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UNICORN™ 5.31 User Reference Manual Chapter 12 to 15

1. r Quaeitation table Addition component Added amount E Sid Ribo x Riboructease A z 0 3 mg C Geiba Personal r Select peak table Source 1 Peak table s Peak toble s EES chiomstogram Addition chromatogram fi uanitaleOO1 1 UV1 28001 PEAX idi 430 uarhitateOO1 1 UVI 280reni01 PEAK Se Se ect Global or Personal quantitation tables ect a quantitation table on the Quantitation table droplist Note Only external standard quantitation tables will be shown Se on Se left ect the chromatogram that contains the unspiked sample peak table the Source chromatogram droplist ect the unspiked sample peak table from the Peak table s list to the Repeat step 2 to select the peak table for the spiked sample on the Ad dition chromatogram fields Select the component that was added prior to the sample preparation on the Addition component droplist Type the injected amount of this component in the Added amount field Click the OK button UNICORN 5 31 User Reference Manual 28 9972 39 AA 13 The Analysis module 13 4 How to quantitate the sample 13 4 3 How to calculate the recovery factor How to view the recovery factor calculation results The recovery factor calculated by the software is placed at the bottom of the peak table in the Evaluation module You need to scroll to the end of the table to see it No Recovery Factor Component name Chym
2. Note In the Options dialog box in the UNICORN Manager you can select if negative re tentions should be displayed or not The default selection is that negative retention is not displayed Sigma formula The formula below is used to calculate Sigma n Sigma m gt x 7 Xymax y i 1 A peak Where 602 UNICORN 5 31 User Reference Manual 28 9972 39 AA B Evaluation functions and instructions B 3 Peak table column components e nis the number of data points e xis the volume or time value Xymox is the volume or time value at the maximum amplitude value Apeak is the area of the peak Note The peak width for a Gaussian peak is 4 x Sigma Peak resolution algorithms The peak resolution is calculated with one of the following three algorithms 1 V2 Vri Wyo Wp 2 2 V9 Vra Sigma Sigma x 2 3 Veo Vga 2 x Wyo Whal 2 354 Where Vp7 Wb2 Sigma and W are the retention width Sigma and width at half height of the previous peak Ves Wp Sigma and W are the retention width Sigma and width at half height of the current peak Note The Resolution algorithm variable in the Options dialog box in the UNICORN Manager determines which of the three algorithms is used If this variable has the value 1 2 or 3 then the algorithm with the corresponding number in the list above is used The default value is 3 How to change the peak resolution algorithm The t
3. You are unable to perform Quit or Logoff from UNICORN for a connection You might be running a Scouting method or a MethodQueue These functions require a control mode connection in order to start subsequent cy cles correctly Action Stop the Scouting method or MethodQueue before you quit or log off UNICORN 5 31 User Reference Manual 28 9972 39 AA 583 A Troubleshooting A 3 Methods and method runs Monitor signals do not appear in the Curves panel in System Control The table below describes a problem and its solution Problem description Solution Monitor signals donot e Choose System Settings in System Control appear in the Curves pane in System Control Result The System Instructions dialog box opens e Choose the Curves group in the Instructions field e Set the Store option to ON Signals for which Store is set to ON can be selected from the View Properties Curves dialog box in System Control Error message Couldn t create result file Destination path could not be found The table below describes a problem and its solution Problem description Solution If you receive the error message Couldn t This may happen if the specified folder create result file Destination path could is on the network server and network not be found at the end of a method the communication has been lost The re local computer was unable to access the sult file is saved in the
4. STEP_RESPONSE_IN Sets the level intervals for the STEP_RESPONSE_TEST TERVALS STEP_RE The relative amplitude is calculated at the specified retentions SPONSE_TEST The 0 and 100 amplitudes are used for reference The calculated relative amplitudes are checked against the specified error margins The 0 level amplitude is verified to be within the specified interval from the absolute 0 level TEST_CURVE_AMPLI Verifies that the curve amplitude has changed more than or TUDE_CHANGE equal to the value of the Delta parameter between the de fined to and from retention points A print parameter may be set to On to generate printed results TEST_CURVE_AMPLI Verifies that the curve amplitude is stable between the de TUDE_STABLE fined to and from retention points The actual curve value is compared to a set amplitude parameter If the difference exceeds a set Delta value the test is failed A print parameter may be set to On to generate printed results UNICORN 5 31 User Reference Manual 28 9972 39 AA 617 B Evaluation functions and instructions B 4 Procedure instructions Instruction Description TEST_INFO Adds selected information items to the output file e g system name UNICORN version etc Also a specified free text can be added A print parameter may be set to On to generate printed results TEST_LOG Verifies if a specified text is present in the logbook curve be BOOK_EVENT tween the defined
5. is entered followed by text e g Enter a file name as File name a full search path must be entered in answer to the question If all parame ters 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_METHOD_XLS Exports a method to the file defined in Export to file in XLS format If is entered as File name the current Re sult file will be used If is entered followed by text e g Enter a file name as File name a full search path must be entered in answer to the question 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_MUL TI_CURVES_ASCII Exports multiple curves previously defined with EX PORT_SEL_CURVES instructions in ASCII format to the file defined in Export to file If is entered as File name the current Result file will be used If is entered fol lowed by text e g Enter a file name as File name a full search path must be entered in answer to the question EXPORT_MUL TI_CURVES_WKS Exports multiple curves previously defined with EX PORT_SEL_ CURVES instructions in WKS format to the file defined in Export to file If is entered as File name the current Result file will be used If is entered fol lowed by text e g Enter a file name
6. 13 The Analysis module 13 4 How to quantitate the sample 13 4 3 How to calculate the recovery factor 1343 Howto calculate the recovery factor How to prepare for the quantitation The table below briefly describes how to prepare for the quantitation Step Action 1 Prepare a quantitation table for the components of interest Note An external standard quantitation must be used Internal standard quantitation tables cannot be used Perform a sample run with the unspiked sample and a run with the spiked sample Peak integrate the sample curves to produce the peak tables for the unspiked and the spiked samples Note The sample curves must use the same X axis base unit as the standards during the integration Time is the recommended unit for highest reliability e Check that the integration is correct e Optimize the integration if necessary e Open one of the sample result files e Use File Open Peak Tables to copy the other peak table to that result file Select File Save to save the result UNICORN 5 31 User Reference Manual 28 9972 39 AA 527 13 The Analysis module 13 4 How to quantitate the sample 13 4 3 How to calculate the recovery factor How to calculate the recovery 528 The table below describes how to calculate the recovery factor Step Action 1 Select Quantitate Calculate Recovery Result The Calculate Recovery Factor dialog box opens Calculate Recovery Factor
7. It is possible to determine the coordinates of any point on a curve and to 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 Measurement options Coordinates can be obtained in two ways e Through direct measurement e From peak table data UNICORN 5 31 User Reference Manual 28 9972 39 AA 443 12 Evaluation 12 1 Peak integration 12 1 8 Measurements How to make direct measurements The table below describes how to make direct measurements in a chromatogram Step Action i Right click in the chromatogram and select Marker Result A vertical line is set in the chromatogram A text box in the top left corner of the chromatogram displays the X axis and Y axis values of the curve at the point where the vertical Marker line crosses the curve See the illustration below uo uo Note The color of the Marker is the same as the selected curve 2 Move the Marker with your mouse to display the peak data 3 Click the curve name legend above the chromatogram to change to another curve Result The Y axis is changed to the one corresponding to the new curve 4 Right click and select Marker again to de select the function 444 UNICORN 5 31 User Reference Manual 28 9972 39 AA 12 Evaluation 12 1 Peak integration 12 1 8 Measurements How to set a reference point The table describes how to set a referen
8. a 294 10 46 HOW toSROW partot CURVES sissies cise ccacuctsnszenssibasacdcceinsnicasesbenqgonisosceceibeedscbetie dyes obs haeih 296 10 4 7 Howto change the size of Fraction Injection ANd Logbook MALKS eeeesssssssssssssssseseee 299 10 5 How to print active Chromatograms ccssssssssssessssssccsssssssssssessssscesssssssssussees 300 10 6 How to create and print reports 302 10 6 1 How to create and print a customized report 303 10 6 2 How to create and print a standard report eeeeeeeesssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssst 320 10 6 3 How to edit an existing report format eeseecssssscsssssssssssssssssssssccssssssssssssssssssesesssssssssssusesssseseesee 324 10 7 RU NCIO CUI SECA sess cBiceecacepsnsa vente aa E 327 11 How to edit results cscs essa cthciarashatabadsi as hades eaaethcecesachadentabianickstieionevesdiaasy 331 11 1 How to reduce noise and remove ghost peaks sscssssssssesssscccsssssssssssseessssscssssssssssssseseseesees 332 11 2 How to Subtract a blank PUM CUFVE ceececscsssssssssssssssssesssssesseseeeessesesseeeseeseseessseeseeseesssseseesssseessssessest 334 11 3 How tO add CUIVES neeeeeeceecccscsssssssssesssseseeeessseessesecesssesssssssssse ss 337 11 4 Howto enter and edit text in the chromatogram 338 UNICORN 5 31 User Reference Manual 28 9972 39 AA 5 Table of Contents 11 5 GW tO DOOM fractions enosn ananin a n a ARAR 340 11 6 How to match protein activity t
9. losd sample loop Flowpath Mode C AlaimstMon Vat Authorize z C Fiac Sound Default sound z C Watch Other The illustration below shows the text instruction for the message described above IAM TID Mul Saas WREST Geass biedhsn i TI Tur Andian DOIS Neman AnA RNAn eulien hia TIO Rare INUN Qs D WW Nieda en R HARTL Mal Saas Leen Gas hiedh UNICORN 5 31 User Reference Manual 28 9972 39 AA 661 F Method examples F 6 Messages Note The message instruction must be followed immediately by the Pause instruction as shown above 662 UNICORN 5 31 User Reference Manual 28 9972 39 AA F Method examples F 7 Quality control procedure F 7 Quality control procedure Introduction When a series of runs is performed irregularities in samples or in system or column performance can produce errors that will make the results inaccurate A quality control procedure can be added to a method to be used for a test run during the series of runs The control procedure can ensure that the results remain within ac ceptable limits If the result from the test run is unacceptable the system can be paused so that the error is not repeated in subsequent runs How to create the quality control procedure The easiest way to create the quality control procedure is to edit an existing procedure that includes a peak integration The table below describes how to do this Step Action 1 e Choose Procedures Ed
10. 1 e Choose Edit Chromatogram Layout or e Click the Chromatogram Layout icon m Result The Chromatogram Layout dialog box opens 408 UNICORN 5 31 User Reference Manual 28 9972 39 AA 12 Evaluation 12 1 Peak integration 12 1 2 How to perform a peak integration Step Action 2 Click the Curve Style and Colour tab 3 Select one or more of the following labelling options in the Peak label field e Number Result The peaks will be numbered sequentially e Peak Name Result Peak names will be displayed See Section 12 1 6 How to edit the peaks on page 427 for information about how to name the peaks e Retention Result The retention volume or time will be displayed e Click OK UNICORN 5 31 User Reference Manual 28 9972 39 AA 409 12 Evaluation 12 1 Peak integration 12 1 3 How to optimize the baseline with a morphological algorithm 1213 How to optimize the baseline with a morphological algo rithm Introduction The first choice when you want to optimize the peak integration is to change the baseline parameters This section describes how to optimize the baseline with a morphological algorithm The Morphological algorithm The Morphological algorithm can be described as a line that follows the chromatogram parallel to the X axis Data points for the baseline are created whenever the line touches the curve and the points are joined at the end to create a baseline The Morphological algorithm gives the be
11. 2 e Select the UV curve you want in the Source chromatogram list e Click the First order radio button e Click OK Result The differentiated curve opens in the chromatogram How to measure the slope values Sometimes the differentiated curve must be filtered to reduce noise and ghost peaks before the measurements See section Section 12 2 1 Peak purity and peak identification on page 448 452 The table below describes how to measure the slope values on the differentiated curve Step Action 1 Click the name of the differentiated curve above the chromatogram window to select the curve 2 Use the zoom function to magnify the curve over an appropriate area 3 Right click and select Marker from the short cut menu Result A vertical cursor bar opens in the chromatogram UNICORN 5 31 User Reference Manual 28 9972 39 AA 12 Evaluation 12 2 Other evaluations 12 2 2 How to find slope values Step Action 4 Place the Marker at the beginning of a peak where you want the Watch conditions to be fulfilled i e where the slope becomes higher 5 Read the actual slope value in the active Marker text box in the top left corner of the chromatogram window Note The unit for the differentiated curve is mAU min or AU min Any Y axis value for the differentiated curve is the UV curve slope at the selected retention point Peak fractionation for AKTAdesign If your system is an AKTA design system measure the slope
12. Code no Typical loading range Mol weight range kDa Scan rate Spectra sec Save As Cancel Help UNICORN 5 31 User Reference Manual 28 9972 39 AA 631 D The Column list D 1 How to edit the Column List How to add a new column The table below describes how to add a new column to the Column List Step Action il e Choose Edit Column List in the Method Editor Result The Column List dialog box opens Note Select a column from the list to display the parameters in the field to the right Most column parameters are displayed in the Normal Parameters tab Additional parameters for special columns may be displayed in the Ad vanced Parameters tab e Click the New button Result The New Column dialog box opens e Select the appropriate parameter tab e Type the desired parameter values e Click the Save as button Note Mandatory parameters are labelled mand The column cannot be saved unless all mandatory parameters are filled in Result The Save as dialog box opens e Type the name of the new column e Click the Save as global checkbox if the column should be available to other users Note You must have Edit global lists authorization to save a column for global use A global column cannot have the same name as a personal col umn e Click OK Result The new column is added to the Column List Note See column instruction to determine the back pressure over the system and the c
13. I I UNICORN 5 31 User Reference Manual 28 9972 39 AA Index How to optimize the chro matogram workspace 281 How to set a reference point 282 Evaluation logs How to export 393 Evaluation procedures How to delete 168 How to edit 169 How to rename 168 Explained variance Definition 627 External standard quantitation How to perform 482 F File Navigator How to change preference settings for Recent Runs 273 How to close 274 How to locate files from the Files list 270 How to open 269 How to open a recent run 272 How to open result files 86 How to use Find to locate a file 271 Files and folders Copy to external 93 How to copy 92 How to copy from exter nal 93 How to move 92 Flow Scheme pane Description 247 How to view manual instruc tions 247 Stretch to fit screen 247 Folders How to create a user specif ic 84 FPLCdirector How to import data 389 Frac 950 Defining the number of available tubes 183 How to select the last tube 183 671 Index 672 Fraction Histogram How to create a curve 459 Fractions How to calculate protein amounts and concentra tions in pooled frac tions 344 How to create a pooled fraction curve 342 How to determine the vol ume of a pooled fraction with a specific concentra tion 344 How to pool fractions 341 How to set up the Frac unit 183 How to use the Pool Frac tion print list 344 How to view the contents of a
14. Peak integration results The peak table is displayed underneath the active chromatogram The start point and end point of each peak are marked by vertical marks drop lines in the chromatogram The peaks are automatically labelled according to what is selected in the Curve Style and Color tab of the Chromatogram Layout dialog box This is an illustration of the results after a peak integration Crabsation Geample Result00Zi1 _ v UNICORN Local fil Niklas result ample ResukOUZ namol Result 002res kee el rte Edt View Integrate GREED Procedures Window Hap les Pla aa oas 535 po poo jso 776 7 hi 62 No Peak naxe Retention min Area mAU min Height mAV 2 Peak A 0 2 6 53 34 6083 95 763 7 06 20 4377 125 560 7 76 36 9911 147 623 Note Peak tables can be copied from one chromatogram to another with the Ed it Copy command However to display the table you must right click in the chromatogram choose Properties and then select the new peak table on the Peak Table tab of the Chromatogram Layout dialog box UNICORN 5 31 User Reference Manual 28 9972 39 AA 405 12 Evaluation 12 1 Peak integration 12 1 2 How to perform a peak integration How to display peak characteristics The peak retention times and several other peak characteristics are calculated automat ically The table below describes how to display other peak characteristics Step Action 1 e Right c
15. Tape 104 E UVI IONOS DASEMT iz 1982 302 Noise window Sometimes you get too many peaks after the peak integration usually because noise on the baseline is erroneously detected as peaks The solution to this is to increase the Noise window parameter However this can result in peak limits too high up on the peak slopes Note You can also use the Reject peaks function in the Integrate dialog box to reduce the number of peaks based on the total number of accepted peaks or the minimum peak height 412 UNICORN 5 31 User Reference Manual 28 9972 39 AA 12 Evaluation 12 1 Peak integration 12 1 3 How to optimize the baseline with a morphological algorithm 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 If you increase the Minimum distance between points value fewer points will be collected on the slopes The illustration below is an example of a baseline A that is created with the Minimum distance between points parameter set at a low value The number of data points is reduced when the Minimum distance between points parameter is set to a higher value B UNICORN 5 31 User Reference Manual 28 9972 39 AA 413 12 Evaluation 12 1 Peak integration 12 1 4 How to optimize the baseline with a classic algorithm 1214 How to opt
16. The table below describes how to create a new procedure with instructions Step Action 1 Choose Procedures Edit New Result The Procedure Editor opens in Edit mode 2 e Select an instruction from the Instruction list e Type the appropriate parameters in the Parameter field e Click Insert 3 Repeat step 2 until the procedure is complete 4 Choose File Save 5 Type a procedure name and click OK 6 Click the Close button in the Procedure Editor UNICORN 5 31 User Reference Manual 28 9972 39 AA 463 12 Evaluation 12 3 Automated evaluation procedures 12 3 2 How to edit a procedure 1232 Howto edit a procedure Introduction Evaluation operations are represented by instructions in the Procedure Editor dialog box The instructions can be modified to suit other specific evaluation needs and be saved for later use This section describes how to use the Procedure Editor to edit a procedure How to edit a procedure The table below describes how to edit an existing procedure Step Action 1 Select Procedures Edit Open Result The Open Procedure dialog box opens Select the procedure from the list and click OK Result The Procedure Editor opens in Edit Mode Select an instruction in the procedure window Result The instruction parameters are displayed in the Instruction and Pa rameter fields A short definition of the selected instruction is displayed at the bottom left corner Type new
17. as File name a full search path must be entered in answer to the question EXPORT_MUL TI_CURVES_XLS Exports multiple curves previously defined with EX PORT_SEL_ CURVES instructions in XLS format to the file defined in Export to file If is entered as File name the current Result file will be used If is entered followed by text e g Enter a file name as File name a full search path must be entered in answer to the question EXPORT_NOR MALISE_RETENTION Normalizes retention when exporting multiple curves UNICORN 5 31 User Reference Manual 28 9972 39 AA B Evaluation functions and instructions B 4 Procedure instructions Instruction Description EXPORT_PEAK Exports the peak table in Peak table source to the file TABLE_ASCII defined in Export to file in ASCII format If is entered as File name the current Result file will be used If is entered followed by text e g Enter a file name as File name a full search path must be entered in answer to the question EXPORT_PEAK Exports the peak table in Peak table source to the file TABLE_WKS defined in Export to file in WKS format If is entered as File name the current Result file will be used If is entered followed by text e g Enter a file name as File name a full search path must be entered in answer to the question EXPORT_PEAK Exports the peak table in Peak table source to the file TABLE_XLS defined in Export
18. consecutive order of decreasing or increasing concentration e Click the Current button at any time to return to the chromatogram that was active before you activated Quantitate e Highlight unwanted tables on the list and click Remove e Click OK to finish the selection Result The Define Component s dialog box opens Continue to Step 2 How to select and define components below this table 500 UNICORN 5 31 User Reference Manual 28 9972 39 AA 13 The Analysis module 13 3 How to prepare for quantitation 13 3 2 How to create a quantitation table Standard concentration levels It is useful to think of each level as an alias for a specific 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 The Define Component s dialog box The components that will be used to produce the calibration curves are selected in the Define Component s dialog box Quantitate must be able to identify these components on all levels This dialog box is used to set the criteria by which peaks are identified The illustration below shows the Define Component s dialog box Show curve for Jevel fia x Double click on a peak to select t 4 0 4 Ba Peaks in level 1 1 Black peak markers UNICORN 5 31 User Reference Manual 28 9972 39 AA 501 13 The Analysis
19. structions before saving the method UNICORN 5 31 User Reference Manual 28 9972 39 AA A Troubleshooting A 3 Methods and method runs Print screen does not send a copy of the screen to the printer The table below describes how to solve a printing problem Problem description Solution The Print screen command only If you want to print the view on the screen makes a copy of the screen to the press the lt Print Scrn gt key and paste the im clipboard and not to the default age from the clipboard into an appropriate printer program such as Microsoft Paint and then print out the image UNICORN 5 31 User Reference Manual 28 9972 39 AA 587 A Troubleshooting A 4 Evaluation A 4 Evaluation In this section This section describes how to solve the following evaluation problems e Incorrect date and time in the result file e valuation procedure aborts Incorrect date and time in the result file The table below describes a problem and its solution Problem description Solution The result file shows incorrect Check the system clock setting date and time The date and time recorded in the result file are taken from the PC system clock setting Evaluation procedure aborts The table below describes a problem and its solution Problemdescription Solution The evaluation proce Instructions in an evaluation procedure refer to curves by dure aborts identification number irrespective of the curve nam
20. 3 How to prepare for quantitation 13 3 2 How to create a quantitation table Step Action 7 e Specify if the table is to be globally accessible to any user or restricted to your personal user ID The default is global e Type a name in the Quantitation table name field e Click the OK button How to select an Internal Standard The table below describes how to select an internal standard in the Quantitation table dialog box Step Action i Click the IS and Table settings button Result The IS and Table Settings dialog box opens The illustration below shows the IS and Table Settings dialog box with an Internal standard selected VS Ss ads Sa Sih 2 Type the amount and concentration multipliers in the General field Note These values are normally set to 1 See remarks below UNICORN 5 31 User Reference Manual 28 9972 39 AA 509 13 The Analysis module 13 3 How to prepare for quantitation 13 3 2 How to create a quantitation table Step Action 3 Select the internal standard component on the Internal standard peak droplist Note The default option is Not selected which is used for external standard quantitation and measurements of the recovery factor 4 Type the injected internal standard amount for the standard and sample runs in the IS amount text box 5 Select if the quantitation will be based on Area default or Height in the Quantitation peaks field Note Select Height if the peaks are no
21. Action il e Click the Edit peaks icon a e Click the peak in the curve or in the peak table to select the peak UNICORN 5 31 User Reference Manual 28 9972 39 AA 433 12 Evaluation 12 1 Peak integration 12 1 6 How to edit the peaks Step Action 2 e Right click and select Join Left or Join Right from the shortcut menu or e Select Edit Join Left or Edit Join Right Result The original intervening drop line is removed and all peaks are renumbered How to add peak names The table below describes how to add names in the Edit Peak Table dialog box to iden tify the peaks Step Action 1 e Click the Edit peaks icon a e Click the peak in the curve or in the peak table to select the peak e Right click and select Peak Name from the shortcut menu or e Choose Edit Peak name or e Double click the peak in the peak table or the curve Result The Edit Peak Name dialog box opens The number and retention of the selected peak is displayed Type a name in the Peak name textbox and click OK 434 UNICORN 5 31 User Reference Manual 28 9972 39 AA 12 Evaluation 12 1 Peak integration 12 1 6 How to edit the peaks How to adjust peak areas with drop lines The table below describes how to move the drop lines to adjust the peak area in the Edit Peak Table dialog box Step Action 1 e Click the Edit peaks icon m e Click the peak in the curve or in the peak table to select
22. Failed folder on folder specified in the result file path the local station 584 UNICORN 5 31 User Reference Manual 28 9972 39 AA A Troubleshooting A 3 Methods and method runs The Method System Connection dialog box keeps appearing The table below describes a problem and its solution Problem description Solution If the Method System Connection dialog box keeps ap Connect the method s pearing you have some method s which is not connected to the appropriate sys to a system tem Reason Most likely you have imported some method s with the command File Copy from External in the UNI CORN Manager The Method Editor window does not fit on the screen The table below describes a problem and its solution Problem description Solution The Method Editor windowdoes e The display screen resolution may be set to not fit the screen and has scroll 1024x768x65536 with Large fonts You bars need to install the Small fonts This requires Reason The incorrect font size that you have the Windows xP or Windows might be installed 7 DVD ROM that was shipped with your computer e Insert the CD ROM and follow the directions on the screen Note Always install the latest service pack after you have installed something from the Windows XP Windows 7 DVD ROM UNICORN 5 31 User Reference Manual 28 9972 39 AA 585 A Troubleshooting A 3 Methods and method runs There are red instruction
23. H Wto export aimethod siennas iniiai diis 212 7 SCOUTING ees E RE 213 7 1 HOW to Set UP a Scouting SCHEME woereecccssssscsssssssssssssessssscssssssssssssuesssssessessssssusssueseessessecesssnssseesees 214 7 2 How to define different columns FOr SCOUTING sssssssseessssscsssssssssssssesesssesccssssssssuusesssesesssssssss 220 UNICORN 5 31 User Reference Manual 28 9972 39 AA Table of Contents 8 WU esse sh shee esac deed ueectestas hedaeancbiateans beat laiecsculadacaseeuendaaaay 221 8 1 How to create a NEW Method Queue ooiveeceeccccccsscsssesscsssessssssseessssessssssecessssuessssesessssuessssseessssssess 222 8 2 HOW to Edit A MeEthOd QUEUE wae eeeecsssescssssesscsssesssssesssssessssssesssssussesssuesessssssessssstessssusessssuessssseess 227 9 HOW to perform method TUNS iasscsassidesccssisasies cacasonsentsacecaessastesdeniasscaateastiasecaviods 229 9 1 How to start a method run 230 9 2 How to monitor a Method run eee 234 9 2 1 How to customize System Control PANES eessssssssssssssssscscsssssssssssssssssssssssssssssssssesssssseesessssssess 235 922 TheRun Data GING ssc szacicsdasbussccobisascctscCanvusssastiscosasebondisdbion sgnssecobaiesvndessgseldidesghutudhestelelsttectessouabye 237 9 2 3 The Curves pane 240 9 2 4 The Flow Scheme pane 247 9 2 5 The Logbook pane wu 249 9 3 AMG UaL SYSTEM COMO os sacvascsssssnsscscasszcssavssassassssnscceeletsassasisassusnsnnccovtsseiea
24. J User defaut Intemal standeed peak Date 471172002 Intemal standard amount Last updated 4 11 2002 Reference peak Table Ext_Std_Ribo Nominal reference retention Injection volume Not used Update reference retention Absolute retention and Highest peak m dmum replicates Ribonuclease A mie 7390 emmy 0614 CS maT a 8004 2 28 4709 14 0643 Out of limit 1 Features The list below describes some features of the dialog box e Components that will not be updated are shown in the column Updated area or Updated ratio if an internal standard is used with the text Out of limit e 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 e Nominal reference retention shows the retention for the reference peak in level 1 1 e Update reference retention shows the retention for the reference peak in the new peak table UNICORN 5 31 User Reference Manual 28 9972 39 AA 517 13 The Analysis module 13 3 How to prepare for quantitation 13 3 3 How to edit and update a quantitation table How to rename a quantitation table The table below describes how to rename an existing quantitation table Step Action iL Select Quantitate Edit Quantitation Table Rename Result The Rename quantitation table dialog box opens 2 e Select Personal to display the quantitation t
25. Table dialog box opens e Click the Peak Window icon Result Two vertical cursor lines are displayed e Drag the cursor lines to the beginning and the end of the selected part of the curve Q tdt Pesk Table ox Fie Edt Baseline Integrate AREA Example FeO 1 vt 2160 Eeantle Rena DOr UVI EEDI PASEM 63 1658 3 Peak A 0 5 6 52 6 26 6 34 44 0967 3 Penk b 0 5 7 08 6 84 2 36 60 5619 4 Peak 0 5 774 7 36 3 68 115 5646 No penx pena Retention min peax start min Peak end min Area mA0rmin 4 Internal sti Note All operations described below will only affect the selected part of the curve UNICORN 5 31 User Reference Manual 28 9972 39 AA 441 12 Evaluation 12 1 Peak integration 12 1 7 How to integrate part of a curve and how to exclude or skim peaks Step Action 3 If desired change the integration parameters Reject peaks e Choose Integrate Settings Result The Reject Peaks dialog box opens e Change the settings as desired and click OK Skim peaks e Choose Integrate Peak Skim Result The Peak Skim dialog box opens e Select the Skim Peaks checkbox and type a ratio e Click OK 4 e Choose Integrate Peak Integrate Result The selected part of the curve is peak integrated based on the changed parameters 442 UNICORN 5 31 User Reference Manual 28 9972 39 AA 12 Evaluation 12 1 Peak integration 12 1 8 Measurements 1218 Measurements Introduction
26. The selected peak is affected on all levels Note More than one peak can be selected to produce calibration curves for several components 5 Highlight the component name and type a new name 4 Double click the internal standard peak if applicable and type a new name 5 Continue to Step 3 How to identify the peaks below this table 502 UNICORN 5 31 User Reference Manual 28 9972 39 AA 13 The Analysis module 13 3 How to prepare for quantitation 13 3 2 How to create a quantitation table The Define component s peak table columns The peak table within the Define Component s dialog box has three columns e The absolute Retention value of the component in level 1 1 e The width of each component s window If you change the width of the window by adjusting the cursor lines this is reflected in the Window column e The Component name with the currently selected component highlighted Component name Possible to change 0 53 Ribonuclease A 9 74 0 56 Cytochrome C 11 01 1 33 Lysozyme Step 3 How to identify the peaks Description When a component is selected vertical cursor lines show the current identification window The software uses this window to search for peaks on other levels and in the sample runs 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 sym metrically so that the limits center on
27. When the component selection and identification settings are completed see Step 3 the Quantitation table dialog box is opened Quantitation table x Enter amour of concentration for every level gt Curve model or 14 8827 C Linear through oegn C Quadratic 14 5968 Causes trou ong Explained variance 98 504 Correlation 0 9363 Seve F Help JS and Table settings Define componerks Ei Update resort The table below describes how to enter data for the standards and create a quantitation table and a calibration curve Step Action 1 e Click the IS and Table settings button if you want to use an internal standard or base the calibration curve on peak height see How to select an Internal Standard below this table 2 e Verify that the selected components in the Components list are correct Ifan internal standard is used the related component is labelled IS If relative retention has been used the reference component is la belled Ref e Click the Define components button to change the components UNICORN 5 31 User Reference Manual 28 9972 39 AA 507 13 The Analysis module 13 3 How to prepare for quantitation 13 3 2 How to create a quantitation table 508 Step Action 3 e Select the first component at the top of the Components list Note Do not select an internal standard component if available as the amount for this
28. You must have Edit global list s rights to be able to delete global tables UNICORN 5 31 User Reference Manual 28 9972 39 AA 551 13 The Analysis module 13 6 How to measure molecular size 13 6 2 How to determine the molecular size How to calculate the molecular size 552 The table below describes how the molecular size curve is used to calculate the molec ular sizes of the components in the sample Step Action 1 Perform a sample run and peak integrate the curve to produce a peak table Note The sample curve must use the same X axis base unit as the standards Use volume for molecular size calculations Select Mol Size Calculate Mol Size in the Evaluation module Result The Molecular Size dialog box opens AAS Ce Nis S AE Bsa C Re N SEAN ON Sega Snasssrgee e Select Global or Personal according to the location of the molecular size table e Select the molecular size table on the Molecular size table droplist e Select a chromatogram on the Source chromatogram droplist e Select a peak table on the Peak table s list and click OK Result The results of the molecular size calculation are shown in the Mol size peak table column See illustration below UNICORN 5 31 User Reference Manual 28 9972 39 AA 13 The Analysis module 13 6 How to measure molecular size 13 6 2 How to determine the molecular size The Mol size peak table column The illustration below shows th
29. also increased Note 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 1 Autoregressive The table below describes the process when the Autoregressive smoothing algorithm is used Stage Description 1 The first data point in the source curve is copied to the processed curve 2 For each subsequent data point the previous processed point is multiplied with the parameter value and added to the current source data point UNICORN 5 31 User Reference Manual 28 9972 39 AA 591 B Evaluation functions and instructions B 1 Smoothing algorithms Median 592 Stage Description 3 The result is then divided by the parameter value plus 1 according to the following formulae t 5 t p t nat Sn p 1 Where t Current processed point ty 1 Previous processed point S Current source point p smoothing parameter value Note If you increase the parameter value the smoothing effect is also in creased Note The filter algorithm only accepts integer parameter values between 1 and 25 The table below describes the process when the Median smoothing algorithm is used Stage Description 1 For each data point in the source curve the processed curve is calculated as the median of the data points within a window centered on the source data point e The width of the window is determined by the parameter
30. and click OK to return to the Define buffer substance dialog box Type appropriate values in the Value cells for each pKa and dpKa dT param eter Note All values must fall within the stated Range limits Up to three values can be entered for each buffering component When the component has less than three pKa values the other values should be set to zero A dpKa dT value of zero means that the pKa does not change with temperature e Type the number of acidic protons for the buffer substance in the form that it is actually weighed in Example The number is 2 for NaH PO 1 for Na HPO and 0 for Tris e Type the charge of the completely de protonated ion This will be a negative value for an acid and zero for a base Example The value is 3 for NaHPO and 0 for Tris e Click OK Result The new buffer substance is added to the list of available substances How to define a new salt Before you can define a new salt you must ensure that the new salt is inert i e a salt with no buffering properties The table below describes how to define a new salt Step Action 1 Choose Edit BufferPrep Recipes and click the New button UNICORN 5 31 User Reference Manual 28 9972 39 AA 643 E How to create and edit BufferPrep recipes E 1 How to create a BufferPrep recipe Step Action 2 Click the Salt button in the New Recipe dialog box Result The Define salt dialog box opens ASSESSES SU N wO OA Sasa es
31. batch run reports Evaluation procedures combined with batch runs can be a useful tool to produce printed documentation simultaneously for many result files e g for a number of scouting runs The table below describes how to create a procedure to batch run reports Step Action 1 2 Choose Procedures Record On to record a procedure Choose File Report Result The Generate Report dialog box opens Choose a report format Click the Print button as the final instruction Choose Procedures Record Off UNICORN 5 31 User Reference Manual 28 9972 39 AA 469 12 Evaluation 12 3 Automated evaluation procedures 12 3 3 How to run a procedure Step Action 6 Save the procedure Note A printing procedure can also be saved with a method to produce automatic prints at the end of a run Note When for example a batch run is performed the latest version of the procedure will be used However procedures that are saved with a method are not affected if the original procedure is edited at a later time How to add procedures to the menu 470 You can add up to 15 created evaluation procedures to the Procedures menu in the Evaluation module The table below describes how to add procedures to the menu Step Action 1 Select Procedures Menu Result The Edit Procedures Menu dialog box opens 2 e Select the checkboxes of the procedures you want to display on the menu e Click OK Result The selected proced
