Ettan microLC - GE Healthcare Life Sciences
Contents
1. x Instructions p Parameters G Pump Flow Inlet omen FlowSplitterCompensation C Flowpath Gradient Ot C On EZA C AlarmstMon FlowBetweenRuns Inlet 2 ZS Off C On Close C Other rei Help ZS Off C On ES InletB2 Off C On 12 If for example you want to change the flow rate select Pump and then Flow Enter a new flow rate under Parameters and then click Execute The new flow rate will be used until the end of the run or until a new flow rate instruction is reached in the method Close the box by clicking Close All manual interactions are recorded in the Logbook 13 If you want to stop the run before it is finished click on the End button at the top Run Pause Continue End as Ettan microLC User Manual 18 1162 25 Edition AB 27 7 Viewing and printing the result gt 28 Viewing and printing the result If you are satisfied with the automated print out obtained after the run if selected you need not alter anything described in this section However if you want to alter the chromatogram layout this section will teach you the basics of the evaluation module 7 1 Viewing 1 After a run you can view the result Click the UNICORN menu icon Double click on a result file icon in the list to the right The Chromatogram window is opened automatically in the Evaluation workspace when you open a result file The Chromatogram window contains all the curves Note that the2. Method Information l Result Name Start Protocol Variables Scouting Notes Questions Gradient Columns ReferenceCurves Evaluation Procedures Initial Eluent_Conditions Eluent_A_Inlet Al kd Eluent_B_inlet B1 Ka Start_ConcB B 0 00 0 00 100 00 System Flow Flow Rate ul min 100 0 2000 Column Equilibration Equilibrate With CV 5 000 0 000 999999 000 System_Flush Flush System With ul 2000 000 0 000 999999 000 Autosampler Iniection Sample D ID Alarm Pressure MPa 35 00 0 00 35 00 Sample Flow ul min 25 0 2000 QuantitationType S h Autosampler_Row Autosampler_Column 1 1 24 E Empty Loop With ul 115 000 0 000 999999 000 Wash Volume wash Column With CV 2 000 0 000 333333 000 Gradient Segment 1 Target Conch 1 B 100 00 0 00 100 00 Length of gradient 1 CV 20 000 0 100 9999 000 Gradient Segment 2 Target ConcB 2 B 100 00 0 00 100 00 Length of gradient 2 CV 0 100 0 100 9999 000 Gradient Segment 3 Target ConcB 3 B 100 00 0 00 100 00 Length of gradient 3 CV 0 100 0 100 3999 000 IV Show details T Show unused variables IV Display tooltip for extended variable cells To ensure complete sample application onto the trap column the sample volume value must be appropriate for the autosampler loop used Check that the variable Empty loop with in the block Autosampler Injection is set to five times the volume of the sample loop In the template Autosampler Injection Spl
3. BLOCKS USED IN THE METHOD All variables are cited within quotation marks The method notes contain comments and information about the method e g how sample injection and elution are performed 12 Select File Save The Save As dialog opens Enter a name Store the method in the directory of your choice by double clicking on a directory Click OK Comment The method name followed by two consecutive numbers starting with 01 will then be used as default name for the result file of your method after runs 13 Vou have now created a method 14 Click on the UNICORN menu icon at the bottom of the screen Your saved method appears in the window to the left Now you are ready to start a run Go to chapters 4 and 5 You can also goto chapter 8 to learn how to vary any variables systematically and automatically in repeated runs This is know as scouting and is a convenient easy to use function Ettan microLC User Manual 18 1162 25 Edition AB Creating a method 3 3 1 Template naming conventions All method templates except the Installation test are named according to its application The template name consists of three parts where each part describes the use of a specific type of application Example Autosampler Injection Splitter Trap Tupe of Description Example description Injection Specifies used sample Manual Injection the sample loop is application method filled manually with a syringe Autosampler Inject
4. arun 6 6 Viewing a run The progress of the run is shown in the System Control window O vemo system Control 1 Systemi Method LEE Server strategy buk AE Autosameler injection met DS Continue End a c 000 ui Fun stared For Pel press PE pe Wa x Ir Corbi Dy dim Carmen When the system pump is running the text Run is shown in the Run Status panel in the Run Data window 1 Choose View Windows Check Rundata Curves and Logbook Click OK 2 The Run Data window at the top shows current values for running parameters Ettan microLC User Manual 18 1162 25 Edition AB 25 6 Viewing a run 26 Position the cursor in the Run Data window Click the right mouse button and select Properties The Properties dialog opens Now you can select which parameters you want to see in the Run Data window For example select Acc time Block time Flow and Pressure Click OK Yaris Curve Style and Colour FlowScheme Logbook Run Data Groups Run Data Colour De e Instruments Y Connection Run Status Scouting No MAcc Volume MRun Data New Group Edit Group Delete Group The Curves window shows the curves during the run Position the cursor in the Curves window Click on the right mouse button and select Properties The Properties dialog opens Here you can select which curves to show during the run All
