ÄKTAdesign - GE Healthcare Life Sciences
Contents
1. Method Information Resukinomaion StatProtoco Settings Caltrain Logbook EvalusionLog Rerik Name P a rese F Alam Warning 0 00 mi Method Fun 2X2 0411 MC NM Example Result Result v Niklas Ex 0 00 mi Batch ID T eH a Raay srt 8 ini Mono SHR am M Am Perae be 500MPa 000MPa 000 mt Wareena 3 215 nm 280 nm OFF 0 00 mi BufferPrep_pH 5 400 82 mi QuarttationD ata Sample 8 83 ml Quantitat Into Rc Re Dr M 8 83 ml Quariitate Info Type 0u No Note Bate SameAsMain Gradent 80 00 8 8 000CV tactionation 18mm Doce Hall Volume 72 Printing and making a report 1 To print the chromatogram select File Report The Generate Report dialog opens 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 AKTAreic Making Your First Runs 18 1140 80 Edition AF 33 7 Viewing and printing the result 3 Click Preview to view the report on the screen Page Toll Preview Mode Add information to the report 1 Click Edit Mode to enable changes in 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 Free text Picture Text method Chromatogram Documentation Evaluation log 4 Move the mouse pointer into the page area of the window You will notice that the mouse pointer has an addition2. 40 Setting SEEME s ast nc federe ota ee Ra RRi 40 Scouting variables iet natn ierit teer ati te etate debe EUH 439 Statt DFOIOGCOlN etatem nerit tiet m Rudi 218 23 iem 28 Stant NICO RN eese cseteris n Rosita RUE AR EUN 10 switch on the Chromatography system sees 8 system control Shout ULC OM esea e aE mentiti et ann 11 sustem descriptiORiuiennocct tta tetas tet e RE e RE 7 U USER eodera ordre p tatud and daa dd A E ic IEE leid sr 10 UV flow cell TOW OIE p 8 9 V Wc Variables page AKTAre c Making Your First Runs 18 1140 80 Edition AF 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 E Elanders i Uppsala 2008 Drop Design KTA AKTArptc re c and UNICORN are trademarks of GE Healthcare companies GE imagination at work and GE monogram are trademarks of General Electric Company 1999 2007 General Electric Company All rights reserved First published March 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
3. to activate this dialog Curve Style and Colour l Edi Texts Layout Library Header CuveNames YAxs x Cuve Peak Table Highlight curves to view UT Example Result001 1_UV1_215nm pem heat 2E 7 m One 7103 Example Resuk001 1_UV3_Onm v 04 Example Result001 1_Cond 05 Example Resuk001 1_Cond v 06 Example Result001 1 Conc 107 Example Resulit001 1 pH 08 Example ResuR001 1 Pressure 09 Example Result001 1 Flow J10 Example Resuit0O1 1 Temp 11 Example Result001 1 Fractions v 12 Example Result001 1_Inject 113 J14 1 S16 V17 Example Result001 1_UV1_215nm 01 BASEM 5 Highlight the curves to view under Curve Curves are named as Resultfile001 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 To zoom in a 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 AKTAreic Making Your First Runs 18 1140 80 Edition AF 31 7 Viewing and printing the result 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 chang
4. Method Queue that is running Method Queues are used to link several methods together 12 AKTAre c Making Your First Runs 18 1140 80 Edition AF 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 Alternativelu 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 Index for a specific topic In any dialog click on the Help button to get help on how to use the current active dialog AKTAreic Making Your First Runs 18 1140 80 Edition AF 13 3 Creating a method 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 xl in the Method Editor module If required choose which system you want to use and click OK The Method Wizard window appears Select technique Select column To get detailed help texts click Help Click Next Note You can restore all settings to default values by clicking Set Default only possible in this dialog 2 Select a chromatographic technique for example Anion Exchange 3 Select the column you intend to use The
5. the separation equipment of AKTAretc 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 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 AKTAreic Making Your First Runs 18 1140 80 Edition AF 5 1 About this guide 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 AKTAreic read all the safety information in AKTArpic System Manual 12 Typographical conventions Menu commands and dialog box prompts 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 6 AKTAre c Making Your First Runs 18 1140 80 Edition AF The system and the software 2 2 The system and the software 231 General AKTAretc is a fully automated liquid chromatography system designed for protein purification in research applications The system has two main instrument components stacked on the base platform They are e Pump P 920 a high perform