32. be correctly selected The default settings will be satisfactory in many cases but the integration results have to be checked for all chromatograms e The standard addition technique assumes a linear through the origin relationship between the amount of component and peak size This is a good approximation for small quantities under normal conditions e Standard addition has no way of compensating for changes that are made between the runs However if losses during sample preparation are constant between the two runs they may be accounted for by spiking the sample prior to the sample preparation e A spike amount which is of the same order of magnitude as the sample must be used to maximize precision e All the runs must be performed consecutively to reduce systematic errors and thereby maximize precision UNICORN 5 31 User Reference Manual 28 9972 39 AA 491 13 The Analysis module 13 2 Quantitation overview 13 2 5 Recovery calculation 1325 Recovery calculation General information Recovery calculation is used to determine losses that can occur during the sample preparation process Recovery can also be used to determine the recovery factor of a preparative purification or a chromatographic process The recovery factor can only be determined for a single component A calibration curve is produced using a concentration series of an external standard The calibration range must cover the amount in both the sample and the spike
33. by size exclusion chromatography A molecular size calibration curve must first be created with compo nents of known molecular size The retention is inversely related to the molecular size This section describes how to measure the molecular size In this section This section contains these sub sections Section See page 13 6 1 Overview of molecular size determination 544 13 6 2 How to determine the molecular size 546 UNICORN 5 31 User Reference Manual 28 9972 39 AA 543 13 The Analysis module 13 6 How to measure molecular size 13 6 1 Overview of molecular size determination 1361 Overview of molecular size determination How to create a molecular size curve The table below is a brief description of how to create a molecular size curve Step Action 1 Perform a run with one or more standards to create a standard curve Note The standards should contain a number of components of known molecular size and these should extend beyond the size limits that are ex pected in the test sample 2 Peak integrate the standard curve to produce a peak table 3 Use the peak table from the standard to produce a molecular size table Each peak is represented by a retention value 4 Select the relevant peaks and input data for the corresponding molecular sizes Result The software plots these values as a molecular size curve This curve has an inverse relationship between the logarithm of the molecular size and retention
34. chromatogram displayed in the Source chromatogram field UNICORN 5 31 User Reference Manual 28 9972 39 AA 499 13 The Analysis module 13 3 How to prepare for quantitation 13 3 2 How to create a quantitation table Step Action 2 e Double click a result file in the Select peak table list if you want to select a source chromatogram from another result file If desired the standard can be expressed in Concentration instead of in Amount e Click the Concentration checkbox and edit the injection volume in the Inj volume field Note The software will always calculate both amount and concentration for the sample e Highlight the standard peak table of level 1 on the Peak table s list and click the Select button Note This should be the table for the highest or lowest concentration of the standard Result The peak table is added to the Level Peak table s list 3 e The level is automatically copied onto the list if it already was set in the method If so continue with step 4 e Ifa level has not been set the Select Level dialog box opens Select 1 on the Level menu and click OK 4 e Click another result file in the Results field and select the new source chromatogram Result The peak tables associated with this chromatogram are displayed on the Peak table s list 5 e Repeat steps 3 and 4 until all the standard peak tables have been select ed Note Increase the level number for each new standard concentration in
35. command 141 How to add an instruc tion 137 How to change 140 How to delete instruc tions 139 nstruction markings 135 nstructions at the same breakpoints 192 Move an instruction to an other breakpoint 143 Move an instruction within the same breakpoint 143 Pause Hold and Hold_until instructions 138 Undo delete 139 MethodQueue File handling 227 Folder handling 227 How to create a new 222 How to display and edit pending and running MethodQueues 263 How to edit a Method Queue 228 How to perform a Method Queue run 262 How to use several systems in a queue 225 Relative timing of steps 225 Start when the system is busy 263 673 Index 674 Temporary hold when sys tem is busy 226 Unattended execution 226 Unattended operation 262 Method runs Curves pane in System Control module 240 Flow Scheme pane descrip tion 247 How to define methods as menu commands 231 Queue run 262 when the system is busy 232 How to start an Instant Run 231 col 231 f network communication fails 265 Logbook pane descrip tion 249 Run Data pane descrip tion 237 Scouting runs 260 Start from System Con trol 230 Start from the UNICORN Manager 230 Methods Different method editing operations 116 Gradient instruction param eters 199 Hold_until instruction de scription 197 Hold instruction descrip tion 197 How to create using a wiz ard 99 How to ex
36. curve point immediately before the curve point you want to connect to e Click the Draw straight to next point button Result The curve is adjusted so that it is drawn as a straight line between the two points 458 UNICORN 5 31 User Reference Manual 28 9972 39 AA 12 Evaluation 12 2 Other evaluations 12 2 5 How to use the Fraction Histogram 1225 Howto use the Fraction Histogram Introduction The Fraction Histogram dialog box in the Evaluation module can be used to create a curve for the average fraction absorbance How to create a Fraction Histogram The table below describes how to create a Fraction Histogram curve Step Action 1 Select Operations Fraction histogram Result The Fraction histogram dialog box opens 2 e Select the desired UV curve Note The fractions curve should already be selected on the middle list If you have previous pooled fractions and created a pooled fraction curve select the desired fraction curve 3 e Click OK Result The average fraction absorbance values are displayed as a new curve in the chromatogram UNICORN 5 31 User Reference Manual 28 9972 39 AA 459 12 Evaluation 12 3 Automated evaluation procedures 12 3 Automated evaluation procedures Introduction An evaluation procedure is a recorded sequence of interactive operations in the Evalu ation module which can be executed for automated data evaluation and report gener ation The concept is similar to the m
37. error 579 U Unconditional call Description 121 Unconditional method instruc tions Base instruction 189 UNICORN Manager Limited access to 29 V Variables Breakpoints or gradient lengths 146 General description 145 How to change method variable values 146 How to define new method variables 147 How to remove a method variable 148 How to rename a method variable 148 Identification in text instruc tions 145 Variable names 147 W Warnings Description 558 Watch instructions Air sensors 204 Delta_Base settings 206 Delta_Peak settings 205 Description 121 How to insert an instruc tion 202 Parameter options 203 Permanent settings 204 Standard Watch condi tions 202 Watch Stable_baseline 206 Wizard How to create a method 99 Y Y axis How to choose the Y axis scale 280 Z Zero baseline Definition 402 Zoom function How to enlarge parts of a curve 296 UNICORN 5 31 User Reference Manual 28 9972 39 AA For local office contact information visit www gelifesciences com contact GE Healthcare Bio Sciences AB Bj rkgatan 30 751 84 Uppsala Sweden www gelifesciences com unicorn imagination at work GE imagination at work and GE monogram are trademarks of General Electric Company U Al Al th e company within GE Hea ICORN Ettan FPLCdirector OligoPilot KTA AKTAxpress AKTAbasic AKTAFPLC TApilot AKTApurifier and KTAexplorer are trademarks o
38. eter decides how many samples wide the filter is SMOOTH_MEDIAN Smooths the source curve with a median filter and stores the result in target curve position The Filter width parameter de cides how many samples wide the filter is SMOOTH_SG Smooths the curve with the Savitzky Golay algorithm SUB Subtracts two curves to produce a third curve which is the difference of the two curves The two source curves must have the same Y axis unit and not be fraction or injection curves UNICORN 5 31 User Reference Manual 28 9972 39 AA B Evaluation functions and instructions B 4 Procedure instructions Instruction Description TDIV Divides two curves to produce a third curve which is the quo tient of the two curves The two source curves can have any Y axis unit The threshold values are used to avoid division of numbers close to zero At those points where source curve 1 has an amplitude less than Threshold1 or the source curve 2 has an amplitude less than Thresholda the result of the division is defined to be 1 0 Integration The table below contains a list of instructions for integration Instruction Description CALCULATE_BASE Calculates a baseline from the source curve The baseline is LINE stored in the target curve position DEFAULT can be selected in the Baseline parameters which will then calculate default baseline parameters for each new curve CALCULATE_BASE Calculates a baselin
39. level Set the level slightly lower than the value that you measured in step 2 e Click OK Example of a correct baseline The illustration below is an example of a correct baseline after the Max baseline level has been changed 422 UNICORN 5 31 User Reference Manual 28 9972 39 AA 12 Evaluation 12 1 Peak integration 12 1 5 How to edit the baseline manually 1215 How to edit the baseline manually The Edit Baseline dialog box You can edit the baseline manually in the Edit Baseline dialog box in the Evaluation module e Select Integrate Edit Baseline to display the dialog box The Edit Baseline dialog box displays the baseline and the curve it was calculated from The baseline points are marked with green squares Hold the cursor above the baseline point to display its coordinates See the illustration below Q Edi Baseline Lof x 4 800 Example sexult 003 t_UV Example tenult 003 1_UV O1 BASEM Example iep 003 1_EditecBareline AU B50 Rs0 NSO 100 s0 o J o O g g g g CJ a O O C g CH o 100 200 200 wo 600 000 700 300 min 17 232 min 2H ele 537 976 mAU Drow straight to pent point ceca Hem How to use the zoom function The table below describes how to use the zoom function in the Edit Baseline dialog box Step Action 1 e Click the Zoom icon Result The cursor is changed into a magnifying glass UNICORN 5 31 User Reference Manual 28 9972 39 AA 423 12 Evaluation 12 1
40. module is an optional extra module that adds functionality to the regular UNICORN Evaluation module Basically the Analysis module is used e to determine the absolute quantity or concentration of a component e to determine the molecular size of a component Module menus The Analysis module is accessed in the Evaluation module After the installation two new Evaluation module menus are added e Quantitate e Mol Size Note The menus are only available when a result file is open in the Evaluation module Quantitate The Quantitate function provides a wide range of techniques for quantitative analysis e External standard quantitation e Internal standard quantitation e Standard addition e 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 474 UNICORN 5 31 User Reference Manual 28 9972 39 AA 13 The Analysis module 13 1 General information about the module Molecular Size The Molecular Size Mol Size function determines the molecular size of components in a sample The function uses a molecular size curve prepared from one or more standards Term definitions The table below lists definitions for some terminology that is used in this chapter Term Definition Amount This specifically refers to the injected amount In most cases the word amount is used as an abbreviati
41. ob jects 309 How to add the Evaluation log 313 How to change the page setup 306 How to create a blank cus tomized report 303 How to create a Standard report 320 How to edit a customized report 326 How to edit a standard re port 324 How to include a peak ta ble 310 How to include a pool ta ble 310 How to include chro matograms 310 How to include Frac 950 data 314 How to include Method ob jects 311 How to include Quantitate and Mol Size data 313 UNICORN 5 31 User Reference Manual 28 9972 39 AA Index How to print 317 How to print a standard re port 322 How to save the report for mat 319 How to save the report in PDF format 318 Toolbar icons in Report Edit Mode 315 Result file name Name options 180 Serial numbers 181 Unique identifier 181 Result files Automated printing of 166 Electronic signature 395 How to open in the Evalua tion module 267 How to open in the File Navigator 86 How to open in the UNI CORN Manager 85 How to open in UNICORN Manager 267 How to save 397 Specify folder for stor ing 182 Run Data pane Description 237 How to change text color or background 238 How to change the appear ance 237 How to set pressure units 238 How to view and edit manu al instructions 239 Run Setup Authorized questions 157 Batch ID 181 Blue variable values 154 BufferPrep description 174 BufferPrep correction fac tors 176 Chromatogram ques tions 157
42. procedure How to perform a batch run The table below describes how to perform a batch run 468 Step Action 1 Choose Procedures Batch run Result The Open Procedure dialog box opens Select the procedure from the list and click OK Result The Batch Run dialog box opens Use the Browse button to find and select the folder to search for result files and chromatograms Note The search will only be performed in the selected folder You can use standard wildcard characters and define restricting search criteria for the Result and Chromatogram fields Up to 10 user defined search filters can be saved in the drop menus Click the Search button Result A list of found chromatograms is displayed Select the chromatograms you want to perform the run on e The Select All button selects all chromatograms e The Clear button removes all chromatograms from the list Click the Run button Result The batch run is performed and any created curve or peak table will automatically be saved in each result file UNICORN 5 31 User Reference Manual 28 9972 39 AA 12 Evaluation 12 3 Automated evaluation procedures 12 3 3 How to run a procedure The Batch Run dialog box The illustration below is an example of search results in the Batch Run dialog box Ta Batch Run Report_Chromatogram c5 Defaults Seach MVBaseline example 1 V Baseline example 2 MBaseline example 3 How to
43. sample preparation See illustration below mau Sample 250 unspiked 17 0 Sample with Standard addition spiked Area corresponding to the sample Area corresponding to the added amount 3 Perform peak integration on both curves in the Evaluation module to produce a peak table for both the spiked and the unspiked sample Result The difference in peak area between the spiked and the unspiked sample represents the peak area from the added amount 4 With the assumption of a linear proportionality between the peak area and amount and with the added amount known the software calculates the amount of the component of interest in the sample Peak area from unspiked sample Unspired sampla amoun SAMSUN aidad Peak area spiked sample Peak area unspiked sample 490 UNICORN 5 31 User Reference Manual 28 9972 39 AA 13 The Analysis module 13 2 Quantitation overview 13 2 4 Standard addition quantitation Reliability 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 extrapola tion Below are factors that determine if standard addition can be used with reliable re sults e The component of interest must be completely resolved from all other components in the chromatogram Overlapping peaks will produce unreliable results e The peak integration parameters baseline settings must
44. save a copy under a new name Use this copy for editing purposes 472 UNICORN 5 31 User Reference Manual 28 9972 39 AA 13 The Analysis module 13 The Analysis module Introduction This chapter describes how to use the Analysis module This module is an optional feature that must be ordered separately and installed after the regular UNICORN installation The Analysis module is accessed in the Evaluation module The Analysis module uses functions in the Evaluation module that are presented in the previous chapters It is recommended that you are familiar with the contents of those chapters before you begin with this chapter In this chapter This chapter contains these sections Section See page 13 1 General information about the module 474 13 2 Quantitation overview 477 13 3 How to prepare for quantitation 496 13 4 How to quantitate the sample 520 13 5 Automated quantitation 530 13 6 How to measure molecular size 543 UNICORN 5 31 User Reference Manual 28 9972 39 AA 473 13 The Analysis module 13 1 General information about the module 13 1 General information about the module Introduction This section is an overview of the Analysis module including e Definitions of terminology that will be used in this chapter e A description of how to install the Analysis module e A description of the new procedure instructions that become available when the Analysis module is installed Module functions The Analysis
45. the Convolution Savitsky Golay Method Analytical Chemistry 1990 Volume 62 570 573 for more information on the Savitzky Golay algorithm UNICORN 5 31 User Reference Manual 28 9972 39 AA 593 B Evaluation functions and instructions B 2 Baseline calculation theory B 2 Baseline calculation theory Overall process The table below describes the overall process of a baseline calculation Stage Description 1 The baseline segments are defined 2 The baseline points are selected 3 The baseline is drawn Baseline segment definition Baseline parameters are used to find the baseline segments The default values for the parameters are determined from the source curve The baseline segments are found by different parameters that are based on the type of algorithm that is selected Note The parameters can be displayed in the Evaluation module if you choose Inte grate Calculate baseline function You can also click the Baseline settings button in the Integrate Peak integrate dialog box Morphological algorithm The Morphological algorithm searches for all parts of the source curve where e The curve parts come into contact at both ends of a horizontal line of the length defined in the Structure width parameter The default value of this parameter is based on the widest detected peak in the curve The horizontal line is moved along the curve up the peak until it reaches the contact points The curve parts below the horizo
46. the Calculate baseline instruction Note In addition to blank run curves it is also possible to select any curve from the current chromatogram as the baseline e g an edited baseline UNICORN 5 31 User Reference Manual 28 9972 39 AA 401 12 Evaluation 12 1 Peak integration 12 1 1 Baseline calculation Zero baseline To use a Zero baseline means that there is no baseline subtraction at all Reuse an existing baseline To reuse an existing baseline for the selected curve is the default alternative whenever there is an existing baseline available The option Correlated baseline is selected if this is the case 402 UNICORN 5 31 User Reference Manual 28 9972 39 AA 12 Evaluation 12 1 Peak integration 12 1 2 How to perform a peak integration 1212 Howto perform a peak integration How to perform a peak integration The table below describes how to perform a basic peak integration Step Action 1 Open a result file in the Evaluation module 2 e Choose Integrate Peak Integrate or e Click the Peak Integrate toolbar icon po Result The Integrate dialog box opens 3 e Select a source curve e Select a baseline or a calculation method from the Baseline list e Click OK to integrate with the default selections or e Proceed with steps 4 to 6 to change the default selections Note See also Section 12 1 3 How to optimize the baseline with a morpholog ical algorithm on page 410 and Section 12 1 4 How to optimiz
47. the Message instruction is inserted in a conditional block it will only be displayed if the conditions of the block for example a Watch is fulfilled All messages are erased when the system reaches the End status This also includes Authorize messages UNICORN 5 31 User Reference Manual 28 9972 39 AA F Method examples F 6 Messages Protecting a method run with a message A message can be set up in the beginning of a method to protect the method run from unauthorized interference Once the message is issued the system is locked from inter action by any user unless the user provides an authorization signature The only command that is available without authorization is Pause The illustration below shows the text instruction for the message described above Wa WA Bee OA DLR EENNREK 4 SEE stess Pausing a method run for a manual sample injection A message can be set up to pause the method until a sample has been injected manu ally If a message requiring an authorization is followed by a Pause instruction the system will be paused until the message is acknowledged and signed No other interaction with the system is available to the user The operator will see a screen with a reminder to inject the sample before the method run proceeds The illustration below shows the selected message instruction in the Instruction box and the parameters for the message described above Instructions M Parameters M Pump Var
48. the component peak The window should be set wide enough to include peaks on the other levels despite minor variations in retention volumes However the window should also be narrow enough to exclude unwanted peaks that will interfere with the quantitation Instruction The table below describes how to adjust the window width for the best results Step Action 1 Drag the cursor lines to set the window to a suitable width 2 e Use the Show curve for level menu to display all levels and check that the width is suitable the window width is the same on all levels e Click the lower green or black triangle to display the actual retention for a peak 3 Repeat steps 1 and 2 for all selected peaks Note Overlapping windows are not allowed UNICORN 5 31 User Reference Manual 28 9972 39 AA 503 13 The Analysis module 13 3 How to prepare for quantitation 13 3 2 How to create a quantitation table Step Action 4 If necessary click the Identification settings button to edit the settings See How to adjust the identification settings below this table 2 e Click the OK button to accept the default identification settings Result The Quantitation table dialog box opens 6 Continue to Step 4 How to create a calibration curve and a quantitation table below this table Identification settings The criteria by which peaks are identified are set in the Identification Settings dialog box The criteria are valid for all
49. the corresponding recipe in the method will not be updated automatically When you open the method you will be asked if you want to update the parameters in the method recipe The recommendation is that you answer Yes Note The question will not appear if you only change the Correction factors The Cor rection factors in the method recipe will not be updated How to determine if the Correction factors need to be changed 646 Correction factors can be set to fine tune a recipe around a specific pH to obtain high pH accuracy The table below describes how to run the BufferPrep manually at 0 and 100 in the System Control module to determine if the Correction factors need to be changed Step Action 1 Choose Manual Other Result The System Other instructions dialog box opens e Select the recipe from the Recipe Name droplist and click the Execute button Result The recipe instruction is added e Click the Pump radio button and select BufferPrep_pH e Set the pH value in the pH parameter box and click the Execute button Result The BufferPrep pH value is added and the run starts e Select Flow e Set the flow rate in the FlowRate parameter box and click Execute Result The new flow rate is added Check the pH reading when it is stable in the BuffPre_pH meter in the Run Data pane Note At least 30 ml of eluent must pass through before the reading stabilizes To display the BuffPre_pH meter right click and se
50. the setting value in the Parameters field Result The parameter is updated with the new value in the list Click the Set Selected Parameter To Strategy Default Value button to return to the default value if necessary Result The default setting that was defined in the system strategy is restored Only the selected parameters will be restored UNICORN 5 31 User Reference Manual 28 9972 39 AA 14 System settings 14 1 General information about system settings Step Action 5 Click OK Limits for monitor signals in methods If the system strategy allows limits for certain monitor signals can be set in the method These limits will only work locally in the method and override the global settings as long as the method is in operation This feature can be used to set the pH warning threshold to one value during the process operation and another during the system cleaning UNICORN 5 31 User Reference Manual 28 9972 39 AA 557 14 System settings 14 2 Alarms 14 2 Alarms Introduction This section is a description of the Alarms system settings Alarms and Warnings The Alarms settings define the upper and lower Alarm and Warning limits for process monitor signals The table below describes the difference between Alarms and Warnings If the signal exceeds then the Alarm limits e an alarm sounds e analarm message is displayed e the process is paused i e the method execution is suspended and all pumps
51. the short cut menu to delete the point e Click a data point and drag the point to a new position to move the baseline Note Accept negative peaks must be selected before the peak integration if you want to be able to drag a data point to move the baseline above the curve How to calculate a new baseline The baseline can be recalculated in the Edit Peak Table dialog box The table below de scribes how to do this 428 Step Action 1 e Select Baseline New Calculate or e Right click and select New Calculate from the shortcut menu Result The Settings dialog box opens 2 e Select an algorithm Morphological is default 3 e Adjust the Baseline parameters as desired or e Click the Default Values button for the default values UNICORN 5 31 User Reference Manual 28 9972 39 AA 12 Evaluation 12 1 Peak integration 12 1 6 How to edit the peaks Step Action 4 e Click OK Result The baseline is recalculated Note Select Baseline New Zero Baseline to replace the calculated baseline with a zero baseline The Edit Peak Table dialog box The illustration below shows the Edit Peak Table dialog box Q Eda Peak Table iS x Fle Ect Bareine Jntegiate Pjg i Example repult 003 1_UV Example 1esult 003 4_UV O1 BASEM UNICORN 5 31 User Reference Manual 28 9972 39 AA 429 12 Evaluation 12 1 Peak integration 12 1 6 How to edit the peaks How to delete a peak The table below describes h
52. to and from retention points The test can be defined to be passed either if the text is present or not A failed or passed text will be added to the output file A print parameter may be set to On to generate printed results UV_RESPONSE_IN Sets the level intervals for the UV_RESPONSE_TEST TERVALS UV_RESPONSE_TEST The amplitudes for the 0 and 100 levels are calculated and the difference between the values are calculated The results of 1 Curve2_Difference Curvel1_Difference and 2 Curve2_Difference Curve3_Difference are calculated The calculated points are checked if they are outside the defined limits from the 50 level 618 UNICORN 5 31 User Reference Manual 28 9972 39 AA C Curve fit models and statistics Appendix C Curve fit models and statis tics Introduction The Analysis module optional is used to produce calibration curves and molecular size curves for analytical purposes The quality of the curve fit model determines the accuracy of the curves This appendix describes e The available curve fit models e The statistical measurements in the Analysis module In this appendix This appendix contains these sections Section See page C 1 Curve fit models 620 C 2 Statistics 626 UNICORN 5 31 User Reference Manual 28 9972 39 AA 619 C Curve fit models and statistics C 1 Curve fit models C 1 Curve fit models Calibration curve models The Analysis module provides a comprehensi
53. to file in XLS format If is entered as File name the current Result file will be used If is en tered followed by text e g Enter a file name as File name a full search path must be entered in answer to the question EXPORT_PEAK Exports the peak table in Peak table source to the file TABLE_XML defined in Export to file in XML format If is entered as File name the current Result file will be used If is entered followed by text e g Enter a file name as File name a full search path must be entered in answer to the question EXPORT_SEL_CURVES Selects a curve for subsequent export using the EX PORT_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 param eter for example every fifth point EX Exports the documentation in the current result file in PORT_DOC_400_ASCII ASCII format to the file defined in Export to file If is entered as File name the current Result file will be used If is entered followed by text e g Enter a file name as File name a full search path must be entered in an swer to the question If all parameters to this function are OFF then no documentation is exported If at least one of them is ON then the documentation will be exported and the corresponding parts will be included in the export ed file UNICORN 5 31 User Reference Manual 28 9972 39 AA 61
54. volume Column volume unt Technique Size_Exchusion ve ao Vo 15 Max pressure 03 Defauk flowrate 10 0 Max flowrate Typ peak width at base pH High value longteim PH Low value longterm HiLoed_16 60_Superdex_30_prep_grade Global pH High value shoitleimn HiLoad_1 60_Superdex_75_prep_giade Global pH Low value shoelterm HiLoad_26 10_PhersA_Sepherose_HP Global Average parlicle diam wn HiLosd_26 10_Q_Sepherose_FF Global Code no 17 1408 01 Typical loading range 011 5 mi Mol weight range kDa Sean rate Spectra sec 630 UNICORN 5 31 User Reference Manual 28 9972 39 AA D The Column list D 1 How to edit the Column List How to print the column list The table below describes how to print the column list data Step Action 1 e Click the print button Result The Print Column List dialog box opens 2 e Select to print a global or your personal column list e Click OK Result The column list is printed on the default Windows printer The New Column dialog box The illustration below shows the New Column dialog box LM x Column name Normal Parameters Advanced Parameters Parameter Vawe U Height mand cm Diameter mand Column volume Column volume unit mand Technique mand Max pressure mand Default flowrate mand Max flowrate Typ peak width at base PH High value longterm PH Low value longterm pH High value shortterm pH Low value shortterm Average particle diam
55. width at nl ul ml or e Not mandatory base liter e Used to set averaging time for UV detec tor e used to set peak fractionation parame ters pH high value e Not mandatory longterm e Used to give a warning when saving or starting the method if the BufferPrep_pH value is higher than the set value PH low value e Not mandatory longterm e Used to give a warning when saving or starting the method if the BufferPrep_pH value is lower than the set value pH high value e Not mandatory shortterm e Used to give a warning when saving or starting the method if the BufferPrep_pH value is higher than the set value PH low value e Not mandatory shortterm e Used to give a warning when saving or starting the method if the BufferPrep_pH value is lower than the set value Average particle Hm e Not mandatory diameter e Information only Code no e Not mandatory e Information only 634 UNICORN 5 31 User Reference Manual 28 9972 39 AA D The Column list D 1 How to edit the Column List Parameter Unit Comment Typical loading mg e Not mandatory range e Information only Mol weight range kDa e Not mandatory e Information only Scan rate spectra sec e Not mandatory e Information only Note 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 t
56. with problems in the dialog box and click the Edit button Check that the strategy computer name and the control number are correct according to the instal lation at the local station which is physically con nected to the system See the Administration and technical manual System definitions e Ifyou connect remotely to a system check that the local station which is physically connected to the system is turned on check that the network is functioning at both the remote and the local station e Check that the limit of eight connections to the system has not been exceeded 582 UNICORN 5 31 User Reference Manual 28 9972 39 AA A Troubleshooting A 3 Methods and method runs A 3 Methods and method runs In this section This section describes how to solve the following method and method run problems Cannot perform Quit or Logoff Monitor signals do not appear in the Curves pane in System Control Error message Couldn t create result file Destination path could not be found The Method System Connection dialog box keeps appearing The Method Editor window does not fit on the screen There are red instructions in a method After Windows logout and login you cannot get a system connection The Print screen command does not send a copy of the screen to the printer Cannot perform Quit or Logoff The table below describes a problems and its solutions Problem description Solution
57. 20 Standard 3 levels Use the peak tables from the standard runs to produce a calibration curve for each component This curve shows the relationship between amount and peak size Below is a calibration curve for one of the components Tea i Thann Perform a run with the sample and peak integrate the curve Identify the components of interest by the peak identification settings from the sample peak table Use the peak size s to calculate the concentration and amount from the calibration curve s UNICORN 5 31 User Reference Manual 28 9972 39 AA 483 13 The Analysis module 13 2 Quantitation overview 13 2 2 External standard quantitation Illustration how to use the calibration curve The illustration below describes how the calibration curve is used to determine the amount based on the sample peak area Amount Reliability External standard quantitation normally produces accurate results and is fairly simple The following reliability factors are specific to the technique e Precision is limited by changes that may take place between the runs for example column degradation and mobile phase variations e There is no compensation for losses of sample during the sample preparation process prior to analysis 484 UNICORN 5 31 User Reference Manual 28 9972 39 AA 13 The Analysis module 13 2 Quantitation overview 13 2 3 Internal standard quantitation 1323 Internal standard quantitation General inform
58. 25038 mAU min 2 Select the Source Chromatogram and the curve the simulated peak frac tionation is to be generated for 3 If necessary select a Destination Curve and type a new Curve name 4 Type new values in the Parameters text boxes 5 Click OK Result The simulated peak fraction curve is displayed on the chromatogram 454 UNICORN 5 31 User Reference Manual 28 9972 39 AA 12 Evaluation 12 2 Other evaluations 12 2 4 How to create curves 1224 Howto create curves Introduction You can draw a curve of your own in the Evaluation module This section describes how this is done Note The right to create and rename curves is defined in the user access rights and may be restricted How to create curves step 1 The table below describes how to set up a chromatogram window to create a curve in the Evaluation module Step Action 1 2 Open a result file Select Operations Create Curve Result The Create Curve dialog box opens COS Sas Y rE NES ES SON NIRERSES NEES NINAS Xe RSS SNS ASSN SS J SOC SSRN MA EiS Select one or more Help curves UNICORN 5 31 User Reference Manual 28 9972 39 AA 455 12 Evaluation 12 2 Other evaluations 12 2 4 How to create curves Step Action 4 Select e minimum and maximum values for the Y axis e appropriate units from the Unit list Note The help curve determines the minimum and maximum values for the X axis 5
59. 3 B Evaluation functions and instructions B 4 Procedure instructions Instruction Description EXPORT_DOC_400_WKS Exports the documentation in the current result file in WKS format to the file defined in Export to file If is entered as File name the current Result file will be used If is entered followed by text e g Enter a file name as File name a full search path must be entered in an swer to the question If all parameters to this function are OFF then no documentation is exported If at least one of them is ON then the documentation will be exported and the corresponding parts will be included in the export ed file EXPORT_DOC_400_XLS Exports the documentation in the current result file in MS Excel XLS format to the file defined in Export to file If is entered as File name the current Result file will be used If is entered followed by text e g Enter a file name as File name a full search path must be entered in an swer to the question If all parameters to this function are OFF then no documentation is exported If at least one of them is ON then the documentation will be exported and the corresponding parts will be included in the export ed file EXPORT_DOC_WKS Exports the documentation in the current result file in WKS format to the file defined in Export to file If is entered as File name the current Result file will be used If is entered followed by text e g Enter a file name
60. 3 3 How to edit and update a quantitation table The Update Quantitation Table dialog box The illustration below shows the Update Quantitation Table dialog box Update Quantitation T able AT2001Apr1 Sn0001 LL AT2001May16n0001 Baseline example 1 LL Baseline example 2 ine example 3 How to prepare the calibration curve for updating The table below describes how to open the function and prepare the calibration curve for updating Step Action 1 Perform a peak integration for the new run and save the result 2 Select Quantitate Edit Quantitation Table Update Result The Update Quantitation Table dialog box opens 3 e Select the Personal radio button if the table is located in your personal folder e Select the quantitation table that is to be updated in the Quantitation table field UNICORN 5 31 User Reference Manual 28 9972 39 AA 513 13 The Analysis module 13 3 How to prepare for quantitation 13 3 3 How to edit and update a quantitation table Step Action 4 e Double click the result file in the Select peak table list to access the new data e Click the Current button if you want to use the result file that is open in the Evaluation module 2 e Select the chromatogram on the Source chromatogram list e Select the peak table that contains the new data in the Peak table s list e Select the level you wish to update on the Level list e Ifthe selected quantitation table is based on
61. 31 User Reference Manual 28 9972 39 AA F Method examples F 7 Quality control procedure Step Action e Choose File Save or e Click the Save icon Result When the method run is performed the quality control procedure will create a second chromatogram If the controlled value is outside the accept able range the system will be paused UNICORN 5 31 User Reference Manual 28 9972 39 AA 667 Index Index 668 A Alarms Description 558 Alarms and warnings Description 259 Effects on the system 259 Analysis External standard quantita tion 482 How to calculate molecular size 552 How to create a molecular size table 548 How to create a quantita tion table 499 Molecular size determina tion overview 544 Quantitation reliability fac tors 495 Recovery calculation de scription 492 Analysis module How to install 476 Automated quantitation Automated update with Av erage for scouting runs 541 Automatic update with Aver age 536 Automatic update with Re place 535 Basic conditions 531 How to perform updates in scouting runs 538 How to prepare the quanti tation table 531 How to set up the sample runs 534 B Baseline Calculation options 401 Definition of a segment 594 How to adjust the baseline graphically 428 How to edit manually 424 Parameters 595 Reuse existing 402 The Calculate function 401 BatchID Logbook illustration 249 Batch run How to perform 468 Blank curve Ca