5. autho rized representative of the manufacturer for information con pem cerning the decommissioning of equipment WARNING This is a Class A product In a domestic environment this product may cause radio interference in which case the user may be required to make adequate measures Contents 10 Contents About this guide LI PRO DIE 5 12 Typographical conventions eerte 5 The system and the software Sch Gelekal qnos ee netu e pente te eese Rete up eene 6 22 UNICORN OVEM EW ati ira LO 2 3 WHOM Em 13 Creating a method 3 1 Template naming conventions tts Preparing the system for a run 4 1 System connection es 4 2 General system preparation s Starting a FU iet Viewing q rUn tud Viewing and printing the result LL VIEWINd WEE 4 2 Pppntipe opd makinga report 17 Contents 1 Ettan microLC User Manual 18 1162 25 Edition AB About this guide 1 About this guide This guide is written for users who are not familiar with UNICORN software and Ettan microLC Here you will learn the basics of UNICORN and how to operate Ettan microLC from UNICORN UNICORN is a software package for control and supervision of the Ettan microLC chromatography system It runs on a PC under Windows and includes hardware for interfacing the controlling PC to the chromatography liquid handling parts of Ettan microLC In this guide you will learn how to e Create methods e Prepare the syste
6. cannot be changed there To change a variable value position the cursor in the value field and double click with the left mouse button To add a column for next run click Add A second column appears with the values from the previous run copied Change the values as required If you want to insert a new run column after a specific column in the scouting scheme position the cursor in the column and click Insert A new column with identical values appears directly after the selected column Repeat the previous step until you have defined all the runs you require If necessary use the horizontal scroll bar to see more runs Click on Run1 Run2 etc at the top of the scheme with the right mouse button to toggle between Run and Excluded for the different runs Those marked Excluded will not be run A scouting scheme is now defined Select the Start Protocol page To stop the system between each run for e g refilling the sample loop check the All runs box Otherwise the system will not stop until all runs are finished Choose File Save When the method is started all the runs in the scheme will be performed automatically and the set flow for each run will be prepared automatically Each run in the scouting scheme will generate a separate result file which are all stored in a special scouting directory Prepare the system and start the run as described in chapters 4 and 5 When filling the inlet tubing with the correct solutions us
7. column and click OK Comment A special template is not required for scouting Scouting can be performed with any template for any technique 3 Choose View Run Setup in the Method editor may already be checked 4 Selectthe Scouting page Method Information l Start Protocol l Result Name Variables Scouting Notes Questions Gradient Columns ReferenceCuves Evaluation Procedures Variable Runt Sample ID n Quanttationtype Rea Vache E Eluent_B_inlet n autosampler Column Start_Conc8 FlowBetweenRuns O Flow Rate O Equilibrate With O Sample_Flow V QuantitationT ype v Autosampler_Row M Autosampler Column Wash Column With sl EEN Pan 4 Autosampler_Injection FlowBetweenRuns Show details Show unused variables Cancel J Help IV Display tooltip for extended scouting scheme cells Defining other Define Clear i Delete imet asi co EdtVarabie Heb scouting variable 5 Alistofallthe variables appears Select the block Autosampler_Injection and the variable you wish to alter Ettan microLC User Manual 18 1162 25 Edition AB 33 8 Scouting 10 11 12 13 14 15 34 Click OK The selected scouting variables will appear to the left with their default values inserted Comment Values for variables selected for scouting are dimmed on the Variables page and
8. microLC User Manual 18 1162 25 Edition AB Viewing and printing the result 7 7 2 Printing and making a report 1 To print the chromatogram choose File Report 2 Select standard format Global Chromatogram This will create a report containing the chromatogram and the questions on one page Generate Report From result Chromatogram Customised Report Format From result Chromatogram_Peaks From result Chromatogram Quant From result Chromatogram Report From result Full Report Global BP Chromatogr Report Global BP Full Report Global Chromatofacusing Global Chromatogram Peaks Global Chromatogram Quant Global Chromatonram Benart 3 tomise Report 4 Click Edit Mode to enable changes in the report 5 To include more information in the report click Add Page A new page is added to the report Ettan microLC User Manual 18 1162 25 Edition AB 31 7 Viewing and printing the result 6 Under Insert click on the item to include Items available are e Free text e Chromathogram e Text method e Documentation e Evaluation log e Quantitate and molsize optional 7 Move the mouse pointer into the page area of the window You will notice that the mouse pointer has an additional symbol according to the item type you selected to insert 8 Press the left mouse button and drag out a box to the desired size Release the mouse button A dialog is displayed specific to the type of item i
9. term chromatogram is used here when talking about the whole window containing all the different curves Evaluation HypersilBDSC821a301 1 sk File Edt View Integrate Operations Procedures Adviser Window Help EIER Ojala S a Dj EA HygeisilBDSC8212301 1 UV1 216nm HypeisilBDSC8212301 1 Cond HypessllBD SC82 1301 1 Coni HypersilBDSCH212301 1_Inject Hr TS ken ben D 2b ab eb 8b 100 120 140 180 180 200 220 240 280 280 min Ready NUM MStart Efrain Shop 3 System Co 3 System Co 4 System Co lt MethodEd 1 System Co JL Evatuati GSUNICORN sa Control Pa EM Microsott 240 PM The result file from a run holds a complete record ofthe run including method system settings curve data and run log Comment Original raw data curves can never be modified renamed or deleted from a result file Maximize the Chromatogram window by clicking on the larger square in the upper right corner Ettan microLC User Manual 18 1162 25 Edition AB Viewing and printing the result 7 4 All changes regarding the presentation of the curves are done in the Chromatogram Layout window Position the cursor in the Chromatogram window Click on the right mouse button and select Properties or select Edit Chromatogram layout to activate this window Chromatogram Layout 1 x Curve Style and Colour l Edit Tests l Layout Library He
