ÄKTAexplorer, making your first runs
Contents
1. WasteF1 Outlet Valve Il a En For Help press F1 e The flow scheme is a graphic representation of the chromatography system End Block Controlled By template The logbook shows all executed instructions and events during the run KTAexplorer Making Your First Runs 18 1140 78 Edition AF Viewingarun 6 6 Viewing a run When the system pump is running the text Run is shown in the Run Status panel in the Run Data pane 1 To choose which panes to display select View Windows In the Customize panes dialog select for example Rundata Curves and Logbook Click OK 2 To customize the pane s display after your own needs you can choose parameters in the Properties dialog In the respective pane select the right click command Properties and click the requested tab 3 The Run Data pane at the top shows current values for running parameters Under the Run Data Groups tab select the parameters you want to display and click OK Properties KTAexplorer Making Your First Runs 18 1140 78 Edition AF 33 6 Viewing arun 4 The Curves window shows the curves during the run All curves are stored in the result file Under the Curves tab select which curves to show during the run Click OK Properties 5 Normally the curves are scaled with auto scaling i e the scale is adjusted continually to the highest and lowest values for each cur2. c Default System Size Type Modified Created Prev Folder Prev Folder WINCORNLAB 647KB Method File 2003 04 28 17 2003 04 28 17 az Example files User Folder 2003 03 04 10 13 2003 03 04 10 13 WINCORNLAB 658KB MethodFile 2003 04 29 11 2003 04 29 11 Manual runs System Contr User Folder 2003 04 30 14 23 2003 04 30 14 23 i inst001 701KB Result File 2003 04 28 17 10 2003 04 28 17 10 u inst002 700KB Result File 2003 04 28 17 15 2003 04 28 17 15 Li Wizard Generated001 357KB Result File 2003 04 28 16 56 2003 04 28 16 56 Li Wizard Generated002 354KB Result File 2003 04 28 17 04 2003 04 28 17 04 Lu Wizard Generated003 376KB Result File 2003 04 28 17 07 2003 04 28 17 07 i Wizard Generated004 359KB Result File 2003 04 28 17 12 2003 04 28 17 12 6 The Main Menu window is the central part of the UNICORN displays It is mainly used for file handling From this window you can navigate through the control system In the Methods pane 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 pane 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 KTAexplorer Making Your First Runs 18 1140 78 Edition AF 15 2 The system and the software 2 4 UNICORN overview gt Syste i Re
3. LT 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 Method Editor opens the Method editor with a dialog window for creating new methods System control opens a dialog window for controlling the system and running your methods Evaluation opens a dialog window for evaluating your results To swap between the module windows click their respective button in the task bar at the bottom of the screen lt S UNICO 4Metno 4 Syste gE valuation Additional buttons are provided in the toolbar These are nstant run opens a dialog where you directly can choose a method to run This is handy for starting routine runs instantly Logon Logoff opens a dialog to control the logon logoff process Method Queue opens a dialog window for defining a new Method Queue Existing Method opens a dialog window for showing the Method Queue Queue that is running 1 Method Queues are used to link several methods together 16 KTAexplorer Making Your First Runs 18 1140 78 Edition AF The system and the software 2 2 5 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 curr
4. In the Save layout dialog enter a name of the layout and click OK Note The saved layout settings can be applied to any result file Click OK at the bottom of the Chromatogram Layout dialog to execute all the changes Click the View Documentation button 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 KTAexplorer Making Your First Runs 18 1140 78 Edition AF 39 7 Viewing and printing the result 7 2 Printing and making a report example click the Notes and Logbook tabs to check the contents Close the Documentation window by clicking on the X in the upper right corner Documentation A lt lt lt 7 2 Printing and making a report 1 Toprintthe chromatogram select File Report The Generate Report dialog opens Generate Report Global BP Chromatogr Report Customised Report Format 2 Select for example format Global BP Chromatogram This will create a report containing the chromatogram and the questions on one page 40 KTAexplorer Making Your First Runs 18 1140 78 Edition AF Viewing and printing the result 7 3 Click Preview to view the report on the screen Cum fle CS Wem ine Lo Mep EE pr Pz one Par zum ue er frere ce Be Deh teli Tao Ge ssi Add information to the report 1 Click Edit Mode to enable changes in
5. 10 KTAexplorer Making Your First Runs 18 1140 78 Edition AF The system and the software 2 Outlet Valve V4 Flow Direction Valve V7 PV 908 INV 907 standard component only in KTAexplorer 100 and KTAexplorer 100 Air Buffer Valve V6 IV 908 Column Selection Valve V2 PV 908 Box 900 Gas e ie Ta Monitor pH C 900 ___ D Monitor UV 900 Li D Sample Valve V5 PV 908 Pump P 900 CT P 901 alt P 903 Column Selection Valve V3 PV 908 Mixer M 925 Valve door Mains switch Sample Pump P 960 UV cell Switch Valve standard component only in SV 903 Conductivity cell KTAexplorer 100 KTAexplorer 100 Air KTAexplorer 10 S Column Selection Valve V2 PV 908 Injection valve V1 INV 907 Column Holders Outlet Valve V4 PV 908 Sample Valve V5 PV 908 pH electrode Flow restrictor To Fraction A Collector Column Selection Valve V3 PV 908 Sample Tubing Holder Sample Tube Holder Sample Tray Only in AKTAexplorer 100 and AKTAexplorer 10S KTAexplorer Making Your First Runs 18 1140 78 Edition AF 11 2 The system and the software 2 1 General 12 Comment The flow path between the different modules and components in the separation unit is shown and described below It is not necessary to go through this in detail to make your first runs Open the valve door if you want to follow the description 1 Buf