62. 4 Perform the evaluation steps that the procedure is to contain Result The steps are recorded in the order that they are performed 5 Stop the recording e Choose Procedures Record Off or e Restore the minimized Procedure Editor dialog box and click the Stop button or e Restore the minimized Procedure Editor dialog box and select Con trol End Record 6 Choose File Save or File Save As in the dialog box Result The Save As dialog box opens f e Type a name for the new procedure in the Procedure name text box e Select the Global procedure checkbox if desired see further information below e Click OK Result The procedure is saved and available for future use 8 Click the Close button to close the dialog box How to create a Global procedure 462 You can choose to save the new procedure as a Global procedure This makes the pro cedure available to all users The procedure will have Global before the name to desig nate that it is available to all users You must have Edit global list s authorization to be able to save Global procedures UNICORN 5 31 User Reference Manual 28 9972 39 AA 12 Evaluation 12 3 Automated evaluation procedures 12 3 1 How to create a new procedure How to build a procedure with instructions You can select instructions in the Procedure Editor dialog box to build a complete pro cedure step by step The procedure instructions are described in Section B 4 Procedure instructions on page 607
63. 72 39 AA 615 B Evaluation functions and instructions B 4 Procedure instructions Other instructions The table below contains a list of instructions for other operations Instruction Description BASE Sets the X axis base that the following calculations will be made in If the value of the 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 evaluation procedure otherwise it will have no effect at all Comment Inserts a comment below the marked instruction ENDLOOP Marks the end of a LOOP statement 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 MOLSIZE Calculates the molecular sizes from a molecular size curve A Mol size column will be added to the Peak table QC_TEST Performs a QC test QUANTITATE Calculates the concentration and amounts in the sample from a quantitation table Amount and Concentration columns will be added to the Peak table REPORT Prints a report with the specified named report layout and title If Title is then the title in the report layout is used If Report Layout is then a default layout is used RUN_PROGRAM Starts a program as a separate process The Program name string cont
64. 9 Procedure instructions Chromatogram func tions 615 Curve operations 607 Export functions 611 File operations 610 Integration 609 Miscellaneous 616 Test instructions 617 Procedures Global procedures 462 How to add instruc tions 465 How to batch run 468 How to build a procedure with instructions 463 How to delete 471 How to edit 464 How to record 461 How to rename 471 675 Index 676 How to run a single proce dure 467 Protein activity Match to UV curve 348 Protein amounts Calculation formula 344 Protein concentrations Calculation formula 343 Q Quality control How to add a control proce dure to a method 666 How to create a control procedure 663 Quantitation Automated in scouting runs 538 Automated sample runs 534 Automated update with Av erage for scouting runs 541 Automatic update with Aver age 536 Automatic update with Re place 535 Calculation error signs 523 External standard reliabili ty 484 General description 478 General reliability fac tors 495 How to adjust peak identifi cation settings 504 How to calculate the amount and concentra tion 522 How to calculate the recov ery 528 How to create a calibration curve 507 How to create a quantita tion table 499 How to delete a quantita tion table 518 How to enter standard da ta 507 How to open a table for editing 512 How to prepare for 521 How to prepare for recovery ca
65. AJ Click the New button Result The New component dialog box opens Type a name for the new salt and click OK to return to the Define salt dialog box e Type the appropriate charge of anion value in the corresponding Value cell Example The value for CI is 1 The value for 50 27 is 2 e Type the appropriate charge of the cation value in the corresponding Value cell Example The value for Na is 1 The value for Mg is 2 e Click OK Result The new salt is added to the list of available salts 644 UNICORN 5 31 User Reference Manual 28 9972 39 AA E How to create and edit BufferPrep recipes E 2 How to edit a BufferPrep recipe E 2 How to edit a BufferPrep recipe Introduction This section describes how to edit a BufferPrep recipe in the Method Editor How to edit a recipe The table below describes how to edit a BufferPrep recipe Step Action 1 Choose Edit BufferPrep Recipes Result The BufferPrep Recipes dialog box opens 2 Select a recipe and click the Edit button Result The Edit Recipe dialog box opens 3 Change the substances and parameters as desired and click the Save button or the Save as button to save the new recipe UNICORN 5 31 User Reference Manual 28 9972 39 AA 645 E How to create and edit BufferPrep recipes E 2 How to edit a BufferPrep recipe Changes to recipes in methods If a recipe has been selected and saved in a method and the recipe is later changed
66. Click OK Result The Create Curve chromatogram window opens How to create new units In the Create Curve dialog box you can also create new units for the curve The table below describes how this is done Step Action 1 Click the New unit button Result The Create New Unit dialog box opens 2 Type a new unit name and a number of decimal places 3 Click OK Result The Create New Unit dialog box is closed The new unit is now avail able in the Create Curve dialog box How to create curves step 2 The new curve is created in the Create Curve window The table below describes how to work in this window Step Action 1 Click the Set Curve Points icon E E 456 UNICORN 5 31 User Reference Manual 28 9972 39 AA 12 Evaluation 12 2 Other evaluations 12 2 4 How to create curves Step Action 2 e Click to insert curve points in the chromatogram e Add more points to draw the curve Result A green square marks the new curve point The curve is drawn from the previous point Hold the cursor over the inserted point to see the coordi nates displayed Curve mode e The regular spline mode draws the curve as a smooth line near but not through every point e Click the Spline through checkbox to draw the curve through all of the curve points 3 Move a point e Select the point and drag it to the new position Result The curve is redrawn 4 Delete a curve point e Double click the curve point
67. Columns tab 173 Description 30 Evaluation Procedures tab 166 677 Index 678 Gradient change X axis base 162 Gradient hatch marks 163 Gradient marker line 162 Gradient tab descrip tion 161 Gradient zoom function 162 How to add reference curves 171 How to change a detail variable into a regular vari able 155 How to change a variable into a detail variable 155 How to change variable values 153 How to create a BufferPrep method 175 How to delete variables 154 How to edit method proce dures 169 How to edit questions 160 How to export values 187 How to import evaluation procedures in the method 167 How to insert a ques tion 159 How to rename vari ables 154 How to search for text in the method notes 165 How to write method notes 164 Mandatory questions 157 Method Information tab description 178 Notes description 164 Questions answer types 158 Questions tab descrip tion 157 Question status alterna tives 157 Reference curves 171 Result file serial num bers 181 Result file unique identifi er 181 Result Name tab descrip tion 180 Run Setup from the Method Wizard 100 Scouting tab 156 Scouting tab buttons 215 Start Protocol contents 185 Tabs description 151 Variable tab view op tions 153 S Scouting Change variable values 214 Changing variables 214 How to change a variable into a detail variable 218 How to copy v
68. GE Healthcare Life Sciences UNICORN 5 31 User Reference Manual Chapter 12 to 15 Appendices Table of Contents Table of Contents 1 Introducing UNICORN lammeyierpiiet pee lire leer iplnit ener iti naets trae pene nere rerreterreresienrnr ener tit 9 1 1 About UNICORN Ss 10 1 2 About this manual on eeccccccsccssssessseesesseescccssseensee D 14 1 3 About the UNICORN user documentation weceecccssccsssssssseesessssscsssssssssussssessesssssssssssssessssceesees 17 UNICORN concepts 20 2 1 Concept definitions 21 2 2 The UNICORN user interface 25 2 2 1 UNICORN Manager u s i 26 222 FRE Method Editor Moduleremssnasnaceainiii i n na nA A 30 223 TheSystem Control MOD Css sssssscasevsssessscssssssasssessduvsveseasierendsiessvasibossastesesticndvbsansesssaacesbest vane 36 2 2 4 The Evaluation module 41 2 2 5 Search FUNCTIONS u 44 2 2 6 Help functions and manuals a 47 ZT SHOP SOUS aininn i e A EEEE EOT OE OEO EA E 50 2 3 QUICK Start GUI Cc scccsccsss ssesssazsvsi5sccoseccdstanssbscsusio seuss nannan i a E ANR A a 54 Ge neral system ODEROUIONG sssiireissiisikiirscnseiissesisnansiiio kee eia da biiecodo kadrai okinak 55 3 1 Log ON routines and log off routines s sssssssississssssissssrssrsssistssssrsssrssrsstsrntrssnentrssnrsntrnnrnnnnnnes 56 3 2 HOW TO CreAte C NEW USET o ceeccsssscsssessssssssnsessssecssseesssccesssessnsecssnscssssessscessssecsrseessassssssess
69. ICORN 5 31 User Reference Manual 28 9972 39 AA 12 Evaluation 12 1 Peak integration 12 1 7 How to integrate part of a curve and how to exclude or skim peaks 1217 Howto integrate part of a curve and how to exclude or skim peaks Introduction There are several possibilities to improve the results if the peak integration is unsatisfac tory This section describes e How to select only part of a curve for integration e How to exclude peaks e How to skim peaks These operations can be performed both in the Integrate dialog box in preparation for the peak integration or in the Edit Peak Table dialog box to adjust an unsatisfactory peak integration This section describes both alternatives UNICORN 5 31 User Reference Manual 28 9972 39 AA 437 12 Evaluation 12 1 Peak integration 12 1 7 How to integrate part of a curve and how to exclude or skim peaks How to select part of a curve The table below describes how to select only a part of a curve for peak integration in the Integrate dialog box Step Action 1 e Choose Integrate Peak Integrate Result The Integrate dialog box opens e Click the Peak Window button Result The Peak window dialog box opens Leit PS aime pa OK caret He 2 e Type new X axis values for the Left limit and the Right limit or e Drag the vertical cursor lines to define the limits 3 Click OK Result The baseline will be calculated from the whole curve but the calcula tion of the peak ar
70. Manual 28 9972 39 AA 13 The Analysis module 13 2 Quantitation overview 13 2 6 General reliability factors for the quantitation techniques 1326 General reliability factors for the quantitation techniques Reliability factors The following factors are valid for all quantitation techniques except for Standard addi tion Quantitation requires that the components of interest are completely resolved from all other components in the chromatogram Overlapping peaks will produce 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 All integrations must be performed using the same X axis base unit For highest reli ability time is the recommended unit 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 limits precision Precision is improved by the appropriate choice of the concentration range of the standard The range should extend across the presumed amount in the sample Use of the most appropriate curve model will maximize precision Accuracy is improved if several runs are performed at each level All the runs should be performed consecutively to reduce
71. Note If there already are Correction factors the measured pH deviation should be added to the old factors UNICORN 5 31 User Reference Manual 28 9972 39 AA 647 F Method examples Appendix F Method examples Introduction This appendix contains practical method examples that can be applied in typical situa tions The examples cover three different topic groups e Watch instructions e Messages e Quality control Watch instructions allow the progress of a method run to be determined by the events during the method run for example 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 The system strategy includes Watch instructions for each monitor defined in the system These instructions are used to survey method runs and instruct the system 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 Messages can be used in a method to provide information to the operator but also for interaction between the system and the operator A Quality contro procedure in a method can be used to ensure that the quality of the results remain consistent in a series of runs In this appendix 648 This appendix contains the following sections Section See page F1 Simple equilibration 650 F 2 Equi
72. O QA CUFVE veceeccssssssssssssssssssessssssscssssssssssssssssssssssssssssussessees 347 11 7 Howto rename chromatograms curves and peak tables o cccsessssessessscscccsssesssseneeees 349 11 8 How to import and compare different rUnS weeccccccccsssssssseesssssccssssssssnusssssesscssssssssnuseessseee 350 11 8 1 Howto use the Multifile Peak Compare wizard ssssssssssssssssscsssssssssssssssssssesssssssssssssssssssessees 351 11 8 2 Howto import and compare chromatograms 367 11 8 3 HOW to import and COMPATE CUEVES neceessssssssssssssssssssssssssssssssessesseseesecessseseesesessseesssssssesessesssssese 371 11 8 4 How to stack and Stretch CUVES oeceeccssssssssssssssssssssssssssssssssssssssessseeseesesessessessesesssesessssssssessessssssee 380 11 8 5 HOW to produce a Mirror IMAGE ecccsssssssssssssssssssssssssssssssssssssuseessssssssssssssssssssssssssesssssssssssssessssseesees 385 11 9 How to import and export results ceeeccccssssssseseessescccssssssssussssssssscsssssssssssssecssssessessssssssnsesesseesee 387 ILOT OW TO MIPORE resultSet ei eeii E A 388 119 2 HOW to export resul S nenoaia eea EEEN R 390 11 10 How to sign results electronically ecceeeccccssssssssssessssscccssssssssusssessssscssssssssssssssssssesssssssssssssesessesees 395 11 11 How to save results and exit the Evaluation module o cceeecccecccsssssssssssscsssseecsseececeeceecceeeeeeees 397 12 Evd oM essnee trensrhirennirenr arenes terre trt et 399 12 1 Peak integration cccsssssssccsssssssss
73. OPEN name and stores it in the Resulting peak table If is entered as File name the current Result file will be used The File name parameter may include a path from the current users root folder UNICORN 5 31 User Reference Manual 28 9972 39 AA B Evaluation functions and instructions B 4 Procedure instructions Export The table below contains a list of instructions for export operations Instruction Description EXPORT_CURVE_AIA Exports the curve in AIA format EXPORT_CURVE_ASCII Exports the Source curve to the file defined in Export to File in ASCII format If is entered as File name the current Result file will be used If is entered followed by text e g Enter a file name as File name a full search path must be entered in answer to the question In the part of the source curve limited by the Left limit and Right limit every lt n gt sample is exported EXPORT_CURVE_WKS Exports the source curve to the file defined in Export to File in WKS format If is entered as File name the current Result file will be used If is entered followed by text e g Enter a file name as File name a full search path must be entered in answer to the question In the part of the source curve limited by Left limit and Right limit every lt n gt sample is exported EX Exports an evaluation log in ASCII format to the file de PORT_EVAL_LOG_ASCII fined in Export to file If is entered as File name the cu
74. Peak integration 12 1 5 How to edit the baseline manually Step Action 2 e Press and hold the left mouse button e Drag the cursor over the area you want to zoom in on e Release the mouse button Result The area is enlarged Right click and select Reset zoom to restore the full view How to edit and insert data points The table below describes how to edit and insert baseline data points Step Action 1 Select Integrate Edit Baseline Result If there are more than one baseline available the Select Baseline to Edit dialog box opens If not proceed to step 2 e Select the baseline you want to edit from the list e Click OK Result The Edit Baseline dialog box opens 2 e Click the Set Curve Points icon pco Result The cursor is changed into a cross 3 Add a data point e Click the left mouse button to place a new baseline point in the chro matogram Result Anew point is created marked by a green square The baseline curve is redrawn as a spline function based on the old and the new points The baseline is guided by the points but does not necessarily pass through them 424 UNICORN 5 31 User Reference Manual 28 9972 39 AA 12 Evaluation 12 1 Peak integration 12 1 5 How to edit the baseline manually Step Action 4 Delete a data point e Double click the data point or e Click the data point to select it and click the Delete button or e Right click the data point and select Delete Poin
75. Ratio an A B 3 03 25 OF 06 Az70nm a A255 nm 03 Ratio A270nm A255 nm 02 01 0 0 0 1 2 3 4 Time units Peak identification The absorbance ratio can be used for peak identification Different compounds have a specific ratio between absorbancies at different wavelengths The illustration below shows a simulated chromatogram of two components with differ ences in their absorbance spectra 448 UNICORN 5 31 User Reference Manual 28 9972 39 AA 12 Evaluation 12 2 Other evaluations 12 2 1 Peak purity and peak identification SSAA Qe RAS L Tse SERS How to divide the curves Both curves must have a baseline close to zero AU before they can be divided This is achieved with baseline subtraction The table below describes how to subtract the baseline from an earlier integration and divide the curves Step Action 1 2 Create a baseline for each UV curve Select Operations Subtract Result The Subtract dialog box opens e Select the UV curve in the first list of curves e Select its baseline in the second list of curves e Click OK Note You can also subtract corresponding blank runs if there are blank runs available Repeat steps 2 and 3 for the second UV curve Select Operations Divide Result The Divide dialog box opens e Select the first result curve from the subtractions in the first list of curves e Select the second result curve from th
76. Result The Maintenance manager dialog box changes into edit mode and the text boxes are activated UNICORN 5 31 User Reference Manual 28 9972 39 AA 565 15 System maintenance and error reporting 15 1 System maintenance functions Step Action 3 e Type new text if necessary e Click the Reset button Result The Reset parameters dialog box opens Reset parameters f J xj No of method runs completed 0 EN Next time out 2002 10 11 Reset OK Cancel Help 4 e Click one or both of the Reset buttons to reset the counters e Click OK How to acknowledge a warning Once a specific Periodicity parameter has been reached a warning message will be displayed The table below describes how to acknowledge the warning Step Action 1 The Warning dialog box opens Lamp check Replace the UV lamp 566 UNICORN 5 31 User Reference Manual 28 9972 39 AA 15 System maintenance and error reporting 15 1 System maintenance functions Step Action 2 e Click the Acknowledge button if you have corrected the problem e Click the Ignore button if you haven t corrected the problem Note You will be reminded later about the unsolved problem if you click the Ignore button UNICORN 5 31 User Reference Manual 28 9972 39 AA 567 15 System maintenance and error reporting 15 2 How to generate problem reports 15 2 How to generate problem reports Introduction UNICORN contains a Generate Report Wizard for
77. Retention 30 025 05 0 75 h 125 log Mol size 544 UNICORN 5 31 User Reference Manual 28 9972 39 AA 13 The Analysis module 13 6 How to measure molecular size 13 6 1 Overview of molecular size determination How to calculate the molecular size in the sample The table below is a brief description of how to use the molecular size table to calculate the molecular size of the components in the sample Step Action 1 Use the sample peak table to obtain retention values for each of the compo nents of interest mau 400 Sample 200 20 0 25 0 min 13 5 2 Use the molecular size curve to obtain the molecular sizes of the components in the sample The molecular sizes are presented in the peak table Retention 30 20 025 05 075 1 125 Molecular size of sample component log Mol size UNICORN 5 31 User Reference Manual 28 9972 39 AA 545 13 The Analysis module 13 6 How to measure molecular size 13 6 2 How to determine the molecular size 1362 Howto determine the molecular size Introduction This section describes the technique for measuring molecular size in detail Before you start Before you create the molecular size curve you need to do the following e Perform chromatographic runs with an appropriate standard with components of known molecular size The standard should contain components of sizes that extend over the range that is expected in the sample If you are using many components
78. System settings 14 3 Curves Setting Function Time between sam This setting determines with which frequency curve data ples is recorded It does not affect the reading frequency of the actual monitor Default value is the shortest possible time between samples UNICORN 5 31 User Reference Manual 28 9972 39 AA 561 15 System maintenance and error reporting 15 System maintenance and error re porting Introduction This chapter describes the system maintenance and error reporting functions In this chapter This chapter contains these sections Section See page 15 1 System maintenance functions 563 15 2 How to generate problem reports 568 562 UNICORN 5 31 User Reference Manual 28 9972 39 AA 15 System maintenance and error reporting 15 1 System maintenance functions 15 1 System maintenance functions Introduction Some strategies support the capacity to view system information for the components in a chromatography unit The system information can be used to issue maintenance warnings for the components This section describes the system maintenance functions How to open the Maintenance manager The system maintenance functions are controlled in the Maintenance manager dialog box in the System Control module e Select System Maintenance Result The Maintenance manager dialog box opens with the Info tab selected The connected chromatography system is scanned for its components After a while
79. Text window 0 00 Block ELUTION Elution 0 00 Base SameAsMain 0 00 Gradient 100 0 20 00 base 0 00 Watch_UV1 Greater_Than 100 mAU Peak_1 Peak_1 0 00 Base SameAsMain 0 00 OutletValve F3 0 00 Watch_UV1 Less_Than_Or_Valley 100 mAU Waste Waste 0 00 Base SameAsMain 0 00 OutletValve WasteF1 0 00 Watch_UV1 Greater_Than 100 mAU Peak_2 Peak_2 0 00 Base SameAsMain 0 00 OutletValve F4 0 00 Watch_UV1 Less_Than 100 mAU End_collect End_collect 0 00 Base SameAsMain 0 00 OutletValve WasteFl 0 00 End_Block 0 00 End_Block 0 00 End_block 0 00 End_Block 20 00 End_Block UNICORN 5 31 User Reference Manual 28 9972 39 AA 655 F Method examples F 4 Collection of absorbance peaks Illustration The illustration below shows peaks collected by the method in the example above STH DT a gt D T ai This is what happens In this example one or two absorbance peaks are collected through outlets F3 and F4 respectively with waste fractions collected through outlet valve F1 waste Each called block except End_collect resets the Watch condition so that the method reacts correctly to subsequent changes in the UV absorbance Invalid Watch instructions The design of a method of this kind with several Watch instructions for the same mon itor is important The construction in the following three lines appears simpler but is in correct 0 00 Watch_UV Greater_tha
80. UNICORN 5 31 User Reference Manual 28 9972 39 AA 549 13 The Analysis module 13 6 How to measure molecular size 13 6 2 How to determine the molecular size Linear logMw Theoretically this is the best choice e Quadratic e Quadratic logMw e Point to point e Point to point logMw Molecular size Statistics With the exception for the two point to point models the molecular size curves can be expressed as mathematical expressions The expressions and related items can be viewed in the Statistics dialog box e Click the More button in the Statistics field of the Molecular size table to open the dialog box The expression is shown at the top of the window followed by the values for the constant that it contains Statistical reference values e The correlation value only for linear models should be as close to 1 00 as possible e The explained variance value should be as close to 100 as possible Note Explained variance values are usually high A value of 90 indicates a very poor model How to open an existing table 550 The table below describes how to open an already existing molecular size table for editing in the Evaluation module Step Action al Select Mol Size Edit Mol Size Table Open Result The Open molecular size table dialog box opens 2 e Select a molecular size table from the Molecular size table s list Note By default the list will show the molecular size tables that are g
81. _Or_Valley 4 75 mAU Waste Waste 0 00 Base SameAsMain 0 00 OutletValve Feed 0 00 Watch_UV1 Greater_Than 5 mAU Peakl Peak1 UNICORN 5 31 User Reference Manual 28 9972 39 AA 657 F Method examples F 5 Collection of three absorbance peaks 0 0 0 0 0 0 0 Base SameAsMain 0 OutletValve Feed 0O Watch_UV1 Less_Than_Or_Valley 4 75 mAU wastel Wastel 0 0 0 0 0 0 0 Base SameAsMain 0 OutletValve Feed 0O Watch_UV1 Greater_Than 5 mAU Peak2 Peak2 0 0 0 0 0 0 0 Base SameAsMain 0 OutletValve Feed 0O Watch_UV1 Less_Than_Or_Valley 4 75 mAU Waste2 Waste2 OO O 7O Or OS O O Oo O O OD O O OG GO O 0 Base SameAsMain OutletValve WasteF1 End_block End_block End_block End_block End_block End_block 0 0 0 0 0 0 0 0 End_block This is what happens The table below describes what happens in the above example Stage Description 1 658 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 UNICORN 5 31 User Reference Manual 28 9972 39 AA F Method examples F 5 Collection of three absorbance peaks Stage Description 3 This process is repeated twice for the next two peaks so that when the UV reading rises ab
82. _UV1 Less_than 100 mAU END_BLOCK oO 0 0 5 00 Watch_Off UV1 5 00 Message The Condition was never reached Screen No n sound 5 00 End_Block This is what happens This is what happens in the above example The column is equilibrated 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 652 UNICORN 5 31 User Reference Manual 28 9972 39 AA F Method examples F 3 Equilibration with extra safeguard F 3 Equilibration with extra safeguard Introduction This section contains an example of how a Watch instruction for extra safeguard can be inse rted into a method Example instruction This is an example instruction as it would be presented in the Text pane 0 00 Equi 0 00 0 00 Block EQUILIBRATE librate Base SameAsMain Block COND_LESS_THAN Cond_less_than 00 00 00 00 00 Oo oT oY oF O Base SameAsMain Watch_Cond Less_ Pause INFINITE End_Block than Block COND_STABLE Cond_stable 0 00 Base SameAsMain 5 mS cm END_BLOCK 00 Message Low conductivity not reached Screen No sound inutes 0 00 WatchPar_Cond 0 500 mS cm 2 mS cm 0 00 Watch_Cond Stable Baseline 5 Minute
83. a quantitation and the procedure instructions for quantitation that are added when the Analysis module is installed About quantitation 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 stan dard The relationship can be linear quadratic or point to point Quantitation is usually based on a number of test 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 Note Quantitation should only be performed on chromatograms that have been inte grated and saved Time is the recommended base unit for quantitation and it must be used for all integrations Quantitation steps 478 The table below describes the general steps in quantitation The steps are described in detail in the sections about the different quantitation techniques Step Action 1 Run the different concentration levels of the standard 2 e Integrate the curves to produce peak tables e Check the integration 3 Identify the components for which calibration curves will be produced 4 Enter the known concentrations for the different standards to produce a calibration curve for each selected component 5 Run the s
84. able below describes how to change the peak resolution algorithm in the UNICORN Manager Step Action 1 e Choose the Administration Options menu item Result The Options dialog box opens UNICORN 5 31 User Reference Manual 28 9972 39 AA 603 B Evaluation functions and instructions B 3 Peak table column components Step Action 2 e Select the desired algorithm number described as described in Peak resolution algorithms above in the Resolution algorithm droplist Result The dialog box closes and the peak resolution algorithm is changed Note You must repeat the peak integrations after the change to update the values based on the new algorithm Capacity factor formula The formula below is used to calculate the Capacity factor _ Va Vt 1 k V 604 UNICORN 5 31 User Reference Manual 28 9972 39 AA B Evaluation functions and instructions B 3 Peak table column components Where e Vp retention volume e V total liquid volume Kav formula The formula below is used to calculate Kav Ve Vo Vor Vo Where e Vp retention volume e Vo void volume Vg column volume Asymmetry formula The formula below is used to calculate the Asymmetry Asymmetry B A Where e Aisa partial peak width measured at a percentage of the peak height for the leading part of the peak e Bisa partial peak width measured at a percentage of the peak height for the tailing part of the p
85. able that was used by selecting Quantitate Edit Quan tita tion Table View Current If the amount cannot be calculated if th e amount cannot be calculated one of the following signs is shown in the peak table Amount column Sign Function gt This means that the value is higher than the highest value in the calibration curve i e outside the valid range of the calibration curve This means that the value is lower than the lowest value in the calibration curve i e outside the valid range of the calibration curve This means that the value cannot be calculated For example this sign might indicate that the peak could not be identified UNICORN 5 31 User Reference Manual 28 9972 39 AA 523 13 The Analysis module 13 4 How to quantitate the sample 13 4 2 Standard addition quantitation 1342 Standard addition quantitation Stages in standard addition Standard addition is performed in five stages Stage Description 1 2 Perform two runs Copy the curves into one result file Integrate the curves to produce the peak tables Select the component to be used Evaluate the amount of a component in the sample How to prepare for the Standard addition quantitation The table below briefly describes how to prepare for the quantitation Step Action 1 Perform a sample run with the unspiked sample and a run with the spiked sample Open one of the two result fil
86. ables that are restricted to your own user ID if needed e Select the quantitation table you wish to rename on the Quantitation table s list e Click in the Quantitation table name text box and type a new name e Click the Rename button e Click the Close button Note You must have Edit global list s rights to be able to rename a global quantitation table How to delete a quantitation table The table below describes how to delete an existing quantitation table Step Action 1 Select Quantitate Edit Quantitation Table Delete Result The Delete quantitation table dialog box opens 2 e Select Personal to display the quantitation tables that are restricted to your own user ID if needed e Select the quantitation table you wish to delete on the Quantitation ta ble s list e Click the Delete button e Click the Yes button to confirm e Click the Close button 518 UNICORN 5 31 User Reference Manual 28 9972 39 AA 13 The Analysis module 13 3 How to prepare for quantitation 13 3 3 How to edit and update a quantitation table Note You must have Edit global list s rights to be able to delete a global quantitation table UNICORN 5 31 User Reference Manual 28 9972 39 AA 519 13 The Analysis module 13 4 How to quantitate the sample 13 4 How to quantitate the sample Introduction This section describes how to use calibration curves to quantitate samples Calibration curves are applicable to external an
87. acro facilities in other programs This section de scribes how to work with automated evaluation procedures In this section This section contains these sub sections Section See page 12 3 1 How to create a new procedure 461 12 3 2 How to edit a procedure 464 12 3 3 How to run a procedure 467 12 3 4 How to rename and remove procedures 471 460 UNICORN 5 31 User Reference Manual 28 9972 39 AA 12 Evaluation 12 3 Automated evaluation procedures 12 3 1 How to create a new procedure 1231 Howto create a new procedure Introduction You can use the Procedure Editor to record or create a new procedure The Procedure Editor can also be used to view and edit the instructions within a procedure This section describes how to use the Procedure Editor to record new procedures The Procedure Editor dialog box The illustration below shows the Procedure Editor in Record mode amp Procedure Editor Untitled Fie Con lelp BASE TIME E v e 2 v v How to record a procedure The table below describes how to record a new procedure Step Action 1 Open the result file in the Evaluation module 2 Choose Procedures Record On Result The Procedure Editor dialog box opens in record mode UNICORN 5 31 User Reference Manual 28 9972 39 AA 461 12 Evaluation 12 3 Automated evaluation procedures 12 3 1 How to create a new procedure Step Action 3 Minimize the Procedure Editor dialog box
88. ains the program name and parameters to start it with UPDATE Updates a Quantitation table with new data from one standard concentration level The default Limit value of 12 5 will be used 616 UNICORN 5 31 User Reference Manual 28 9972 39 AA B Evaluation functions and instructions B 4 Procedure instructions Test instructions The Instruction field also contains a group of test instructions These instructions are only available for the UNICORN software development team Instruction Description AUTOSAM Sets the area intervals for the AUTOSAMPLER_PEAK_TEST PLER_PEAK_INTER VALS AUTOSAM Locates the first peak in the peak table Compares the area PLER_PEAK_TEST of the peak in the peak table with the specified maximum and minimum areas EXPORT_TEST_RE Finishes the current result and saves the output file as an SULT_TO_FILE ASCII file in a destination and with a file name specified in the variable DestFilename txt A complete search path may be incluede in the file name GRADIENT_TEST_IN Sets the level intervals for the GRADIENT_TEST TERVALS GRADIENT_TEST The theoretical straight line between the 0 and 100 levels are calculated The deviation between the curve and the ideal straight line is compared in both directions from the center position 50 until the deviation exceeds the defined maximum deviation The calculated deviation points are checked against the defined limits
89. ample and integrate the curve UNICORN 5 31 User Reference Manual 28 9972 39 AA 13 The Analysis module 13 2 Quantitation overview 13 2 1 General information about quantitation Step Action 6 Let the program calculate the concentration and amount of the components of interest in the sample Note The steps above do not apply to Standard addition See Standard addition quantitation below Illustration of the work flow The quantitation work flow is illustrated below Area Comporest 3 Area Component 2 AreaComponent 1 Quartitated san ple Calikration curves Standards at different concentration levels The four quantitation techniques The Analysis module provides four different quantitation techniques e External standard quantitation e Internal standard quantitation e Standard addition quantitation e Recovery calculation Each technique is described below UNICORN 5 31 User Reference Manual 28 9972 39 AA 479 13 The Analysis module 13 2 Quantitation overview 13 2 1 General information about quantitation External standard quantitation One or several component s of interest are run to produce a calibration curve The amount and concentration of the component in the sample is then determined from the calibration curve This technique is fairly simple and usually produces accurate results Internal standard quantitation Peak areas of the components of interest are related to the peak
90. and their solutions Problem description Solution You have forgotten your password Ask the system administrator to supply a new password Username and password not accepted e Restore the file USERS30 MPM from the latest back up copy You cannot log on although you use your or correct username and password e reinstall the default user Reason The file USERS30 MPM in the folder UNICORN SERVER FIL could be corrupt No user names Remote station Make sure that the computer is logged Both these conditions must apply on to the network before you start UNI CORN Note A remote station accesses the user list directly from the network server e The Username drop down box in the Logon dialog box is empty e Youare trying to log on froma remote station in a network installation No user names Local station Make sure that the computer is logged The user list on a local station ina network on to the network before starting UNI installation is not up to date CORN Note The user list is stored locally on a local station and is updated automati cally from the network server if the computer is logged on to the network 578 UNICORN 5 31 User Reference Manual 28 9972 39 AA Error message Strategy file error A Troubleshooting A 1 Logon The table below describes some problems and their solutions Problem description Solution Stand alone installation If you r
91. any of the windows includes more than one peak The second droplist in the Peak identification field of the Identification Settings dialog box offers the following options e Highest peak maximum default e Closest to retention i e closest to the center of the window see the retention column in the peak table e Maximum peak area Examine the nature of the peaks enclosed by the window and select the option that differs between the wanted and the unwanted peaks Use Closest to retention if there are large peaks from components that are not going to be quantitated Note The selection applies to all peaks even the internal standard and reference if used Absolute and Relative window width 506 When the Peak identification is set to Absolute retention the peak window width can be displayed as Absolute or Relative Select the appropriate button in the Identification Settings dialog box e Select Absolute to show the window width of each peak in minutes or the base volume unit e Select Relative to display the width of each component as a percentage of its reten tion If Peak identification is set to Relative retention Window is set automatically to Relative except for the reference peak UNICORN 5 31 User Reference Manual 28 9972 39 AA 13 The Analysis module 13 3 How to prepare for quantitation 13 3 2 How to create a quantitation table Step 4 How to create a calibration curve and a quantitation table
92. are stopped e the alarm is noted in the logbook The situation must be acknowledged and corrected before the process can be continued the Warning limits e a warning message is displayed e the process continues e the warning is noted in the logbook Note The message text in an Alarm dialog box and the corresponding text in the logbook are both color coded in red Warning texts are color coded in orange both in the dialog box and in the logbook The text in the logbook is changed into black when the Alarm or Warning is acknowledged Note The Alarms are not active unless the mode is set to Enabled 558 UNICORN 5 31 User Reference Manual 28 9972 39 AA 14 System settings 14 2 Alarms Alarms in a network Alarms and warning messages are displayed on all stations with a connection to the concerned system This is regardless of the activity that is currently performed in UNICORN and regardless of the identity and access rights of the current user Alarms and warnings can only be acknowledged from the station that is connected in control mode The hysteresis setting The hysteresis setting not available for AKTAdesign systems for a warning determines to which extent the signal can oscillate up or down from the warning limit threshold without re activating a warning After the signal has activated a warning the warning will not be repeated as long as the signal remains within a window defined by the hysteresi