10. 00 0 000 999999 000 System Flush Flush System With ul 2000 000 0 000 999999 000 Autosampler Injection Alarm Pressure MPa 35 00 0 00 35 00 Sample Flow ul min 0 2000 QuantitationType Autosampler_Row Autosampler_Column TransportVial IniectionVolume ul 0 1 495 5 Wash Volume Wash Column With Cv 2 000 0 000 99999H 000__w Show unused variables Iv Display tooltip for extended variable cells Click here to see the next part ofthe Variables page 7 Run Setup consists of a number of pages You will only look at Variables and Notes now You select a page by clicking on the respective tab at the top of the Run Setup screen 8 Onthe Variables page the method is presented by a number of Blocks The Blocks represent the typical steps in a chromatographic run Start conditions Column equilibration e Autosampler injection e Wash out unbound sample Ettan microLC User Manual 18 1162 25 Edition AB 15 3 Creating a method 16 Gradient e Clean after gradient e Re equilibration Each block contains a number of Method variables with suitable default values The values are easily changed to suit your requirements Some ofthe variables are normally hidden but can be shown by checking the Show details box Click on the scroll bar to see the next part of the Variables page 10
11. 04 27 10 12 1999 04 08 1040 1999 09 03 13 42 Ee 0 3 E 189912090807 199912090807 gt z 200004281106 19991208 08 06 200004281106 19991208 08 05 Srnie oaa de Alger Ges F Ready I JUser default Asta lt gt UNICORN Method Edit 1 System Cont _2 System Cont Evaluation Control Panel i Paint Shop iD 11 08 The Main Menu window is the central part of UNICORN From this window you can navigate through the whole control system The Main Menu window is mainly used for file handling In the Methods window to the left in Main menu all method files that you create are displayed A method file contains a series of instructions for controlling a run In the Results window to the right all result files are displayed A result file is the result from a run including all documentation e g the method used and the generated chromatogram Ettan microLC User Manual 18 1162 25 Edition AB 11 2 The system and the software In general UNICORN consists of 4 different modules of which the Main menu is one The other modules are represented by icons in the Toolbar These modules are A e New method e System control di Evaluation d Ces We e opens a dialog window for creating new methods opens a dialog window for controlling the system and running your methods opens a dialog window for evaluating your results A module is opened by clicking on its icon Addition
12. A1 and B1 respectively Set the start concentration of eluent B default 0 B The flow rate is set equal to the default value of the selected column System Flow The default Flow rate value is set to 100 ul min To get the desired flow rate for the selected column calibrate the flow rate from System Settings Special e Column Equilibration The number of column volumes CV required to equilibrate the column is set here An autozero of the UV monitor is done at the end of equilibration e System Flush Washes the mixer with set B buffer concentration Default value is 2000 ul for the 0 2 ml mixer e System Volume Compensation Make sure that the correct mixture of A and B buffer reaches the column by compensating for the dead volume in the system Pressure delay Allows the pressure to decrease before injecting the sample e Autosampler Injection Injects the sample from a selected vial in the autosampler To apply all the sample onto the trap column empty the sample loop with a volume 5 times the volume of the sample loop additional 15ul If using the default autosampler loop 20 pl The method uses scouting to facilitate automatic injection of several samples or standards Set the following variables on the Scouting page Sample ID Quantitation_Type Autosampler Row and Autosampler Column Replace the default text ID in Sample ID to allow individual identification of all runs Select the vial that contains the sample Fo
13. En Sul A A 6 V Variables page VIGUWIDQ O TU seien ade ses Viewing and printing the result sccacsesacssascssoosassssstassesssssossesandscdsseissaccasstsstsbassansasadbeesensessossiec 28 W Wash out Unbound Sample block entes 17 Z el El EEN 29 Ettan microLC User Manual 18 1162 25 Edition AB Short instructions The following short instructions are intended as a guide for users who are fully familiar with the safety precautions and operating instructions described in this manual The instructions assume that the unit is installed according to the installation instructions 1 Choose File New Method in the main menu or click on 2 Select System Technique Template and Column Click OK 3 Choose File Save in the Method editor and give the method a name Click OK 4 Clickon 5 Choose File Run Select the method and click Run 6 Thestart protocol will appear Check the method on the Variables page and change values as you require 7 Clickthe Next button and go through all the other pages 8 On tbe Evaluations procedures page select Print Chromatogram to get a print out automatically after the run 9 Click the Start button on the last page and the run will start For contact information for your local office please visit www gelifesciences com contact www gelifesciences com GE Healthcare Bio Sciences AB Bj rkgatan 30 751 84 Uppsala Sweden imagination at work GE imagination at wor
