same for all the runs If the method is created from a Wizard or template for AKTAdesign systems you may select the correct standard concentration level in the variable Quantitation_Type You can also set the level after the run has been performed see Editing and updating a quantitation table on page 334 Each level is an alias for a specific concentration of the standard Level 1 should be selected for the standard with the highest concentration The levels must be set in consecutive order of decreasing concentration of the standard All runs with the same concentration must be given the same level 2 Perform at least one run for each concentration level of the standard Preferably the standard series should include standard concentrations which extend beyond the lower and upper limits of the sample amount If internal standard is being used the internal standard must be added in the same concentration in all standards 3 Using the Evaluation module peak integrate the curves to produce a peak table for each of the standard curves When integrated all standards must use t
426. un Click on the View Windows icon or choose View Windows from the menu to select which windows to display Rundata Curves Flow Scheme Logbook M M M M F Cancel Help Fig 6 3 Dialogue for choosing windows to display in the System Control workspace 6 2 1 General window techniques Windows in the System Control are always displayed over the full width of the workspace The split bar between the displayed windows can be moved by selecting and dragging up or down to change the size of a specific window Any window can be maximized to the full view or restored to its original size by selecting the Maximize or Restore toggle command respectively in the shortcut menu To hide a window from view select Hide from the shortcut menu 6 2 2 Run data The Run Data window displays the current values for selected run parameters Values are updated at least every 5 seconds the actual interval is defined in the system strategy e p145 ED Performing a run Instruments Connection Run Status Acc Volume Block Volume Acc Time Block Time Fig 6 4 The Run Data window Run data layout The general display for the Run Data window can be selected using layouts by doing the following 1 Select View Properties or select Properties from the right click pop up menu The Properties dialogue is displayed for all windows in System Control 2 Select the Run Data Groups tab Properties x YArs Run Data G
427. ure 0 00 End_method Fig 11 39 Evaluate instruction inserted in the method By defining evaluation procedure as a variable different procedures can be selected on the Scouting tab for different scouting runs De 5 Analysing results CH Select View Run Set up Select the Scouting tab click on Define and edit the scouting variables list to include Procedure Sample_ID Vial_Number Injection_volume and Quantitation_Type Note The Procedure variable will appear in the beginning of the list of variables even though the Evaluate instruction is inserted at the end of the method 6 Set up the scouting scheme First all the standards must be run in the scouting scheme Select the Update_Quantitation procedure on the Scouting tab for all the standard runs Make sure that Quantitation_Type is set to the correct standard level for each run The quantitation table will now be updated with new values after each run Note that you can only perform one run at each level since the quantitation table will be updated by replacement of the old points Method Information l Result Name Start Protocol Frac 950 Variables Scouting Notes Questions Gradient Columns Reference Curves Evaluation Procedures Block Variable Run1 Run2 Run3 Run4_ Excluded Autosampler_Injection_Partial Vial Number 1 1 1 1 fl Injection_Volume ul 50 50 50 50 50 Quantitation_Type Sample Sample Sample Sample Sample Sample_ID Sample_ID Samp
428. ure and averaging time as variables The Methodbase instruction Volume or column volume base is calculated from the flow rate of the SystemPump or the SamplePump selected with the instruction Methodbase If no Methodbase instruction is included in the method the default setting SystemPump will be used 5 10 2 Instructions at the same breakpoint Instructions placed at the same breakpoint in a block are executed simultaneously with the exception of successive Block instructions which are executed in the sequence in which they are written This can have important consequences in some situations For example the instruction sequence e p105 Editing methods Note an 18 1138 73 e p106 0 00 Block WASH 0 00 FractionCollector 5 m1 0 00 Block ELUTE will start the fraction collector when the Wash block is completed 5 10 3 Block and method length The length of a block is determined by the breakpoint of the last instruction in the block A block in which all breakpoints are at 0 will take no time or volume during a run e g Initial_Eluent_Conditions_BP 0 00 Base SameAsMain 0 00 BufferPrep_pH 7 BufferPrep_pH 0 00 Buffer alyeA1 411 Buffer_Inlet 0 00 Flow 5 Flow_rate ml min 0 00 Gradient 0 00 Start_ConcB B 0 00 base 0 00 End_block 0 00 End_block To extend the length of a block without performing any other operation set the breakpoint of the End_block instruction appropriately e g