6. 30 KTAexplorer Making Your First Runs 18 1140 78 Edition AF Startingarun 5 9 Click Next The Result Name page appears Name the result file and define in which directory the result should be stored A default name the method name followed by 001 and a directory are suggested To change the result name and directory click Browse Result Name KTAexplorer Making Your First Runs 18 1140 78 Edition AF 31 5 Starting arun 32 10 Click START The run starts You will view the run in the System Control module The Curves pane shows curves during the run Method lt gt DEMO System Control 1 systuml File View Manual System Adviser The Run Data pane shows current values for running parameters Result Run Hold 300 200 100 Curves A2 Buffer 2 LIE Valve PumpA Aig f AL 3 mis F f ve une Al Be PumpB his umpB ALS nlet 0 00 min Base CV 0 96 m RESOURCE_Q_1_ml 0 05 min Block Start_Congitions 0 05 min Base SameAstfain 0 05 min ColumnPositiof Position 0 05 min Wavelength 780 nm OFF OFF 0 05 min AveragingTimeUY 1 28 sec NNS min Alarm Presfure Fnahled 15NnMPa_NNNMPa 0 00 min Run 1999 11 04 0916 53 Method Manual Injection FP Segmented Result c Default Manual Injection FP Segmentedl1 res x l x3 x4 xs x6 7 xB West 7 LA Li i N ji ji ji Em 1 0 1 2 a 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
7. A Example Result001 1_UV1_215nm 01 PEAK1 E ean er ra 44 0967 3 Peak B 0 5 60 5619 4 Peak c 0 5 115 9646 ae Ready FRE A The result file from a run contains a complete record of the run including method system settings curve data and run log KTAexplorer Making Your First Runs 18 1140 78 Edition AF 37 7 Viewing and printing the result 71 Viewing Note Original raw data curves can never be modified renamed or deleted from a result file 3 Maximize the Chromatogram window by clicking on the larger square in the upper right corner 4 _ All changes regarding the presentation of the curves are done in the Chromatogram Layout dialog Right click in the Chromatogram window and select Properties or select Edit Chromatogram layout to activate this dialog Curve Style and Colour Edit Texts Layout Library Header Curve Names Y Anis Axis Curve Peak Table Select curves to display Highlight curves to view NT Example Result001 1_UV1_215nm 04 Example Result001 1_Cond 05 Example Result001 1_Cond 06 Example Result001 1_Conc 07 Example Result001 1_pH 08 Example Result001 1_Pressure 7109 Example Result001 1_Flow 10 Example Result001 1_Temp 11 Example Result001 1_Fractions 12 Example Result001 1_Inject T Apply to all chromatograms Cance
8. Change the flow rate on the Variables page Continue through the start protocol by clicking Next and then start the run You do not need to change the method in the Method Editor Go through the Variables page to check that the method is OK this is not necessary if this was done in the Method Editor 28 KTAexplorer Making Your First Runs 18 1140 78 Edition AF Startingarun 5 5 Click Next For example the Notes page appears You can write your own comments in the Start Notes tab 6 Click Next For example the Questions page appears Type the answers on the questions The answers will be saved in the result file Questions KTAexplorer Making Your First Runs 18 1140 78 Edition AF 29 5 Starting arun 7 Click Next For example the Evaluation Procedures page appears Evaluation procedures are automated evaluation operations that are performed after the run For instance select Print_Chromatogram and the chromatogram will automatically be printed after the run Integrate_Full Report MReport_Chromatogram Mintegrate_and_Print Integrate_BP_Report BP_Report_Chromatogram JUpdate_Quantitation Quantitate_Sample 8 Click Next For example the Method Information page appears Here you see information about the run Under the Method Duration tab the approximate volume of buffer used A B is shown as well as how long time the method will take Method Information
9. E coli proteins 2 in the Method Handbook Purifying synthetic peptides 3 in the Method handbook Purifying oligonucleotides 4 in the Method Handbook Different sample applications KTAexplorer Optional Configurations User options Manual Different fraction collection KTAexplorer Optional Configurations User options Manual and AKTAexplorer System Manual BufferPrep details KTAexplorer System Manual Columns and recommended KTAexplorer System Manual tubing Changing tubing kits KTAexplorer System Manual Calibrating monitors and pumps UNICORN User Manuals Comparing chromatograms UNICORN User Manuals Integrating curves UNICORN User Manuals Measuring HETP and resolution UNICORN User Manuals Exporting curves and data to UNICORN User Manuals other programs Finding information about a Click on Help button in the dialog box that certain menu instruction in appears or look in the index in UNICORN UNICORN 5 11 User Manuals Controlling Pump P 900 Monitor KTAexplorer System Manual to unlock the UV 900 and Monitor pH C 900 dials Chapter 3 in the User Manual for from the dials on the instruments each instrument found in the binder themselves AKTAdesign Components Details about each component See each individual manual in the binder AKTAdesign Components Security features UNICORN User Manuals Controlling the system from a UNICORN User Manuals remote computer KTAexplorer Making Your First Runs 18 1140 78 Edition AF 47 10 G