14. GE Healthcare Ettan microLC Making Your First Runs User Manual Ettan Important user information All users must read this entire manual to fully understand the safe use of Ettan microLC WARNING The WARNING sign highlights instructions that must be fol lowed to avoid personal injury It is important not to proceed until all stated conditions are met and clearly understood CAUTION The Caution sign highlights instructions that must be followed to avoid damage to the product or other equipment It is important not to proceed until all stated conditions are met and clearly understood Note The Note sign is used to indicate information important for trouble free and optimal use of the product CE Certifying This product meets the requirements of applicable CE directives A copy of the corresponding Declaration of Conformity is available on request The CE symbol and corresponding declaration of conformity is valid for the instrument when it is used as a stand alone unit or connected to other CE marked GE Healthcare instruments or connected to other products recommended or described in this manual and used in the same state as it was delivered from GE Healthcare except for alterations described in this manual Recycling This symbol indicates that the waste of electrical and electronic equipment must not be disposed as unsorted municipal waste and must be collected separately Please contact an
15. UNICORN overview 1 Switch on the computer Log in to Windows by first pressing Ctrl Alt Del and then clicking on OK After a while the Windows desktop appears 2 Start UNICORN by double clicking on the UNICORN icon UNICORN 3 401 An information window appears during start up 4 When the Logon dialog appears select a user from the Users list and enter the password If you log in for the very first time select user default and enter the password default Click OK Note You should enter users and individual passwords before starting using Ettan microLC on a regular basis x EE UNICORN logon User name default m Password ee Cancel Help 10 Ettan microLC User Manual 18 1162 25 Edition AB The system and the software 2 Eventually the UNICORN Main Menu window appears on the screen At delivery a Toolbar guide is displayed providing a quick guide on how to use the toolbar items The Toolbar guide isinhibited by unchecking the square at the bottom left Click Close Ele View Administration Tools Window Help Our CEOs EX NDelsul Fiter on system WEE stem Modified Created x WINCORN LAB ATIKE 1999 04 08 10 36 1999 09 03 13 42 1999 03 30 11 20 1999 09 03 13 42 x 8 1999 04 08 10 39 1999 09 03 13 42 Begin your work directly by using the toolbar buttons 1999 09 90 23 45 1999 10 04 09 34 1999 10 01 15 41 1999 10 04 09 34 2000 04 27 0953 2000 04 27 09 52 2000 04 27 10 13 2000
16. ader CuveNames Y axis XAxis Cuve PeskTable Select curves to display Highlight curves to view 101 skrap001 1_UY1_280nm 302 skrap001 1_UW2_Onm 303 skrap001 1_UW3_Onm 04 skrap001 1_Cond 05 skrap001 1_Cond 4 06 skrap001 1_Conc 307 skrap001 1_pH 08 skrap001 1_Pressure 09 skrap001 1_Flow 110 skrap001 1_Temp 11 mie 13 skrap001 1_SamplePres 14 skrap001 1_SampleFlow Apply to all chromatograms Cancel Help 5 Highlight the curves to view under Curves Curves are named as Resultfile 1_ curve where a curve can be for example UV_wavelength Cond pressure etc De select all curves except for example the UV Cond and Conc curves Click OK at the bottom ofthe Chromatogram Layout window 6 You can easily zoom in on the curves Place the cursor in the chromatogram press the left mouse button and drag a rectangle When you release the mouse button the part within the rectangle will be enlarged You can zoom further on the enlarged part Click the right mouse button and choose Undo or Reset zoom to return to the complete chromatogram 7 Click on the Y axis scale to change to a scale for another curve The style and colour of a curve its Y scale and its X scale can all be changed 8 Open the Chromatogram Layout window again Click on the Y axis and X axis tabs to set the scale for the different curves Normally t
17. al buttons are provided in the Toolbar These are e nstant run e Logon Logoff e e about UNICORN e Method Queue A e Existing Method lt E Queue 12 opens a dialog window where you directly can choose a template method to run This is handy for starting routine runs instantly opens a dialog window to control the logon logoff process opens an information window about the UNICORN version and how to contact GE Healthcare via the world wide web internet opens a dialog window for defining a new Method Queue opens a dialog window for showing the Method Queue that is running Ettan microLC User Manual 18 1162 25 Edition AB The system and the software 2 2 3 Help Comprehensive on line help is available To get help about an instruction or module place the cursor on the instruction module and press the F1 key Alternatively click on the Help menu in the upper right corner of each module and select Help for to get general help about the current instruction or module and find new help topics or Help Index for a specific topic Double click on green text to get more help In any box click on the Help menu to get help on how to use the current active box Ettan microLC User Manual 18 1162 25 Edition AB 13 3 Creating a method 3 Creating a method UNICORN is supplied with a number of almost ready made methods called method templates The method templates can be run as they are or you can easily mod