93. 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 the separation runs and is therefore the technique that can produce the highest precision if a suitable internal standard can be selected Standard addition quantitation 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 compo nent can be quantitated Compared to other techniques results can be obtained more quickly when you are performing a small number of sample runs with standard addition However the precision is limited Recovery calculation Recovery is used to determine the losses that can occur during the sample preparation process The sample is spiked with a known amount of 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 Analysis procedure instructions The table below describes the new procedure instructions for quantitation that become available when the Analysis module is installed 480 Instruction Description QUANTI The instruction calculates the concentration an
94. ariable con tent 219 How to define columns 220 How to delete a vari able 218 How to edit a Scouting Scheme 216 How to perform a Scouting run 260 How to rename vari ables 218 How to set up a Scouting Scheme 216 Result files 260 Specify folder for storing re sults 182 Start Protocol settings 186 Usage 214 Scouting runs Change of variables during arun 261 Searches General functions 44 Security Backup 96 Set_Mark How to issue 196 Usage 196 Slope values How to measure 452 Usage 451 SMART Manager How to import data 389 UNICORN 5 31 User Reference Manual 28 9972 39 AA Smoothing algorithms Autoregressive 591 Median 592 Moving average 591 Savitzky Golay 593 Snapshots How to add a text instruc tion 109 How to view 50 Standard addition quantitation How to identify sample peaks 525 How to perform 489 How to select the compo nent 525 Reliability 491 Start Protocol How to use 231 Statistics Correlation 626 Explained variance 627 Stock solutions Description 175 Strategy How to display the strategy instructions 108 System Control module Alarms and warnings 259 Description 36 How to customize the panes 236 How to save manual re sults 258 How to select the displayed panes 236 Manual instructions 256 Manual instructions during a method run 256 Overview 235 Toolbar buttons 252 System Control status bar Description 255 Watch status 255 System Co
95. as File name a full search path must be entered in an swer to the question If all parameters to this function are OFF then no documentation is exported If at least one of them is ON then the documentation will be exported and the corresponding parts will be included in the export ed file EXPORT_DOC_XLS Exports the documentation in the current result file in XLS format to the file defined in Export to file If is entered as File name the current Result file will be used If is entered followed by text e g Enter a file name as File name a full search path must be entered in answer to the question If all parameters to this function are OFF then no documentation is exported If at least one of them is ON then the documentation will be exported and the corresponding parts will be included in the exported file 614 UNICORN 5 31 User Reference Manual 28 9972 39 AA B Evaluation functions and instructions B 4 Procedure instructions Instruction Description EXPORT_DOC_ASCII Exports the documentation in the current result file in ASCII format to the file defined in Export to file If is entered as File name the current Result file will be used If is entered followed by text e g Enter a file name as File name a full search path must be entered in an swer to the question If all parameters to this function are OFF then no documentation is exported If at least one of them is ON then the documentati
96. asesesseesenseess 61 33 HOW tO assign user properties cceccsssssssescccsssssseccscssssusecessssssessessssuusccesssssssecssssssuecsecsssssnseeee 65 3 4 How to change your passwords and user attributes 69 3 5 How to connect to the chromatography system 72 3 6 How to back up and restore system data P 78 3 7 How t set up Q ITEM sssssscsnsdacsecogessedeeddascenssenssonasssgagnevsssdsjssntacnesedpsgsddesttcheconnsneecdcezennseogetesaitononna 82 Files and folders in UNICORN ssssssesssssssssessssesssseeesssseesessssessesssessessoressesessssesessese 83 4 1 HOW to Create folders s ssssssssses11ssss1rsssssrrreseerrreserrreseerreseenrrsseerrrsseenissneenrsssettrssnnensnnntnsn sneer annern ane 84 4 2 How to open and preview files 85 4 3 HOw to arrange and locate your files o ecescssssssssssssssssssscssssssssessesseseecessssssssusssssesccsssssssssunseeses 88 44 How to copy delete rename and backup files and folders woc eeeesssssssssesesssssccssssessssasiees 92 How tocredte a Method ssi csczcssasecasccicantsces seanstedciuedsnssackeseesacderacessceracelasiaicoutietics 97 5 1 Howt sethe Method WIZ o ccssssssssssessssssccssssssssssssessssscessssssssssssesscsessesessssssssessssssseessssesssees 98 52 HOW to USE THE Meth Oden Plt asisssscscisdesssscasssc casensvassanseecesesesstvaaseasisan bsbotoacsvsivastia tosreebooees 103 5 3 HOW LO USE TEXT IMSERU CIOS areena n A ENN ARN 106 54 HOW tosin th Method sessssicssssssss
97. assssssesscossnssssaoassssceconsventozsasassosescoossevssssuniass ass aachecibibesssacionntanenivads 110 How to Sdit MetNOdS oriisgin A N 111 6 1 The Method Editor interface o cscsssssssesssssssssssssssssssssesssssessssssssssssussessessecssssssssunssssessecsesssssssunseesess 112 611 Method Editor mod le ssrisrenasnimnusnieonnan nak enia 113 6 1 2 Text Instructions editor seisssssissssssississeeseesresessississesrsteneeniestesississtissizinstastinersnisiessnaiosterianennaenisnei 115 E A NE 110 Aoa EE E AE EE PEEN E E PEET E NEAN 118 6 2 1 How to view method blocks ssesscssssssssacscssccscasecsasssctisioanagsssccssscsocabvssisisansnnnssancacbossstisarsusnbenenetsscnsss 119 UNICORN 5 31 User Reference Manual 28 9972 39 AA 3 Table of Contents 6 2 2 How to call method blocks vaeceeccsesesscsssssscsssssssssssessssssesssssssessssssesessssesessssesesssssesssssseesssseesssasecsssseess 121 6 2 3 How to add method blocks vaveecsseessssssssssssssesscssssescssssesssssssessssssesssssusssssssesessssesesssssesssuseessssssessssseess 122 624 Howtodel te method DIOCKS sierenininirsansanaae an i i R 126 625 HOW tO FENAME method DIOCKS ressiasuisunrtuiennoiiiiaiiiinn i Aa 129 6 2 6 How to find copy and move method blocks eeeecsssssssssssssssssssssssssssssssssssssecssssssssssssssessseeeees 130 6 2 7 How to import method blocks wecceccccccsssssssseseeeeees n 132 6 3 Method INstructiOns oi ceccccssescssssescssssssssssssssssssscsssecsss
98. at the beginning and the end of the smallest flattest peak of all the peaks of interest and use these values Illustration Slope value measurement The illustration below shows a measurement of the slope limit after differentiation 1d196Level 1 to 4002 4_UV1_2a0nm id100Level 1 to 4002 4_Inject 1d188Level 1 to 4002 1_UV1_2a0nm o1 BASEM id 130Level 1 to 4002 1_UV1_Z3OnmM OL BASEMS idt6OLevel 1 to 9002 1_UV1_2B0nm O1 14 ee wee ee em eee ee a UNICORN 5 31 User Reference Manual 28 9972 39 AA 453 12 Evaluation 12 2 Other evaluations 12 2 3 How to simulate a peak fractionation 1223 Howto simulate a peak fractionation Introduction You can create a curve that simulates a peak fractionation to test the outcome before the actual peak fractionation is run This section describes how this is done How to simulate a peak fractionation The table below describes how to simulate a peak fractionation in the Evaluation module Step Action 1 Choose Operations Simulate Peak Fractionation Result The Simulate Peak Fractionation dialog box opens Simulate Peak Fractionation 05 idl 48Quantitate001 1_Cond 08 id148Qusntitate001 1_Cone O08 id148Quantitale001 1_Pressuie OS id148Quaniitate001 1_Flow 10 id148Quaniitate001 1_Temp 17 id148Quantitate001 1_UV1_280nm 01 BASEM Curve name UV1_220ren o1 SIMPF Fraction size f m Min width fp min Start slope 92782 m min End slope 8
99. ation Internal standard quantitation uses peak tables prepared from the standard similar to the External standard quantitation However a fixed quantity of an additional component is added to every separation run including the sample The peak sizes of the standards and the sample are then related to the peak size of the internal standards to compensate for any changes that may have occurred between the runs General assumption Advantages The internal standard technique relies on the assumption that any changes in the injected amount of the componentis of interest e g due to sample preparation losses correspond to equal changes in the injected amount of the internal standard component Internal standard quantitation reduces errors that 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 that are injected What is a suitable internal standard A suitable internal standard must meet the following conditions e t must be well separated from the components in the sample not just from the components of interest e It must not be present naturally in the sample s e It must have similar chemical properties to the components of interest To be able to compensate for losses during the sample preparation all the standard concentra
100. atogram section to the right to select the addition peak table for the spiked sample 4 e Edit the default unit mg in the Unit label field if necessary e Type the amount of the component that was added as the spike in the Added amount field e Click OK Result The Identify Peak dialog box opens 5 To locate and select the peak of the unspiked sample do the following e Click its triangle marker black or select its reference in the Source table 6 e Repeat step 5 to select the spiked sample The triangle color is blue Use the Addition table e Click the OK button UNICORN 5 31 User Reference Manual 28 9972 39 AA 525 13 The Analysis module 13 4 How to quantitate the sample 13 4 2 Standard addition quantitation The Identify Peak dialog box The illustration shows the Identify Peak dialog box described in the table above 8 6 4 2 0 2 4 84 10 12 14 16 18 20 Source table Addition table Retention Retention min min 5 84 5 85 653 6 53 778 How to view the quantitation results The amount of the component of interest is displayed in the peak table Amount columns of the Evaluation module NUENA usd Su SLRS USS TER Ss ES AL RRRA nR TakEs B Ss BESS Ses SERIN DS NAL IRE Su TAS a E SSSA AE SSL SSUES QEN SSL BASSA Dm SS Ss ENASESS SSSA TS Se SALE AO TOV ESS QE EST ERIAN QE SESE CULT 526 UNICORN 5 31 User Reference Manual 28 9972 39 AA
101. baseline with a classic algorithm 414 12 1 5 How to edit the baseline manually 423 12 1 6 How to edit the peaks 427 12 1 7 How to integrate part of a curve and how to exclude or skim peaks 437 12 1 8 Measurements 443 400 UNICORN 5 31 User Reference Manual 28 9972 39 AA 12 Evaluation 12 1 Peak integration 12 1 1 Baseline calculation 1211 Baseline calculation Introduction The first step when you integrate peaks is to calculate a baseline A correct baseline is crucial for accurate calculation of the peak areas This section describes the options for how to calculate baselines in the Integrate dialog box Baseline options UNICORN offers several options for how to create an accurate baseline e To use the automatic Calculate baseline function e To create a baseline based on a blank curve e To use a Zero baseline e To reuse an existing baseline The Calculate baseline function The Calculate baseline instruction provides automatic calculation of the baseline In most cases the measurement is very accurate The calculation can be performed using the Morphological algorithm or the Classical algorithm Baselines based on a blank curve A blank curve can be used as the baseline for peak integration e Youcan use a blank curve with the same chromatographic conditions as the corre sponding sample or e You can subtract the blank run from the source curve and then perform peak inte gration on the resulting curve with
102. ble for the components of interest See Section 13 3 2 How to create a quantitation table on page 499 for further information Perform a sample run Note If internal standard quantitation is 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 on the standard levels Open the sample result file and peak integrate the sample curve to produce a peak table Note The sample curve must use the same X axis base unit as the standards during the integration Time is the recommended unit for highest reliability Select File Save to save the peak table UNICORN 5 31 User Reference Manual 28 9972 39 AA 521 13 The Analysis module 13 4 How to quantitate the sample 13 4 1 External and internal standard quantitation How to calculate the amount and concentration The table below describes how to calculate the amount and concentration in the sample Step Action i Select Quantitate Calculate amount and conc Result The Calculate Amount and Concentration dialog box opens Calculate Amount and Concentration 2 Select a quantitation table on the Quantitation table droplist Select the chromatogram that contains the sample curve on the Source chromatogram droplist Select the sample peak table from the Peak table s list Check the Injection volume value and type a new value if necessary Note For internal standard qua
103. can be selected on the Scouting tab for different scouting runs 6 Proceed with the instructions on how to set up the scouting runs for the standards see table below How to perform automated update in scouting runs step 3 The table below describes how to set up the scouting runs for the standards Step Action 1 Select View Run Setup and click the Scouting tab 2 Click the Define button Result The Scouting Variables dialog box opens UNICORN 5 31 User Reference Manual 28 9972 39 AA 539 13 The Analysis module 13 5 Automated quantitation 13 5 3 How to perform automated update Step Action 3 Edit the scouting variables list to include e Procedure e Vial_Number e Injection_volume e Sample_ID e Quantitation_Type Note The Procedure variable will appear at the beginning of the list of vari ables even though the Evaluate instruction is inserted at the end of the method Set up all the standard runs in the scouting scheme e Select the Update_Quantitation procedure e Ensure that Quantitation_Type is set to the correct standard level for each run Result The quantitation table will now be updated with new values after each run Since the runs will be performed with the Replace the default se lection option you can only perform one run at each level Proceed with the instructions on how to set up the scouting runs for the samples in step 4 see table below Note The quantitation
104. ccurred All the problems errors that have oc curred together with help texts are automatically recorded and included in the report Tip If you select a specific error in the Description dialog box the appropriate help text is shown in the error message box Add the following information in the Description dialog box e A short description of the problem e The circumstances under which the problem occurs e The consequences of the problem Click the Next button Result The Reproducibility dialog box opens UNICORN 5 31 User Reference Manual 28 9972 39 AA 573 15 System maintenance and error reporting 15 2 How to generate problem reports 15 2 2 How to generate a problem report from the System Control Step Action 4 Specify whether the problem is reproducible or not Select one of these al ternatives e Yes Provide a short description in the text box of how the problem can be reproduced e No e Unknown Click the Next button to proceed to attach example files see table below Step 2 How to attach a file You can attach method files and or log files to the problem report The table below describes how to attach a file Step Action 1 The Attachments dialog box is displayed Attachments 574 UNICORN 5 31 User Reference Manual 28 9972 39 AA 15 System maintenance and error reporting 15 2 How to generate problem reports 15 2 2 How to generate a problem report from the System Control Step Act
105. ce Manual 28 9972 39 AA 547 13 The Analysis module 13 6 How to measure molecular size 13 6 2 How to determine the molecular size How to create and save a molecular size table The table below describes how to create and save a molecular size table in the Evaluation module Step Action 1 Open a result file and select Mol Size Edit Mol Size Table New Result The New Molecular Size Table dialog box opens New Moleculas Size Table Peak table s hd 68 uaniesteOOT 1_UVI_celrm 0l PEAR id1 230 uatan UVI Zare PEAK 2 e Double click the result file in the Select peak table s list e Select the source chromatogram on the Source chromatogram droplist 3 Highlight a peak table that was prepared from the standard in the source Peak table s list and click the Select button 4 Repeat step 3 to select more peak tables Note The runs must all have been made under the same conditions 5 To deselect a table highlight the table in the Peak table s list to the right and click the Remove button 6 Repeat steps 2 to 4 to select peak tables from other result files 548 UNICORN 5 31 User Reference Manual 28 9972 39 AA 13 The Analysis module 13 6 How to measure molecular size 13 6 2 How to determine the molecular size Step Action 7 e Type the appropriate size measurement unit in the Molecular size unit label field default kDa e Click OK when the Peak table s list to the right contains all the req
106. ce Manual 28 9972 39 AA Manual instructions in System Control Column prompt 257 During a method run 256 Functions of buttons 257 How to save results manual ly 258 Measurements How to make direct 444 Messages How to add a message in struction to a method 660 How to issue 195 Usage 195 Method blocks Base instructions descrip tions 189 Block length 193 Blocks in the Block pane 120 Blocks in the Gradient pane 120 Blocks in the Text pane 119 Calls 121 Conditional calls 121 Description 119 Fields of the New Block dia log box description 124 How to copy a block 130 How to delete unused blocks 128 How to find text strings 130 How to import 132 How to import general infor mation 132 How to move a block 131 How to rename blocks 129 How to show or hide instruc tions 119 How to use the Delete Block command 127 Unconditional calls 121 Use the Delete Block dialog box 126 Use the Instruction box to add 122 Use the New Block dialog box to add 123 Method Editor Icon descriptions 113 UNICORN 5 31 User Reference Manual 28 9972 39 AA Index Log Format 193 Modes 30 Text instructions display panes 31 The Block pane 32 The Flow Scheme pane 32 The Gradient pane 33 The Instruction box 34 The Text pane 33 Method files How to connect a method to a system 95 How to open in the UNI CORN Manager 85 Method instructions Difference in function be tween Change and Replace
107. ce point Step Action 1 Right click in the chromatogram and select Set Marker Ref Point to define a reference point for the marker position 2 When the marker is moved from the reference point the X axis and Y axis values for the new position are displayed together with e the new position in relation to the position of the reference point e the minimum maximum and average values for the curve interval be tween the reference point and the new position How to record a Snapshot The table below describes how to record a Snapshot of the current curve values Step Action 1 e Right click in the chromatogram and select Snapshot from the shortcut menu Result The Snapshot dialog box opens 2 The dialog box displays all the curve data that was current at the moment the snapshot was taken e Click the Save to file button to save the snapshot as an Excel file e Click the Print button to print the snapshot UNICORN 5 31 User Reference Manual 28 9972 39 AA 445 12 Evaluation 12 1 Peak integration 12 1 8 Measurements How to select peak table data 446 The retention time and amplitude of any peak can be viewed directly in a peak table after an integration This data and more is selected in the Chromatogram Layout dialog box The table below describes how to select peak table data Step Action 1 Click the Chromatogram Layout icon Result The Chromatogram Layout dialog box opens 2 Click th
108. colors UNICORN 5 31 User Reference Manual 28 9972 39 AA 589 B Evaluation functions and instructions Appendix B Evaluation functions and instructions Introduction This appendix describes the functions that are implemented in the Evaluation module In this appendix 590 This appendix contains these sections Section See page B 1 Smoothing algorithms 591 B 2 Baseline calculation theory 594 B 3 Peak table column components 600 B 4 Procedure instructions 607 UNICORN 5 31 User Reference Manual 28 9972 39 AA B Evaluation functions and instructions B 1 Smoothing algorithms B 1 Smoothing algorithms Introduction This section describes how the smoothing functions are calculated Choose Opera tions Smooth in the Evaluation module to view and edit the options Moving Average The table below describes the process when the Moving Average smoothing algorithm is used Stage Description 1 For each data point in the source curve the processed curve is calculated as the average of the data points within a window centered on the source data point e The width of the window is determined by the parameter value expressed as number of data points 2 When the source point is less than half the window size from the beginning of the end of the curve the average is calculated symmetrically round the source point over as many data points as possible e Ifyou increase the window width the smoothing effect is
109. columns are imported and available in the column list Note Select Import as global to import the columns to the global column list UNICORN 5 31 User Reference Manual 28 9972 39 AA 637 E How to create and edit BufferPrep recipes Appendix E How to create and edit BufferPrep recipes Introduction The BufferPrep function is available for some AKTAdesign systems This appendix de scribes how to create and how to edit the recipes for BufferPrep In this appendix This appendix contains these sections Section See page E 1 How to create a BufferPrep recipe 639 E 2 How to edit a BufferPrep recipe 645 638 UNICORN 5 31 User Reference Manual 28 9972 39 AA E How to create and edit BufferPrep recipes E 1 How to create a BufferPrep recipe E 1 How to create a BufferPrep recipe About BufferPrep recipes New BufferPrep recipes are created in the Method Editor The list of recipes is not linked to a specific method Which recipe to use in a certain method is selected on the Buffer Prep tab in the Run Setup How to create a recipe The table below describes how to create a new BufferPrep recipe in the Method Editor Step Action pi Choose Edit BufferPrep Recipes Result The BufferPrep Recipes dialog box opens The illustration below shows the BufferPrep Recipes dialog box with a recipe selected BulfeiPrep Recipes 0M tap ie ooe UNICORN 5 31 User Reference Manual 28 9972 39 AA 639 E How
110. concentration verify or edit the Inj Volume field e Click OK Result The Update Calibration Curve dialog box opens See How to update a calibration curve below The Update Calibration Curve dialog box Data on the selected components for the curve to be updated are shown in the Compo nent name table When a component is highlighted its calibration curve is displayed above in the Calibration curve before update field The calibration curve to be updated is shown without taking the new point into consid eration 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 514 UNICORN 5 31 User Reference Manual 28 9972 39 AA Update Calibration Curve E3 m Level no 2 Calbration curve before update te Update by ea Average Replace Deviation and limit as 13 The Analysis module 13 3 How to prepare for quantitation 13 3 3 How to edit and update a quantitation table Amount Area Deviation Limit 7 eean _ Component name gant nt maU Ribonuclease A 40 8619 0 0614 5 1077 Cytochrome C 112 5148 28 4709 14 0643 How to update a calibration curve Peak size deviation The Deviation column of the Update Calibration Curve dialog box shows how much the peak size for the proposed new p
111. cribes how to export a column Step Action 1 Choose Edit Column List Result The Column List dialog box opens 2 Click the Export button Result The Export Column dialog box opens 3 e Click the checkbox for each column you want to export e Click Export Result The Export Column to file dialog box opens 4 e Select the desired folder in the navigation window e Type anew file name if neccessary e Choose the type of file to export column file or Excel file e Click the Save button Result The column file is saved and the dialog box closes 636 UNICORN 5 31 User Reference Manual 28 9972 39 AA D The Column list D 1 How to edit the Column List Note If a column is selected in the Column List when the Export Column dialog box is opened this column will automatically be selected in the Export Column dialog box How to import a column The table below describes how to import a column Step Action 1 Choose Edit Column List Result The Column List dialog box opens 2 Click the Import button Result The Import Column dialog box opens 3 e Click the Browse button to locate the column file Result The Import Column from file dialog box opens 4 e Select a column file e Click Open Result The Import Columns dialog box opens e Select the columns to import from the list e Select Import as global to add the columns to the global column list if desired e Click Import Result The selected
112. ction of absorbance peaks onn eessssssssessesssssccsssssssssusseessescesssssssssussssssesscessssssssnssuessessseseesesssess 655 F5 Collection of three absorbance peaks oun cccssssssssssssesssssccssssssssssssessessessesessssssssesecsesseesesseasssesees 657 F 6 Messages srunsa 660 F 7 Quality control procedure 663 DOER ee ne ee eer er ve R renee eee anne ee eee es neers 668 8 UNICORN 5 31 User Reference Manual 28 9972 39 AA 12 Evaluation 12 Evaluation Introduction This chapter describes e How to evaluate results with the focus on how to integrate peaks e How to automate evaluation operations e How to export data and curves In this chapter This chapter contains these sections Section See page 12 1 Peak integration 400 12 2 Other evaluations 447 12 3 Automated evaluation procedures 460 UNICORN 5 31 User Reference Manual 28 9972 39 AA 399 12 Evaluation 12 1 Peak integration 12 1 Peak integration Introduction Peak integration is used to identify and measure a number of curve characteristics in cluding peak areas retention time and peak widths This section describes e How to perform peak integrations e How to optimize peak integrations In this section This section contains these sub sections Section See page 12 1 1 Baseline calculation 401 12 1 2 How to perform a peak integration 403 12 1 3 How to optimize the baseline with a morphological algorithm 410 12 1 4 How to optimize the
113. d amounts in the TATE sample from a quantitation table Amount and concentration columns will be added to the peak table UNICORN 5 31 User Reference Manual 28 9972 39 AA 13 The Analysis module 13 2 Quantitation overview 13 2 1 General information about quantitation Instruction Description UPDATE The instruction updates a quantitation table with new data from one standard concentration level The default Limit value of 12 5 will be used The quantitation table will not be updated if the peak area or peak height of the new and the previous results differ more than the Limit value Note Either peak area or height is selected for the Limit value Default values The DEFAULT value for the injection value will be taken from the injection volume report ed by the Autosampler A 900 from the method DEFAULT can only be used when the injection is performed by the autosampler The DEFAULT value for the concentration level for the standard will be taken from the level entered in the QuantitationData instruction in the method UNICORN 5 31 User Reference Manual 28 9972 39 AA 481 13 The Analysis module 13 2 Quantitation overview 13 2 2 External standard quantitation 13 22 External standard quantitation General information 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 c
114. d internal standard quantitation and to recovery factor measurement Standard addition measurements are also described In this section This section contains these sub sections Section See page 13 4 1 External and internal standard quantitation 521 13 4 2 Standard addition quantitation 524 13 4 3 How to calculate the recovery factor 527 520 UNICORN 5 31 User Reference Manual 28 9972 39 AA 13 The Analysis module 13 4 How to quantitate the sample 13 4 1 External and internal standard quantitation 134 1 External and internal standard quantitation Introduction This section describes how to perform quantitation in the Evaluation module using either an external standard or an internal standard The processes involved in both external standard and internal standard quantitation of a sample are very similar The procedural differences mainly concern the creation of the quantitation tables A quantitation table is specific to either external standard or internal standard quantitation Method for the sample runs The method that is used for the sample runs must be the same as for the standard runs If the method is created from a wizard or a template for AKTAdesign systems select Sample in the variable Quantitation_Type on the Variables tab in the Run Setup How to prepare for the quantitation The table below describes briefly how to prepare for the quantitation Step Action 1 Prepare a quantitation ta
115. d sample Two runs are performed one with the sample and a second with the sample that was spiked prior to the sample preparation with a known amount of the component of interest Quantitation of the data from the two sample runs allows the recovery factor of the sample preparation to be calculated Note The recovery is measured as the recovery for the sample preparation not for the separation during the chromatographic analysis The recovery factor The recovery factor can 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 How to perform Recovery calculation 492 The table below describes briefly how Recovery calculation is performed Step Action 1 Perform a run with each level of the standard 2 Peak integrate the curves to produce a peak table for each level 3 Use the data from the peak tables to produce a calibration curve Note This is the same process that is used in the External standard quanti tation 4 Spike a portion of the sample with a known amount of the component of interest prior to the sample preparation 5 Run both the spiked and an unspiked sample UNICORN 5 31 User Reference Manual 28 9972 39 AA 13 The Analysis module 13 2 Quantitation overview 13 2 5 Recovery calculation Step Action 6 Peak integrate both samples to produce peak tables for the unspiked sample and th
116. e Use this copy for editing purposes UNICORN 5 31 User Reference Manual 28 9972 39 AA 12 Evaluation 12 3 Automated evaluation procedures 12 3 3 How to run a procedure 1233 How to run a procedure Introduction You can run the saved procedures either for a specific chromatogram or as batch runs How to run a single procedure The table below describes how to run a procedure for a specific chromatogram Step Action 1 Open a result file 2 Select Procedures Run Result The Run Procedure dialog box opens 3 Select the procedure from the list and click OK Result The procedure is executed Note You can also open the procedure in the Procedure Editor dialog box and choose Control Run or click the Play button Batch runs Itis possible to apply an evaluation procedure to a designated batch of result files if they are not open in the Evaluation module An open file will not run and an error message will be displayed The batch run is performed in the background of the Evaluation module and the results of the run are not seen with the exception of prints and documentation that are defined as steps in the procedure For example batch runs are useful e to perform integration with the same parameter settings on many results e to printa number of results with the same settings UNICORN 5 31 User Reference Manual 28 9972 39 AA 467 12 Evaluation 12 3 Automated evaluation procedures 12 3 3 How to run a
117. e Import as text box e Click the Import button e Click the Close button Result The new procedure is added to the list of Evaluation Procedures Select the new procedure and click the Edit button Result The Procedure Editor dialog box opens Highlight the existing Update instruction Use the scroll bar in the Parameter field to locate the Average or replace point droplist e Select the AVERAGE option e Click the Replace button UNICORN 5 31 User Reference Manual 28 9972 39 AA 541 13 The Analysis module 13 5 Automated quantitation 13 5 3 How to perform automated update Step Action 6 Select File Close in the Procedure Editor dialog box to return to the Run Setup rs Select the Update_Average procedure and click the Quantitate button Result The Quantitation table dialog box opens 8 Select the quantitation table and click OK 9 Deselect all the procedures on the Evaluation Procedures tab otherwise they will be run twice 10 Click the Scouting tab e Select the Update_Average procedure for the second and all following runs at each standard level concentration Note The Update_Quantitation procedure Update by Replace should still be used for the first run at each level 542 UNICORN 5 31 User Reference Manual 28 9972 39 AA 13 The Analysis module 13 6 How to measure molecular size 13 6 Howto measure molecular size Introduction The molecular size of components in a sample can be determined
118. e Mol size peak table column QRaacsseFAsE WEE SSL ZAI Svs When the result file is saved it includes the molecular size table that was used for the molecular size determination You can view the table that was used by selecting Mol Size Edit Mol Size Table View Current If the molecular size cannot be calculated If the molecular size cannot be calculated one of the following signs is shown in the peak table Mol size column Sign Function gt This means that the molecular size is larger than the largest size in the molecular size curve i e outside the valid range of the curve lt This means that the molecular size is smaller than the smallest size in the molecular size curve i e outside the valid range of the curve This means that the retention value is negative Molecular size procedure instruction The table below describes the new procedure instruction for molecular size measurement that becomes available when the Analysis module is installed Instruction Description MOLSIZE The instruction calculates the molecular sizes from a molecular size curve A Mol size column will be added to the peak table UNICORN 5 31 User Reference Manual 28 9972 39 AA 553 14 System settings 14 System settings Introduction This chapter describes some of the general UNICORN system settings In this chapter This chapter contains these sections Section See page 14 1 General i
119. e Peak Table tab 3 e Select the checkboxes on the Select peak table columns list for all items that you want to display in the table e Click OK UNICORN 5 31 User Reference Manual 28 9972 39 AA 12 Evaluation 12 2 Other evaluations 12 2 Other evaluations Introduction This section describes how the results can be used for other types of evaluations In this section This section contains these sub sections Section See page 12 2 1 Peak purity and peak identification 448 12 2 2 How to find slope values 451 12 2 3 How to simulate a peak fractionation 454 12 2 4 How to create curves 455 12 2 5 How to use the Fraction Histogram 459 UNICORN 5 31 User Reference Manual 28 9972 39 AA 447 12 Evaluation 12 2 Other evaluations 12 2 1 Peak purity and peak identification 1221 Peak purity and peak identification Introduction Ratios between UV curves measured at different wavelengths give useful information about peak purity or peak identity Peak purity The absorbance ratio can be used to check peak purity If the peak is pure the absorbance spectra are the same over the whole peak and the ratios should therefore remain con stant The peak is probably not pure if the absorbance ratio is not the same over the whole peak The illustration below shows a simulated chromatogram of two co eluting components with differing absorbance spectra and a small difference in retention time Co eluting peaks AU ae
120. e automatically integrates the first UV curve with default baseline settings and uses the selected quantitation table to quantitate the sample The amounts and concentrations are then printed 4 Click the Quantitate button Result The Quantitation table dialog box opens 5 Select the quantitation table from the Global or Personal folder and click OK Result The quantitation table is copied into the Quantitate_Sample proce dure Note The procedure cannot be executed if a quantitation table has not been selected Time must have been selected as the X axis base unit 6 Save the method with a new name 7 Perform the run s Result The amount and concentration of the components in the samples will be printed automatically after each run 534 UNICORN 5 31 User Reference Manual 28 9972 39 AA 13 The Analysis module 13 5 Automated quantitation 13 5 3 How to perform automated update 1353 Howto perform automated update Introduction This section describes how to update quantitation tables automatically also in scouting runs See also Section 13 3 3 How to edit and update a quantitation table on page 512 How to perform automated update with the Replace option The table below describes how to automatically update a quantitation table with the Replace option default Step Action 1 2 Open a method in the Method Editor e Click the Scouting tab in the Run Setup e Click the Clear All button to clear the
121. e below describes how to generate and save the report Step Action i By default the report is saved in the folder Unicorn Reports If you want to save the report at another location select a folder in the tree structure 2 You also have these options e Click the Preview button to open the report in Notepad e Click the Print button to print the report without any preview 3 Click the Finish button to generate and save the report 572 UNICORN 5 31 User Reference Manual 28 9972 39 AA 15 System maintenance and error reporting 15 2 How to generate problem reports 15 2 2 How to generate a problem report from the System Control 152 2 Howto generate a problem report from the System Control Introduction The Generate Report Wizard is used to generate problem reports When an error mes sage appears in System Control you can activate the report wizard from the error message dialog box The Generate Report Wizard can also be activated anytime if you choose System Report Step 1 How to create the report When an error message appears in System Control follow the instructions in this table to activate the Generate Report Wizard and create a report Step Action 1 e Click the Report button in the error message dialog box or e Choose System Report The first step is a Welcome screen Click the Next button Result The Description dialog box is displayed and shows a list of the problems errors that have o
122. e from the curve crvSrc using a morpho LINE_MORPH logical method DEFAULT can be selected in the Baseline parameters which will then calculate default baseline param eters for each new curve The baseline is stored in curve crvDst CLEAR_PEAKTABLE Clears the peak table in Peak table source from the computer memory COPY_PEAKTABLE Copies a peak table from Peak table source to Resulting peak table NEGATIVE_PEAKS Controls the baseline behavior in subsequent baseline calcu lations If ONOFF is ON then the baseline can be drawn above the curve and negative peaks can be detected by PEAK_IN TEGRATE If ONOFF is OFF then the baseline is never drawn above the curve PEAK_INTEGRATE Performs a peak integration on the source curve and stores the resulting peak table in Resulting peak table It is assumed that the baseline is subtracted PEAK_WINDOW Specifies which part of the source curve that will be integrat ed Peaks between retention Left limit and Right limit will be detected if the ONOFF parameter is set to ON If ONOFF is set to OFF the whole curve will be used for integration UNICORN 5 31 User Reference Manual 28 9972 39 AA 609 B Evaluation functions and instructions B 4 Procedure instructions Instruction Description REJECT_PEAKS Any combination of conditions is allowed If all parameters are OFF then every detected peak is included in the peak table SET_COL UMN_HEIGHT Sets the colum
123. e number of largest peaks to detect has a default value of 20 and it may be helpful if this is set to a smaller value 498 UNICORN 5 31 User Reference Manual 28 9972 39 AA 13 The Analysis module 13 3 How to prepare for quantitation 13 3 2 How to create a quantitation table 133 2 How to create a quantitation table Introduction The quantitation table contains all the necessary data such as the calibration curves that are needed to quantitate one or several components in a sample This section de scribes how the quantitation table is created How quantitation tables are created Quantitation tables are created in the same way 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 Four process steps The creation of the quantitation table can be divided into four steps ni 2 3 4 Standard data input Component selection and definition Peak identification Calibration curve and quantitation table creation Step 1 How to input the standard data The table below describes how to input the standard data in the Evaluation module Step Action 1 Select Quantitate Edit Quantitation Table New on the menu bar Result The New Quantitation Table dialog box opens with the name of the active