429. ure width When optimising the baseline parameters using a Morphological algorithm changes to Structure width will in most cases give the best improvement in results Structure width determines the length of the straight line see above which is set to a default value of the widest peak on the chromatogram multiplied by 1 5 In situations with drifting baseline for a curve the morphological baseline follows the curve faithfully Subsequent subtraction of the baseline thus creates a curve with the baseline at a more even level Evaluating results 10 Baseline example 1 1 Mi E ATJMF006 1_UV_215nm_Morphological Fig 10 9 Fluctuating curve with a morphological baseline that follows the bases of the peaks at the different levels in the curve In some circumstances the default setting for the Structure width should be increased since a too low setting may result in a baseline that reaches too high up in the peaks of the curve see figure below This situation may arise when for example a wider peak is not recognised due to it containing a cluster of smaller peaks The default Structure width is therefore set to a default value according to the largest width of the identified narrower peaks Baseline I Baseline example 2 1 UU i m UV1_216nm_Morphological_default mapp104 1_UV1_215nm_Morphological_Axdefault Fig 10 10 Chromatogram displaying a curve with two baselines A default morpholog
430. urves The default layout can be changed by the user see Saving and applying a layout on page 195 f id186Level 1 to 4004 1_UV1_280nm id186Level 1 to 4004 1_Inject id186Level 1 to 4004 4_UV1_280nm 01 BASEM mau 8 27 10 48 11 71 po Fig 9 2 Displayed chromatogram in a newly opened result file Each curve is automatically assigned a default colour and style with default information about each curve displayed in the key above the curves This information includes the result file name chromatogram name and curve name Each curve has a correspondingly coloured Y axis To choose the appropriate Y axis scale click on the Y axis until the desired scale is displayed or simply click on the name of the curve of interest Optimising the workspace Chromatograms can be minimised in the desktop by clicking on the minimize button in a chromatogram window Icons can be neatly arranged in the workspace by selecting Window Arrange icons To restore a window click on the restore button for the iconised window or select the chromatogram name from the Window menu You can also maximise a chromatogram window to fit in the whole Evaluation desktop by clicking on the maximize button To view several chromatogram windows side by side select Window Tile Alternatively Window Cascade will stack all of the open windows like a deck of cards e p187 ED Presenting results Note an 18 1138 73 e p188
431. urves panel For monitor signals to be displayed in system control they must be set to STORE ON in system settings Signals for which STORE ON is set can be chosen from the View Curve contents dialogue box for display in the curves panel Error message Couldn t create result file If you receive an error message Couldn t create result file Destination path could not be found at the end of a method the local computer was unable to access the folder specified in the result file path This may arise if the specified folder is on the network server and network communication has been lost The result file is saved in the FAILED folder on the local station The Method System Connection dialogue keeps appearing If the Method System Connection dialogue keeps appearing you have some method s that you have not connected to a system most likely from imported methods using the Copy from external function in the Main Menu see Copy files from external on page 27 Connect the method s to the appropriate system and the dialogue disappears The method editor window does not fit on the screen If the Method Editor window does not fit on the screen and has scroll bars you may have the incorrect font size installed For pre installed Windows NT 4 0 the display screen resolution is set at 1024x768x65536 with Large fonts You need to install the Small fonts which requires that you have available the Windows NT Workstation 4 0