18. curves are stored in the result file x YAris Curve Style and Colour FlowScheme Logbook Run Data Groups l Run Data Colour Curves Anis r Display curves Select All Clear All Cancel Help By clicking the different tabs in the Curve Properties window you can set the properties for the different curves Normally the curves are scaled with auto scaling i e the scale is adjusted continually to the highest and lowest values for each curve Ettan microLC User Manual 18 1162 25 Edition AB Viewing arun 6 7 To fix the Y axis scale for a curve mark the curve click on Y axis click on Fixed and enter the max and min values You can repeat this for other curves Click OK 8 Tomaximize the Curve Data window position the cursor in the Curve Data window Click the right mouse button and select Maximize Go back to normal size by clicking on Restore 9 Click on the Y axis scale or click on the curve name at the top of the Curve Data window to shift to a scale for another curve The colour of a curve its Y scale and its name are always the same Click on the X axis to shift between time and volume 10 The Logbook is shown ot the bottom The Logbook shows exactly when the instructions in the method are executed during the run The Logbookis stored in the result file 11 You can make manual changes during the run Select Manual Pump The Pump Instructions dialog opens
19. e installed i d 75 um or 150 um See Chapter 2 in the Ettan microLC System Manual for an overview over columns with recommended tubing kits Comment If tubing with too large inner diameter is used the peaks will become broader than necessary If tubing with too small inner diameter is used the backpressure from the tubing might become higher than the maximum pressure for the column and the run will stop immediately after it is started Ettan microLC User Manual 18 1162 25 Edition AB Preparing the system for a run 4 2 Immerse inlet tubing A1 lor A2 if you changed this in the method in buffer A and inlet tubing B1 in buffer B 3 Checkthatthe outlet tubing from the UV cell is put into a waste bottle or connected to an MS unit if used 4 If there is air in the inlet tubing or if you suspect air in the pump purge the pump with a syringe as described in Pump P 905 User Manual 4 2 2 Connecting the columns 1 Connect the trap column if used between the flow direction valve port 3 and the injection valve port 1 Use the S7 capillary and 1 16 1 16 male union or the tubings delivered with the trap column 2 Connect the separation column between the conductivity cell and the top of the UV flow cell Conductivity cell UV flow cell m me axi a T If the 300 um column is equipped with a male connector attach it directly to the conductivity cell Follow tubing recommendations as follows Column Tubing i d Le
20. e the instruction PumpWash in System Control Manual Pump Select ON for the inlet tubings that should be filled and select OFF for the nonused tubings Click Execute to fill the inlet tubing When the PumpWash is finished click Close Ettan microLC User Manual 18 1162 25 Edition AB Going further 9 9 Going further Once you are used to the system and software you may want to learn more about it and its capabilities Below is a list of operations and descriptions that you may find of interest they are cross referenced to other manuals in the Ettan microLC manual package To learn about Read Columns and recommended tubing Changing tubing kits Calibrating monitors and pumps Comparing chromatograms Intergrating curves Measuring HETP and resolution Exporting curves and data to other programs Finding information about a certain menu instruction in UNICORN Controlling Pump P 905 and Monitor UPC 900 from the dials on the instruments themselves Details about each component Security features Controlling the system from a remote computer Ettan microLC System Manual Ettan microLC System Manual UNICORN 5 0 User Manuals CORN 5 0 User Manuals NICORN 5 0 User Manuals CORN 5 0 User Manuals U U U UNICORN 5 0 User Manuals Click on Help button in the dialog box that appears or look in the index in the UNICORN 5 0 User Manuals Ettan microLC System Manual to unlock the dials The User Manual for each in
21. he curves are scaled with auto scaling i e the highest and lowest values for each curve set the scale 9 Tofixthe Y axis scale mark a curve click Fixed and enter the max and min values for that curve You can repeat this for other curves Ettan microLC User Manual 18 1162 25 Edition AB 29 7 Viewing and printing the result 30 10 11 12 13 To fix the X axis scale click Fixed in the X axis field and enter the min and max values for the X axis Click OK Click OK at the bottom of the Chromatogram Layout window to execute all the changes Comment When you have made the necessary changes in the Chromatogram layout box they can be saved as a layout Click Save as at the top of the Chromatogram layout dialog to save the layout Give the layout a name and click OK Layouts can be selected in Apply layout at the top of the box and all your saved selections will apply Saved layouts can be applied to any result file Minimize the chromatogram window by clicking on the smaller squares in the upper right corner Click View Documentation A number of pages appear as in the Run Setup in the Method Editor All documentation about the run is stored here e g the method answers to questions variables logbook etc For example click on the Notes and Logbook pages to check the contents Close the Documentation window by clicking on the X in the upper right corner Documentation Iv a M v M Ettan