124. e settings 560 Default curve names 287 Draw a straight curve be tween selected points 458 Export options 390 Fraction text alignment op tions 289 How to add 337 How to add a second Y ax is 292 How to align with Nor malise 380 How to apply a layout 295 How to change and fix the X axis 293 How to change and fix the Y axis 292 How to change the color and style 289 How to choose the Y axis scale 280 How to compare peaks in different curves 351 How to copy curves into one chromatogram 377 How to copy into the Tempo rary chromatogram 276 How to create 455 How to create a fraction histogram 459 How to cut a curve and store as new 297 How to delete unwanted curves 397 How to differentiate 452 How to divide 449 How to export 391 How to export in AIA for mat 392 How to filter logbook infor mation 290 How to import 388 How to import a blank run curve 335 How to import using File Open 376 How to move using the Shift function 383 How to produce a mirror image 385 How to reduce noise 332 450 How to remove ghost peaks 332 450 How to rename 349 How to save a layout 294 How to set a hatched back ground 291 How to shift a mirror im age 386 How to stretch or shrink us ing Multiply 383 How to subtract a blank curve 336 How to use the Open to compare command 372 How to use the zoom func tion 296 Logbook text alignment op tions 289 Manual p
125. e spiked sample 7 The amounts for unspiked and spiked sample are calculated from the cali bration curve The difference between these amounts provides the apparent amount of the addition See illustration below mau Sample unspiked 240 200 180 100 16 5 Sample with addition Area corresponding spiked to the sample Area corresponding to the addition 15 0 15 5 16 0 16 5 17 0 min 8 The ratio of this apparent amount compared to the amount actually added to the sample determines the recovery of the system Apparent amount added Recon ee Actual an ount added Apparent amount added Am ount of spiked sample Amount of unspiked sample 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 recov ery factor would then be 0 8 UNICORN 5 31 User Reference Manual 28 9972 39 AA 493 13 The Analysis module 13 2 Quantitation overview 13 2 5 Recovery calculation Reliability 494 Below are some specific factors that determine if the recovery factor result is reliable A spike amount that is of the same order of magnitude as the sample must be used to maximize the precision It is assumed that the recovery is the same for both the sample and the spiked sample However if the recovery varies according to the amount of the component of interest the results are unreliable UNICORN 5 31 User Reference
126. e subtractions in the second list of curves UNICORN 5 31 User Reference Manual 28 9972 39 AA 449 12 Evaluation 12 2 Other evaluations 12 2 1 Peak purity and peak identification Step Action 7 Click the checkbox for Threshold and type values for each curve This results in the following e The quotient is set to 1 0 if either of the sample values is closer to zero than the threshold value Very high quotient values are prevented if divi sion is performed with values close to zero Very low quotient values are also prevented Note Default Threshold values are entered by UNICORN The values can be changed 8 Click OK How to filter the result curve 450 The resulting curve can be filtered to reduce noise and to remove ghost peaks The table below describes how to filter the curve Step Action 1 Select Operations Smooth Result The Smooth dialog box opens 2 e Select the Source Chromatogram e Select a Filter Type Note The Median filter is recommended to remove noise that appears as spikes or occurs in a small area of the curve e Click OK UNICORN 5 31 User Reference Manual 28 9972 39 AA 12 Evaluation 12 2 Other evaluations 12 2 2 How to find slope values 1222 Howto find slope values Introduction With AKTAdesign systems it is possible to only collect peaks during fractionation The way to find suitable slope values for a particular run is described in this section Where to use s
127. e the baseline with a classic algorithm on page 414 4 e Click the Baseline settings button to change the calculation algorithm in the Settings dialog box The default algorithm is Morphological e Change the selections or values e Click OK 5 e Click the Peak window button to edit the peak window limits if necessary e Click the Reject peaks button to set the parameters for peak rejection if necessary e Edit the Column height or Column V values if necessary UNICORN 5 31 User Reference Manual 28 9972 39 AA 403 12 Evaluation 12 1 Peak integration 12 1 2 How to perform a peak integration Step Action 6 e Click OK to integrate and close the dialog box or e Click Save and Edit Peak Table to save the integration and open the in tegrated curve for editing See Section 12 1 5 How to edit the baseline manually on page 423 See Section 12 1 6 How to edit the peaks on page 427 See Section 12 1 7 How to integrate part of a curve and how to exclude or skim peaks on page 437 Illustration This is an illustration of the Integrate dialog box Integrate 05 125200101 1_Cond 06 125200101 1_Cone 07 125200101 1_pH 08 125200101 1_Pressure 09 125200101 1_Flow 10 125200101 1_Temp 13 125200101 1_SamplePres 14 125200101 1_SampleFlow Column height 404 UNICORN 5 31 User Reference Manual 28 9972 39 AA 12 Evaluation 12 1 Peak integration 12 1 2 How to perform a peak integration
128. eak UNICORN 5 31 User Reference Manual 28 9972 39 AA 605 B Evaluation functions and instructions B 3 Peak table column components How to change the Asymmetry Ratio The Asymmetry Ratio is selected in the Options dialog box in the UNICORN Manager The table below describes how to select a value Step Action 1 e Choose the Administration Options menu item Result The Options dialog box opens 2 e Type a ratio value in the Asymmetry Ratio at text box e Click OK Result The ratio value is changed and the dialog box closes Note You must repeat the peak integrations after the change to update the values based on the new asymmetry ratio The default ratio is 10 HETP formula 606 The formula below is used to calculate the HETP value HETP L N N 5 54 x Vp w assuming a Gaussian peak Where e N no of theoretical plates e L bed height in cm e Vp peak retention elution volume or time e Wy peak width at half height expressed in the same units as Vp UNICORN 5 31 User Reference Manual 28 9972 39 AA B Evaluation functions and instructions B 4 Procedure instructions B 4 Procedure instructions Introduction This section contains lists of procedure instructions with descriptions These instructions are used in the Procedure Editor Choose Procedures Edit New in the Evaluation module to view the Instruction list Curve operation The table below contains a list of instructio
129. eak end time or volume base A and G in the diagram above UNICORN 5 31 User Reference Manual 28 9972 39 AA 601 B Evaluation functions and instructions B 3 Peak table column components Parameter Description Peak name Name of the peak Percent of total area Peak area as a percent of the total area under the curve above the baseline Time or volume base Note This value can differ in time and volume base if the flow rate is not constant throughout the method Percent of total Peak area as a percent of the sum of all integrated peaks peak area Note This value can differ in time and volume base if the flow rate is not constant throughout the method Resolution Peak resolution See definition below this table Retention Retention at the peak maximum time or volume base C in the diagram above Sigma Standard deviation for a Gaussian shaped peak See definition below this table Type of peak limits Identifies the criteria for peak start and peak end as either the baseline intersection or dropline to the baseline or skim line Width Difference in retention between the peak end and peak start time or volume base G A in the diagram above Width at half Calculated by taking the maximum height of the peak above height the baseline then determining the peak width at half this value above the baseline Time or volume base B D in the diagram above where BD bisects CF
130. eak identifica tion 358 Molecular size curve mod els 620 Monitor signals stored as curves 560 Multifile Peak Compare Wiz ard 351 Peak labels 289 Run curves default appear ance 280 Simulated peak fractiona tion 454 Curves pane in System Control Description 240 How to change curve colors and styles 241 How to change scale of the X axis 243 How to change scale of the Y axis 242 How to display a vertical marker 241 UNICORN 5 31 User Reference Manual 28 9972 39 AA x ow to display complete ogbook information 246 ow to select curve pres ure units 244 ow to select curves to be monitored 240 How to select text align ment 245 How to set a reference point 241 How to zoom in regions of the pane 244 Reduce scale of zoom 244 D Delete files and folders 96 Delete Method blocks How to delete unused blocks 128 How to use the Block Delete Block command 127 How to use the Delete Block dialog box 126 Documentation Documentation tabs de scription 328 How to export 393 How to view 327 Result information 329 E Electronic signature How to sign a result 395 Evaluation Chromatogram window shortcut menu 281 Chromatogram window views 278 How to display a vertical marker 282 How to display chro matogram header informa tion 279 How to display peak table information 279 How to exit the module 398 How to make chro matogram layout changes general 286
131. eas is only performed on the selected section 438 UNICORN 5 31 User Reference Manual 28 9972 39 AA 12 Evaluation 12 1 Peak integration 12 1 7 How to integrate part of a curve and how to exclude or skim peaks How to exclude peaks You can define criteria to exclude peaks from integration The table below describes how to define peaks to be excluded in the Integrate dialog box Step Action 1 Click the Reject peaks button Result The Reject Peaks dialog box opens e Select the appropriate checkboxes and type values for height width and area e Define how many of the largest peaks you want to include e Click OK How to include negative peaks Select the Accept negative peaks checkbox of the Integrate dialog box to include neg ative peaks in the integration Result The negative peaks will be reported as negative areas in the peak table By default negative peaks are not included in the integration Peak skimming vs drop lines The area under a peak can be calculated either using separating drop lines or peak skimming e Drop lines are vertical marks that split two peaks at the valley Drop lines are used mostly for peaks of relatively similar size When a peak has a shoulder splitting with drop lines will cause the first peak to lose too much of its area to the peak that forms its shoulder e The Peak skim option can be used to skim off the smaller peak with a straight line that starts in the valley betw
132. eased by 50 in this example 416 UNICORN 5 31 User Reference Manual 28 9972 39 AA 12 Evaluation 12 1 Peak integration 12 1 4 How to optimize the baseline with a classic algorithm Slope limit A changed Slope limit will often improve the baseline calculation The Slope limit sets the maximum slope of the curve to define when a peak is recognized A too high Slope limit will cause the up slopes of the peaks to be recognized as baseline segments The example above was improved by the shorter baseline segments but the high slope of the short segments in the region between the second and the fourth peak still makes the baseline unacceptable In the example below the Slope limit is increased by a factor of 2 5 which produces a correct baseline Too high slope limit A too high Slope limit value can cause peak limits too high up on the peaks This can be the case when the chromatogram includes a very large flow through or solvent peak The large peak affects the calculation of the default parameters and leads to too high values for the Slope limit Note A too high value for the Noise window can have the same effect and be caused by the same situation often also in combination with a high Slope limit Peak limits are defined on peaks in the example below due to the high Slope limit UNICORN 5 31 User Reference Manual 28 9972 39 AA 417 12 Evaluation 12 1 Peak integration 12 1 4 How to optimize the baseline with a classic algo
133. eature Description Equation y Ax BK C Mathematical model The constants A B and C are determined by linear least squares regression Minimum number of required points 3 at least 6 points recommended Measuring range for the calibration Within the highest and lowest values for the curve points Note A variant of this model is available for the production of a molecular size curve This uses the logarithm of the molecular size as the x value in the expression above The illustration below is an example of the statistical information for an applied Quadratic curve model UNICORN 5 31 User Reference Manual 28 9972 39 AA C Curve fit models and statistics C 1 Curve fit models Statistics Explained variance 96 990 Number of point s 4 The Quadratic through origin model The table below describes the features of the Quadratic through origin curve fit model Feature Description Equation y Ax BX Mathematical model The constants A and B are determined by lin ear least squares regression Minimum number of required points 2 at least 4 points recommended Measuring range for the calibration From the point with the highest value down curve to the origin The illustration below is an example of the statistical information for an applied Quadratic through origin curve model UNICORN 5 31 User Reference Manual 28 9972 39 AA 623 C Curve fi
134. eccecssssscsssssssssssescssssssssssssessssssessssssessssssssssssessssssesssssuesssssseessssseees 178 6 5 12 The Result Name ta sss sssssssssssessssseressssersssssrrsssssrrsssstrrsssserrsstorssssttrssseerssssttntssnetrasssteraseterssnntna 180 6 5 13 The Frac 950 tab 183 6514 ThesStart Protocol FAD arenneren aaa AERE 185 6 5 15 How to export the values in the Run Setup s ss ss1ss1sssissrisrrssrssrrssrsssrssrissrsserrsnrssnrsrrnnrne 187 6 6 How to use Selected method instructions s ssss ssssssrssssssresessresesrresserrrissesrrssserrrssseeesssseressse 188 6 6 1 Base IAStrUC UO aseenaan a NIE ee eea TEED PaE Eaa ddera iE ett 189 6 6 2 Instructions at the same breakpoint 192 6 6 3 Block and method length essssssssesssssssssccssssssssvsssesssssccsssssssssssssssessecsessssssssssssssssseceesesssssssssesssseseeeesee 193 664 Messages Ond Set Marks irsisnsiiinremiomnianiieiiii E a A AR A 195 6 6 5 Howto CEIGY GC methot asiensaneinaiieeniniaieniiir EEA 197 666 Linear TOW LOBOS eena EA ANA AA A ARE AA 198 6 6 7 Gradients and eluent concentrations 199 6 7 Standard Watch conditions wiceeecccseccsssseessssseessssssessssseesssseessessuesssssusessssutesssssssssuussssuesssuneesssuee 202 6 8 How to save or delete a Method template on ecccssscsssssssssneessesseccssssssssessseessessssessssennees 208 69 TOW LO pim a Method cacescassthcasssscasstsstesstasastestssedeccesssssuss deeb nenscencvonssati aiscscedhsbthsnrsanaaniesssesata ht 210 610
135. eceive the error message Strategy file error in a stand alone installation the strategy file is probably corrupt Reinstall the strategy as described in the Administration and technical man ual Install selected software compo nents after the initial installation Network installation In a network installation the error message Strategy file error may appear if you try to create a method for a system not physi cally 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 UNICORN 5 31 User Reference Manual 28 9972 39 AA 579 A Troubleshooting A 2 UNICORN access A 2 UNICORN access In this section This section describes how to solve the following UNICORN access problems e Unable to access certain UNICORN functions e Connection problems Connections are not available System is not available Error message in a network installation You cannot control the system e Run data Connection in System Control displays a NO 1 NO 2 or NO 3 Unable to access certain UNICORN functions The table below describes an access problem and its solution Problem description Solution UNICORN functions to which you do Choose Administration User Setup in the not have access appear grey in the UNICORN Manager to change the user profile menu and canno
136. ed update with the Average option The table below describes how to automatically update a quantitation table with the Average option 536 Step Action 1 Open a method in the Method Editor 2 Click the Scouting tab in the Run Setup dialog box Click the Clear All button to clear the scouting scheme Double click each Quantitation_Type table cell and select the correct concentration level for the standards Click the Evaluation Procedures tab Select the Update_Quantitation procedure Click the Quantitate button Result The Quantitation table dialog box opens 4 Select the quantitation table from the Global or Personal folder and click OK Result The quantitation table is copied into the Update_Quantitation pro cedure 5 Click the Edit button on the Evaluation Procedures tab Result The Procedure Editor dialog box opens See illustration below 6 Select the existing UPDATE instruction UNICORN 5 31 User Reference Manual 28 9972 39 AA 13 The Analysis module 13 5 Automated quantitation 13 5 3 How to perform automated update Step Action 7 Use the scroll bar in the Parameter field to locate the Average or replace point droplist e Select the AVERAGE option e Click the Replace button to the right of the scroll bar 8 Select File Close in the Procedure Editor dialog box to return to the Run Setup 9 Save the method and perform the runs Result The quantitation table will be updated automaticall
137. ed update so that you can assess its viability See illustration below 7 e Click the Print button for a print out of the Update report and or e Click Save or Save as to save the updated table Update by Average The Average option means that 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 the 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 516 UNICORN 5 31 User Reference Manual 28 9972 39 AA 13 The Analysis module 13 3 How to prepare for quantitation 13 3 3 How to edit and update a quantitation table Update by Replace The Replace option means that 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 The Update Report dialog box The illustration below shows the Update Report dialog box r General
138. edures tab 3 Select Update_Quantitation and click Quantitate Result The Quantitation table dialog box opens 4 Select the quantitation table and click OK 5 Deselect the Update_Quantitation procedure on the Evaluation Procedures tab 6 Repeat steps 3 to 5 for the Quantitate_Sample procedure Note Make sure that both procedures are deselected after this is completed Otherwise they will be run twice 7 Proceed with the instructions how to edit the instructions see table below How to perform automated update in scouting runs step 2 The table below describes how to edit the text instructions Step Action 1 Click the Text Instructions icon 2 Select the last instruction in the method in the Text pane 3 e Click the Other radio button in the Instructions field of the Instruction box e Select Evaluate on the Instructions list 538 UNICORN 5 31 User Reference Manual 28 9972 39 AA 13 The Analysis module 13 5 Automated quantitation 13 5 3 How to perform automated update Step Action 4 e Select Update_Quantitation in the Procedure droplist of the Parameters field Li Coe moanf EE FA fe i e Click the Var button in the Parameters field Result The Variable Name Definition dialog box opens 5 Type a variable name for example Procedure and click OK Result The Evaluate instruction is inserted in the method By defining the evaluation procedure as a variable different procedures
139. een the peaks and ends at the other side of the smaller peak at the point where the skim line and the curve slope are equal The illustration below is an example of how a drop line A and a skimmed peak B affects the area under the main peak and the peak shoulder The peak shoulder area is marked in gray UNICORN 5 31 User Reference Manual 28 9972 39 AA 439 12 Evaluation 12 1 Peak integration 12 1 7 How to integrate part of a curve and how to exclude or skim peaks Drop line How to skim peaks The table below describes how to select a ratio to skim peaks in the Integrate dialog box Step Action 1 Select the Peak skim checkbox 2 Determine the ratio when peak skimming should be applied based on the relationship in the illustration below hp hy gt skim ratio hp2 hy Note The default ratio value is 10 5 Type the ratio value in the text box 440 UNICORN 5 31 User Reference Manual 28 9972 39 AA 12 Evaluation 12 1 Peak integration 12 1 7 How to integrate part of a curve and how to exclude or skim peaks How to integrate part of a curve Part of a curve can be selected in the Edit Peak Table dialog box and integrated with settings that differ from the rest of the curve The table below describes how to do this Step Action 1 e Choose Integrate Edit Peak Table Result The Select Peak Table to Edit dialog box opens e Select the peak table to edit and click OK Result The Edit Peak
140. elected file is added to the tab in the Attachments dialog box Note To remove a file select the check box and click the Delete button 3 To include more information in the report select the appropriate check boxes in the System information field By default all options are checked Computer amp operating system information Asummary of the computer and operating system information for example type of processor processor speed RAM hard disk capacity and printer AKTA hardware information Asummary of the specific AKTAdesign hardware for example the instrument and PROM version for every instrument that is connected Integrity check When UNICORN is installed a checksum calculation is performed on the stationary files dll and exe for the system An integrity check means that a new checksum calculation is performed for the same files in their folders This new calculated value is compared with the checksum value obtained during installation The results of the comparison are presented in the report and any deviations are included Click the Next button Result The Generate report dialog box is displayed 4 Proceed to Step 3 How to generate and save the report below UNICORN 5 31 User Reference Manual 28 9972 39 AA 571 15 System maintenance and error reporting 15 2 How to generate problem reports 15 2 1 How to generate a problem report from the UNICORN Manager Step 3 How to generate and save the report The tabl
141. eline segment e The height of the box corresponds to the maximum level of noise on the baseline segments This is referred to as the Noise window e The box is allowed to be tilted with a maximum slope corresponding to the Slope limit e The box is not allowed to move up above the Max baseline level UNICORN 5 31 User Reference Manual 28 9972 39 AA 595 B Evaluation functions and instructions B 2 Baseline calculation theory Baseline parameters illustration The illustrations below shows the baseline parameters graphically Shortest baseline segment C _ Noise Window Max baseline level Slope limit 596 UNICORN 5 31 User Reference Manual 28 9972 39 AA B Evaluation functions and instructions B 2 Baseline calculation theory Baseline segment identification The table below describes the baseline segment identification process Stage Description 1 The box is virtually moved along the source curve in steps of one third of the Shortest baseline segment length to look for baseline segments 2 A baseline segment is found whenever the currently examined part of the source curve fits completely within the box 3 The found baseline segments are joined by connecting adjacent segments provided that the slope of the joining lines does not exceed the Slope limit Baseline points Classic algorithm When the baseline segments have been defined and joined they are replaced by baseline points at
142. en the controlled value is out of the acceptable range Example PAUSE Message text Free text message that is displayed when the controlled value is out of the acceptable range Example Retention out of range Note All values must be included before the instruction can be inserted UNICORN 5 31 User Reference Manual 28 9972 39 AA 665 F Method examples F 7 Quality control procedure How to add the quality control procedure to a method The table below describes how to add the quality control procedure to a method 666 Step Action 1 e Open the method in the Method Editor e Click the Run Setup icon Result The Run Setup for the method opens 2 e Select the Evaluation Procedures tab e Click the Import button Result The Import dialog box opens 3 e Select the quality control procedure you created and saved Example QC_test in the Select field e Click the Import button Result The quality control procedure is added to the available evaluation procedures e Click the Close button 4 e Click the check box to de select the quality control procedure Note If the quality control procedure is selected it will initiate a new manual run at the end of the method run 5 e Click the Text Instructions icon e Select the last instruction in the method e Select Other Evaluate in the Instructions field e Select the quality control procedure in the Procedure list e Click the Insert button UNICORN 5
143. es Make sure that the curves processed when the procedure is exe cuted 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 588 UNICORN 5 31 User Reference Manual 28 9972 39 AA A Troubleshooting A 5 AKTAdesign system specific problems A 5 AKTAdesign system specific problems In this section This section describes how to solve the following problems e Connected to a system but no system contact e Flow scheme not displayed properly Connected to a system but no system contact The table below describes a problem and its solution Problem description Solution You are connected to a system but have no e Check that the system is turned system contact on Indications In the System Control e Check that all the cable connec e the option Connection in the Run data tions are intact pane says Yes e Ifthe above actions do not help e the option Instruments says Scanning try to restart both the computer and the system e there is no contact with the system after a period of waiting Flow scheme not displayed properly The table below describes a problem and its solution Problem description Solution The flow scheme is not displayed Choose Settings Control Panel Display Settings properly in the Windows Start menu to check that you have selected 65536
144. es Use File Open Curves to copy the second curve to the opened result file Peak integrate the sample curves to produce the peak tables for the unspiked and the spiked samples Note The sample curves must use the same X axis base unit Time is the recommended unit for highest reliability e Check that the integrations are correct e Optimize the peak integration if necessary Select File Save to save the peak table 524 UNICORN 5 31 User Reference Manual 28 9972 39 AA 13 The Analysis module 13 4 How to quantitate the sample 13 4 2 Standard addition quantitation How to select the component and identify the sample peaks The table below describes how to select the component to be used for the standard addition and how to identify the sample peaks Step Action 1 Select Quantitate Standard addition Result The Standard Addition dialog box opens Select pesk table Soca chromatogram Addition chromstogam Added amount _ ee On Peak labke Peak tablets Una label WS1GELovel 1 to 4004 1_UVI_220nen 01 FEAR d1BELevel 10 4004 1_UVI_Z80eem O1 PEAK rg id106Level 1 to 4004 1_UVI_280nnG01 PEAKI W10GLevel 1 10 4004 1 UVI 200rmE0 PEAKI iR Cancel Hee 2 e Select the chromatogram that contains the peak table for the unspiked sample in the Source chromatogram droplist e Select the unspiked sample peak table from the Peak table s list to the left 3 Repeat step 2 in the Addition chrom
145. f GE Healthcare companies third party trademarks are the property of their respective owners 2005 2011 General Electric Company All rights reserved First published May 2011 goods and services are sold subject to the terms and conditions of sale of thcare which supplies them A copy of these terms and conditions is available on request Contact your local GE Healthcare repre sentative for the most current information U CORN Any use of this sofi ware is subject to GE Healthcare Standard Software End User License Agreement for Life Sciences Software Products GE M unzinger Strasse 5 D 791 GE Healthcare UK Limited Amersham Place Little Chal GE Healthcare Bio Sciences 800 Centennial Avenue P O Box 1327 Piscataway NJ 08855 1327 USA GE Healthcare Europe GmbH 1 Freiburg Germany ont Buckinghamshire HP7 9NA UK Corp Healthcare Japan Corporation Sanken Bldg 3 25 1 Hyakunincho Shinjuku ku Tokyo 169 0073 Japan 28 9972 39 AA 05 2011
146. fits the data better than a linear model This would be confirmed by a higher explained variance value Note The explained variance is not calculated for curve models drawn through the origin UNICORN 5 31 User Reference Manual 28 9972 39 AA 627 C Curve fit models and statistics C 2 Statistics Explained variance calculation The explained variance is equal to Re adjusted for degrees of freedom The illustration below shows the mathematical model SSresduals l k 1 SSreail n 7 D Explained variance 100x l Where SSosiduats gt 9Y Residual Sum of Squares i l Soa 2 yY Total Sum of Squares i l eF is the average of all y values e i is a function value using the fitted model For example 3 Ax B C en is the number of points xy pairs ek is the number of x terms in the model For example 1 for Linear and 2 for Quadratic Undefined value for explained variance 628 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 an explained variance value UNICORN 5 31 User Reference Manual 28 9972 39 AA D The Column lis
147. fraction 340 Show only the pooled frac tion curve 343 G Generate Report Wizard How to generate a report from the UNICORN Manag er 569 Gradient Effects of Change and Re place on gradient length 142 Gradients Define length as a vari able 201 Gradient breakpoints 200 Instruction parameters 199 Step gradient instruc tion 200 Text instructions 200 Instant Run How to start 231 Internal standard quantitation How to perform 486 Reliability 488 Suitable components 485 L Linear flow rates Description 198 Logbook How to display an overlay in the chromatogram win dow 283 How to display an overlay in the Curves pane in Sys tem Control 246 How to filter the informa tion 290 Logbook pane Autoscroll function 249 Description 249 How to filter the con tents 250 Search function 250 Log on and log off routines How to log on 57 How to start the pro gram 56 Log off alternatives 57 Log off and set a password for a running process 58 Quit UNICORN after log off 60 Unlock the system 59 M Maintenance How to reset warning coun ters 565 How to set up a mainte nance warning 564 How to use the Generate Report Wizard from the System Control 573 How to use the Generate Report Wizard from the UNICORN Manager 569 How to view maintenance information 563 How to view warning param eters 564 Manual direct commands Buttons in System Con trol 252 UNICORN 5 31 User Referen
148. gn a new value to a system setting below Like the default values the new value can be changed temporarily in a method Note You must have System settings authorization to assign a new value to an actual system setting UNICORN 5 31 User Reference Manual 28 9972 39 AA 555 14 System settings 14 1 General information about system settings How to assign a new value to a system setting 556 The table below describes how to assign a new value to a system setting in the System Control module Step Action 1 Select System Settings Result The Instructions dialog box for the connected system opens The il lustration below shows the dialog box opened with the Alarms group of settings selected m Instructions Alam Piesswe Paraenetets C Alarms Mode Enabled pon b HighSlsem 10 00 MPa C Disabled Enabled C Speciats Low lam 0 00 MPa 5 Wath Alam Pressure High lsma 0 00 10 00 Ciono Fightlsen 1 00MPa how jro Aloim_SamplePressure Lonia 10 00 5 00 Mode Enabled Com ERREA a Set Selected Parametes To Strategy Default Value Corea Heb 2 Click the radio button to select one of the following instruction groups e Alarms e Specials e Monitors e Curves Result The instructions for the group are displayed The parameters are listed below each instruction The title bar of the dialog box shows the select ed instruction group 3 e Select a parameter from the list e Change
149. h of the standard curves Note When integrated all standards must use the same X axis base unit Time is the recommended unit for the highest reliability 4 Check that each integration is correct and consistent 5 Select File Save to save all the peak tables Concentration levels The standard series should include standard concentrations that extend beyond the lower and upper limits of the sample amount If an internal standard is used the internal standard must be added in the same concentration in all standards Methods created from a wizard If the method is created from a wizard 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 Each level is an alias for a specific con centration of the standard The list below describes how the levels are applied e Level 1 should be selected for the standard with the highest or lowest concentration e The levels must be set in consecutive order of changing concentration of the standard e All runs with the same concentration must be given the same level UNICORN 5 31 User Reference Manual 28 9972 39 AA 497 13 The Analysis module 13 3 How to prepare for quantitation 13 3 1 Preparations before quantitation Reject irrelevant peaks If many small irrelevant peaks are detected it may be an advantage to re integrate after adjusting the Reject peaks criteria Th
150. has already been entered and does not change between the levels e Highlight the Amount Concentration for Level 1 e Type the amount or concentration of the component in the standard at this level Note This is the amount corresponding to the injected volume not the total amount used when the standard level was prepared e Repeat this for the other levels for this component Click the Curve model radio button for the best curve model e Linear recommended e Linear through origin e Quadratic e Quadratic through origin e Point to point Result The curve is displayed in the Calibration curve window Each com ponent level is labelled with crosses If more than one run has been per formed for any level all points in that level will be shown The average of these points is calculated and this value is used to produce the calibration curve Repeat steps 3 and 4 for all the remaining components Result The quantitation table is complete with a calibration curve for each component e Save the quantitation table and click Close or e Click the Save as button Result The Save quantitation table dialog box opens Note The Save button is used to save updates in an existing quantitation table However this will overwrite the original table You might prefer to use Save as and create a new name for the edited table to preserve the original UNICORN 5 31 User Reference Manual 28 9972 39 AA 13 The Analysis module 13
151. he corresponding instructions are available as vari ables How to edit column parameters The table below describes how to edit column parameters in the Method Editor Step Action 1 Choose Edit Column List Result The Column List dialog box opens 2 Select a column and click the Edit button Result The Edit Column dialog box opens 3 Select the desired parameters and change the value settings 4 e Click the Save button or e Click the Save as button to save the column under a new name Note If a column has been selected and saved in a method and the parameters for the column are changed later the column in the method will not be updated automatically When you open the method you will be asked if you want to update the parameters The recommendation is that you answer Yes UNICORN 5 31 User Reference Manual 28 9972 39 AA 635 D The Column list D 1 How to edit the Column List How to delete a column The table below describes how to delete a column Step Action 1 Choose Edit Column List Result The Column List dialog box opens 2 Select a column and click the Delete button Result The Delete Column dialog box opens 5 e Click the checkbox for each column you want to delete e Click OK Result The selected columns are deleted How to export a column The column information for a system can be transferred to another by using the export and import functions in the column list The table below des
152. hieve a high positive correlation coefficient A value of 1 indicates a perfect fit of all the data to the straight line A molecular size curve has a negative slope so the aim is towards a correlation coefficient of 1 Too few data points If you only have 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 indicates too few data points In these cases the Statistics table will display a symbol instead of the correlation value 626 UNICORN 5 31 User Reference Manual 28 9972 39 AA C Curve fit models and statistics C 2 Statistics Correlation calculation The correlation is derived as follows D 4 9 Correlation se Where eX is the average of the x value ey is the average of the y value For the molecular size model Linear log Mw ex is the average of the logarithms of the molecular sizes Explained variance Explained variance provides a measurement of how much of the variation in the data points xy pairs is due to the model The remaining variation can be attributed to noise i e random errors or to the fact that an inappropriate model has been selected This makes it possible to use the explained variance value for model selection e g to decide if a quadratic model
153. ification Settings dialog box opens Identification Settings Eg r Peak identification vj and Highest peak maximum Absolute retention Identify peaks on Window widthas Relative Absolute m Reference peak for relative retention Component z Window fO min Cancel Help See How to identify peaks within a window below 3 Select Relative retention on the Identify peaks on droplist See Absolute and Relative window width below 4 Scroll down the Component menu and select the component to be used as the reference peak 5 e Type the window width for the reference peak an absolute value Note Set the width fairly wide to accommodate a larger drift in the retention value Make sure that there are no other large peaks within the window e Click OK Result A column for the relative retention is added in the peak table Ret Ref The column displays the 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 UNICORN 5 31 User Reference Manual 28 9972 39 AA 505 13 The Analysis module 13 3 How to prepare for quantitation 13 3 2 How to create a quantitation table How to identify peaks within a window Quantitate must be advised of how the peaks are to be identified if
154. imize the baseline with a classic algorithm Introduction The first choice when you want to optimize the peak integration is to change the baseline parameters This section describes how to optimize the baseline with a classical algorithm What is the Classic algorithm The Classic algorithm searches for all parts of the source curve that are longer than a defined minimum baseline segment and fall within limiting parameters Together the parameter values define the limits for a rectangular box A part of the source curve must fit entirely inside this rectangular box to be identified as a baseline segment The Classic algorithm is particularly useful when you need to integrate curves with negative peaks and when quantitative data from negative peaks are important Classic algorithm parameters The parameters for the Classic algorithm are e Shortest baseline segment e Noise window e Max baseline level e Slope limit See more information about the parameters below How to set a Classic baseline The table below describes how to set a Classic algorithm and define a baseline Step Action il Click the Baseline settings button in the Integrate dialog box Result The Settings dialog box opens 414 UNICORN 5 31 User Reference Manual 28 9972 39 AA 12 Evaluation 12 1 Peak integration 12 1 4 How to optimize the baseline with a classic algorithm Step Action 2 e Select the Classic algorithm e Change the Baseline pa