10. Run and Excluded for the different runs Those marked Excluded will not be run A scouting scheme is now defined 9 To save the scouting method select File Save 10 Prepare the system and start the run as described in chapters 4 and 5 When the method is started all the runs in the scheme will be performed automatically and the set flow rate 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 In scouting samples must be loaded several times Use the sample pump in KTAexplorer 100 to accomplish this automatically In AKTAexplorer 10 sample application must be carried out manually Use a large sample loop or Superloop to apply the samples Empty a section of the sample loop for each scouting run In AKTAexplorer 10S sample application is done automatically by the sample pump In AKTAexplorer 10XT an autosampler is used for performing automated multiple sample injections 46 KTAexplorer Making Your First Runs 18 1140 78 Edition AF Going further 10 10 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 KTAexplorer manual package To learn about Read manual section Purifying
11. might be required to take appropriate measures WARNING All repairs should be done by personnel authorized by GE Healthcare Do not open any covers or replace parts unless specifically stated in the instructions CE certification This product complies with the European directives listed below by fulfilling corresponding standards A copy of the Declaration of Conformity is available on request e 73 23 EEC Low Voltage Directive e 89 336 EEC EMC Directive The CE logo 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 inthe same state as it was delivered from GE Healthcare except for alterations described in this manual Note The Declaration of conformity is valid only for systems that are marked with the CE logo CE About this guide 1 1 About this guide This guide is written for users who are not familiar with UNICORN software and AKTA explorer Here you will learn the basics of UNICORN and how to operate AKTAexplorer from UNICORN UNICORN is a software package for control and supervision of the AKTAexplorer chromatography system It runs on an IBM compatible PC under Windows and includes hardware for interfacing the controlling PC to the chromatography liquid handl
12. the report 2 To add an empty page to the report click Add Page 3 Select from the Insert menu the item to include Items available are e Free text e Picture e Text method e Chromatogram e Documentation e Evaluation log e Quantitate and molsize optional e Frac 950 optional 4 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 5 Click and drag to create a box of the desired size Release the mouse button A dialog is displayed specific to the type of item inserted Make the appropriate selections in the dialog and then click OK to view the inserted item KTAexplorer Making Your First Runs 18 1140 78 Edition AF 41 7 Viewing and printing the result 7 2 Printing and making a report Change page layout 1 If you want to change the page layout select Edit Page Setup The Page Setup dialog opens 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 Page Setup Coturent user MRun user Current date amp time MRun date amp time MRepoit title MResult file name MMethod file nam Page number UNICORN Report 2 Toprint the report click Print 42 KTAexplorer Making Your First Runs 18 1140 78 Edition AF Buffer prep 8 8 Buffer prep The BufferPrep function allow
13. F ON InletB2 C OFF C ON 2 Select the instruction PumpWashExplorer 3 Select the inlet A11 for Inlet A1 4 Set Inlet A2 to OFF Inlet B1 to ON and Inlet B2 to OFF 5 Click Execute to start filling the tubing The injection valve will automatically switch to waste during the pump wash 6 When finished click End in the System Control toolbar eS Run Continu End aloe ee 7 Inthe Pump Instructions dialog click Close to close the dialog KTAexplorer Making Your First Runs 18 1140 78 Edition AF 25 4 Preparing the system fora run 4 2 General system preparation 26 4 2 2 1 Filling the sample loop Check that the correct loop is mounted between port 2 and 6 on the injection valve Connect an injection fill port or a union luer female 1 16 male to port 3 on the injection valve and apply the sample manually with a syringe KTAexplorer Making Your First Runs 18 1140 78 Edition AF Startingarun 5 5 Starting a run 1 Open the System Control module 2 Select File Run Select the method to start Click OK the method will not start yet A Start protocol appears consisting of a number of Run Setup pages The pages that are displayed depending on your selections in the Method Editor 3 If using a Frac 950 the Frac 950 page can be the first page that appears In the Frac 950 page you set up the Frac 950 fraction collector Define the order of fractionation and if desired set up the las