22. he end of this chapter 4 Selectthe column you intend to use The correct column volume the recommended flow rate and the correct pressure limit for that column will then be automatically implemented in the method 14 Ettan microLC User Manual 18 1162 25 Edition AB Block names Creating a method 3 Comment If you do not find your column in the list you can add one See the UNICORN User Manual If you manually alter the default values and thereby exceed the recommended values for the selected column you will get a warning when saving your method If you want to perform a test run without a column select Any from the column list to get suitable default parameters in the method Then in the method use a piece of tubing to replace the column 5 Click OK The method template will now be opened as an untitled method 6 Choose View Run Setup may already be checked Click here to select page Jain N Result Name Start Protocol Variables otes V Scouting N Questions Gradient Columns Reference Curves Evaluation Procedures Main Column ul Start_Conditions LIV Averaging Time Pump Wash A1 Off Pump_Wash_42 Off Pump_Wash_B1 Off Pump_Wash_B2 Off Initial Eluent_Conditions Event A Inlet Al Eluent_B_inlet B1 Start ConcB B 0 00 0 00 100 00 System Flow Flow Rate ul min 4 0 2000 Column Equilibration Equilibrate with CV 5 0
23. ify them to design your own method in a very short time The following example shows how to create a method when using autosampler injection the flow splitter and a trap column 1 Click on in the Main menu Toolbar The New Method window will appear New Method For system Select system System 1 Template C Method Editor Template selection Technique Method notes Select technique nr e TEMPLATE Autosampler Injection Splitter Trap version1 00 06 Template STRATEGY EULC_100 Autosampler Injection Autosampler Injection Splitter Autosampler Injection Splitte Autosampler Injection Trap T Manual Injection Manual Injection Splitter Manual Injection Splitter Trap Manual Injection Trap configuration only Select template before method start Gradient techniques For column Select column Any s NOTE This template is designed for micro flow NOTE The flowsplitter has to be calibrated to 4 l min Injection Autosampler injection with InjectionFlushed Elution Linear segmented or step gradient Press OK 2 Select a system Then select RPC as chromatographic technique 3 Alist of available templates will appear By clicking on a template an explanation for the template appears to the right in the Method Notes field Select the template Autosampler Injection Splitter Trap Comment The other templates are briefly described at t
24. in the separation unit is shown and described below It is not necessary to go through this in detail to make your first runs Ettan microLC User Manual 18 1162 25 Edition AB The system and the software 2 System pump Mixer Flow direction Autosamler Injection valve Conductivity e monitor SC RPC column UV monitor 1 Thepump has 4 pump heads two for pump A and two for pump B Pump A is the one closest to the front 2 Thebuffersolutions are pumped through switch valves and further to a mixer Inlet A1 and B1 are placed in buffer A and B respectively 3 The flowpath continues from the pump to the mixer and forward via an on line filter to the flow direction valve 4 The flowpath passes through the flow splitter which is connected between ports 1 and 2 on the flow direction valve 5 Atrapcolumnis connected between the flow direction valve and the injection valve 6 Asample loop is connected between ports 2 and 6 on the injection valve The sample loop is filled manually using a syringe For manual filling connect a fill port to port 3 on the Injection valve 7 After the injection valve the flow is directed to the separation column via an autosampler and the flow direction valve and then forward to the conductivity cell and the UV cell 8 The flowpath finally continues to waste or to an MS instrument optional Ettan microLC User Manual 18 1162 25 Edition AB 9 2 The system and the software 2 2
25. ion automatic sample application with an autosampler Splitter Specifies use of a flow Splitter splitter Trap Specifies use of a trap Trap column Ettan microLC User Manual 18 1162 25 Edition AB 19 4 Preparing the system for a run 4 20 Gr n e Preparing the system for a run 4 1 System connection 1 Click on the 1 System Control button in the Task bar at the bottom of the monitor 2 If the text NO is written in the Connection panel in the Run Data window continue below If it says YES go directly to General system preparation Comment Before you can start a run you must always connect to the system Connecting means that the System Control window is set up for a particular system If you are not connected the text NO is written in the Connect panel in the Run Data window Once you are connected the text changes to YES 3 Select System Connect The System connect dialog appears System Connect x Cancel Help Select a system from the list If you are not connected to a network only one system will be shown Click OK When connected the text YES is written in the Connect panel in the Run Data window You only have to connect once If you do not select System Disconnect you will be automatically connected to the system the next time you login to UNICORN 4 2 General system preparation 4 2 1 Checking the tubing 1 The correct tubing kit for the column you intend to use must b
26. itter Trap the default value is set to 115 ul which is an appropriate value for the default autosampler loop 20 ul x 5 4 15 ul The length of a gradient segment is limited to 9999 volume or time units ul or min To run longer gradients than 9999 units use additional gradient segments so that each segment does not exceed 9999 units Define the additional segments on the Variables page Enter desired values in the variables Length of Gradient respective Target ConcB in the block Gradient Segment Ettan microLC User Manual 18 1162 25 Edition AB Creating a method 3 Note If using the flow splitter SplitterFlow as Method Base the gradient segment limit is decreased according to how much the flow splitter reduces the system flow For example if the system flow is 100 ul min and the split flow is 4 ul min the gradient segment limit will be 399 ul or min 9999 25 Comment Each method template is unique but they are all built up with the same principle Below is a description of all the blocks in the Autosampler Injection Splitter Trap template Main The column selected is shown here Start Conditions Defines the general settings during a run Wavelengths not used are set to zero In the column list typical peak width and max pressure are pre defined UV averaging time and column pressure limit are automatically set to a default value for the column selected Initial Eluent Conditions Select the eluent inlets default