155. ion 2 e Depending on the character of the file to be attached select the appro priate tab Result Method System log or Global log e Attach a file Click the Add button Selecta file in the dialog box and click the Attach or OK button Result The selected file is added to the tab in the Attachments dialog box Note To remove a file select the checkbox and click the Delete button 3 To include more information in the report select the appropriate check boxes in the System information field By default all options are checked Computer amp operating system information Asummary of the computer and operating system information for example type of processor processor speed RAM hard disk capacity and printer AKTA hardware information Asummary of the specific AKTAdesign hardware for example the instrument and PROM version for every instrument that is connected Integrity check When UNICORN is installed a checksum calculation is performed on the stationary files dll and exe for the system An integrity check means that a new checksum calculation is performed for the same files in their folders This new calculated value is compared with the checksum value obtained during installation The results of the comparison are presented in the report and any deviations are included Click the Next button Result The Generate report dialog box is displayed 4 Go to step 3 below UNICORN 5 31 User Reference Man
156. ion is fulfilled Method execution continues issuing a Watch_UV command Again the method is put on Hold until the Watch condition is fulfilled Note Even though the line 650 UNICORN 5 31 User Reference Manual 28 9972 39 AA F Method examples F 1 Simple equilibration Watch_Cond Less_than 5 mS cm Continue is in the method placed before 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 that will be executed at its breakpoint while Continue is not an instruction but rather an action for the Watch instruction Evaluation of the method 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 ap pendices Section F 2 Equilibration with simple safeguard on page 652 and Section F 3 Equilibration with extra safeguard on page 653 UNICORN 5 31 User Reference Manual 28 9972 39 AA 651 F Method examples F 2 Equilibration with simple safeguard F 2 Equilibration with simple safeguard Introduction This section contains an example of how a Watch instruction for simple safeguard can be inserted into a method Example instruction This is an example instruction as it would be presented in the Text pane 0 00 Block EQUILIBRATE Equilibrate oO 0 Base SameAsMain co 0 Watch
157. it Open in the Evaluation module Result The Open Procedure dialog box opens 2 e Select the procedure Global Integrate_and_Print e Click the OK button Result The Procedure Editor opens with the procedure displayed 3 e Select the REPORT instruction in the procedure e Choose Other and QC_TEST in the Instruction field 4 e Type appropriate values in the Parameter field See QC_TEST Parameter descriptions below e Click the Replace button Result The REPORT instruction is replaced by the QC_TEST instruction UNICORN 5 31 User Reference Manual 28 9972 39 AA 663 F Method examples F 7 Quality control procedure Step Action 5 e Choose File Save As Result The Save As dialog box opens e Type a name for the procedure for example QC_test e Select the Global procedure check box if the procedure is to be available to all users Note If you select File Save to save the procedure it will replace the Global Integrate_and_Print procedure Illustration The Procedure Editor The illustration below shows the Procedure Editor with the QC_TEST instruction displayed amp Procedure Editor Integrate_and_Print Fie BASE TIME REJECT_PEAKS OFF OFF OFF OFF 20 NEGATIVE_PEAKS OFF CALCULATE_BASELINE_MORPH 01 17 DEFAULT DEFAULT DEFAULT SUB 01 17 47 PEAK INTEGRATE 47 4 OC TEST 4 0 100 RETENTION 1 RETENTION 10 11 PAUSE Retention out of range QC_TEST parameter descripti
158. it may be better to split them into two or more standard runs e Peak integrate the curves to produce peak tables The standard curves must all use the same X axis base unit during the integration Volume is the recommended unit for molecular size determination e Save the results The Molecular size table dialog box This dialog box is used to select the peaks that will be used to produce the molecular size curve Each curve and its peak table name is color coded All the available peaks for all the curves are listed together in the Retention Mol size table riangles show that a peak has been selected The name of its source peak table is shown above the curve window This is useful when you wish to know which peak has been selected of two closely spaced peaks from different peak tables The illustration below shows the Molecular size table dialog box 546 UNICORN 5 31 User Reference Manual 28 9972 39 AA 13 The Analysis module 13 6 How to measure molecular size 13 6 2 How to determine the molecular size Peak table id 231 wizard size exchssion001 10_UV1_215nemn 01 PEAK1 Double click on a peak lo select it 12 400 1 355 0 243 C Linear Linear log Mw Quetatic Quadratic log Mw Poink to point Paint to point flog Mw r Stalistics Explained variance 98 800 Correlation 0 9952 log Mol size me Print Save Saveas_ Help UNICORN 5 31 User Referen
159. l peaks are detected if the Noise window is decreased see example below Note Missing peaks can also be caused by improper settings for Reject peaks in the In tegrate dialog box or Filter peaks in the Chromatogram layout dialog box 420 UNICORN 5 31 User Reference Manual 28 9972 39 AA 12 Evaluation 12 1 Peak integration 12 1 4 How to optimize the baseline with a classic algorithm When to change the Max baseline level In rare cases the top of a broad flat peak can be incorporated as a baseline segment This is one of the very few situations where it is useful to change the Max baseline level The illustration below is an example How to set the Max baseline level The table below describes how to set the Max baseline level Step Action 1 Right click in the chromatogram and select Marker Result A vertical line is set in the chromatogram A text box in the top left corner of the chromatogram displays the X axis and Y axis values of the curve at the point where the vertical Marker line crosses the curve 2 e Move the Marker with your mouse e Measure the height of the peak you want to exclude from the baseline 3 Choose Integrate Calculate baseline UNICORN 5 31 User Reference Manual 28 9972 39 AA 421 12 Evaluation 12 1 Peak integration 12 1 4 How to optimize the baseline with a classic algorithm Step Action 4 e Select the Classic checkbox as the Chosen algorithm e Type anew value for Max baseline
160. l the system that is the Manual menu commands in the System Control are grey Check that no other user has a control mode connection Check that you have sufficient access rights to control the system manually Note The Method Wizard can be used on a local system even if the network connection is not established UNICORN 5 31 User Reference Manual 28 9972 39 AA 581 A Troubleshooting A 2 UNICORN access The Connection field in System Control displays a NO X The table below describes some connection problems and their solutions Problem Description Solution The Connection fieldin e Check that the UNICORN PC Control board is config the Run data pane in ured according to the settings made during the instal System Control says lation of the program The same Control unit number NO 1 or NO 2 Address and IRQ must be set at the Control board see the Administration and technical manual Hardware installation e The communication may also fail if there is a conflict between the UNICORN PC Control board configura tions and other boards in the PC If so select a free Address and a free IRQ during UNICORN installation and at the Control Board see the Administration and technical manual Hardware installation The Connection fieldin e Choose Administration System Setup in the UNICORN the Run data pane in Manager System Control says NO 3 Select the system
161. lculate baseline based on 401 BufferPrep Buffer concentration 641 Description 174 How to adjust the correction factors 176 How to change correction factors 647 How to create a method 175 How to create a recipe 639 How to define a new buffer substance 642 How to define a new salt 643 How to edit a recipe 645 How to select the pH range 641 How to verify correction factors 646 Stock solutions 175 C Calibration curve How to update 515 Chromatogram Layout Curve tab 287 Default curve names 287 How to choose curve name appearance 287 The Curve Style and Color tab description 289 Chromatograms Commands to import curves from result files 371 Description 276 UNICORN 5 31 User Reference Manual 28 9972 39 AA How to add annota tions 338 How to add a second Y ax is 292 How to apply a layout 295 How to change and fix the X axis 293 How to change and fix the Y axis 292 How to change the size of fraction marks 299 How to change the size of injection marks 299 How to change the size of logbook marks 299 How to copy curves into a new 377 How to cut a curve and store as new 297 How to display several simul taneously 370 How to edit annotation text 338 How to import curves with File Open 376 How to import curves with File Open to compare 372 How to make layout changes general 286 How to open several to compare 367 How to open several with the File Open com ma
162. lculation 527 How to prepare for stan dard addition 524 How to rename the ta ble 518 How to select an Internal Standard 509 How to select table compo nents 502 How to select the standard addition component 525 How to update a calibration curve 513 How to use an external standard 482 How to use an internal standard 486 How to use recovery calcu lation 492 How to use standard addi tion 489 How to view the results 523 Internal standard descrip tion 485 Peak identification 503 Peak identification crite ria 506 Preparations before 497 Procedure instructions 480 Process steps 478 Recovery calculation de scription 492 Recovery calculation relia bility 494 Reject peaks 498 Relative retention 504 Standard addition descrip tion 489 Standard addition reliabili ty 491 Standard addition stages 524 Standard concentration levels 497 Statistics 510 Quick View How to preview result files 87 UNICORN 5 31 User Reference Manual 28 9972 39 AA R Recovery calculation How to perform 492 Reliability 494 Recovery factor calculation Error signs 529 How to calculate the recov ery 528 How to prepare for recovery calculation 527 Reference curves In Run Setup 171 Rename files and folders 95 Reports Edit mode toolbar but tons 304 How to add documenta tion 312 How to add free text 308 How to add objects to a re port 307 How to add or delete pages 305 How to add picture
163. lect Properties Select BuffPre_pH on the Run Data Groups panel and click the OK button UNICORN 5 31 User Reference Manual 28 9972 39 AA E How to create and edit BufferPrep recipes E 2 How to edit a BufferPrep recipe Step Action 6 e Select Gradient in the Instructions list e Type 100 in the Target parameter box 0 in the Length parameter box and click Execute Result The gradient instruction is added 7 Check the pH reading when it is stable at 100 See How to change the Correction factors below How to change the Correction factors If the readings described in the instruction above are acceptable at both 0 and 100 the Correction factors do not need to be changed If the Correction factors do not produce an acceptable result they must be adjusted in the Method Editor module The table below describes how to change the Correction factors Step Action 1 Choose Edit BufferPrep Recipes Result The BufferPrep Recipes dialog box opens 2 Select the recipe from the Recipe droplist and click the Edit button Result The Edit Recipe dialog box opens 3 Click the Correction factors button Result The Correction Factors dialog box opens e Type the deviation at 0 and 100 Example If the pH is set to 7 0 and the actual pH is 7 1 the Correction factor is 0 1 If the actual pH is 6 9 the Correction factor is 0 1 e Click OK e Click the Save button or the Save as button to save the recipe
164. libration with simple safeguard 652 F 3 Equilibration with extra safeguard 653 F 4 Collection of absorbance peaks 655 F 5 Collection of three absorbance peaks 657 F 6 Messages 660 UNICORN 5 31 User Reference Manual 28 9972 39 AA F Method examples Section See page F 7 Quality control procedure 663 UNICORN 5 31 User Reference Manual 28 9972 39 AA 649 F Method examples F 1 Simple equilibration F 1 Simple equilibration Introduction This section contains an example of how a Watch instruction for simple equilibration can be inserted into a method Example instruction This is an example instruction as it would be presented in the Text pane 0 00 0 0 0 0 00 0 00 0 10 0 10 10 O Block EQUILIBRATE Equilibrate Base SameAsMain Watch_Cond Less_than 5 mS cm CONTINUE Hold Watch_UV1 Less_than 100 mAU CONTINU Gl Hold End_Block If you are not using AKTA instruments If you are not using AKTA instruments a delay should be added after the Hold Pause instruction so that the following instruction will not be executed simultaneously with the Hold Pause instruction This is what happens The table below describes what happens in the above example Stage Description 1 The Watch is started on the conductivity signal and the method is then put on Hold Continue is issued and Watch_cond is turned off automatically when the Watch_cond condit
165. lick in the active chromatogram e Select Properties from the shortcut menu Result The Chromatogram Layout dialog box opens 2 Click the Peak Table tab 3 e Select options from the Select peak table columns list e Click OK Result The selected items will be displayed in the peak table How to filter peaks from view Peaks can be removed from display in a peak table The table below describes how to filter the peaks Step Action 1 e Right click in the active chromatogram or peak table e Select Properties from the shortcut menu Result The Chromatogram Layout dialog box opens 2 Click the Peak Table tab 3 e Click the check boxes in the Filter Peaks field to select the filter criteria e Specify filter values e Click OK 406 UNICORN 5 31 User Reference Manual 28 9972 39 AA 12 Evaluation 12 1 Peak integration 12 1 2 How to perform a peak integration To filter peaks vs to reject peaks The table below describes the major differences in the effect of filtering peaks compared to excluding the peaks by rejection Filter peaks Reject peaks excludes the peaks from display permanently excludes peaks from the in tegration does not exclude the peaks from the excludes the peaks from the calculation calculation of the total peak area of the total peak area can be reversed cannot be reversed Peak labels Peaks can be labelled with their retention sequentially numbered or be
166. lid operation when the procedure is run Any subsequent instructions in the procedure will not be executed Address the right curves Curves are identified only by their storage position An instruction can become invalid if it addresses the wrong curve Example e The instruction ADD 01 02 03 will try to add curve 01 to curve 02 and store the result in position 03 e Acurve in position 03 that is not a raw data curve will be overwritten e Araw data curve in position 03 cannot be overwritten and the procedure will be stopped at that point UNICORN 5 31 User Reference Manual 28 9972 39 AA 465 12 Evaluation 12 3 Automated evaluation procedures 12 3 2 How to edit a procedure Default values for classic baseline instructions When a classic or morphological algorithm is used to calculate a baseline UNICORN will suggest default values for the four control parameters 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 param eters Example e CALCULATE_BASELINE 01 06 XXX XXX XXX XXX Can be changed to e CALCULATE BASELINE 01 06 DEFAULT DEFAULT DEFAULT DE FAULT Global procedures 466 It is not advisable to edit existing global procedures Open the global procedure instead and save a copy under a new nam
167. llation The Analysis module in a network One or several computers in a network may have the Analysis module installed The module does not need to be installed on all network computers that run UNICORN All installations must be made in accordance with the license agreement UNICORN 5 31 User Reference Manual 28 9972 39 AA 13 The Analysis module 13 2 Quantitation overview 13 2 Quantitation overview Introduction Quantitation is used to determine the amount or concentration of components in a sample This section is an overview over quantitation in general and the four quantitation techniques that the Analysis module provides The section also contains information about the reliability of quantitation In this section This section contains these sub sections Section See page 13 2 1 General information about quantitation 478 13 2 2 External standard quantitation 482 13 2 3 Internal standard quantitation 485 13 2 4 Standard addition quantitation 489 13 2 5 Recovery calculation 492 13 2 6 General reliability factors for the quantitation techniques 495 UNICORN 5 31 User Reference Manual 28 9972 39 AA 477 13 The Analysis module 13 2 Quantitation overview 13 2 1 General information about quantitation 13 21 General information about quantitation Introduction This section is a brief presentation of the quantitation techniques that the Analysis module provides The section also contains an outline of the steps in
168. lobally available Click the Personal radio button to display the tables that are re stricted to your own user ID e Click OK Result The Molecular size table dialog box opens UNICORN 5 31 User Reference Manual 28 9972 39 AA 13 The Analysis module 13 6 How to measure molecular size 13 6 2 How to determine the molecular size How to rename a molecular size table The table below describes how to rename an existing molecular size table Step Action 1 Select Mol Size Edit Mol Size Table Rename Result The Rename molecular size table dialog box opens 2 e Select Personal to display the tables that are restricted to your own user ID if needed e Select the molecular size table you wish to rename in the Molecular size table s list e Click in the Molecular size table name text box and type a new name e Click the Rename button Note You must have Edit global list s rights to be able to rename global tables How to delete a molecular size table The table below describes how to delete an existing molecular size table Step Action 1 Select Mol Size Edit Mol Size Table Delete Result The Delete molecular size table dialog box opens 2 e Select Personal to display the tables that are restricted to your own user ID if needed e Select the molecular size table you wish to delete in the Molecular size table s list e Click the Delete button e Click the Yes button to confirm Note
169. lope values The slope values can be used in the Method Editor e as StartSlope and EndSlope values in the Peak_FracParameters instruction e as parameters for the Watch instruction Using slope values for Watch instructions The slope values can be used in the Method Editor as parameters for the Watch instruc tion Conditional Watch instructions can be set up to let the progress of a run be determined by the events during the run e g start to collect fractions when the first peak emerges The slope of the curve can be set as a condition to satisfy a Watch condition in the method during the run It is important to use accurate slope values for the specific Watch instruction parameter Asample run You must first make a separation run with the sample you intend to purify The result from this separation run is then used to find the slope values Retention scale Time should be used as the X axis scale for retention Step Action 1 Click the Chromatogram Layout icon UNICORN 5 31 User Reference Manual 28 9972 39 AA 451 12 Evaluation 12 2 Other evaluations 12 2 2 How to find slope values Step Action 2 e Click the X axis tab e Select Time e Click OK How to differentiate the curve The slope values are measured on a differentiated curve The table below describes how to create a differentiated curve Step Action 1 Select Operations Differentiate Result The Differentiate dialog box opens
170. lowest values for the points Note A variant of this model is availab e for the production of a molecular size curve This uses the logarithm of the molecular size as the x value in the expression above The illustration below is an example of the statistical information for an applied Linear curve model Model F A x B Model function values 4 4 92378E 001 B 7 49572E 001 Explained variance 99 989 Correlation 1 0000 Number of point s 4 Help The Linear through origin model The table below describes the features of the Linear through origin curve fit model Feature Description Equation y Ax Mathematical model The constant A is determined by linear least squares regression Minimum number of required points 1 at least 2 points recommended UNICORN 5 31 User Reference Manual 28 9972 39 AA 621 C Curve fit models and statistics C 1 Curve fit models Feature Description Measuring range for the calibration From the point with the highest value down curve to the origin The illustration below is an example of the statistical information for an applied Linear through origin curve model Statistics x Model F A Model function values A 2 22192E 000 Correlation 0 9949 Number of point s 4 Help The Quadratic model 622 The table below describes the features of the Quadratic curve fit model F
171. marked with specific identification names See table below for an instruction on how to display peak labels The label type can be selected on the Curve Style and Colour tab in the Chromatogram Layout dialog box De select all label options to hide the labels e g for presentations The illustration below shows the Chromatogram Layout dialog box with the Curve Style and Colour tab opened UNICORN 5 31 User Reference Manual 28 9972 39 AA 407 12 Evaluation 12 1 Peak integration 12 1 2 How to perform a peak integration Chromatogram Layout 1 x Header CurveNames YAss XAxis Curve PeakTable Curve Style and Colour Edit Texts Layout Library Select curve to modify colour and linestyle for Colour Line style 01 id1 48Quantitate001 1_UV1_280nm 02 id148Quantitate001 1_UV 2_Onm 03 id 48Quantitate001 1_UV3_Onm 04 id148Quantitate001 1_Cond Be DS id148Quantitate001 1_Cond E T DE id 48Quantitate001 1_Cone 07 o 08 id1 48Quantitate001 1_Pressure A idi AN ee ih he UIT A ee xl E rm Peak label m Fraction text alignment p Logbook text alignment Text alignment I Number Vertical Vertical Vertical M Peak Name i Horizontal C Horizontal Horizontal M Retention l C Fly Over C Flg Over Filter T Apply to all chromatograms Cancel Hep How to display peak labels The table below describes how to display peak labels Step Action
172. module 13 3 How to prepare for quantitation 13 3 2 How to create a quantitation table Examine the components The Define Component s dialog box initially displays the components from level 1 1 that is the peak table from the highest or lowest concentration of the standard The Show curve for level list is used to examine the curve for each standard run The size of the components are reduced or increased progressively as you select levels further down on the list which reflects the decreasing or increasing concentration of the stan dard If an internal standard has been incorporated its peak remains about the same size on each level Peaks detected during the peak integration Each component peak that was detected during the peak integration i e that is present in the peak table is identified by a lower triangle black in level 1 1 green in other levels There may be different peaks detected for different levels Upper triangles will later identify the peaks that are selected for quantitation Step 2 How to select and define components The table below describes how to select and define the components Step Action 1 Select level 1 1 in the Show curve for level list and click a peak Result The peak is highlighted in the table e Double click the peak or e Click the Include button Result The peak is selected for quantitation marked with an upper triangle and component no is listed as the Component name
173. n 100 mAU Peak_2 0 00 Watch_UV Less_than 100 mAU End_collect 0 00 End_block Here the second Watch instruction will annul the first since a signal can only be watched for one condition at a time 656 UNICORN 5 31 User Reference Manual 28 9972 39 AA F Method examples F 5 Collection of three absorbance peaks F 5 Collection of three absorbance peaks Introduction This section contains an example of how to collect three absorbance peaks through outlets F3 F5 and F7 with waste fractions through outlets F4 F6 and F8 The maximum number of peaks collected in this example is three due to the limited number of positions on the outlet valve Recommendations Waste container needed 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 Condition for UV threshold The UV threshold for collecting the waste fraction must be below the threshold for col lecting the peak fraction so that the waste condition will not be fulfilled simultaneously or immediately after peak collection Example instruction This is an example instruction as it would be presented in the Text window 0 00 Block Eluate_Fractionation Eluate_Fractionation 0 00 Base SameAsMain 0 00 Watch_UV1 Greater_Than 5 mAU Peak Peak 0 00 Base SameAsMain 0 00 OutletValve Feed 0 00 Watch_UV1 Less_Than
174. n height for the peak integration calculation of the HETP value The Column height parameter is the height of the column in centimetres If Column height is OFF then the HETP value is not calculated for the following inte grations SET_COLUMN_VO Sets void volume for Kav peak integration calculation SET_COLUMN_VT Sets the total liquid volume for peak integration calculation of the capacity factor SET_SKIM_SIZE_RA TIO Sets the Skim size ratio to be used in the following peak in tegration s WINDOW_PEAK_IN TEGRATE Integrates the curve within the peak window All curve parts outside the peak window remain unchanged File operation The table below contains a list of instructions for file operations Instruction Description 610 CURVE_OPEN Opens the curve specified in the Result file defined in File name and stores it in target curve position If is entered as File name the current result file will be used The File name param eter may include a path from the users root folder IMPORT_CURVE Imports a curve to the current chromatogram from another chromatogram in the current file and stores it in the target curve position IMPORT_PEAK Imports a peak table to the current chromatogram from another TABLE chromatogram in the current file and stores it in the target curve position PEAK Opens the specified Peak table in the Result file defined in File TABLE_
175. nd 369 How to print active chro matograms 300 How to rename 349 How to save a layout 294 How to set a reference point 445 Temporary chro matogram 276 The command File Open to compare 368 Chromatogram window How to display a vertical marker 282 How to display header infor mation 279 UNICORN 5 31 User Reference Manual 28 9972 39 AA Index How to display the Logbook overlay 283 How to optimize the workspace 281 Shortcut menu 281 Classic algorithm Definition 414 595 How to measure baseline segments 598 How to measure noise lev el 598 How to measure the slope imit 599 How to set 414 How to set Max baseline evel 421 Missing peaks 420 Noise window 419 Parameters 414 Shortest baseline seg ment 416 Slope limits 417 When to change the Max baseline level 421 Columns Column prompt in manual instructions 257 How to add a new 632 How to delete 636 How to edit parame ters 635 How to export 636 How to import 637 Normal column parame ters 633 Concentration levels Levels in quantitation defini tion 501 Conditional call Description 121 Correlation Explanation 626 Curve fit models Linear 621 Linear through the ori gin 621 Point to point 624 Quadratic 622 Quadratic through ori gin 623 669 Index 670 Curves Calibration curve mod els 620 Commands to import curves into a chromatogram 371 Curv
176. nformation about system settings 555 14 2 Alarms 558 14 3 Curves 560 554 UNICORN 5 31 User Reference Manual 28 9972 39 AA 14 System settings 14 1 General information about system settings 14 1 General information about system settings System settings The system settings e define settings for alarms and warnings e select the data that will be stored in result files When to change the system settings Each system has a set of default settings e Changes to the default settings should be made when the system is installed Certain system settings may need to be adjusted in the following cases e If system components are changed e g the alarm and warning limits e For specific separation runs e g the monitor and curve settings Note Only the settings for the selected components will be shown for strategies where you select the system components How to change the default settings The table below describes the two different ways to change the default system settings Change Effect To assign a new value to a parameter within a method The specific change is valid only until End in the method After End the parameter returns to its default setting Note Only some parameters can be changed in the method To assign a new value to the system setting The new value is valid for all runs and remains until you change the value again or return the setting to its de fault value See How to assi
177. njection dialog box 2 Proceed with the following dialog boxes in the Method Wizard and click the Finish button on the last dialog box Result The Run Setup opens UNICORN 5 31 User Reference Manual 28 9972 39 AA 531 13 The Analysis module 13 5 Automated quantitation 13 5 1 How to set up for automated quantitation 532 Step Action 3 Click the Scouting tab See illustration below e Select the Quantitation_Type variable from the Scouting Variables dia log box e Select other scouting variables of interest e g Sample_ID Vial_Number etc 4 e Double click the Quantitation_Type variable table cell e Select the correct standard concentration level Note This corresponds to the text instruction QuantitationData You can also set this level after the run has been completed For more information about scouting see Section 7 1 How to set up a Scouting Scheme on page 214 5 Click the Evaluation Procedures tab e Select the Integrate_and_Print procedure Result This procedure will automatically use default baseline settings and integrate the first UV curve 6 Save the method 7 Perform all the standard runs 8 In the Evaluation module select Quantitate Edit Quantitation Table New 9 Create a quantitation table manually from the standard runs See Sec tion 13 3 2 How to create a quantitation table on page 499 UNICORN 5 31 User Reference Manual 28 9972 39 AA 13 The Analysis module 13 5 Automated quantitation 13 5 1 How
178. non fraction curve source curve 1 and a fraction curve source curve 2_frac and stores the result in the target curve position If source curve 2 is nota fraction curve arun 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 integration of the source curve and stores the result in a Result curve This instruction is not the same as Peak integrate which performs a real peak integration POOL_FRACTIONS Pools fractions from the source curve and stores the result in the target curve position The fractions are pooled from the first selected fraction to the last selected fraction If the source curve is not a fraction curve or First or Last is not an existing identi fication a run time error will occur LATE_PEAK_FRAC RET_MUL Multiplies the retention of the source curve by the Multiplication factor and stores the result in the target curve position RET_SHIFT Shifts the retention of the source curve by the Shift factor and stores the result in the target curve position SIMU Simulates Peak Fractionation SMOOTH_AR Smooths the source curve with an autoregressive filter and stores the result in the target curve position The Filter param eter decides the strength of the filter SMOOTH_MA Smooths the source curve with a moving average filter and stores the result in the Resulting Curve The Filter width param
179. ns for curve operations Instruction Description ADD Adds two curves to produce 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 AMP_MUL Multiplies the amplitude of the source curve by the multiplica tion factor and stores the result in the target curve position AMP_SHIFT Shifts the amplitude of the source curve by the shift factor and stores the result in the target curve position CLEAR Clears the specified curve from the working memory of the computer COPY Copies the source curve to the target curve position CUT Cuts out the part of the source curve between the Left and Right limits and stores the result in the target curve position DERIVATE Differentiates the source curve first or second order and stores the result in the target curve position The Y axis of the target curve position will be a normalized scale without unit DIV Divides two curves to produce a third curve which is the quo tient of the two curves The two source curves can have any Y axis unit The Y axis of the target curve position will be a nor malized scale without unit UNICORN 5 31 User Reference Manual 28 9972 39 AA 607 B Evaluation functions and instructions B 4 Procedure instructions Instruction Description HISTOGRAM Creates a histogram from any
180. nt on the calibration curve See illustration below mau 240 200 Standard Internal standard 150 we One of three levels ad N 150 15 5 16 0 6 5 17 0 min Ares Standard Area ntemal Standard Area Standard Area Interna Standard Amount sndard Amount intemal amp ndard UNICORN 5 31 User Reference Manual 28 9972 39 AA 487 13 The Analysis module 13 2 Quantitation overview 13 2 3 Internal standard quantitation Step Action 6 e Prepare data from the sample in the same way as the data from the standard runs to produce peak sizes relative to the internal standard peak size e The resulting relative value is applied to the calibration curve to determine the amount and concentration of the component of interest See illustration below intemal stan dard Area component of interest Area Interna Standard Amount component of interest Amount Internal Standard Reliability Internal standard quantitation is potentially the most reliable of the quantitation tech niques However if the internal standard component is not selected carefully the relia bility will probably be worse than with the external standard technique There are some specific factors that can affect the reliability e There is an increased risk of overlap when the extra component the internal standard is added if the sample contains many peaks e The addition of the internal standard must be accurate in both the standard
181. ntal line and the line will now form a curve with a plateau The center point in the plateau formed by the horizontal line will be the data point for the baseline e The data points fulfil the Minimum distance between data points This parameter reduces the total number of data points that are created from a curve 594 UNICORN 5 31 User Reference Manual 28 9972 39 AA B Evaluation functions and instructions B 2 Baseline calculation theory Classic algorithm The Classic algorithm searches for all parts of the source curve where e The curve parts are longer than the Shortest baseline segment This parameter determines the minimum length for a part of the source curve to be considered a possible baseline segment e The curve has no point outside the Noise window The noise window is defined as a rectangular corridor parallel to the slope of the curve and centered on the first and last points within the currently inspected segment e The slope is less than the Slope limit This limits the maximum slope of the baseline to differentiate baseline segments from peaks e The curve parts are lower than the Max baseline level This parameter determines the highest acceptable signal level for the baseline Baseline parameters The baseline parameters can be illustrated as a rectangular box that the source curve has to fit into in order to be identified as a baseline segment where e The length of the box corresponds to the Shortest bas
182. ntitation the injection volume must be the same as used for the standard runs Click the OK button Result The peak table is updated UNICORN 5 31 User Reference Manual 28 9972 39 AA 13 The Analysis module 13 4 How to quantitate the sample 13 4 1 External and internal standard quantitation How to view the quantitation results The results of the quantitation are shown in the Concentration and Amount peak table columns of the Evaluation module The Peak Names are shown in the table and the type of quantitation is also listed See illustration below ID 26500210 UV2_215nm 02PEAKI No Peak nare Retention min Area mwAU tmin Height mat Cone mg ml Amount mg 1 8 49 106 7160 584 031 ene i 2 Component 2 9 24 75 4914 466 665 7 957 0 398 3 Cowponent 3 9 77 98 1309 593 788 7 939 0 397 _4 Component 4 10 45 175 9518 694 142 7 900 0 395 5 6 Tota nurber of detected peaks 60 7 Total area mAU win 886 0878 8 Acea in evaluated peaks mAU min 456 2401 9 Ratio peak area total area 0 514893 10 Tovat peak width min 2 49 11 Column height cm 5 00 12 Calculated from ID 265002 10_UV2_215nm 13 Basetine ID 265002 10_UV2_21Snr 02 BASEN 14 Peak rejection on 15 Maximum number of peaks 20 16 Ine standard applied 17 The quantitation table used for the quantitation When the result file is saved it includes the quantitation table that was used for the quantitation You can view the t
183. ntrol Toolbar Manual command buttons availability 252 Manual command buttons functions 253 System Access buttons 254 Windows buttons 254 UNICORN 5 31 User Reference Manual 28 9972 39 AA Index System data How to backup 78 How to restore backup da ta 80 System operation Automated workstation lock or logoff 59 Connection modes 73 How to connect to a sys tem 72 How to disconnect a sys tem 74 How to leave control of the system 74 Unlock system locked by other users 59 Unlock the system 59 System settings How to assign a new val ue 556 How to change default set tings 555 System summary 75 T Temporary chromatogram Description 276 Text instructions Gradient breakpoints 200 Gradient instructions 200 Hold_until instruction 197 Hold instructions 197 How to form a step gradient instruction 200 How to insert a Watch in struction 202 Linear flow rates 198 Message instruction 195 Pause instruction 197 Set_Mark instruction 196 Watch instructions 202 Watch parameter op tions 203 Text Instructions Editor How to edit instruc tions 106 How to select panes 115 Save a new method 108 When to use 106 Toolbar icons 679 In the System Control mod ule 38 Troubleshooting Access problems 580 AKTAdesign system prob lems 589 Connection problems 580 Evaluation procedure aborts 588 Incorrect time and date 588 Logon problems 578 Method problems 583 Strategy file
184. oint differs from the existing size The Limit column displays the set limit for the deviation The default value is 12 5 of the existing peak size You can edit the Limit value Use the Deviation and limit as radio buttons to specify if both of these columns are expressed in Absolute or Relative units Instruction The table below describes how to use the Update Calibration Curve dialog box for cali bration curve updates Step Action 1 Choose to update by Average or by Replace The same selection applies to all components See explanations for the options below this table UNICORN 5 31 User Reference Manual 28 9972 39 AA 515 13 The Analysis module 13 3 How to prepare for quantitation 13 3 3 How to edit and update a quantitation table Step Action 2 Select each component table rows in turn and check that the new point falls within acceptable limits 3 Click the Statistics after update button Result The Statistics after update dialog box opens 4 e Use the statistical data to check the curve model Note 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 e Click OK to close the Statistics after update dialog box 5 Repeat steps 2 4 for each component 6 Click OK Result The Update report dialog box opens This report provides a summary of the propos
185. ollowing information in the dialog box e a short description of the problem e the circumstances under which the problem occurs e the consequences of the problem Click the Next button Result The Reproducibility dialog box opens UNICORN 5 31 User Reference Manual 28 9972 39 AA 569 15 System maintenance and error reporting 15 2 How to generate problem reports 15 2 1 How to generate a problem report from the UNICORN Manager Step Action 5 Specify whether the problem is reproducible or not Select one of these al ternatives e Yes Provide a short description in the text box of how the problem can be reproduced e No e Unknown Click the Next button to proceed to attach example files see table below Step 2 How to attach a file You can attach result files method files and or log files to the problem report The table below describes how to attach a file Step Action 1 The Attachments dialog box is displayed Attachments 570 UNICORN 5 31 User Reference Manual 28 9972 39 AA 15 System maintenance and error reporting 15 2 How to generate problem reports 15 2 1 How to generate a problem report from the UNICORN Manager Step Action 2 e Depending on the character of the file to be attached select the appro priate tab Result Method System log or Global log e Attach the file Click the Add button Selecta file in the dialog box and click the Attach or OK button Result The s