432. usted by forcing it equal to the source curve in every position where the difference between the baseline and the source curve is small enough If the Accept negative peaks option see Including negative peaks on page 266 is off the baseline will be forced down to the level of the source curve whenever the created baseline goes above the source curve e p373 AY Evaluation functions and instructions an 18 1138 73 p374 A 2 4 Estimating the baseline parameters from the source curve for Classic algorithm The baseline parameters can sometimes be difficult to set Rough estimates can be found by analysing the source curve Measuring the Shortest baseline segment using curve co ordinates If you are uncertain of the length of the Shortest baseline segment you can try to measure it directly on your chromatogram Locate the shortest segment of the curve that you consider as a part of the baseline and measure the length of the segment using the marker box on the chromatogram see Optimising the baseline parameters using a classic algorithm on page 269 Insert this value as the Shortest baseline segment value Measuring the noise level using curve co ordinates As for measuring the Shortest baseline segment curve co ordinates can be used in exactly the same manner to measure noise levels on the source curve First use the Zoom function to select a part of the curve representative of the baseline noise Measure the Y ax
433. utomatically assigned a colour and line style To reassign the colour and or style of a specific curve do the following 1 In the Chromatogram Layout dialogue select the Curve Style and Colour tab 2 To change the colour and or line style of a curve select the curve of interest from the list 3 Select the desired colour and or style Chromatogram layout 1 xi Header CurveNames YAxis X Axis Curve Peak Table Curve Style and Colour Edit Texts Layout Library Select curve to modify colour and linestyle for Select colour Select line style 01 ATJMEO06 1 UV1 215m 02 ATJMFO06 1_UV 2_254nm 03 ATJMFOO6 1_UV3_280nm 04 ATJMFOO6 1_Cond 05 ATJMFO06 1_Cond 06 ATJMFOOB 1_Cone 07 ATJMFOOB 1_pH 08 ATJMFO06 1_Pressure Sere esses 09 ATJMFOO6 1_Flow ae in Peak label T Fraction text alignment r Logbook text alignment 3 Text alignment Retention C Verti C Verti Di C Vartical Vertical Vertical C Number p Horizontal Horizontal Horizontal C Peak Name C Fly Over C Fly Over No Peak Label Filter T Hatch IT Apply to all chromatograms Cancel Help Fig 9 6 Curve Style and Colour dialogue 9 2 7 Defining and positioning curve text The Curve Style and Colour dialogue also allows for variable forms of peak labelling as well as alignment of Fraction and Logbook text Peaks may be labelled according to Retention times the default label by seque
434. ve The ratio can be used to check peak purity If the peak is pure the absorbance spectra are the same over the whole peak and therefore the ratios should remain constant If the absorbance ratio is not the same over the whole peak then the peak is probably not pure Co eluting peaks A B E Z Ratio 3 A270nm A255 nm Ratio 270 nm A255 nm 2999990990900 ON w EAD go a gt 2 3 Time units Fig 10 30 Simulated chromatogram of two co eluting components with differing absorbance spectra and a small difference in retention time The resulting ratio can also be used for peak identification as different compounds have a specific ratio between absorbencies at different wavelengths e p285 ED Evaluating results Separated peaks gt Ratio B Ratio 270nm A255 nm eesscoces So N Ww Ein D USS 2 3 Time units Fig 10 31 Simulated chromatogram of two components with differences in their absorbance spectra 10 2 2 Simulate Peak Fractionation To create a curve that simulates a peak fractionation select Operations Simulate Peak Fractionation Simulate Peak Fractionation 13 14 15 16 17 id186Level 1 to 4002 1_UV1_ 18 id186Level 1 to 4002 1_UV1 06 id186Level 1 to 4002 1_Conc 07 id186Level 1 to 4002 1_pH id186Level 1 to 4002 1_Pres Sy wy N ANNAA FL T Fig 10 32 The Simulate Peak Fractionation dialogue an 18 1138 73 p286 Evaluating r
435. ve from the list for which you want to fix the scale Click on the Fixed option 3 Type in the desired minimum and maximum values for the Y axis If you click on All with this unit other curves that have the same Y axis units as the current scaled curve will be similarly scaled Click on OK Note Note Presenting results 9 For pressure curves Y Axis units may be changed by clicking on the appropriate Pressure unit MPa psi bar The default Pressure unit is From strategy which is the unit defined in the original run strategy Curve Style and Colour Edit Texts Layout Library Header Curve Names Axis Curve Peak Table Select curve to modify axis scale for 01 ATJMFOOG 1 UV1 21 5nm 02 ATJMFOO6 1_UV 2_254nm 03 ATJMFOO6 1_U 3_280nm 04 ATJMFOO6 1_Cond 05 ATJMFOO6 1_Cond 06 ATJMFO06 1_Cone 07 ATJMFOO6 1_pH 08 ATJMFOO6 1_Pressure 09 ATJMFOO6 1_Flow 10 ATJMFOO6 1_Temp 17 ATJMFOO6 1_UV1_215nm 01 BASEM 18 ATJMFOO6 1_UV1_215nm 01 BASEC I Apply to all chromatograms Fig 9 8 Chromatogram Layout dialogue Y Axis tab C Auto Pressure unit C MPa Min Max Fixed 5 225 mAU All with this unit bar C psi From strategy Cancel Help All with this unit will only be applied to existing curves It will not be applied to new curves created after this function was last used New curves are automatically scaled X Axis 1 Cl