6. variables systematically and automatically in repeated runs This is know as scouting and is a convenient easy to use function Ettan Pump P 905 User Manual 18 1145 36 Edition AC 17 4 Preparing the system for a run 4 Preparing the system for a run 18 gusce 4 Syste 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 If you are not connected the text NO is shown in the Connection panel in the 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 monitor 2 To connect to a system Select System Connect The System connect dialog appears 3 Select a system 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 AKTAre c Making Your First Runs 18 1140 80 Edition AF Preparing the system fora run 4 4 2 General system preparation 1 Immerse inlet tubing A in buffer A and inlet tubing B in buffer B 2 Check that the tubing marked G5 from the FR 902 Flow restrictor outlet is connected correctly Check that the three waste tubings are put into w
7. 80 Edition AF 9 2 The system and the software 10 2 2 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 2 Start UNICORN by double clicking on the UNICORN icon 3 Aninformation window appears during start up 4 In the Logon dialog 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 AKTAretc on a regular basis 5 The four UNICORN modules open and their respective short cut button is placed in the task bar at the bottom of the screen The UNICORN Main Menu window appears on the screen AKTAre c Making Your First Runs 18 1140 80 Edition AF The system and the software 2 UNICORN Main Menu Fie View Administration Took Window Help WINCORNLAB 647KB MethodFie 2003042017 20030428 17 20030304 10 13 WINCORNLAB 656KB MethodFie 2003042911 2003 04 29 11 200304301423 20030430 1423 i 200204281710 20030429 1710 200204281715 20030428 1715 200204281656 20030428 16 56 2003 04 28 17 04 2002 04 28 17 04 20030428 1707 20030429 17 07 200204281712 20030428 1712 J User dela f a 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 navi
8. AKTAre c Making Your First Runs 18 1140 80 Edition AF Short instructions 10 10 Short instructions The following short instructions are intended as a guide for users who fully familar 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 2 Select File Method Wizard in the Method Editor module or click NJ s If neccessary select a system and click OK Go through the selections on the Method Wizard pages click Next to go to next page Click Finish on the last page Select File Save in the Method Editor module and give the method a name Click OK Z Click the System Control button in the tase bar 1 syste Select File Run Select the method and click Run The start protocol will appear Check the method on the Variables page and change valus as you require Click Next a few times On the Evaluations procedures page select Print_Chromatogram to get a printout automatically after the run 10 Click the Start button on the last page the run starts AKTAreic Making Your First Runs 18 1140 80 Edition AF 39 Index A add a column Scouting page eterne 40 air i theinlet tablbig noe titre etcetera 20 iri dbi pfi cese repeteret e ect btes EAER 20 C calibrate the pH monitor sseseeetttttttttttttttttttnntnnnnnnnnnn 20 change a variable value ttt ttttttttttttntn
9. Bio Sciences AB Bj rkgatan 30 751 84 Uppsala Sweden 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 1140 80 AF 12 2007
10. GE Healthcare AKTAdesign Making your first runs Begin here with AKTArpic AKTA Important user information All users must read this entire manual to fully understand the safe use of AKTAretc WARNING The WARNING sign highlights instructions that must be followed 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 pzd electrical and electronic equipment must not be disposed as unsorted municipal waste mmmm ond mu