186. olumn 632 UNICORN 5 31 User Reference Manual 28 9972 39 AA The normal column parameters The table below is a list of all the available normal column parameters D The Column list D 1 How to edit the Column List Parameter Unit Comment Height cm e Mandatory e Calculation of N m Diameter cm e Mandatory Column volume nl ul ml or e Mandatory liter e Automatically calculated from Height and Diameter e User cannot set this parameter directly Column volume nl pl ml or e Not mandatory unit liter e The column volume is calculated in the set unit Technique e Mandatory e Decides which technique the column should be available for Vt nl ul ml or e Not mandatory liter e Total liquid volume e Used to calculate the capacity factor after an integration Vo nl ul ml or e Not mandatory liter e Void volume e Used to calculate Kay after integration Max pressure MPa e Mandatory e Used for setting pressure limit in a method automatically Default flowrate nl min e Mandatory ul min ml min or e Used to set the flowrate in a method au liter min tomatically UNICORN 5 31 User Reference Manual 28 9972 39 AA 633 D The Column list D 1 How to edit the Column List Parameter Unit Comment Max flowrate nl min Not mandatory ul min ml min or e Used to give a warning if a higher flowrate liter min is chosen when saving or starting a method Typ peak
187. on for concentration or amount Both concentration and injected amount can be used to produce the calibration curve When analyzing the sample both amount and concentration are calculated Calibration curve The relationship between amount and peak size of a component The relationship can be shown as a curve and as a mathematical expression Level A known amount or concentration of a standard The levels are numbered 1 20 in decreasing or increasing order of concentration Molecular size curve The relationship between molecular size and retention volume for a number of components The relationship can be shown as a curve and as a mathematical expression Molecular size table Allnecessary data required to determine the molecular size of one or several components in a sample The molecular size table con tains the molecular size curve Peak size Used generally as a common term for peak area or peak height Peak table 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 Quantitationta All necessary data required to quantitate one or several compo ble nents in a sample The quantitation table contains calibration curve s and peak identification settings Sample A sample with an unknown concentration of the comp
188. on will be exported and the corresponding parts will be included in the export ed file Chromatogram functions The table below contains a list of instructions for chromatogram functions Instruction Description ATE_NEW_CHROM COPY_CHROM Creates a copy of the specified chromatogram If is used as source then the current default chromatogram is used If is used as destination then a default name will be cre ated for the copy CRE Creates a new chromatogram with the given name If is used for the chromatogram name a default name will be generated and used Note It is a recommendation not to use only numbers as names for new chromatograms DELETE_CHROM Deletes the named chromatogram If trying to delete the current default chromatogram a run time error will be caused OPEN_CHROM Opens the specified chromatogram from the specified file RENAME_CHROM Renames the specified chromatogram If is used as From then the current default chromatogram is used RESTORE_DESTINA TION_ CHROM Resets the destination for the subsequent curve and peak table operations to the default chromatogram Used in pair with the SET_DESTINATION_CHROM instruction SET_DESTINA TION_CHROM Opens the named chromatogram as destination for the subsequent curve and peak operations Used in pair with the RESTORE_ DESTINATION_CHROM instruction UNICORN 5 31 User Reference Manual 28 99
189. onents of interest The concentration is determined by Quantitation For molecular size calculations the sample contains a component or several components of unknown molecular size UNICORN 5 31 User Reference Manual 28 9972 39 AA 475 13 The Analysis module 13 1 General information about the module Term Definition Sample run A chromatographic sample run of a sample to be analyzed Spiking The addition of a known quantity of the component of interest to the sample prior to the sample preparation for the run Standard A defined concentration of one or several components The con centration does not have to be the same for all components in the standard One or several standards are used to produce a calibra tion curve For molecular size calculations the standard contains components of known molecular size Standard run A chromatographic standard run of a specific concentration level of a standard How to install the Analysis module 476 The table below describes how to install the Analysis module Step Action ii Close all other applications 2 Insert the installation CD in the CD drive 3 Open My Computer 4 Double click the CD drive icon Result The file window opens 5 Double click Setup exe 6 Follow the instructions on the screen 7 Remove the CD after the installation is complete Note See the license agreement for information on the legal aspects of the insta
190. ons The table below describes the parameters for the QC_TEST instruction The example values are used in the illustration above 664 UNICORN 5 31 User Reference Manual 28 9972 39 AA F Method examples F 7 Quality control procedure Parameter Description Peak table source The peak table indicated in the PEAK_INTEGRATE instruc tion Example A Left limit The retention value where the control instruction will begin Example 0 Right limit The retention value where the control instruction will end Example 100 Note The control instruction will be applied to the run up to the sequence in the method where the control instruc tion is inserted e The controlled part of the run will end at the Right limit if this retention value is reached before the con trol instruction is reached in the method e If not the controlled part of the run will end when the control instruction is reached in the method Peak selection on The criteria for peak identification Example RETENTION Order number The sequential order number of the peak Example 1 Peak table parameter The peak table parameter that will be tested by the control instruction Example RETENTION Less than Values less than the parameter value will be out of the acceptable range Example 10 Greater than Values greater than the parameter value will be out of the acceptable range Example 11 QC Action The action the system will take wh
191. or e Select the point and click the Delete button or e Select the point right click and choose Delete Point from the shortcut menu 5 e Click the Zoom icon to focus on details in the curve Note Right click and select Reset zoom to return to the full view e Right click in the chromatogram window and select Marker e Position the Marker bar over peaks in the help curve to measure the coordinates Result The coordinates are displayed in the Marker text box in the top left corner of the chromatogram Note Click the Marker text box to display the coordinates for the created curve Click again to return to the help curve coordinates 6 Click OK Result The Save Curve dialog box opens A Type a new name if desired and click OK UNICORN 5 31 User Reference Manual 28 9972 39 AA 457 12 Evaluation 12 2 Other evaluations 12 2 4 How to create curves Curve example The illustration below is an example of a curve created by using the Draw Spline com mand in the Create Curve chromatogram window Create Curve O t o0 1d148 QuantitateOO1 1_UV1_280nm i41 QuandtateO0t 1_CrestesCurve ough How to force the curve through points In cases where you have created a curve and not selected the Spline through option you may want the curve to pass through some of the points that are outside the created curve The table below describes how to force the curve through these points Step Action 1 e Select the
192. otrypsinogen A Calculated from id 16501 1_UV1_280nm Baseline id 18501 1_UV1_280nn 01 BASEC Peak rejection on Maximum number of peaks Recovery applied Current peak filter settings Maximum number of peaks Note The checkbox Do not show global peak table data must be de selected in the Peak Table tab of the Chromatogram Layout dialog box If the recovery cannot be calculated If the recovery cannot be calculated one of the following signs is shown in the peak table Amount column Sign Function gt This means that one of the amounts concentrations is higher than the highest value in the calibration curve i e outside the valid range of the calibration curve lt This means that one of the amounts concentrations is lower than the lowest value in the calibration curve i e outside the valid range of the calibration curve This means that the recovery factor cannot be calculated For example this sign might indicate that the peak could not be identified in both runs UNICORN 5 31 User Reference Manual 28 9972 39 AA 529 13 The Analysis module 13 5 Automated quantitation 13 5 Automated quantitation Introduction Some method wizards designed for quantitation are available for AKTAdesign systems supplied with Autosampler A 900 or A 905 These can be used to quantitate a sample automatically or to update a quantitation table The procedures described in this chapter are designed for
193. ove 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 UNICORN 5 31 User Reference Manual 28 9972 39 AA 659 F Method examples F 6 Messages F 6 Messages When to use a message Messages are used to inform the operator of the progress of the run Messages can also be used for interaction between the operator and the system when necessary A message can be for information in a screen only or it can require a signature before the user can control the system The messages are all added to the logbook text This appendix de scribes how to add a message to a method The appendix also gives two examples of how a message can be used How to add a Message instruction 660 The table below describes how to add a Message instruction to the method Step Action 1 e Select Other in the Instructions field of the Instruction box e Select Message in the instructions list 2 Type a message in the Message text box in the Parameters field 3 Select one of the display options on the Mode menu e Screen i e only a text message is displayed e Noscreen i e the message will not be displayed but only inserted into the logbook e Authorize i e the message will require a signature from the user before the user can interact with the system again 4 e Select a sound on the Sound menu if desired e Click the Insert button Note If
194. ow this table Step Action 1 e Select Integrate Edit Peak Table Result If there are more than one peak table available the Select Peak Table to Edit dialog box opens The name of the baseline on which the peak table was based is displayed at the bottom of the panel 2 e Select the peak table from the list and click OK e Select one or more Help Curves to be displayed for reference if necessary Result The Edit Peak Table dialog box opens Note The Edit Peak Table dialog box will be opened immediately if you select Save and Edit Peak Table as the last step of the peak integration 3 Perform the changes described in the instructions below 4 Click OK Result The Save Edited Peak Table dialog box opens The dialog box displays a suggested name and location for the peak table 5 Confirm the name and location and click OK UNICORN 5 31 User Reference Manual 28 9972 39 AA 427 12 Evaluation 12 1 Peak integration 12 1 6 How to edit the peaks How to adjust the baseline The baseline can be adjusted graphically see also Section 12 1 5 How to edit the baseline manually on page 423 in the Edit Peak Table dialog box The table below describes this Step Action 1 e Click the Set Curve Points icon Result The cursor is changed into a cross 2 Perform the operations below as desired e Click to insert a new data point e Double click on a data point or right click the point and select Delete Point from
195. ow to delete a peak in the Edit Peak Table dialog box Step Action 1 e Click the Edit peaks icon e Click the peak in the curve or in the peak table to select the peak 2 e Right click and select Delete Peaks from the shortcut menu or e Select Edit Delete Peaks Result The peak is deleted and the remaining peaks are renumbered How to add color to a peak The table below describes how to add a fill color and a pattern to a peak in the Edit Peak Table dialog box 430 Step Action 1 e Click the Edit peaks icon m e Move the cursor over the peak you want to edit Result The cursor is changed into a larger arrow e Click to select the peak UNICORN 5 31 User Reference Manual 28 9972 39 AA 12 Evaluation 12 1 Peak integration 12 1 6 How to edit the peaks Step Action e Right click and select Fill Peak from the shortcut menu or e Select Edit Fill Peak Result The Color and Pattern dialog box opens Color El Ce Bee sie fae ADETE E Imi f oe E E E E im EEE EEE ee EEE OT Pattern e Select a color and a pattern e Click OK Result The peak is filled according to the selections Note The color and pattern selections will override the general Fill settings that can be selected for all peaks on the Peak Table tab in the Chromatogram Layout dialog box UNICORN 5 31 User Reference Manual 28 9972 39 AA 431 12 Evaluation 12 1 Peak integration 12 1 6 How to edit the peak
196. port 212 393 How to print 210 How to sign 110 Linear flow rates 198 ethod variables general description 145 onitor signal limits 557 How to perform a Method How to start a method run How to use the Start Proto How to select a column 190 Pause instruction descrip tion 197 Method templates How to create a method 103 How to create a template from a method 208 How to delete a tem plate 208 Save a new method 105 Template information 104 Method Wizard How to create a method 99 How to save a new method 102 Molecular size calculation Error signs 553 How to calculate the size 552 How to create a size ta ble 548 How to delete a molecular size table 551 How to open an existing ta ble 550 How to rename a molecular size table 551 Overview 545 Preparations before curve creation 546 Procedure instruction 553 Molecular size curve Description 549 Overview 544 Morphological algorithm Definition 594 Description 410 How to set 410 Incorrect structure width 412 Minimum distance between points 413 Noise window 412 Structure width 411 Multifile Peak Compare Wizard How to open saved set tings 366 How to save the set tings 365 UNICORN 5 31 User Reference Manual 28 9972 39 AA P How to select data to com pare 354 How to select the opera tion 351 How to select the Peak Da ta 359 How to select the peaks 356 How to
197. rameters See more information about the parameters below this table e Click OK Note The same settings can be edited in the Calculate Baseline dialog box when a new baseline is created Choose Integrate Calculate Baseline to open the dialog box Test your parameter changes The best way to optimize the baseline is to change the baseline parameters step by step and then check the resulting baseline after each change When the desired effect is ac complished it is best to go back and try a parameter value in between the two last settings to avoid an unnecessarily low or high value How much the values should be changed depends on the cause of the peak integration problem The table below is a general guideline Baseline parameter Recommended initial change Shortest baseline segment 20 50 Noise window 10 30 Max baseline level Usually not necessary to adjust Slope limit 25 50 Note If necessary click the Default button to restore the default values UNICORN 5 31 User Reference Manual 28 9972 39 AA 415 12 Evaluation 12 1 Peak integration 12 1 4 How to optimize the baseline with a classic algorithm Shortest baseline segment If a too high Shortest baseline segment value is set short curve segments between peaks in the middle of the chromatogram are not identified as baseline segments The calculated baseline does not follow the source curve see below The Shortest baseline segment value is decr
198. reate a BufferPrep recipe Step Action 2 Click the Buffer substance button in the New Recipe dialog box Result The Define buffer substance dialog box opens A Pretila Secs RESSAR SSAA WS ASOS KRIAR SBA A SAANEN EONS Rea AA Ae A Es N We Sass ee N Select the buffer component from the Name droplist and note the displayed pKa value Click Cancel to return to the New Recipe dialog box Use the pKa value to determine the pH range Typically a range of 0 5 units around the pKa value is useful Note Check buffer tables for the exact ranges How to define a new buffer substance Note Before you can define a new buffer substance you must ensure that all pKa values are available for the substance The pKa values should be true i e the pKa value at in definite dilution and not apparent pKa values i e measured at a non zero concentration The pKa values should be given at 25 C The table below describes how to define a new buffer substance 642 UNICORN 5 31 User Reference Manual 28 9972 39 AA Step E How to create and edit BufferPrep recipes E 1 How to create a BufferPrep recipe Action Choose Edit BufferPrep Recipes and click the New button Click the Buffer substance button in the New Recipe dialog box Result The Define buffer substance dialog box opens Click the New button Result The New component dialog box opens Type a name for the new component
199. registration of errors or problems that you have detected or that occur during a run The Generate Report Wizard takes you through the steps to generate your report There are two ways of accessing the Generate Report Wizard e From the UNICORN Manager e From the System Control In this section This section contains these sub sections Section See page 15 2 1 How to generate a problem report from the UNICORN Manager 569 15 2 2 How to generate a problem report from the System Control 573 568 UNICORN 5 31 User Reference Manual 28 9972 39 AA 15 System maintenance and error reporting 15 2 How to generate problem reports 15 2 1 How to generate a problem report from the UNICORN Manager 152 1 How to generate a problem report from the UNICORN Manager Introduction The Generate Report Wizard is used to generate problem reports This section describes how to generate a problem report from the UNICORN Manager Step 1 How to create the report The table below describes how to create a report with the Generate Report Wizard Step Action 1 Select Administration Create System Report in the UNICORN Manager module The first step is a Welcome screen e Click the Next button Result The Systems dialog box opens with a list of the available systems for the logged on user Select a system for which the report is to be generated and click the Next button Result The Description dialog box opens Add the f
200. rithm The example below has a much lower Slope limit and a lower Noise window 418 UNICORN 5 31 User Reference Manual 28 9972 39 AA 12 Evaluation 12 1 Peak integration 12 1 4 How to optimize the baseline with a classic algorithm Noise window Sometimes you get too many peaks after the peak integration usually because noise on the baseline is erroneously detected as peaks The solution to this is to increase the Noise window parameter However this can result in peak limits too high up on the peak slopes The illustration below is an example of noise detected as peaks A and the result of a second peak integration with an increased Noise window B TE 2 037 1 715 1 311 6 052 No Peak name Retention min Area mAU min Height mav 2 Internal st 5 85 60 5051 336 828 2 Peak A 0 2 6 53 14 6032 95 761 fre B 0 2 7 06 20 4377 125 560 a loeser noa 77K ar amn 147 eral a Note You can also use the Reject peaks function in the Integrate dialog box to reduce the number of peaks based on the total number of accepted peaks or the minimum peak height UNICORN 5 31 User Reference Manual 28 9972 39 AA 419 12 Evaluation 12 1 Peak integration 12 1 4 How to optimize the baseline with a classic algorithm Missing peaks Sometimes obvious peaks are not detected in the peak integration The probable cause is that the Noise window is set too high See the illustration below NANA Al
201. rrent Result file will be used If is entered followed by text e g Enter a file name as File name a full search path must be entered in answer to the question EX Exports an evaluation log in WKS format to the file defined PORT_EVAL_LOG_WKS in Export to file If is entered as File name the current Result file will be used If is entered followed by text e g Enter a file name as File name a full search path must be entered in answer to the question EXPORT_EVAL_LOG_XLS Exports an evaluation log in XLS format to the file defined in Export to file If is entered as File name the current Result file will be used If is entered followed by text e g Enter a file name as File name a full search path must be entered in answer to the question EXPORT_METHOD_ASCII Exports a method to the file defined in Export to file in ASCII format If is entered as File name the current Result file will be used 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 UNICORN 5 31 User Reference Manual 28 9972 39 AA 611 B Evaluation functions and instructions B 4 Procedure instructions Instruction Description 612 EXPORT_METHOD_WKS Exports a method to the file defined in Export to file in WKS format If is entered as File name the current Result file will be used If
202. s Peak start and end points 432 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 The illustration below shows an example of start and end point drop lines 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 curve at the same point See the illustration below UNICORN 5 31 User Reference Manual 28 9972 39 AA How to split a peak 12 Evaluation 12 1 Peak integration 12 1 6 How to edit the peaks It is possible to split the peak into two new peaks by inserting a drop line The table below describes how to split a peak in the Edit Peak Table dialog box Step Action 1 e Click the Edit peaks icon a e Click the peak in the curve or in the peak table to select the peak e Right click and select Split Peak from the shortcut menu or e Select Edit Split Peaks Result A new drop line is inserted at the middle point between the two ex isting drop lines and the peak is split Note The area under each new peak will not be the same if the symmetry of the original peak was not perfect How to join peaks It is possible to join the areas of adjacent peaks if they are separated by a drop line The table below describes how to join adjacent peaks in the Edit Peak Table dialog box Step
203. s END _BLOCK 10 00 10 00 10 00 0 00 Pause INFINITE End_Block End_Block essage Conductivity not stable Screen No sound Minutes Note If you are not using AKTA instruments a delay should be added after the Hold Pause instruction so that the following instruction will not be executed simultaneously with the Hold Pause instruction UNICORN 5 31 User Reference Manual 28 9972 39 AA 653 F Method examples F 3 Equilibration with extra safeguard This is what happens The table below describes what happens in the above example Stage Description 1 The column is equilibrated until the conductivity is below 5 mS cm 2 If this value is not reached within 6 column volumes the method is paused and a message is displayed 3 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 4 If this condition is not met within 10 column volumes the method is again paused Note At each pause the operator can decide whether to continue or abort the run 654 UNICORN 5 31 User Reference Manual 28 9972 39 AA F Method examples F 4 Collection of absorbance peaks F 4 Collection of absorbance peaks Introduction This section contains an example of how to collect absorbance peaks through outlets F3 and F4 Example instruction This is an example instruction as it would be presented in the
204. s and samples otherwise the precision of the quantitation will be reduced dramatically 488 UNICORN 5 31 User Reference Manual 28 9972 39 AA 13 The Analysis module 13 2 Quantitation overview 13 2 4 Standard addition quantitation 1324 Standard addition quantitation General information Standard addition quantitation is a simple way to obtain measurements of amount in your sample concentration is not calculated It requires only a first sample run and a second sample run which has been spiked with a known amount of the component of interest The technique is straight forward and relatively fast when you are running only a few samples Standard addition can be useful when you want to use the internal standard technique but do not have a suitable internal standard Disadvantages The disadvantages of Standard addition quantitation are e its limited precision compared to the external and internal standard techniques e its lack of ability to measure more than one component How to perform Standard addition quantitation The table below describes briefly how Standard addition quantitation is performed Step Action 1 Perform a sample run UNICORN 5 31 User Reference Manual 28 9972 39 AA 489 13 The Analysis module 13 2 Quantitation overview 13 2 4 Standard addition quantitation Step Action 2 Perform a second run with a sample that has been spiked with a known quantity of the component of interest prior to the
205. s ina method 586 The table below describes some solutions to syntax error problems Problem description Solution There are several actions that you can take Red instructions instructions with a red dot ina method are syntax errors and may be due to the following The method was connected to the wrong system that is the strategy of the system is incompatible with the method The method instructions do not corre spond to the components you have cho sen for your system Check your system components under Administration Sys tem Setup in the UNICORN Manager The Copy function was used instead of Copy from external when a method was imported from a diskette The wrong system may have been select ed in the Save As dialog box in the Method Editor You may also have templates not intended for your system which might be the case for custom designed systems The systems strategy has been updated with a new strategy that differs in the in struction set Check that the method has been connected to the correct system in either of these ways inthe System Method Con nection dialog box when you use the Copy from ex ternal dialog box inthe Save As dialog box in Method Editor If the system is custom de signed open the Method Editor select the red instruction and either delete it or replace it with a corresponding instruction if available from the Instruction box Repeat this for all red in
206. s interpreted as the UV curve slope at the selected retention point e Determine the highest slope value of the baseline non peak part of the curve e Add 10 e Select Integrate Calculate Baseline and use this value as the Slope limit Note If the differentiated curve is very noisy it can be filtered with a light Moving average filter in the Operations Smooth function UNICORN 5 31 User Reference Manual 28 9972 39 AA 599 B Evaluation functions and instructions B 3 Peak table column components B 3 Peak table column components Introduction This section contains a list of peak parameters with explanations and calculation formulae when applicable Peak parameters illustration The diagram below illustrates the peak parameters See the parameter list below for explanations 600 UNICORN 5 31 User Reference Manual 28 9972 39 AA B Evaluation functions and instructions B 3 Peak table column components Peak parameter descriptions The list below contains descriptions of the peak parameters Parameter Description Amount Values calculated by the Analysis module Only available if the Quantitation module is installed Area Calculated as the area between the curve and baseline be tween the peak start and peak end time or volume base Gray area in the diagram above Asymmetry Peak asymmetry indicator of column packing See definition below this table Baseline height Baseline amplit
207. s setting above and below the warning limit This prevents repeated warnings from noisy or oscillating signals close to the warning boundary Warning limit ee WARNING WARNING Note Hysteresis is only relevant for warnings since an alarm puts the system into Pause mode at the first alarm UNICORN 5 31 User Reference Manual 28 9972 39 AA 559 14 System settings 14 3 Curves 14 3 Curves Introduction This section is a short description of the Curves system settings The Instructions dialog box The illustration below shows the Instructions dialog box with the Curves instructions selected System Curves Instructions Time between samples 0 100 s Cond Store ON Time belween samples 0 100 Store ON Time belween samples 0 100 s Cond Store ON Curve settings The curve settings determine which monitor signals that will be stored as curves in the result file Verify that Store ON is set in the Instructions dialog box for all signals that are to be stored Note If a curve is set to Store OFF data from the specific monitor cannot be dis played in the curves window during a process run The data will not be recorded in any way Store and Time between samples The table below describes the function of the two curve settings Setting Function Store OFF ON This setting determines whether the curve data is stored or not 560 UNICORN 5 31 User Reference Manual 28 9972 39 AA 14
208. scouting scheme e Double click each Quantitation_Type table cell and select the correct concentration level for the standards Click the Evaluation Procedures tab e Select the Update_Quantitation procedure Click the Import button to import the Update_Quantitation procedure if it isn t displayed on the list Result This procedure automatically integrates the first UV curve with default baseline settings and updates the selected quantitation table with the new standard An update report is then printed Click the Quantitate button Result The Quantitation table dialog box opens Select the quantitation table from the Global or Personal folder Time must be selected as the X axis base unit Result The quantitation table is copied into the Update_Quantitation pro cedure Note You can only perform one run at each level since the old points in the quantitation table will be replaced after each run Save the method with a new name UNICORN 5 31 User Reference Manual 28 9972 39 AA 535 13 The Analysis module 13 5 Automated quantitation 13 5 3 How to perform automated update Step Action 7 Perform the run s Result The quantitation table will be updated automatically after each run Note The quantitation table will not be updated if the peak area or peak height of the new and the previous results differ more than the Limit value The Limit value is defined either for peak area or height How to perform automat
209. sesssssssssssssssssssssssssssssssssssssssssssssssssssssssstt 546 System Setting Seanca aa Son theresa ena aes 554 14 1 General information about system Settings oecceecccccssssssseesesssssccsssssssssuseesssssssssssssssusssssecesee 555 DAP E T A N E TEESE O E EEEE 558 TAGS CIOS APPIA OE E T NEE E AN E E EE A EA 560 System maintenance and error reporting s ssssssssssssessssessssssesrsssssessesesessss 562 15 1 System maintenance FUNCTIONS wiceeccccccssssssssssssessssssessccsssssssseesesssssessssssusssssessssscsssssssssusessesseesse 563 15 2 How to generate problem reports oicc ecsccssssssssesesssssccsssssssssesssssescssssssssssussessessesssssssssusssessesseesees 568 15 2 1 Howto generate a problem report from the UNICORN Manager essssssssssssssssssssssssseee 569 15 2 2 Howto generate a problem report from the System Control o cceesssssssssssssssssssssssssssssseee 573 TUN CSI eters E E sate lach E EE 577 A 1 LOJO ia EN A AN 578 A 2 UNICORN ACCESS oinnnnneriainna naaa E ATTE ETERA 580 A 3 Methods and method runs aeccacsssessssssssssssssssssssssssssssssssssesessseseesceesencencenceneeeceeeseceeceeneeccccceeceeeceeees 583 V R TESI EE EE EEEE 588 A 5 _ KTAdesign system specific problems sseeessssssssssssssssusssssseesssussssssessesssssssseessussssssseeetussssnee 589 Evaluation functions and instructions s sssseesssessessssessessssssesesessssessesesessess 590 BL Smoothing algorithm a i sctacacessssssesecadebbassnsvvvsbessascacoeceabesshi
210. ssesesssusessssuesesssussesssseessssevessssusessssuessssesesessueess 134 6 3 1 HOW to read method instructions o ceeeccscssscssssssssssssssssssescssssesssssssessssesessssusessssueessssuessssssessssseees 135 6 3 2 How to add method instructions eeeeecssesscsssssssssssesssssseessssssessssssessssssesessssessssssesessssetssssssessssseess 137 6 3 3 How to delete method instructions wocccccccccsssssscsssssssssssessssssesessssessssssesessssessssssseesssseessssssecsssseees 139 6 3 4 Howto change or move method instructions 140 6 4 How to use method variables 145 6 5 RUN SetU snene 149 651 Overview Of RUM SETUD eision ninina ai anini 150 6 5 2 TRE VAIADICS CAD waiceseeeccssessssssessssssessssssssessssssssssusessssssssssssecssssustsssssuesssssuesssssesssssssusessssesessssstessssseess 153 6 5 3 The Scouting tab 156 6 5 4 The Questions tab 157 6 5 5 The Gradient tab 161 G56 THC NOTES AOD sete ices asst a aa a tll NRA ANAN ARN 164 6 5 7 The Evaluation Procedures tab ccccsssscsessssisesssssssssssesvscessssssssnssesossusssasnevesssssssconasisasoonssteecenbedossenuses 166 6 5 8 The Reference Curves tab weeceeeccsesssssssessssssssssssssssssssssssssssessssssssssssssssssssessssesssssuseesssuetessssssessssseess 171 6 5 9 TRE COLUMNS td vaiceeceeeccsssesscsssstssssssssssssssesssssssssssssesssssssssssseessssuusssssuesssssesesssusscssssssessssetessssusessssesess 173 6 5 10 TheBufferPr p tabiina aiia ainiai 174 6 5 11 The Method Information tab i
211. sssessee 489 13 25 Recovery calculdti ON sisinio ias 492 13 2 6 General reliability factors for the quantitation techniques esssssssssssssscccsssssssssseesesseee 495 UNICORN 5 31 User Reference Manual 28 9972 39 AA 14 15 Table of Contents 13 3 How to prepare f r GUAMAN wcceeeeeccsssssssssseesssscssssssssssssssessesssssssssssssssssusssesseccsssssssssusessssseeess 496 13 3 1 Preparations before quantitation 497 13 3 2 Howto create a quantitation table 499 13 3 3 How to edit and update a quantitation table e eeeeeeecesssssssssssssssssssssssssssssssssssssssssssssssssssssesee 512 13 4 How to quantitate the sample woueccccccccsssssssssssesssssccsssssssssssssessessessscsssssssssssessessesssssssssssusesseseeesee 520 13 4 1 External and internal standard quantitation 521 13 4 2 Standard addition quantitation 524 13 4 3 Howto calculate the recovery factor 527 13 5 A tomated quantitati Niseni inanin 530 13 5 1 How to set up for automated quantitation ceeeececcssssssssssssssssssssssssssssssssssssssssssssssssssssssssssseset 531 13 5 2 How to perform automated quantitation 534 13 5 3 How to perform automated update wan 535 13 6 Howto measure molecular size o ceccceceee 543 13 6 1 Overview of molecular size determination o eeeeeeesssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssee 544 13 6 2 How to determine the molecular size o eceeeesessssssssssssssss
212. ssssssssssssssssssesssssssssessessessesssesssessssssssssssssssssseese 459 12 3 Automated evaluation procedures unn eececsssssssssssessssssccssssssssussssssssessssssssssussssesesssecsssssssassnseseseesees 460 12 3 1 HOW to create a new procedure oacceccesssssssssssssssssssssssssssssssssssessessssssssssssssssssssssseeesssssssssssecssssseesees 461 12 3 2 How to edit a procedure ssssssssesssssssssssssssssssssssssssssessssssssssssssssssseseessssssssnisussessssessesssssssssussessseseesee 464 12 3 3 How t r n a Procedure sirsiran eisini 467 12 3 4 HOW to rename and remove procedures sssississsisssissississsrsrississsrsssrssrssrisssresrrsssssnene 471 13 The Analysis module cscsscssssescsscssssessssescssssessessesesssnsacssesseceseeeescensaessesesees 473 13 1 General information about the module oecceeceecccccsccsssssssssssssssesesseeeeseceececeeceeseeceeseeeeeeeseeeseeeessseees 474 132 QUGMUMGTOMOVERVICW riesenie AGE a 477 13 2 1 General information about quantitation woeecccsccscsssssssssssessssssssssssssssssssssssssssscssssssssssssecssssseesee 478 13 2 2 External standdid Q aNttati N ciscascscacisestessesecsssistivacstascsssiescstosssctesrsvresiesies aassensenetaaniandan 482 1323 Internalstandard guanttati h scscccsssscactsceccsascesescscstssesstsactsastacatessescersiesdaimasidgiostonsstiansasnianian 485 13 2 4 Standard addition quantitation oecceeccsssssssssssssssssssssssssssssssssssssssssssssssssssssssssssseessssssssssssueesss
213. st result in curves with drifting baseline and peak clusters The morphological baseline follows the curve faithfully and a curve with a baseline at a more even level can be created by subtracting the morphological baseline The Morphological algorithm does not work well if there are negative peaks or if quantitative data from negative peaks are important in the run Note The Morphological algorithm is the default baseline setting How to set a Morphological baseline The table below describes how to choose a Morphological algorithm and define baseline settings Step Action 1 Select Integrate Peak Integrate Result The Integrate dialog box opens 2 Click the Baseline settings button in the Integrate dialog box Result The Settings dialog box opens 3 e Select the Morphological algorithm e Change the Baseline parameters if necessary See more information about the parameters below this table e Click OK 410 UNICORN 5 31 User Reference Manual 28 9972 39 AA 12 Evaluation 12 1 Peak integration 12 1 3 How to optimize the baseline with a morphological algorithm Note The same settings can be edited in the Calculate Baseline dialog box when a new baseline is created Choose Integrate Calculate Baseline to open the dialog box Morphological algorithm parameters The parameters for the Morphological algorithm are e Structure width e Noise window e Minimum distance between points Structure width Struct
214. systematic errors and thereby maximize precision Further information Refer to statistical reference books for more detailed information about quantitative analysis An example is Statistics for Analytical Chemistry 3rd Edition 1993 J C Miller and J N Miller Ellis Horwood PTR Prentice Hall UNICORN 5 31 User Reference Manual 28 9972 39 AA 495 13 The Analysis module 13 3 How to prepare for quantitation 13 3 How to prepare for quantitation Introduction This section describes how to use peak data from standards to prepare quantitation tables and calibration curves for use with External standard Internal standard and Recovery quantitation In this section This section contains these sub sections Section See page 13 3 1 Preparations before quantitation 497 13 3 2 How to create a quantitation table 499 13 3 3 How to edit and update a quantitation table 512 496 UNICORN 5 31 User Reference Manual 28 9972 39 AA 13 The Analysis module 13 3 How to prepare for quantitation 13 3 1 Preparations before quantitation 133 1 Preparations before quantitation Description The table below describes the preparations before the quantitation Step Action 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 2 Perform at least one run for each concentration level of the standard 3 Peak integrate the curves to produce a peak table for eac
215. t Appendix D The Column list Introduction The Column List includes all available columns and their specific parameters This ap pendix describes how to edit the Column List In this appendix This appendix contains this section Section See page D 1 How to edit the Column List 630 UNICORN 5 31 User Reference Manual 28 9972 39 AA 629 D The Column list D 1 How to edit the Column List D 1 Howto edit the Column List Introduction This section describes how to edit the list of available columns Available columns When you create a new method and select a column certain column specific parameters are automatically copied into the method The list of available columns is found in the For column field of the New Method dialog box The Column List is not linked to a par ticular method although the columns are edited within the Method Editor Columns are either globally available to all users or only personally available It is best not to edit the globally available columns unless you save the changes under a new column name since other users may not appreciate the changes Note It is recommended that only a limited number of users are given access to the right to edit global columns This is essential to avoid unintentional changes LIM oo x Columns Fiter All Columns Data for 2_HiTrap_Desatng Noimal Parameters 2x _HiTsap_Q_HP_1_ml Global Advanced Parametere Height Diameter Column
216. t be used Note Contact the system administrator if you are not authorized to change your user profile Connection problems The table below describes some connection problems and their solutions Problem description Solution The connections are not available e Check the connection between the PC and the chromatography system e Check thatthe power to the chromatogra phy system is turned on 580 UNICORN 5 31 User Reference Manual 28 9972 39 AA A Troubleshooting A 2 UNICORN access Problem description Solution The connections are not available even though e the connection between PC and chromatography system ap pears to be correct e the power is turned on Quit UNICORN Shut down and switch off the computer Switch off the chromatography system Restart the entire system Asystem is not available when you attempt to establish a connection Check that you have access rights to the sys tem Access rights are not automatically as signed for a newly defined system You receive the error message Cannot connect to system in a network installation Check that the local computer to which the system is connected is turned on and logged on to the network Check that the computer where you try to establish a connection is logged on to the network Check that the limit of 8 connections to the system has not been exceeded You can establish a connection but cannot contro