436. vels must be subjected to the same sample preparation procedure as the samples An additional demand on the internal standard is then e it must have similar chemical properties to the component s of interest This also implies that if you have several components of interest they all must be chemically similar An outline of the technique 1 A series of concentration levels is prepared from the standard An additional component the internal standard is added in the same concentration to all the standards and to the sample This addition should be made prior to the sample preparation procedure A run is performed for each standard and the sample 2 The curves are peak integrated to produce a peak table for all standard runs and for the sample Each curve contains a peak from the internal standard Changes in the size of the internal standard peak indicate changes in the system e p311 ED Analysing results mAU Standard 250 Internal Standard 200 level 150 3 levels 100 4 50 12 0 13 0 14 0 15 0 16 0 17 0 18 0 min mAU 250 Sample Internal Standard 7 12 0 13 0 14 0 15 0 16 0 17 0 18 0 min Fig 11 5 Chromatographic curves for sample and standard levels all include the peak for the internal standard 3 All peak sizes are plotted relative to the size of the internal standard peak to produce a calibration curve for each component Standard Internal Standard One of th
437. ver it will reappear each time you perform other copy to from external procedures for method files Method files that have been copied in and connected are displayed in the previously designated folder in the Methods window 3 3 7 Deleting files To delete a file or folder 1 3 Select the item s to be deleted in the Methods or Results window of the Main Menu To select multiple files hold down the lt Ctrl gt key while you click on the file names or icons Highlight the file to be deleted and either select the lt Delete gt key on the keyboard choose File Delete or choose Delete from the shortcut menu Confirm the deletion in the dialogue Home folders cannot be deleted by this method see Section 4 2 of the UNICORN 4 0 Administration and Technical manual Explicit authorisation is required to delete files see Section 4 2 of the UNICORN 4 0 Administration and Technical manual A file that has been deleted cannot be recovered except by restoring a back up copy 3 3 8 Renaming files To rename a file or folder 1 Select a file or folder to be renamed in the Methods or Results window of the Main Menu Select Rename from the shortcut menu or choose File Rename The Rename dialogue is displayed Enter the new name for the file and click on OK e p29 3 Logon and file handling Caution an 18 1138 73 e p30 3 3 9 Backup security To protect important data against accidental deletion or los
438. view in the Chromatogram Layout dialogue Curves can also be scaled individually or all with the same scale using the All with this unit function in the Chromatogram Layout dialogue see Y Axis on page 192 If you stacked the curves and you want to change the stack offset select Shift offset from the shortcut menu and select a new Offset value The individual curves can also be moved see Alternative B Moving a curve using the Shift function on page 228 e p223 ED Presenting results an 18 1138 73 p224 If desired select the Store in new chromatogram box and give the chromatogram a new name Alternative B Importing curves using Open Using the File Open Curves function individual curves may be imported into the active chromatogram Ensure that the destination chromatogram for the imported curve s is active on the screen Open Curves x c Default id2301 Ll Baseline example 3 Lu Baseline example 2 ui id186Level 1 to 4004 i id1 6Level 1 to 4003 ai Peak skim example Ui id1S6Level 1 to 4002 buJid186Level 1 to 4001 Lu Baseline example 1 r Select curves Chromatogram Selected curves fi w ATJMFOO6 1_UV1_215inm ATJMFO06 1_Cond Available ATJMFOO6 1_U 1_215nm ATJMFOO6 1_UV2_254nm CIATJMFOO6 1_UV3_280nm ATJMFOO6 1_Cond ATJMFOOB 1_Cond ATJMFOO6 1_Cone ATJMFOO6 1_pH K Remove Store in chromat
439. when the watch begins not when the block is activated The block will in fact never be activated if the watch condition is not met during the run Once set a watch remains active until the condition is met or a new Watch instruction is issued for the same monitor The watch is cancelled automatically when the condition is met A watch can also be turned off with the Watch_off instruction See Section 5 11 2 for more details of Watch instructions 5 2 3 Adding blocks To add a Block select the Block instruction in the Instruction box enter a name for the block in the parameter field and click on Insert The block will be inserted after the instruction block that was highlighted in the Text Instruction window You can also select an existing block from the parameter field drop down list epdl Editing methods an 18 1138 73 e p52 Breakpoint r Instructions Parameters Block ms owo Hev aie 3 Var Var Change AlatmstMon Pea C Frac Replace C Watch eats New_Chromatogram Delete Pause Ready Fig 5 8 Instruction box with the Block instruction selected Alternatively to add a new block use the Text Instruction editor and click on the New block icon or select Block New Name m Base Same as main C Time Volume Column volume E 10 ml m Length Length 0 00 CV r Call From M ain gt ar foo cv 0K Cancel Help OK Fig 5 9 New block