14. GE Healthcare AKTAexplorer Making your first runs KTA Contents 1 About this guid nr nn tee 7 1 1 Pre requisit s 55isnstnumennid attenante 7 12 Typographical conventions zususenenesesesenenenenenenenenenennnennnnnnenenene 8 2 The system and the software 9 2 1 General 1 cscs ccsccsscsiecesasisteccesesecesiecetsossecnsudecucastssenduts a at 9 2 2 AKTAexplorer 100 rm 2 3 AKTAexplorer 10 u cccsscssssssssssssssssssssssssssssssssssssssesssssssssssesssessnsessees 2 4 UNICORN overview 25 Hebel rege 3 Creating a m thodes eue 19 4 Preparing the system for a run 23 4 1 System connection esessssesesnenonnnnnnnennsnennnnnnesnenennennnnnnnsnenennnnnnane 23 4 2 General system preparation eeesesesesesesenenenenenenenenenenenenenenenennee 24 4 2 1 Filling the inlet tubing 25 4 2 2 Filling the sample 100p xs 2 9 12114111011011011011511511111s1010rs1rsnrs orn nr stseene 26 5 Starting A FUN one Ras 27 6 Viewingarun eessssessssesnsnennsnsnnnnessnnsnnsnsnnnnsnnsnsnennsnsnnsnnnennnnenen 33 7 Viewing and printing the result 37 Tile VIGWING a eed TR A ee eke 37 7 2 Printing and making a report euesssnsnsnesenenenenenenenenensnsnnenenenenenee 40 8 Buffer PIED dem a eros te ei en un 43 9 SCoUtINg ren nn in ne 45 10 Going f rther u 47 Index tn nn TE 49 KTAexplorer Making Your First Runs 18 1140 78 Edition AF 3 KTAexplorer Making Your First R
15. O Wavelength_3 O BufferValve_A1_lnlet O Column_Position O Start ValveFrac_at Block Variable Sia SS a IE BES Be A Er ee ti i ie a De Ba EZ En Er ee v Define other Scouting variables 3 Alist of all the variables will appear Select the variable Flow_Rate and any other variable you wish to alter e g Peak_Frac_Size 4 Click OK The selected scouting variables will appear to the left with their default values inserted Note Values for variables selected for scouting are greyed on the Variables page and cannot be changed there KTAexplorer Making Your First Runs 18 1140 78 Edition AF 45 9 Scouting 5 To change a variable value position the cursor in the Run value field and double click with the left mouse button Type the new value Flow_Rate ml min 00 6 To add a table column for the 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 7 Repeat step 6 until you have defined all the runs you require If necessary use the horizontal scroll bar to see more runs 8 Click Run1 Run2 etc at the top of the scheme with the right mouse button to toggle between
16. e chromatography system has three main modules which are stacked on the left hand side of a base platform They are e Pump P 900 a family of binary high performance gradient pumps In AKTAexplorer 100 the flow rate is up to 100 ml min and the pressure up to 10 MPa pump designation is P 901 In KTAexplorer 10 the flow rate is up to 10 ml min and pressure up to 25 MPa pump designation is P 903 e Monitor UV 900 a multi wavelength UV Vis monitor for simultaneous monitoring of up to 3 wavelengths in the range 190 700 nm e Monitor pH C 900 a combined monitor for on line conductivity and pH monitoring If installing a fraction collector it should be placed on the right hand side of the system KTAexplorer Making Your First Runs 18 1140 78 Edition AF 9 2 The system and the software 2 1 General Components such as the mixer column and different valves are mounted in the section to the right Open the valve door to view them all Columns are snapped in place on the outside of the valve door The separation unit is controlled from UNICORN software Pump P 900 Monitor UV 900 and Monitor pH C 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
17. e correct solutions use the instruction PumpWashExplorer in System Control Manual Pump WINCORN_LAB2 Pump Instructions x Instructions r Parameters men f FlowRate 0 100 set Gradient ee celal ing gun mimin aa m Alarms amp Mon DirectLoad PumpwashE xplorer Other PumpwashPurifier Fr PumpwashBasic Help 11 Select the correct inlet A11 for Inlet A1 and set Inlet A2 to ON 12 Select ON for both Inlet B1 and Inlet B2 13 Click Execute to fill all the inlet tubing 14 When finished click End at the top of the System Control window Dann Run Continu End 38 8 L 4 15 Inthe Pump Instructions dialog click Close to close the dialog window Note Iftubings A2 and B2 are empty they will have to be primed first See the Pump P 900 User Manual The sample should if possible have a pH close to the highest pH in the run for anion exchange and close to the lowest pH for cation exchange 44 KTAexplorer Making Your First Runs 18 1140 78 Edition AF Scouting 9 9 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 a flow rate scouting 1 Create a new method as described in chapter 3 Creating a method 2 When the Run Setup window appears click the Scouting tab Scouting Variables LE O Column_PressureLimit O Wavelength_1 O Wavelength_2
18. em symbol If you are not connected to a network only one system will be shown Click OK When connected the text YES is shown in the Connection panel in the Run Data pane 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 log on to UNICORN Connection YES KTAexplorer Making Your First Runs 18 1140 78 Edition AF 23 4 Preparing the system fora run 4 2 General system preparation 4 2 24 General system preparation 1 The correct tubing kit for the column you intend to use must be installed System Tubing kit i d mm KTAexplorer 10 0 25 0 50 KTAexplorer 100 0 50 0 75 1 00 Installed at delivery See KTAexplorer System Manual for an overview over columns with recommended tubing kits For most columns the tubing kit mounted at delivery can be used Note Iftubing with too large inner diameter is used the peaks will become broader than necessary If tubing with too small inner diameter is used the back pressure from the tubing might become higher than the max pressure for the column and the run will stop immediately after it is started Buffer Valve V6 Alternative placement for tubing holder 2 Immerse inlet tubing A11 or another inlet tubing if you changed this in the method in buffer A and inlet tubing B1 or B2 in buffer B 3 Check that the waste tubings from