27. k and GE monogram are trademarks of General Electric Company Drop Design Ettan and UNICORN are trademarks of GE Healthcare companies O 2002 2007 General Electric Company All rights reserved First published Dec 2002 All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them A copy of these terms and conditions is available on request Contact your local GE Healthcare representative for the most current information GE Healthcare Europe GmbH Munzinger Strasse 5 D 79111 Freiburg Germany GE Healthcare UK Limited Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK GE Healthcare Bio Sciences Corp 800 Centennial Avenue P O Box 1327 Piscataway NJ 08855 1327 USA GE Healthcare Bio Sciences KK Sanken Bldg 3 25 1 Hyakunincho Shinjuku ku Tokyo 169 0073 Japan 18 1162 25 AB 09 2007 K Elanders sterv la 2007
28. m for runs e Perform runs e Make simple evaluations e Make reports Follow the guide from page to page in front of the computer Your time will be well spent Note To follow the instructions it is not necessary to read the comments written with smaller font containing additional information 11 Pre requisites The system and the software must be installed and functioning and the monitor and pump calibrated as described in the separate Installation guide IMPORTANT Before using Ettan microLC read all the safety information in section 1 2 in Ettan microLC System Manual 12 Typographical conventions Software menu commands dialog box prompts hardware controls and indicators are identified in the text by bold text A colon separates menu levels thus File Open refers to the Open command in the File menu 2 The system and the software 2 The system and the software 21 General Ettan microLC is a fully automated liquid chromatography system designed for analytical applications The separation unit of the chromatography system consists of three main modules They are e Pump P 905 including a flow splitter for gradient formation at low flow rates e Monitor UPC 900 a combined UV and conductivity monitor e Autosampler A 905 for microtiter plates Components such as the mixer the flow splitter and the injection valve are mou
29. ngth 300 um Silica 75 um 450 mm gt 1 0 mm PEEK 150 um 300 mm 4 2 3 Preparing the pump 1 Click System Control to open the System Control window 2 Select Manual Pump The Pump Instructions dialog opens 3 Instructions p Parameters G Pump Flow Kant en FlowSplitterCompensation C Flowpath Gradient Of C On Delete AlamstMon FlowBetweenRuns Inleta2 of C On sons InletB1 le Help of C On Er InletB2 of C On Ettan microLC User Manual 18 1162 25 Edition AB 21 4 Preparing the system for a run 22 Input Micro flow Waste CO O Pump P 905 s A Calibrator Select instruction PumpWash Select ON for the inlet tubing that should be filled Select OFF for the non used inlet tubing Click Execute to fill the inlet tubing with correct solutions The flow valve will automatically switch to waste during the PumpWash When the PumpWash is finished click Close Click End in the System Control window Make sure that the correct compression compensation values are set in Pump P 905 This is described in Pump P 905 User Manual 4 2 4 Calibrating the flow splitter if used 10 11 12 Connect the port 1 on the flow direction valve to the Input port of the splitter Use the tubing marked P3 Connect the enclosed waste tubing to the Waste port Connect the calibrator CAL 100 0 3 included with the system between the Micro fl
30. nserted Make the appropriate selections in the dialog and click OK to view the inserted item 9 Ifyou want to change the page layout choose Edit Page Setup The Page Setup window is opened and you can for example select page size and items to be included in the header and in the footer The information selected here will be printed in the report Click OK at the bottom of the Page Setup window Page Setup x Page Setup First Header Header Footer The selected items will be included in the report m Select Items Logo 3 SIE pu rie Fr Run user Current date amp time Select All Browse C Middle wv Bun date amp time Bight wv Report title v Result file name Geaa Layout VIMethod file nam Iv line over Page number Si 1 aunut name LH Line under Free text EEE Font Report title UNICORN Report Font 10 Click Print to print the report F Ettan microLC User Manual 18 1162 25 Edition AB Scouting 8 8 Scouting Scouting allows any run parameters e g flow rate to be systematically varied automatically in repeated runs Below is a description of how to perform scouting 1 Open the Main menu window Choose File New Method The New Method dialog opens Select system Then select RPC as technique 2 Select the desired template for example Autosampler Injection Splitter Trap Select a
31. nted in the valve box to the right Ettan microLC User Manual 18 1162 25 Edition AB The system and the software 2 The separation unit is controlled from the UNICORN software Autosampler A 905 Monitor UPC 900 Pump P 905 ON OFF button Flow direction valve INV 917 Injection valve INV 917 Column in holder See figure on next page UV flow cell See figure on next page Conductivity cell Valve box see figure on next page Switch valve A Switch valve B SV 903 Pump P 905 and Monitor UPC 900 can also be controlled individually from the modules without UNICORN software In this guide however you will only learn how to operate the chromatography system from UNICORN Switch on the chromatography system with the ON OFF button located on the front of the base platform to the bottom left Ettan microLC User Manual 18 1162 25 Edition AB 2 The system and the software 2 1 1 The valve box The valve box contains the flow direction and the injection valve the flow splitter the mixer and the on line filter Flow direction valve A e n Injection valve D Te Flow splitter S 901 N On line filter From column To e g MS y J The column in holder The UV flow cell 6 Comment The flowpath between the different modules and components