440. will not be used for update The Deviation column shows how much the peak size for the proposed new point differs from the existing size The Limit shows the set limit for the deviation The default value is 12 5 of the existing peak size You can edit the Limit value Both of these columns can be expressed in Absolute or Relative units by use of the Deviation and limit as buttons The update can be made by Average or Replace With Average the average area value is calculated from the old point representing the average of the old points at this level together with the new point The green point represents this new average value and not the position of the point from the new peak table Update by Average may be used if you want to increase the precision of the calibration curve by performing several runs at each level With Replace the old point representing the average of the old points at this level will be replaced with the new point shown in green The data for the old point can then not be recovered Update by Replace may be used to simplify the process of renewing the calibration curve before each analysis Instead of manually producing a new quantitation table you may renew an existing table by running all standard levels again and updating the table with Replace The old data will then be deleted You use this dialogue as follows 1 Select to update by Average or Replace The same selection applies to all
441. will not begin at zero unless designated as one of the limits The original curve is not changed 4 Click on OK A new dialogue is displayed Select whether to save the new cut curve in the Source chromatogram i e the current active chromatogram or in a New chromatogram If you select the latter option you can change the name of the chromatogram Click on OK 5 Ifthe destination of the cut curve was the source chromatogram the cut curve is automatically displayed in the source chromatogram If the destination of the cut curve was a new chromatogram this will be represented as a new open chromatogram window e p199 ED Presenting results an 18 1138 73 e p200 9 3 3 Reducing noise and removing ghost peaks Sometimes the chromatograms may contain curves with a noisy baseline The noise can be caused by several things e g a dirty flow cell air bubbles electrical noise dirty buffers etc The amount of noise can usually be reduced by taking proper precautions e g filtration of buffers and instrument maintenance Smoothing a curve The smoothing function allows background noise to be reduced or removed from any selected curve The type of smoothing function you should choose depends upon the type of noise encountered 1 Select Operations Smooth 2 Select the source curve to be smoothed and its target destination By default smoothed curves are given the suffix SMTH Target chromatogram Source
442. window 48 threshold values 284 time and date incorrect 403 tool bar in System Control workspace 157 toolbar buttons 3 buttons available 18 guide 18 method editor 45 triangle markers peak identifying 323 selected peaks 324 U unconditional calls 51 undo zoom 65 UNICORN access problems 399 cannot quit or logoff 401 concepts 3 control software 3 display of version 18 main menu windows 20 problem re establishing system connection 402 quitting 31 security features 14 security system 2 stand alone PC installation 13 starting 17 summary of functional features 1 user interface 3 unit in standard addition 343 units adjusting in page layout 244 update area ratio 339 by average or replace 337 new point 337 quantitation table 335 reference retention 339 Update report dialogue 338 user interface 3 username entry 17 e 441 um 18 1138 73 e 442 V variable changing default values 69 defining 67 defining and assigning 66 displaying and changing at start of run 96 editing in run setup 70 for scouting 143 format for name and value 8 identifying 66 in run documentation 252 in run setup 70 method 66 naming 68 removing 69 renaming 69 start protocol items 143 variables in methods 8 Variables tab in Run setup 37 vertical cursor line 65 80 150 view log format 106 mode 22 mode connections 11 view all 24 View Windows icon 145 viewing blocks 48 viewing the results of internal external standard quantitation 3
443. window and minimum distance between points see Optimising the baseline parameters using a morphological algorithm on page 266 Evaluating results 10 The Classic algorithm has long been used as the standard for calculating the baseline see Optimising the baseline parameters using a classic algorithm on page 269 The Classic algorithm is particularly useful in integrating curves containing negative peaks Thus the Classic algorithm should be selected if the Morphological algorithm gives poor results from the presence of negative peaks or where quantitative data from negative peaks are important in the run To select the appropriate algorithm and change baseline settings 1 Display the Settings or Baseline Settings dialogue respectively by i clicking on the Baseline settings button in the resulting Integrate dialogue or ii selecting Integrate Calculate baseline These dialogues function identically However in the former case the baseline is immediately used in a peak integration Settings x r Chosen algorithm C Classic M Baseline Structure width 43 996 ml Noise window pa mau Minimum distance between points 33 353 al Default Values Cancel Help Fig 10 4 Settings dialogue 2 Select the appropriate algorithm Classic or Morphological 3 Change the Baseline parameter values as appropriate Excluding peaks It is possible to exclude peaks from integration bas