19. ent instruction or module and find new help topics or Index for a specific topic In any dialog click on the Help button to get help for how to use the current active dialog KTAexplorer Making Your First Runs 18 1140 78 Edition AF 17 2 The system and the software 2 5 Help 18 KTAexplorer Making Your First Runs 18 1140 78 Edition AF Creating amethod 3 3 Creating a method The UNICORN software is supplied with a Method Wizard used for creating new methods The wizard is a number of dialog windows with questions and instructions that help you creating the method To create a method 1 Click the Method Wizard icon N in the Method Editor module If required choose which system you want to use and click OK The Method Wizard window appears Method Wizard for System EXP100F xi r Main Selection Column and BufferPrep Main Selection Select technique ion Exchange x Select column Positi Select column _____ Frositiont Bypass z position I Flexible Flow Rates NOTE Different Flow Rates are Set on Variable Page Select column __ IF Flow Regulation of the System Pump position I BufferPrep To get detailed help texts click Help Click Next Note You can restore all settings to default values by clicking Set Default can only be done in this dialog 2 Select a chromatographic technique for example Anion_Exchange 3 Select the column you intend to use The correct column v
20. fer A is connected to inlet A11 of the buffer valve which is placed at the back of the system Up to 8 different buffers can be connected to the buffer valve Buffer B is connected to inlet B1 Inlet A2 and B2 are used when buffers are prepared automatically by BufferPrep Buffer passes the two switch valves placed between the pump heads as it is drawn to the pump The pump has 4 pump heads two for pump A and two for pump B Pump A is the one closest to the front The flow path continues from the pump to the mixer then to the on line filter and forward to the injection valve A sample loop can be connected between ports 2 and 6 on the injection valve e In AKTAexplorer 100 and in AKTAexplorer 10S the sample loop can be filled manually or automatically using the sample pump Connect the inlet tubing on the sample pump to port 3 on the injection valve The sample pump draws sample from one of the inlets on the sample valve to fill the sample loop e In AKTAexplorer 10 the sample loop can be filled manually using syringe connect a fill port to port 3 on the injection valve After the injection valve the flow is forwarded to the column e In AKTAexplorer 100 the flow first passes a flow direction valve inside the door The flow direction valve allows the flow direction through the column to be selected The flow then continues to the first column valve After the column the flow passes the flow direction valve again and then forward to
21. ing parts of AKTAexplorer In this guide you will learn how to e create methods e prepare the system for runs e perform runs e make simple evaluations e make reports e perform automatic method optimization Scouting e prepare automatically buffers of any pH BufferPrep Follow the guide from page to page in front of the computer The 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 Before using the system see the separate Installation guide e the system and the software must be installed and functioning and e the monitor and the pump must be calibrated as described in the guide IMPORTANT Before using AKTAexplorer read all the safety information in KTAexplorer System Manual KTAexplorer Making Your First Runs 18 1140 78 Edition AF 7 1 About this guide 1 2 Typographical conventions 1 2 Typographical conventions Menu commands and dialog box prompts are identified in the text by bold text Acolon separates menu levels thus File Open refers to the Open command in the File menu 8 KTAexplorer Making Your First Runs 18 1140 78 Edition AF The system and the software 2 2 The system and the software 2 1 General KTAexplorer is a fully automated liquid chromatography system designed for method development and research applications The separation unit of th
22. it is finished click the End button at the top of the System Control module O RT Run Continud End sole Bere ol 36 KTAexplorer Making Your First Runs 18 1140 78 Edition AF Viewing and printing the result 7 7 Viewing and printing the result If you are satisfied with the automated print out obtained after the run if selected you do not need to 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 Open the UNICORN Main Menu Double click on a result file icon in the list to the right 2 The Chromatogram window is opened automatically in the Evaluation module when you open a result file The Chromatogram window contains all the curves Note that the term chromatogram is used here when talking about the whole window containing all the different curves amp Evaluation Example Result001 1 v UNICORN Local fil Niklas result Example Result002 Example Result001 res File Edit View Integrate Operations Procedures Quantitate Mol Size Window Help laj xj BPE 5 bias Example Result001 1_UV1_216nm Example Result001 1_Cond Example Result001 1_Fractions Example Result001 1_Inject Example Result001 1_UV1_215nm 01 BASEM mau 50 we d poo f Z 7 04 6 52 PA 50 nos 2 ob 20