11. Going further 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 AKTArpic manual package To learn about Read manual section Protein purification strategies Different sample applications options Different fraction collection options Columns 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 900 and Monitor UPC 900 from the dials on the instruments themselves Details about each component Security features Controlling the system from a remote computer The Method Handbook AKTArptc System Manual AKTAretc Optional Configurations User Manual AKTArP c System Manual UNICORN User Manuals UNICORN User Manuals UNICORN User Manuals UNICORN User Manuals UNICORN User Manuals Click on Help button in the dialogue box that appears or look in the index in the UNICORN User Manuals AKTAre c System Manual to unlock the dials Chapter 3 in the User Manual for each instrument found in the binder AKTAdesign Components See each individual manual in the binder AKTAdesign Components UNICORN User Manuals UNICORN User Manuals
12. al symbol according to the item type you selected to insert 34 AKTAre c Making Your First Runs 18 1140 80 Edition AF Viewing and printing the result 7 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 Change page layout 1 If you want to change the page layout select Edit Page Setup The Page Setup dialog opens and you can e g 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 mme ran a 2 To print the report click Print AKTAreic Making Your First Runs 18 1140 80 Edition AF 35 8 Scouting 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 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 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 o
13. ance laboratory pump for constant flow delivery e Monitor UPC 900 a high precision on line combined monitor for measuring UV absorption conductivity and pH optional If installing a fraction collector it should be placed on the right hand side of the system Components such as the mixer column and different valves are mounted on a system rack on the right side of the separation unit Pump P 920 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 AKTAre c Making Your First Runs 18 1140 80 Edition AF 7 2 The system and the software Switch on the chromatography system with the ON OFF button located on the front of the base platform to the bottom left Injection valve INV 907 Buffer tray Column holder with column On line filter Monitor UPC 900 Pump P 920 Iw KIo ex Mixer M 925 W Sh with mixer O a chamber DE SYS OZ SiS Sample loop Optical unit UV lamp Mains switch UV flow cell Mounting rack with opening handle Base Platform Flow restrictor Conductivity flow FR 902 cell with holder The system is controlled from UNICORN software 8 AKTAre c Making Your First Runs 18 1140 80 Edition AF The system and the software 2 Comment The flow path between the different modules and com
14. arun 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 xample Flesult 1 1 v MINICORNM ocalMi Niklas vesult VE xample Fiesult 2XE xample Result 01 res Fle Edt View Integate Operations Procedures Quanliae Mol Sie Window Heb A81 xl Example Result001 1 UV1 215nm Example ResulO1 Cond E ampls Pes 1 Cas Example ResultO 1 1 Fracfions Example ResuntOO 1 1 Inject Example ResultQO1 1 UV1 215m 1 BASEM mme 2 Peak 0 5 z 44 0967 60 5619 115 9646 30 AKTAre c Making Your First Runs 18 1140 80 Edition AF Viewing and printing the result 7 The result file from a run contains a complete record of the run including method system settings curve data and run log 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
15. aste bottles 3 If there is air in the inlet tubing or if you suspect air in the pump purge the pump as described in Pump P 920 User Manual 4 Calibrate the pH monitor optional if required Refer to the UNICORN User Manual or the Monitor UPC 900 User Manual 5 Connect the column between port 1 of the injection valve and the UV flow cell Use a suitable length of 0 50 mm PEEK tubing orange supplied with your system 5 mm flow cell 2 mm flow cell Connect from Connect from above below 6 Insert a sufficient number of tubes into the fraction collector 4 2 1 Filling the inlet tubing 1 Select Manual Pump in the System Control module 2 Select the instruction PumpWash The Pump Instructions dialog opens WINCORNPILOT Pump Instructions bx 3 Select ON for PumpA 4 Select ON for PumpB 5 Click Execute to start filling the tubing The injection valve will automatically switch to waste during the pump wash Ettan Pump P 905 User Manual 18 1145 36 Edition AC 19 4 Preparing the system for a run 6 When finished click End in the System Control toolbar E Md Run Pause Continue End lalola LLL ll 7 Inthe Pump Instructions dialog click Close to close the dialog 4 2 2 Filling the Sample loop 1 Check that the correct loop is mounted between port 2 and 6 on the injection valve 2 Connect an injection fill port or a union luer female 1 16 male to por