444. xi Name For system Explorer1 00 w Technique JAnion_Exchange a OCation_Exchange ORPC OHIC Cine Cualhinins zl Templates Chromatofocusing CIP Direct Injection FP BufferPrep Direct Injection FP Isocratic Direct Injection FP Segmented Direct Injection FP Step Direct Injection Y BufferPrep Direct Injection Y Isocratic Direct Injection Segmented Direct Iniection Y Step fad Cancel Help Fig 5 54 Save as Template dialogue 2 Enter a name for the template in the Name field or choose an existing template name from the list 3 Choose the system for which the template is intended in the For system field 4 Check the appropriate Technique from the list 5 The Templates field lists the templates currently available within the chosen system and is useful for reviewing which names are in use 6 Click on OK Note The templates for each system are common for all users Be restrictive in saving methods as templates We recommend that only methods that are useful for all users are saved as templates 5 6 3 Deleting a template You can delete templates if you have Edit global lists authorisation 1 Choose Edit Delete template an 18 1138 73 e p100 Note Note Note2 Editing methods 5 2 Select the system and the template to delete and click on Delete 3 Confirm the action The templates for each system are common for all users Be restrictive in d
445. xtensive facilities for presentation and evaluation of separation results Essential features of evaluation include e Curve manipulation A wide range of operations can be performed on curves such as addition and subtraction of two curves differentiation integration normalization and scaling The original raw data curves are always kept unmodified in the result file e Curve comparisons Curves from different result files can easily be compared in the evaluation module e Peak purity and peak identification Ratios between UV curves measured at different wavelengths give useful information about peak purity or peak identity e Peak integration Peak area calculations are performed with respect to a baseline either set by the operator or calculated by the software Peak data are presented in a tabular form e Fraction histograms If the result file contains fraction marks a curve can be converted automatically to a histogram for collected fractions e Evaluation procedures Operations performed in the evaluation module can be recorded as an evaluation procedure and repeated for other result files with a single menu command Evaluation procedures may be executed either automatically on completion of a method run or interactively from within the evaluation module e Reports Comprehensive reports of the evaluation results can be generated for hard copy documentation of the separation process Generation and printing of reports may be
446. y installed on each workstation in the network and the network is used as the medium of communication to establish control with the chromatography systems e p13 2 UNICORN concepts an 18 1138 73 e p14 EizinlE elajas Ee Chromatography system aT PC with UNICORN Network sever Chromatography system PC with UNICORN Chromatography system is NOT installed 9 G000 PC with UNICORN PC where UNICORN PC with UNICORN anag Chromatography system Fig 2 7 A network installation with 4 chromatography systems and 5 workstations PCs The chromatography systems physically connected to PCs 1 and 5 can be controlled locally Alternatively any of the PCs with UNICORN installed can be used to remotely control any of the chromatography systems via the network In this example PC 4 is connected to the network but it cannot be used to control any chromatography systems since it does not have UNICORN installed Note also that the server does not have UNICORN program files installed and is not involved in the control process per se 2 9 Security Security features in UNICORN include e Access security Use of UNICORN is restricted to authorised users Each user is assigned an access level which defines the functions that the user is permitted to use e Connection security Running systems may only be controlled from one connection Systems may be locked wit