23. l Help 5 Highlight the curves to view under Curve Curves are named as Result001 1_ curve where a curve can be for example UV_wavelength Cond pressure etc Clear all curves except for example the UV Cond and Conc curves Click OK at the bottom of the Chromatogram Layout dialog 6 Tozoomina peak of interest left click and drag to create 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 on the right mouse button and select 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 color of a curve its Y scale and its X scale can all be changed 38 KTAexplorer Making Your First Runs 18 1140 78 Edition AF Viewing and printing the result 7 Open the Chromatogram Layout dialog again Click the Y axis and X axis tabs to set the scale for the different curves Normally the curves are scaled with auto scaling i e the highest and lowest values for each curve setthe scale e To fix the Y axis scale mark a curve click Fixed and enter the Min and Max values for that curve You can repeat this for other curves e To fix the X axis scale click Fixed in the X axis field and enter theMin and Max values for the x axis Chromatogram Layout 1 To save changes in the chromatogram layout click the Layout Library tab Click Save Current layout as
24. oing further 48 KTAexplorer Making Your First Runs 18 1140 78 Edition AF Index A air inthe inlet tubing 24 inthe pump 24 AKTAexplorer 10 13 100 13 B BufferPrep 43 Cc chromatogram print 40 chromatography technique 19 column position 20 select 19 volumes 21 column valve flow path 12 conductivity cell flow path 12 connect to system 23 connection panel 23 create method 19 customize pane display 33 D default setting 19 E evaluation button 16 module 37 procedures 30 F flow path 12 bufferA 12 column valve 12 conductivity cell 12 flow direction valve 12 flow restrictor 12 injection valve 12 outlet valve 12 pH flow cell 12 sample pump 12 switch valve 12 UV cell 12 flow rate KTAexplorer 10 9 KTAexplorer 100 9 KTAexplorer Making Your First Runs 18 1140 78 Edition AF fraction collector placement 9 G gradient 21 H Help 17 I injection valve flow path 12 inlettubing 25 instant run button 16 L ogon UNICORN 14 Windows 14 ogon logoff button 16 M mainmenu 15 manuals 47 method create 19 existing queue 16 queue 16 method editor button 16 method files 15 method wizard 19 mixer 10 modules 9 monitor pH C 900 9 UV 900 9 N notes page 29 O ON OFF button 10 p password 14 prepare system 23 pre requisites 7 pressure KTAexplorer 10 9 KTAexplorer 100 9 print chromatogram 40 49 50 result 40 result 37 pump P 900 9 Ww Q wavelengths 9 que
25. olume the recommended flow rate and the correct pressure limit for that column will then be automatically implemented in the method Note Ifyou manually alter the default values and thereby exceed the recommended values for the selected column you will get a warning when you save your method Note Ifyou want to perform a test run without a column you should still select a column a small one is recommended to get suitable default parameters in the method Then when running the method use a piece of tubing to replace the column Note Ifyou do not find your column in the list you can add one Refer to the UNICORN User Manual KTAexplorer Making Your First Runs 18 1140 78 Edition AF 19 3 Creating a method 4 Select Column Position and if required Flexible Flow rates and or Flow Regulation of the System Pump and or BufferPrep 5 Click Next to go through the subsequent windows In each window select the appropriate parameter values 6 Click Finish in the last window The Run Setup window appears Click here to select page CE el em Vea lor EE eae ee IGS Men Jon Hlosd1610 0 Sephaose FFi lbalm Fon Rae om fun Con Pressue ma ou Pessweling MPa fon um stat moto Teen 2 E 90 700 90 700 avelength_3 nm ufferValve_ amp 1_Inlet Wavelenath_2 nm ufferValve_41_Inlet p ump_ amp _Inlet ump_B_Inlet olumn_Valve olumn_Position u u u u u g lowthrough_Fractionation owthrough_FracSi
26. olumn selection N valve k Switch valves 5 I Buffervalve Waste N To fraction collector IN optional N Column selection valve KTAexplorer Making Your First Runs 18 1140 78 Edition AF 2 The system and the software 2 4 UNICORN overview 2 4 UNICORN overview 1 Switch on the computer Log on to Windows by first pressing Ctrl Alt Del and then clicking OK After a while the Windows desktop appears BB vraconns s 2 Start UNICORN by double clicking on the UNICORN icon 3 Aninformation window appears during start up OUNICORN 5 11 imagination at work COPYRIGHT GENERAL ELECTRIC COMPANY 2006 ALL RIGHTS RESERVED 4 Inthe Logon dialog select a user from the Users list and enter the password If you log on 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 KTAexplorer on a regular basis KK ce Hep KTAexplorer Making Your First Runs 18 1140 78 Edition AF 14 The system and the software 2 5 Eventually the UNICORN Main Menu window appears on the screen 4 UNICORN Main Menu olx File View Administration Tools Window Help Te 3 Methods of xi Results
27. port 4 and 5 of the injection valve and port 1 of the outlet valve are put into a waste bottle If fraction collector Frac 950 is used check that the tubing from the chosen port of the outlet valve is connected to the fraction collector For fraction collector Frac 900 the port 2 of the outlet valve will be selected by default 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 the Pump P 900 User Manual KTAexplorer Making Your First Runs 18 1140 78 Edition AF Preparing the system forarun 4 5 If pH measurement is required calibrate the pH monitor Refer to the UNICORN User Manuals or the Monitor pH C 900 User Manual Mount the pH electrode in the flow cell 6 Connect the column to the two column valves at the position set in the method normally position 2 When using the bypass tubing select position 1 7 Insert a sufficient number of tubes in the fraction collector optional 4 2 1 Filling the inlet tubing 1 Select Manual Pump in the System Control module The Pump instruction dialog opens WINCORNLAB22 Pump Instructions x r Instructions p Parameters Pump Flow InletA1 Insert Gradient Flowpath BufferPrep_pH fant Be c SystemPumpControlMode Alarms amp Mon SystemPIDParameters Inlet amp 2 C Frac Pump ashE xplorer G OFF CON Pump ashPurifier Close Other PumpWashBasic En InletB1 Help C OF