32. ow port on the flow splitter and port 2 on the flow direction valve Connect a measurement syringe to the UV cell outlet tubing If not already open open the System Control window Measure the flow rate after the column ul l _ volumelul Flowrate min time min Choose System Settings The Settings dialog opens Select Special in the Instructions field and then FlowsplitterParameter Enter the desired and the measured flow rates Click OK The calculation takes place When running the pump manually the flow splitter compensation must be switched on in Manual Pump Select On in FlowSplitterCompensation Measure the flow rate again Ettan microLC User Manual 18 1162 25 Edition AB Preparing the system for a run 4 4 2 5 Initializing the microplate The plate type used must be entered in UNICORN 1 Open the System Control window if it is not already open 2 Choose System Settings The Settings dialog opens 3 Select Special in the Instructions field and then Autosampler Parameters PlateType 4 Select the plate type used Ettan microLC User Manual 18 1162 25 Edition AB 23 5 Starting a run 5 Starting a run 1 Open the System Control window if it is not already open 2 ChooseFile Run Select the method to start Click OK The run starts Run E ch ADefault System Size Type Modified Prev Folder Example metho 24 Ettan microLC User Manual 18 1162 25 Edition AB Viewing
33. r automatic quantification set Quantitation Type to Sample or the appropriate standard level Ignore QuantitationType unless the run is intended for quantification e Wash out Unbound Sample To wash out unbound sample with the start eluent set the length of the wash here Gradient Elution Gradient Segment 1 2 and 3 The start concentration of eluent B the end concentration and the length of the gradient is set here A linear gradient is created Three separate gradients can be defined A step gradient is defined by setting the concentration of eluent B and zero length in one segment Ettan microLC User Manual 18 1162 25 Edition AB 17 3 Creating a method 18 Gradient delay Clean after Gradient Set the concentration of eluent B for column cleaning and the duration in column volume units here Flow between runs Allows the flow to continue between the runs 11 Click on the Notes tab TEMPLATE Autosampler Injection Splitter Trap version1 00 02 STRATEGY EULC 100 Gradient techniques without BufferPrep Injection Autosampler Elution Linear segmented or step gradient cl ELUENTS Eluent A Pump A Default inlet A1 Eluent B Pump B Default inlet B1 Prime the System Pumps 4 and B with eluents to be used Prime the inlet tubings 41 B1 with the correct eluents No air may be left in the tubings BufferPrep is not supported by this method SAMPLE LOADING Autosampler FUNCTION OF
34. strument found in the binderEttan microLC Components See each individual manual in the binder Ettan microLC Components UNICORN 5 0 User Manuals UNICORN 5 0 User Manuals Ettan microLC User Manual 18 1162 25 Edition AB 35 10 Index 10 36 Index A Autos ampliar Oia ea as 6 Autosampler Injection Block eerte tnnt Autosampler Injection Splitter Trap template Autosamipl r Injection template eieiei Change the page layout Changes during the run checking the tubing pitt err tee es tive deter te ere EE Chromatogram Layout window ttt tete aai 29 Chromatogr ani WIRGDW seinen 28 Clear after Gradientlbloek uttter ee e ent tier tit le i Rode 18 Column Equilibrationiblock eer 17 Compression compensation values ttt 22 Conductivitu Cel ege eegen 21 Connect thie caliBIatof xcci utu anni nee 22 Connecting Heelung ceret Eelere 21 Dee leie 26 D Documentation WIROOW suede ttn trt cette tree ec Een 30 E EE e 5 Existing Method Quete an essen un 12 F gi PR 13 Faults and ACHOS eege 20 Flow between runs block esent ttti seres 18 Fl widireetion VIVE nennen near 9 lee Flow splitter compesatio s crt WO Flowpath Ettan microLC User Manual 18 1162 25 Edition AB Index 10 G Generalsustem prepat tiOm rca iii 20 En aan 18 Gradient Segment block 17 Gradient SEMEN lenmgtli rnt certe ite aaa nal 16 H Helme In RP 13 l nitial Eluent Conditions block e
35. tes EE e 17 nitializing the mieroplote sesinin 23 WE ele 9 HEI FC UN see een 12 L Belg ebe 35 e 10 A PR 27 M A ON 11 Manual Injection template canina lirica 19 META DEE 12 Methods windouw EE 11 MICH TEE 6 N ew Method window EE 14 LOS OCG 18 P Perform a test run 15 Plate typi 23 a ee Seele EN NEE 17 Printing and making a report 31 AA abdannsetdvetacchsvonsasee 26 e Sit 26 Pump Instructions il tete rere re rer eed 21 Pump P 905 R Ettan microLC User Manual 18 1162 25 Edition AB 37 10 Index 38 S Sample applicato setae reiten iet eter nee 16 SCOUUNG EE 33 Scouting SCHEMES gedeit Eeer 34 a a cra eerte en n ore tr EE poa D iA De EAR 34 SCOUTING ele 33 Select chromatographic technique ttes 14 Select column elsi SPE CGS C X e 4 6 Cege H O ES Start Conditions block inssin tn 17 SUSTEMCONNECLON terca 20 SgstemiFlow blOGK cse retten ttti eere tete ettet teen pedea e rede 17 Sustemiklush block nitent iere ti etse nen 17 System Volume Compensation block ttt 17 T Technique RPG tct oi dass 33 Template Sele eite no td HER 33 Toolbar guide 11 TECIBAGO MERI WEE 9 connect 21 template 19 lege Mee Egeter 20 TUDING EE 20 TUDINGFECOMMEMAGLONS ici ia 21 U Pes
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