217. t completely separated from those of other components 6 Click OK Note The amount and concentration of the sample are multiplied by the multiplier values when the calibration curve is applied to a sample Change the default values if you want to determine the amount or concentration in the starting volume of the sample instead of in the injected volume of the sample Quantitation statistics The Statistics field in the Quantitation table dialog box displays the Correlation and Explained variance values when available 510 Click the More button to open the Statistics dialog box for a complete display of available data Statistics Ea Model AEN Model function values Explaine UNICORN 5 31 User Reference Manual 28 9972 39 AA 13 The Analysis module 13 3 How to prepare for quantitation 13 3 2 How to create a quantitation table Statistical reference values e The correlation only available for linear models should be as close as possible to 1 00 e The explained variance value should be as close as possible to 100 Note that the value is usually rather high even for poor models A value of 90 indicates a very poor model The explained variance is not shown for curve models that are drawn through the origin Note If the point to point curve model is selected no statistics are available UNICORN 5 31 User Reference Manual 28 9972 39 AA 511 13 The Analysis module 13 3 How to prepare for quan
218. t from the shortcut menu Result The data point is deleted and the curve is redrawn A Move a data point e Select the data point and drag it to a new position Result The baseline curve is redrawn 6 Click OK Result The Save Edited Baseline dialog box opens 7 e Confirm the location and type a new name if necessary Result The new baseline is saved Edited baseline The illustration below is an example of a baseline before and after editing UNICORN 5 31 User Reference Manual 28 9972 39 AA 425 12 Evaluation 12 1 Peak integration 12 1 5 How to edit the baseline manually How to draw a straight line The table below describes how to force a straight baseline between two points Step Action i Select the first of the two points in the point list 2 Click the Draw straight to next point button Result The baseline is drawn through the points as a straight line 426 UNICORN 5 31 User Reference Manual 28 9972 39 AA 12 Evaluation 12 1 Peak integration 12 1 6 How to edit the peaks 1216 How to edit the peaks Introduction Once a peak table has been generated based on an appropriate baseline it is 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 values will all be recalculated How to open the peak table for editing The table below describes how open the peak table for editing The editing options are described bel
219. t models and statistics C 1 Curve fit models Statistics 1 33366E 001 B 2 92587E 000 Number of point s 4 The Point to point model The table below describes the features of the Point to point curve model Feature Description Equation No single equation Mathematical model As these curves are not based on a single equa tion no statistical data is available The statistics table only contains information on the number of points in the curve Minimum number of required 2 points Measuring range for the calibra Within the highest and lowest values for the tion curve points The illustration below is an example of the statistical information for an applied Point to point curve model 624 UNICORN 5 31 User Reference Manual 28 9972 39 AA C Curve fit models and statistics C 1 Curve fit models Number of peint s 4 UNICORN 5 31 User Reference Manual 28 9972 39 AA 625 C Curve fit models and statistics C 2 Statistics C 2 Statistics Introduction This section explains the correlation and explained variance calculations that are used by the Analysis module Correlation The Analysis module calculates the correlation coefficient for linear models This shows how well the data are linearly related The correlation is displayed in the Statistics table If you are producing a calibration curve that relates peak area or height to amount or concentration you aim to ac
220. table will not be updated if the peak area or peak height of the new and the previous results differ more than the Limit value The Limit value is defined either for peak area or height How to perform automated update in scouting runs step 4 The table below describes how to set up the scouting runs for the samples Step Action 1 540 Select the Quantitate_Sample procedure UNICORN 5 31 User Reference Manual 28 9972 39 AA Step 13 The Analysis module 13 5 Automated quantitation 13 5 3 How to perform automated update Action Select Sample in the variable Quantitation_Type for all the sample runs Result All samples will be run automatically and the amount and concentra tion of the components of interest will be printed after each run Note The result files will include an additional chromatogram labelled 12 that contains a small part of the curves collected during the execution of the evaluation procedure How to change the scouting runs to be updated by average The table below describes how to change the scouting runs so that the quantitation table is updated by average after each standard run Step Action 1 e Click the Evaluation Procedures tab in the Run Setup Editor e Click the Import button Result The Import dialog box opens e Select the current method from the Method file menu e Highlight Update_Quantitation in the Select field e Type anew name e g Update_Average in th
221. the components are displayed The illustration below shows the Maintenance manager dialog box with the Info tab selected and general information about the pump displayed Maintenance manager Information Warning info D Waning B S System Air sensor Auto Sampler 9 Frac 950 pH Cond Pump General rears Specific H E Pump P 950 w Uv Valve 3 Valve 2 Valve 3 Valve 4 Valve 5 J E Valve 6 pae version 56 3045 944M K318 i oA perea software version 1 03 0 2 8 M odule software version V1 40 pa number 56119594 K4 002102 2 A oeo eg D UNICORN 5 31 User Reference Manual 28 9972 39 AA 563 15 System maintenance and error reporting 15 1 System maintenance functions How to display component information Click a component in the list to display the component information You can choose two different views General e g serial number version number etc Specific e g how many hours a pump has been run etc How to set up a maintenance warning The table below describes how to set up a maintenance warning Step Action 1 2 Click the Warning tab Select a component Choose Warning New or Right click the component and select the New option on the shortcut menu Type the appropriate value in the Periodicity field Type a warning text in the Pop up text box Type a name for the warning
222. the length of the segment Choose Integrate Calculate Baseline and insert this value as the Shortest baseline segment value How to measure noise level Classic algorithm Curve coordinates can also be used to measure noise levels on the source curve The table below describes how to do this Step Action il Use the Zoom function to focus on a part of the curve that is representative for the baseline noise Select an appropriate Y axis scale Measure the Y axis coordinates e Calculate the noise range as the difference between the max and min values e Addanextra 20 e Choose Integrate Calculate Baseline and insert this value as the Noise window value 598 UNICORN 5 31 User Reference Manual 28 9972 39 AA B Evaluation functions and instructions B 2 Baseline calculation theory How to measure the slope limit Classic algorithm The table below describes how to measure the slope at any part of the curve Stage Description al Select Operations Differentiate in the Evaluation module Result The Differentiate dialog box opens e Select the desired source curve e Select the First order calculation option e Click OK Result The differentiated curve will appear in the active chromatogram Select an appropriate Y axis scale right click and select Marker to measure the Y axis values for the differentiated curve with the curve coordinates function Result The Y axis value i
223. the peak Result Two vertical bars become superimposed over the drop lines that delimit the selected peak The area between the bars is filled with a yellow fill pattern 2 Drag the bars to define the new limits for the selected peak Result The drop lines are moved and the peak areas are automatically re calculated Note A drop line can never be moved beyond another drop line or beyond a point where the peak meets the baseline How to use the zoom function The table below describes how to use the zoom function in the Edit Peak Table dialog box Step Action 1 e Click the Zoom icon Result The cursor is changed into a magnifying glass UNICORN 5 31 User Reference Manual 28 9972 39 AA 435 12 Evaluation 12 1 Peak integration 12 1 6 How to edit the peaks Step Action 2 e Press and hold the left mouse button e Drag the cursor over the area you want to zoom in on e Release the mouse button Result The area is enlarged Right click and select Reset zoom to restore the full view The Integrate menu If needed you can use the selections on the Integrate menu to perform a peak integration in the Edit Peak Table dialog box This is useful for example if you want to re integrate the curve using different settings or integrate only part of a curve with different settings See Section 12 1 7 How to integrate part of a curve and how to exclude or skim peaks on page 437 for more information 436 UN
224. the recipe values are unfeasi ble UNICORN 5 31 User Reference Manual 28 9972 39 AA E How to create and edit BufferPrep recipes E 1 How to create a BufferPrep recipe Step Action 9 e Type a name in the dialog box Result The new recipe is added to the recipe list Note It is recommended that restricted access be given to the right to edit global recipes The recipes are either globally available to all users or only personally available It is best not to edit the globally available recipes unless you save the changes under a new recipe name since other users may not appreciate the changes Buffer concentration Use buffer concentrations that are 2 4 times higher than the concentration that is used in the normal preparation When BufferPrep is used the buffer will be diluted 2 10 times depending on the amount of acid base that has to be used to reach the desired pH value Up to five different buffering components can be selected To prevent a too high ionic strength the sum of the concentrations for all selected buffers should be between 0 03 M and 0 2 M typically 0 1 M How to select the pH range The useful pH range depends on the pKa value The table below describes how to deter mine a pH range based on the pKa value Step Action a Choose Edit BufferPrep Recipes and click the New button UNICORN 5 31 User Reference Manual 28 9972 39 AA 641 E How to create and edit BufferPrep recipes E 1 How to c
225. the selected peaks in the Define Component s dialog box These settings also affect the information provided in the peak table in the dialog box How to adjust the identification settings 504 Description 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 Peak identification by absolute retention works well 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 Instruction If you have drifting retention that makes peak identification difficult you can choose to identify peaks according to their position relative to a reference peak The table below describes how to adjust the identification settings in the Define Component s dialog box Step Action 1 Identify a component peak that can be used as the reference Note 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 UNICORN 5 31 User Reference Manual 28 9972 39 AA 13 The Analysis module 13 3 How to prepare for quantitation 13 3 2 How to create a quantitation table Step Action 2 Click Identification Settings Result The Ident
226. the start and end of each segment The line between these is also filled with points Note The baseline points are shown as green squares in the Integrate Edit baseline function of the Evaluation module Baseline drawing 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 However the curve does not necessarily pass through the baseline points The baseline will be a smoothly curved function passing close to or through the points To reduce the effect of noise at the peak integration the created baseline is forced equal to the source curve in every position where the difference between the baseline and the source curve is small enough Choose Integrate Calculate Baseline If the Accept nega tive peaks option is off the baseline will be forced down to the level of the source curve whenever the created baseline goes above the source curve UNICORN 5 31 User Reference Manual 28 9972 39 AA 597 B Evaluation functions and instructions B 2 Baseline calculation theory How to measure the baseline segment Classic algorithm You can try to measure the Shortest baseline segment length directly on your chro matogram The table below describes how to do this Step Action 1 Locate the shortest segment of the curve that you consider a part of the baseline Use the marker box on the chromatogram to measure
227. tion levels must be subjected to the same sample preparation procedure as the samples Note If there are several components of interest they must all be chemically similar UNICORN 5 31 User Reference Manual 28 9972 39 AA 485 13 The Analysis module 13 2 Quantitation overview 13 2 3 Internal standard quantitation How to perform Internal standard quantitation The table below describes briefly how Internal standard quantitation is performed Step Action i Prepare a series of concentration levels from the standard 2 Add an additional component the internal standard in the same concentra tion to all the standards and to the sample 3 Perform a run for each standard and the sample 4 Integrate the curves to produce a peak table for all standard runs and for the sample Result Each curve contains a peak from the internal standard Changes in the size of the internal standard peak indicate changes in the system See illustration below mau 240 200 Standard 150 100 Slevels Internal standard 15 0 15 5 16 0 16 5 17 0 min 486 UNICORN 5 31 User Reference Manual 28 9972 39 AA 13 The Analysis module 13 2 Quantitation overview 13 2 3 Internal standard quantitation Step Action e Plot all peak sizes relative to the size of the internal standard peak to produce a calibration curve for each component e The standard peak area relative to the internal standard peak area is used to produce a poi
228. titation 13 3 3 How to edit and update a quantitation table 1333 How to edit and update a quantitation table How to open an existing table The table below describes how to open an existing quantitation table for editing in the Evaluation module Step Action 1 Select Quantitate Edit Quantitation Table Open Result The Open quantitation table dialog box opens 2 Select a quantitation table from the Quantitation table s list Note By default the list will show the quantitation tables that are globally available Click the Personal radio button to display the tables that are re stricted to your own user ID 3 Click OK Result The Quantitation table dialog box opens Note Quantitate includes an update function that can be used to add new peak size data to an existing quantitation table in a simplified way This function does not allow you to redefine components in the Define Component s dialog box The update function The update function can be used to add new peak size data to an existing quantitation table This enables precision to be improved through the use of data from a number of standard runs It also simplifies the process of renewing the calibration curves before each analysis Note The injection volume must always be the same for the new run as it was for the previous standard runs 512 UNICORN 5 31 User Reference Manual 28 9972 39 AA 13 The Analysis module 13 3 How to prepare for quantitation 13
229. to create and edit BufferPrep recipes E 1 How to create a BufferPrep recipe 640 Step Action 2 Click the New button Result The New Recipe dialog box opens The illustration below shows a complete example of a BufferPrep recipe in the New Recipe dialog box M Buller Notes Stock cone Bulfer substances 0 03 M Hydiogen phosphate al pa j Correction factors 003M fFromae i al ta 75 0 06 M acetate u J z m Acid Base m Sak Component defination CT a Sa 3 Select buffers from the Buffer substances droplists and type stock concen trations in the corresponding Stock conc box See How to define a new buffer substance below if the desired substance is not available 4 Select either HCI acid or NaOH base from the Acid Base droplist and type the required stock concentration typically 0 1 M 5 Select a salt from the Salt droplist and type the maximum outlet concentra tion of the salt for 100 B typically 1 0 M See How to define a new salt below if the desired salt is not available 6 Type the desired pH range minimum and maximum values in the From and To boxes See How to select the pH range below this table 1 e Click the Notes button optional e Type your notes about the recipe in the displayed dialog box e Click OK to return to the New Recipe dialog box 8 Click Save as to save the recipe under a new name Note A warning message will appear if any of
230. to set up for automated quantitation The Scouting tab The illustration below shows the Scouting tab in the Run Setup used to enter standard data before the standard concentration level is defined Evaluation Procedwes l Method Infomation l Start Protocol l Questions Resut Name Fiac 950 Variables Scouting Noles Geden BulePep Columns Reference Curves JV Display tooltip for extended scouting scheme cel Define Clear Al Add Insert Delete Series Edt varisbte _ Help UNICORN 5 31 User Reference Manual 28 9972 39 AA 533 13 The Analysis module 13 5 Automated quantitation 13 5 2 How to perform automated quantitation 1352 Howto perform automated quantitation Instruction The table below describes how to set up sample runs to perform automated quantitation Step Action 1 e Select File Open in the Method Editor module e Select a method that has been used for standard runs in the Open dialog box See Section 13 5 1 How to set up for automated quantitation on page 531 e Click OK 2 e Click the Scouting tab in the Run Setup e Click the Clear All button to clear the scouting scheme e Double click each Quantitation_Type table cell and select Sample for all sample runs 3 Click the Evaluation Procedures tab e Select only the Quantitate_Sample procedure Click the Import button to import the Quantitate_Sample procedure if it isn t displayed on the list Result This procedur
231. type in the Name text box Click the Save button Repeat steps 2 and 3 to set up more warnings Click the Close button How to view the warning parameters and counters The component that has been set up for a maintenance warning is marked by an icon and the name of the warning 564 Select the warning to display the parameters UNICORN 5 31 User Reference Manual 28 9972 39 AA 15 System maintenance and error reporting 15 1 System maintenance functions Counters show the remaining time or number of operations before the next maintenance warning See the illustration below Maintenance manager Information Warning into D Waring a System Air sensor Auto Sampler Frac 950 pH Cond Pump Pump P 950 Uv A Lamp check Valve 1 amp Valve 2 Valve 3 amp Valve 4 Valve5 Valve 6 Lamp check ZE frequency Every 6 months or every 500 method runs whatever occurs m Fa Next time out 2002 10 11 No of method runs completed 0 Cean Eee How to reset the counters The table below describes how to reset the maintenance warning counters Step Action 1 e Select System Maintenance in the System Control module to open the Maintenance manager dialog box e Click the Warning tab 2 Select the warning you want to reset on the component list e Choose Warning Edit or e Right click and select Edit on the shortcut menu
232. ual 28 9972 39 AA 575 15 System maintenance and error reporting 15 2 How to generate problem reports 15 2 2 How to generate a problem report from the System Control Step 3 How to generate and save the report The table below describes how to generate and save the report Step Action i By default the report is saved in the folder Unicorn Reports If you want to save the report in another location select a folder in the tree structure 2 You also have these options e Click the Preview button to open the report in Notepad e Click the Print button to print the report without any preview 3 Click the Finish button to generate and save the report 576 UNICORN 5 31 User Reference Manual 28 9972 39 AA A Troubleshooting Appendix A Troubleshooting Introduction This appendix describes different problems which may arise in UNICORN and how to solve the problems In this appendix This appendix contains these sections Section See page A 1 Logon 578 A 2 UNICORN access 580 A 3 Methods and method runs 583 A 4 Evaluation 588 A 5 AKTAdesign system specific problems 589 UNICORN 5 31 User Reference Manual 28 9972 39 AA 577 A Troubleshooting A 1 Logon A 1 Logon In this section This section describes how to solve the following log on problems e Unable to log on to UNICORN e Error message Strategy file error Unable to log on to UNICORN The table below describes some log on problems
233. uantaarnaavavsucirtiaitevabaiiioes 251 93 1 The toolbar Gnd Status bar somarminen niiin acetate eno RRO 252 9 3 2 Manual instructions 256 9 3 3 Alarms and warnings 259 9 4 How to perform a scouting run 260 9 5 How to perform a MethodQueue run uaccccccsccsssssssssesssssssccsssssssssussssessesesssssssssssesssssecesassssssasseesses 262 9 6 Ifthe network connection fails ee seesesssssssssssssssssesssesssssnessssessstssesesssecesneeneceeceeceeeceeseceeceeeeeeees 265 10 How to view UU acre i a asea iai E a iati 266 10 1 Howto openmaTesult file suisssaisinnatininmnjansannnsiangannahiinimanai 267 10 2 How to use the File Navigator weceeecccccccsssssssssssseesssscsssssssssssssssssessessssssssssssssssssssseccessssssssusesssseceese 269 10 3 Basic presentation of Chromatograms ccssecscssssssssssssssssesesssssscsssssssssusessssssesssssssssssssessssseesees 275 10 3 1 Introduction and temporary chromatograms 276 10 3 2 The chromatogram window ceecesssssssssssssssssssssssssssssssssssssssse 278 10 4 How to optimize the presentation of a chromatogram s s 285 10 4 1 How to make changes in the Chromatogram Layout dialog BOX ecceeeessssssssssssseeeee 286 10 4 2 The Curve tab and Curve Names tab eeeceeesssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssnsssst 287 10 4 3 The Curve Style and Color tab 289 10 4 4 How to change and fix the axes 292 10 4 5 Howto save and apply a layout
234. ude at peak start peak maximum and peak end A F and G in the diagram above Capacity factor The capacity factor will only be calculated when the chro matogram is in volume base The total liquid volume Vt must be entered in the Integrate dialog box for this parameter to be calculated See definition below this table Concentration Values calculated by the Analysis module Only available if the Quantitation module is installed Fraction tube id Fraction number at peak start peak maximum and peak end Height Maximum amplitude above the baseline C F in the diagram above Kav Gel phase distribution constant in gel filtration 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 dialog box for this parameter to be calculated See definition below this table Molecular size Values calculated by the Analysis module Only available if the Quantitation module is installed Plate height HETP Height equivalent to theoretical plate and plates meter The column height must be entered in the Integrate dialog box for this parameter to be calculated See definition below this table Peak endpoint heights Amplitude above the baseline at left A in the diagram above and right peak limits E G in the diagram above Peak endpoint re tention Retention value at peak start and p
235. uired peak tables Result The Molecular size table dialog box opens 8 Use one of the following ways to select a peak e Click the peak in the curve e Click the peak entry in the Retention Mol size table 9 Double click in the Mol size column cell and type the known molecular size from the standard 10 Repeat step 8 and 9 for all components of known molecular size 11 To remove unwanted entries click the peak entry in the table and click the Exclude button 2 Select the appropriate curve model in the Curve model field see The molecular size curve below 13 Click the Save as button Result The Save molecular size table dialog box opens 14 e Choose if the table is to be globally accessible to any user or restricted to your personal user ID The default is global e Type a name in the Molecular size table name field e Click OK The molecular size curve The molecular size curve shows the relationship between molecular size and the corre sponding retention The curve is plotted from the Retention Mol size data that you have typed in the table as described above Before this can be done a curve model is needed which describes the relationship between molecular size and retention 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 selected model Select one of the available models in the Curve model field e Linear
236. ure width determines the length of the straight line that follows the chromatogram The default value is set at the widest peak in the chromatogram multiplied by 1 5 The illustration below is an example of how a morphological baseline follows the peaks at the different levels in the curve ATIMIOOO 1_UVI_ZISAn ATO 1_Uv1_2ISAn GOS BASEM UNICORN 5 31 User Reference Manual 28 9972 39 AA 411 12 Evaluation 12 1 Peak integration 12 1 3 How to optimize the baseline with a morphological algorithm The correct structure width settings Too low settings Too low Structure width settings can result in a baseline that reaches too high up in the peaks of the curve Sometime a wider peak is not recognized because it contains a cluster of smaller peaks The Structure width is then set to a value according to the largest width of the identified narrower peaks and must be increased Too high settings Too high Structure width settings mean that narrower peaks especially in fluctuating curves are not properly followed This happens when an artifact in a curve is identified as the widest peak by the morphological algorithm and then is used to set the default Structure width value The illustration below is an example of baselines using the default morphological algo rithm settings A and a morphological algorithm with an increased Structure width value B apg 104 t_UV1_21Gnm mao 104 1 UVI LIONO BASEC
237. ures are included on the Procedures menu Remove a procedure Open the Edit Procedures Menu dialog box and select the checkbox again to de select and remove a procedure from the menu UNICORN 5 31 User Reference Manual 28 9972 39 AA 12 Evaluation 12 3 Automated evaluation procedures 12 3 4 How to rename and remove procedures 1234 Howto rename and remove procedures Introduction The procedures that you have created can be renamed or removed from the list of available procedures This section describes how this is done How to rename a procedure The table below describes how to rename a procedure Step Action 1 Choose Procedures Edit Rename Result The Rename Procedure dialog box opens 2 Select a procedure Result The procedure name is displayed in the New name text box 3 Type the new name 4 Click OK Result The procedure name is changed How to delete a procedure The table below describes how to delete a procedure Step Action 1 Choose Procedures Edit Delete Result The Delete Procedure s dialog box opens 2 Select a procedure e Click OK e Click the Yes button to confirm Result The procedure is deleted UNICORN 5 31 User Reference Manual 28 9972 39 AA 471 12 Evaluation 12 3 Automated evaluation procedures 12 3 4 How to rename and remove procedures Global procedures It is not advisable to edit existing global procedures Open the global procedure instead and
238. urves are produced to show the relationship between amount and peak size for each component The calibration curves are used to quantitate the components in the sample Note The standard should contain known amounts of all the components that are to be quantitated in the sample How to improve quantitation External standard quantitation can be based on the use of a single standard concen tration level but the calibration curve is then limited to a linear through the origin rela tionship The use of a number of different concentration levels of the standard broadens the range of the calibration curve It also allows the development of non linear calibration curves and improves precision Multiple runs at each level improve precision further The description in this section is based on the use of a standard e that contains two components e which is run at three different concentration levels How to perform External standard quantitation The table below describes briefly how External standard quantitation is performed based on the use of a standard which contains two components and which is run at three different concentration levels Step Action 1 Perform a run for each standard level 482 UNICORN 5 31 User Reference Manual 28 9972 39 AA Step 13 The Analysis module 13 2 Quantitation overview 13 2 2 External standard quantitation Action Integrate the curves to produce a peak table for each run maAU
239. use the 2D data view 361 How to use the 3D data view 363 Manual peak identifica tion 358 The Data View dialog box 360 Peak integration Differences between to filter peaks and to reject peaks 407 How to display peak la bels 408 How to perform 403 How to select part of a curve for peak integra tion 438 Peaks Drop lines description 432 Edit integration for part of a curve 441 How to add a fill color and pattern 430 How to add peak names 434 How to change the As symetry Ratio value 606 How to change the peak resolution algorithm 603 How to delete a peak 430 How to display peak la bels 408 How to exclude before inte gration 439 How to filter from view 406 How to join peaks 433 How to open the peak ta ble 427 UNICORN 5 31 User Reference Manual 28 9972 39 AA Index How to select a skim ra tio 440 How to split a peak 433 nclude negative peaks in integration 439 Labels 407 Peak identification through the absorbance ratio 448 Peak parameters 601 Peak purity 448 Peak skim Compared to drop ines 439 How to select a ratio 440 Peak table How to display informa tion 279 How to export 393 How to rename 349 How to select contents 446 Pool Fraction print list P r How to use 344 oblem reports How to use the Generate Report Wizard from the System Control 573 How to use the Generate Report Wizard from the UNICORN Manager 56
240. use with the systems mentioned above In this section This section contains these sub sections Section See page 13 5 1 How to set up for automated quantitation 531 13 5 2 How to perform automated quantitation 534 13 5 3 How to perform automated update 535 530 UNICORN 5 31 User Reference Manual 28 9972 39 AA 13 The Analysis module 13 5 Automated quantitation 13 5 1 How to set up for automated quantitation 135 1 How to set up for automated quantitation Introduction This section describes how to create a quantitation table for automated quantitation Basic conditions for the quantitation table A quantitation table must be produced from standards before samples can be quanti tated The list below describes the basic conditions for the quantitation table e Thesame method must be used for all standard and sample runs e Each level is an alias for a specific concentration of the standard e All runs with the same concentration must be assigned the same level e Level 1 must be selected for the standard with the highest or lowest concentration e The levels must be set in order of decreasing or increasing concentration of the standard How to prepare the quantitation table The table below describes how to prepare the quantitation table for automated quanti tation Step Action 1 e Use the Method Wizard to create a method e Select Autosampler from the Injection Technique droplist in the Sample I
241. usssssssssecsssssssssssssssssseessssssssssssssssssessessssssssssssucsesssesssssssssunssesssseceese 400 1211 Baseline calculat N edissa itel msanii 401 12 1 2 How to perform a peak integration oeceeeeeesssssssssssssssssssssssssssssssssssssssssssssesssesssessseesssssssssssssssssssse 403 12 1 3 How to optimize the baseline with a morphological algorithm esessssssssssssssssssssssseeeee 410 12 1 4 Howto optimize the baseline with a classic algorithm 414 12 1 5 How to edit the baseline manually cesssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssessessssssssesssssseesens 423 121 6 SHOW toedit the DOOKS ssessstecsscccsccscccapssissascadtaaaasessesccsagaosicastastauegsussseiecsaeerasaantisssee 427 12 1 7 Howto integrate part of a curve and how to exclude OF skim peaks u 437 DATE Measurement Sonsin EEE EES A ONTE 443 122 Other evaluations nissens sotsia E anatetea taap Es SN anatata Ets 447 12 2 1 Peak purity and peak identification essssssssssssssssccscsssssssssssssssssssessssssssssssssssssssccssssssssssseessssseesees 448 1222 Howtofind slope Vales aisssscccsssnssencossossesessstensvescescsoctsnscsotzivocesitecscedossosevisspudaopersonneccasensiniossiounven 451 12 2 3 How to simulate a peak fractionation sseseccssssssscssssssssssssssssssssssssssssssssssessssscessssssssssssesesssseeees 454 1224 HOW CO create CUVEE onierinoierin rati E iata A E eiiiai 455 12 2 5 How to use the Fraction HIStOgram necceessssssssssssss
242. value expressed as number of data points 2 When the source point is less than half the window size from the beginning of the end of the curve the median is calculated symmetrically round the source point over as many data points as possible e Ifyou increase the window width the smoothing effectis also increased e To completely remove a noise spike the window width should in effect be slightly more than twice the width of the spike Note 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 UNICORN 5 31 User Reference Manual 28 9972 39 AA B Evaluation functions and instructions B 1 Smoothing algorithms Savitzky Golay The table below describes the process when the Savitzky Golay smoothing algorithm is used Stage Description 1 he algorithm is based on performing a least squares linear regression fit of a polynominal of degree k over at least k 1 data points around each point in the curve to smoothen the data he derivate is the derivate of the fitted polynominal at each point he calculation uses a convolution formalism to calculate 1st through 9th derivatives he calculation 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 is re reversed afterwards Note See Gorry Peter A General Least Squares Smoothing and Differentation by
243. values in the Parameter text boxes and click the Replace button Result The old parameters are replaced by the new parameters Add a new instruction e Select the instruction in the procedure immediately before where you want the new instruction e Select a type and an instruction in the Instruction field e Type parameter values in the Parameter field e Click the Insert button Result The new instruction is inserted after the selected instruction Remove an instruction Select an instruction in the procedure and click the Delete button to remove the instruction from the procedure Choose File Save and click the Close button to close the dialog box 464 UNICORN 5 31 User Reference Manual 28 9972 39 AA 12 Evaluation 12 3 Automated evaluation procedures 12 3 2 How to edit a procedure Descriptions of the procedure instructions Appendix Section B 4 Procedure instructions on page 607 contains a list of procedure in structions with descriptions How to add instructions to a procedure when recording If you start recording again you can add more instructions to a procedure that is already open in the Procedure Editor e The new instructions will be added to the end of the present procedure or e The new instructions will be inserted after the selected instruction if an instruction has been selected Invalid instructions The procedure will stop and display an error message if an instruction calls for an inva
244. ve range of curve fit models The following models are available for calibration curves e Linear e Linear through origin e Quadratic e Quadratic through origin e Point to point Note The average peak size for all points at a specific level is used to calculate the cali bration curve Molecular size curve models 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 Statistics The Analysis module provides values for the appropriate constants that are used in 620 each curve equation for all models except for the point to point models It also provides statistical data that you can use to assess the quality of fit of the curve to the data Click the More button in the Statistics field of the Quantitation table or Mol size table dialog boxes to view the applied model statistics UNICORN 5 31 User Reference Manual 28 9972 39 AA The Linear model C Curve fit models and statistics C 1 Curve fit models The table below describes the features of the Linear curve fit model Feature Description Equation y Ax B Mathematical model The constants A and B are determined by lin ear least squares regression Minimum number of required points 2 at least 4 points recommended Measuring range for the calibration curve Within the highest and
245. veeresdonsscctsoondesaasigadorlisSeotanssvetseneabsatdistsctauss 591 B 2 Baseline calculation theory ssssississsississsisssissrssssrsrrssnienrsssisssrnstnsntrntsnnrnrnnnannnrntnnnrnntannnrtrnnas 594 B 3 Peak table column components 600 B4 PROCECUIEAIMSERUCHIONS cosssssssssssscisssectescesiascssnaceen ccoieonseaste nei adnndidbadimndanuditedtss 607 C rve fit models ard Statistics Sess nctuesrsacirnrsiecaiettaneier ati diecoemtnicanenistiaeins 619 Gl C rve fit Model Sisco e ss astucn seni ven waitin a na a a aan 620 Ce SHOUISTLCS panigi a E Aa OT EA A 626 The Col nin NE sssini eenei aeaii 629 D 1 How to edit the Column List on eeseessssssssesssssessssssesssssessssssssssssassuntuassintussttunssninusnssnsaeesaeenees 630 UNICORN 5 31 User Reference Manual 28 9972 39 AA 7 Table of Contents E Howto create and edit BufferPrep recipes s es sessessssessssessessssssessssessesesseses 638 E 1 How to create a BufferPrep recipe sssssssssssssscssssssssssusesssssecsssssssssssssessesscsesssssssassuesssssseseesesssess 639 E2 HOW t edita BUPTERPRE ECOG asssssscecscscessstasssasbsssbetiesansvsiclavsbdeoosbovunssuvnssissessensttasossubsianecsibiiheits 645 F Method example S rsicho eia hien NARA RNE EA RARE 648 EL Simpe eguilhibratioN necer aniani 650 F 2 Equilibration with simple safeguard weseeecccccccsssssssssssssssessccssssssssssssessssesssssssssssseeseessesscsesssnsssesees 652 F 3 Equilibration with extra safeguard 653 F 4 Colle
246. y after each run New average values will be calculated from the old points together with the new points Note The quantitation table will not be updated if the peak area or peak height of the new and the previous results differ more than the Limit value The Limit value is defined either for peak area or height The Procedure Editor dialog box The Procedure Editor dialog box is illustrated below amp Procedure Editor Update_Quantitation BASE TIME REJECT_PEAKS OFF OFF OFF OFF 20 NEGATIVE_PEAKS OFF CALCULATE_BASELINE 01 17 DEFAULT DEFAULT DEFAULT DEFAULT SUB 01 17 47 PEAK_INTEGRATE 47 4 PORT Chromatogiam Peaks UPDATE A PERSONAL Ext Std Chym DEFAULT DEFAULT AVERAGE ON ON J ai Replace RT RUN PROGRAM in Update Extemnal Intemal quantitation table UNICORN 5 31 User Reference Manual 28 9972 39 AA 537 13 The Analysis module 13 5 Automated quantitation 13 5 3 How to perform automated update How to perform automated update in scouting runs step 1 It is possible to run both standards and samples in the same scouting run and continu ously update a previously created quantitation table with new values The table below describes how to set up the evaluation procedures for the updates Step Action 1 Open the same method that was used to create the quantitation table from the standard runs and open the Run Setup 2 Click the Evaluation Proc

UNICORN™ 5.31 User Reference Manual Chapter 12 to 15

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UNICORN&trade; 5.31 User Reference Manual Chapter 12 to 15

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UNICORN™ 5.31 User Reference Manual Chapter 12 to 15