28. s a buffer of any pH to be prepared online from four stock solutions The pH can be varied automatically between scouting runs to find the optimal pH for the separation see chapter 9Scouting ApH electrode is not necessary to obtain correct pH using BufferPrep For more details about BufferPrep see KTAexplorer System Manual T o create a method that includes BufferPrep 1 Inthe Method Editor module select File Method Wizard If required select system 2 Select a chromatographic technique for example Anion_Exchange 3 Select BufferPrep A collection of BufferPrep recipes and the pH setting appear Method Wizard for System EXP100F Anion_Exchange Position Bypass E Vv 5 0 9 5 pH AIEX Global v Select recipe and set the desired pH 4 5 Complete the rest of the Method Wizard 6 In Run Setup select the BufferPrep tab 7 The required solutions and the inletsto which they should be connected are displayed to the right on the BufferPrep page Accurate buffer preparation is essential You find the correct method for preparing buffers in the Notes field When the preparation is finished connect the buffers to the correct inlets Note Manual pH adjustment is not necessary KTAexplorer Making Your First Runs 18 1140 78 Edition AF 43 8 Buffer prep 8 Select File Save 9 Prepare the system and start the run as described in sections 4 and 5 10 When filling the inlet tubing with th
29. stions page 29 queue method 16 R report generate 40 print 40 restore allsettings 19 result filename 22 view and print 37 result name page 31 run data 33 setup page 27 start 27 status 33 run setup page 22 S sample loop 12 26 scouting 45 variables 45 software 9 start protocol 22 starting arun 27 switch on chromatography system 10 computer 14 system 9 connection 23 control button 16 modules 9 preparation 24 T tubing kit 24 typographical conventions 8 U UNICORN 14 user list 14 UV cell flow path 12 V valves 10 variables 21 scouting 45 variables page 21 view arun 33 KTAexplorer Making Your First Runs 18 1140 78 Edition AF KTAexplorer Making Your First Runs 18 1140 78 Edition AF 51 For contact information for your local office please visit www gelifesciences com contact GE Healthcare Bio Sciences AB Bj rkgatan 30 751 84 Uppsala Sweden www gelifesciences com imagination at work GE imagination at work and GE monogram are trademarks of General Electric Company rop Design KTA KTAexplorer UNICORN and Superloop are trademarks GE Healthcare companies og Cc NICORN Any use of this software is subject to GE Healthcare Standard oftware End User License Agreement for Life Sciences Software Products gt v third party trademarks are the property of their respective owners 1999 2008 General Electric Company All rights reserved First published No
30. t the start of a method run Reference Curves ME valuation Procedures Method Information 10 Tosave the method select File Save In the Save dialog enter a name Store the method in the directory of your choice by doubleclicking on a directory Click OK In the UNICORN Main Menu module the method appears in the Methods window Note The method name followed by three consecutive numbers starting with 001 will then be used as default name for the result file of your method after runs Now you are ready to start a run Go to chapters 4 and 5 You can also go to chapter 9 to learn how to alter variables systematically and automatically in repeated runs This is known as scouting and is a convenient easy to use function 22 KTAexplorer Making Your First Runs 18 1140 78 Edition AF Preparing the system forarun 4 4 Preparing the system for a run 4 1 System connection Before you can start a run you must connect to the system Connecting means that the System Control module is set up for a particular system Re If you are not connected the text NO is shown in the Connection panel in the EE Run Data window Once you are connected the text changes to YES 1 Click on the 1 System Control button in the task bar at the bottom of the screen 4 Syste 2 To connect to a system Select System Connect The System connect dialog appears System Connect x Cancel Help 3 Select a syst
31. t tube used in the fractionation The system will be paused when the last tube is reached and the fractionation will stop 4 Click Next For example the Variables page appears This is the same page you were working on in the Method Editor Here you can verify and fine tune KTAexplorer Making Your First Runs 18 1140 78 Edition AF 27 5 Starting arun the method before proceeding This is very convenient when repeating runs with minor adjustments Variables FE Main Cobar Flow Rate sd CS Flow_Rate ml min Column Pressure_Limit Column_PressureLimit MPa Start Instructions g Bo ae gt DI fuavoengn 3m BuffeValve At_Inlet JBuffervalve_A1_Inlet Euename Fm ana Eten Bin Fume B iet Dune Posten owthrough_FracSize ml Fractionation luate_Frac_Size ml 1 HiLoad_16 10_Q_Sepharose_FF ml 0 00 100 00 0 00 10 00 190 700 190 700 190 700 oo EA EWEEN osition Bypass 000 000 99999 000 000 39999 000 000 99999 000 0 100 0 000 99999 000 Linear_Gradient arget_ConcB B ength_of_Gradient CV r o m n o a a lt lt vja ala EN als F Se IND pes ala Sha 2 3 H u E GE eak_Frac_Size ml E E 3 E i elep ojo 815 i i i Note When starting run no 2 immediately after run no 1 with the same method but for example a different flow rate you simply Click the Run button in System Control