447. yed for information only and cannot be changed at this stage Questions are data entry fields which are filled in by the operator when the run is started Some questions may be mandatory and some may require authorised confirmation Method notes are shown and start notes can be entered Displays the gradient for the method Displays the recipe selected in the method The correction factors can be changed for the current run It is not possible to change recipe at the start of a run Displays columns selected in the method Select a column and click on View to see the details of the column definition Reference curves which may be displayed in the System Control workspace during the run can be selected here see Reference curves on page 86 e p143 6 Performing a run Note an 18 1138 73 e p144 Evaluation Procedures Method Information Settings Calibration Result Name Changeable Frac 950 Evaluation procedures which will be executed automatically after completion of a run can be selected here see Evaluation procedures on page 83 Information about the method being run Displays the system settings for the run If the settings are not suitable cancel the method start change the settings with the System Settings command see the UNICORN 4 0 Administration and Technical manual and restart the method Displays calibration data for system monitors If the calibration is not accep
448. zards and method templates are Variables Adjust variable values as required for the run Notes Read the method notes and enter start notes if required Questions Fill in answers to the questions Evaluation Procedures Select the Evaluation procedure to be run at the end of the method Gradient If desired click on the X axis to alter the base unit Result Name Set the result file name and path folder as desired The default result file name includes a 3 digit serial number Frac 950 Choose the fractionation order and set the Last Tube 5 The last page of the Start Protocol has a Start button Click on this button to start the run 4 7 Editing text instructions Methods for most purposes can be created by adjusting the method variable values as described above The method is actually programmed as a series of instructions that use these variables as parameters To see and or change the instructions click on the Text Instructions icon on the Method Editor toolbar or select View Text Instructions from the menu To see an overview of the block structure and the gradient in the method select View Windows Block and View Windows Gradient respectively in the Text instruction window see Method instructions on page 57 With the Text instruction editor you have complete facilities for designing and editing your own customised methods You will also use the Text instruction editor for refining and modifying methods based on
449. ze to content 4 Ifappropriate check the This position on all pages box to insert the text box in the same position on all pages This is useful for setting texts in headers and footers 5 Select the font type style colour and size by clicking on the Font button 6 Click on OK once you have made your selections The free text object is inserted e p235 ED Presenting results Picture The Picture dialogue is used to define the settings for picture object This is useful when placing logos pictures or other figures in the report Settings Start on new page J This position on all pages I Size to picture OK Cancel Help Fig 9 49 Picture dialogue 1 Use the Browse button to locate the desired picture file bmp emf jpg tif The currently selected picture is then previewed in the dialogue 2 Check on the desired Settings options to format the picture 3 Click on OK to set the picture in the report Chromatogram The Setup Chromatogram dialogue is used to define the settings for the chromatogram object an 18 1138 73 p236 Presenting results 9 Setup Chromatogram x Selected chromatogram s Active chromatogram ed rm Settings J Thick lines I Landscape Start on new page IV Full page T Original file name Layout l I Current m Fonts Chromatogram Peak table Header text Ok Cancel Help
450. zero the warning parameters You can do this by selecting the warning in the Warning tab followed by selecting Warning Edit from the menu Alternatively click with the shortcut menu on the warning and select Edit from the displayed menu In the edit mode click on the Reset button which displays the Reset parameters dialogue Reset parameters x No of runs completed 1 Reset Next time out 3 29 1999 Reset Cancel Fig 6 23 Reset parameters dialogue You can zero specific counters by clicking on the associated Reset button in the dialogue 6 5 4 Getting a warning When you end a method using a chromatography system for which maintenance warnings have been set up a warning message will be displayed once the specific Periodicity parameter has been reached e p165 6 Performing a run an 18 1138 73 e p166 Fig 6 24 Warning dialogue for a lamp check Select Acknowledge if you have corrected the problem in the Warning dialogue Select Ignore if no correction has been made If Ignore is selected you will be reminded about the unresolved problem s at a later stage Scouting 7 Scouting Scouting is a facility for automatically repeating a run with systematic variation of one or more parameters Some typical situations where scouting is useful are e screening for the best column e finding the optimal pH e testing column capacity sample volume e finding the optimal flow rate for binding an

Unicorn 4 User Manual - GE Healthcare Life Sciences

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