32. the UV cell and the conductivity cell which are placed inside the cell holder on Monitor UV 900 e In AKTAexplorer 10 the flow passes the first column valve The column is connected between the two column valves Up to 7 columns can be connected A bypass tubing is connected to position 1 on the two column valves After the second column valve the flow is forwarded to the UV cell and the conductivity cell which are placed inside the cell holder on monitor UV 900 The flow path continues to the pH flow cell on the outside of the door to the flow restrictor and then to the outlet valve which is used to select fraction collection method and to handle waste and flowthrough The flow path can continue to the fraction collector optional if desired KTAexplorer Making Your First Runs 18 1140 78 Edition AF The system and the software 2 2 2 AKTAexplorer 100 Sample Column Monitoring Fractionation Mixer On line filter Flow direction UV monitor i F Injection valve valve Conductivity monitor Outlet valve pH monitor I Column pecan To fraction collector optional Waste 2 3 AKTAexplorer 10 Buffer Sample Column Monitoring Fractionation System pump Mixer On line filter UV monitor Injection valve Conductivity monitor Outlet valve pH monitor 8 2 Flow restrictor C
33. uns 18 1140 78 Edition AF Important user information All users must read this entire manual to fully understand the safe use of AKTAdesign Important KTAdesign is intended for research use only and should not be used in any clinical or in vitro procedures for diagnostic purposes KTAdesign should not be used in any clinical or in vitro procedures for diagnostic purposes Safety notices This manual contains warnings and cautions concerning the safe use of the product See definitions below WARNING The WARNING symbol and notice highlight instructions that must be followed to avoid personal injury Do not proceed until all stated conditions are clearly understood and met CAUTION The CAUTION notice highlights instructions that must be followed to avoid damage to the product or other equipment Do not proceed until all stated conditions are met and clearly understood Notes Note A Note is used to indicate information that is important for trouble free and optimal use ofthe product 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 authorized representative of the manufacturer for information concerning the decommissioning of equipment WARNING This is a Class A product In a domestic environment it might cause radio interference in which case the user
34. v 1999 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 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 Europe GmbH Munzinger Strasse 5 D 79111 Freiburg Germany GE Healthcare Bio Sciences KK Sanken Bldg 3 25 1 Hyakunincho Shinjuku ku okyo 169 0073 Japan 18 1140 78 AF 05 2008
35. ve For example to fix the Y axis scale for a curve click the Y axis tab Mark the curve click Fixed and enter the Max and Min values You can repeat this for other curves Click OK 34 KTAexplorer Making Your First Runs 18 1140 78 Edition AF Viewingarun 6 Properties pass foen 6 Tomaximize the Curves pane right click in the Curve Data pane and select Maximise Go back to normal size by clicking Restore 7 Toshift to a scale for another curve click on the Y axis scale or click on the curve name at the top of the Curves pane The color of a curve its Y scale and its name are always the same Click the X axis to shift between time and volume 8 The Logbook is shown at the bottom The Logbook shows exactly when the instructions in the method are executed during the run The Logbook is stored in the result file 9 You can make manual changes during the run Select Manual Pump The Pump Instructions dialog opens WINCORN_LAB2 Pump Instructions If for example you want to change the flow rate select Pump and then Flow Enter a new flow rate under Parameters and click Execute The new flow rate will be used until the end of the run or until a new flow rate KTAexplorer Making Your First Runs 18 1140 78 Edition AF 35 6 Viewing arun instruction is reached in the method Close the dialog by clicking Close All manual interactions are recorded in the Logbook 10 If you want to stop the run before
36. ze ml u u u u luent_A_Inlet luent_B_Inlet 000 39999 000 000 99999 000 000 99999 000 uate_Frac_Size ml eak_Frac_Size ml arget_ConcB B ength_of_Gradient CV ractionation inear_Gradient 000 99999 000 ee ZZ ay i E al M Run setup consists of a number of pages You will only look at a few now Select a page by clicking the respective tab at the top of the window 20 KTAexplorer Making Your First Runs 18 1140 78 Edition AF Creating amethod 3 7 Onthe Variables page the method is presented by a number of blocks The blocks represent typical steps in a chromatographic run such as e Start instructions e Column equilibration e Sample injection e Wash out unbound sample e Fractionation e Gradient e Clean after elution e Re equilibration Some of the blocks contain a number of Variables with suitable default values The values can be changed to suit your application Some of the variables are normally hidden but can be shown by checking the Show details box 8 Click the Gradient tab to view the method graphically Gradient Block name 0 00 Main The length of each block is marked at the bottom of the graph Click the x axis to view the method in time volume or column volumes KTAexplorer Making Your First Runs 18 1140 78 Edition AF 21 3 Creating a method 9 Click the Start Protocol tab to decide which of the Run Setup pages to be displayed a
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