16. correct column volume the recommended flow rate and the correct pressure limit for that column will then be automatically implemented in the method Comment If you 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 If you 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 14 AKTAre c Making Your First Runs 18 1140 80 Edition AF Creating a method 3 Comment If you do not find your column in the list you can add one Refer to the UNICORN User Manual 4 If required select Flexible Flow rates and or Flow Regulation of the System Pump 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 E B E essei ki IER C A a hs he Frac 9 000 E sedet m pss a emn 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 Ettan Pump P 905 User Manual 18 1145 36 Edition AC 15 3 Creating a method 7 Onthe Variables page
17. ed 8 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 set the 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 To fix the X axis scale click Fixed in the X axis field and enter the Min and Max values for the X axis Chromatogram Layout 1 9 Tosave changes in the chromatogram layout click the Layout Library tab Click Save Current layout as 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 10 Click OK at the bottom of the Chromatogram Layout dialog to execute all the changes 32 AKTAre c Making Your First Runs 18 1140 80 Edition AF Viewing and printing the result 7 11 Click the View Documentation button A number of pages appear as in Ey 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 the Notes and Logbook tabs to check the contents Close the Documentation window by clicking on the X in the upper right corner Frac 950 Variables Scouting TextMethod Notes Questions BuffePrep Columns Reference Curves Evaluation Procedures
18. gate 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 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 e Method Editor opens the Method editor with a dialog window for creating new methods e System control opens a dialog window for controlling the system and running your methods AKTAreic Making Your First Runs 18 1140 80 Edition AF 11 2 The system and the software aS 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 SD UNICO zi Metho 4 Syste lt a Evaluation Additional buttons are provided in the toolbar These are e Instant run opens a dialog where you directly can choose a method to run This is handy for starting routine runs instantly e Logon Logoff opens a dialog to control the log on log off process Method Queue opens a dialog window for defining a new Method Queue e Existing Method opens a dialog window for showing Queue the
19. n the Variables page and cannot be changed there 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 F ow Rete Flow Rate 0T 36 AKTAre c Making Your First Runs 18 1140 80 Edition AF Scouting 8 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 Rune 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 9 Tosave 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 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 AKTAreic Making Your First Runs 18 1140 80 Edition AF 37 9
20. nnnnnnnnn 39 chromatographic technique jo 15 COUM ET 7 SEEGE SQ 15 conductivity flow cell Vile oro ig semen 9 connect to the system sisse tttttttttttentnnnnnnnnnn 19 connecting thie Gol rmrsu tasti tenetis 20 connection panel nnn 19 create CIMINO zianie eniin uin erna 15 customize the pane s display ttes 29 E evaluation Short GUE DUTTON c evaluation procedures existing method queue Short c t DUON s et EROS 12 F flow path GSS CH PUL Nisen anian ii Ria 9 POW rate SCONO ase tte ettet ete eeepc 39 flow restrictor TOW CUA 9 Frac 950 DOGS e 23 fraction collector placere AEE E EAE 7 G GMO CIC aI AAE E EE RECETA 17 H FG EEEE EASA E AE EEEE 13 40 AKTAre c Making Your First Runs 18 1140 80 Edition AF l injection fill POLE eeeeesessssssssssssscccccccccnsssssssssusnunussssssssessceeceescessessssssnsnustumunssssscseceseeeeeeees 21 injection valve TOW Mero 9 inlet A TOW DIY vs cotto denda eta ena dte gas 9 inlet tubing o 20 nlets B TOW ORIN e 9 L l g on to WIDOOWS nete tete TA eer i eee 10 log on log off SNom cut BULOM NE 12 M Main Menu WINGOW sssssssssssssssssessesessccecccecessessssssnsssn
21. nsssssssseesesseceesecsesssssssssnsnnsnsnees 11 method editor Shortcut DUEEOFIS siae citer eet it Butea atienden 11 method files displayed in the Methods pane 11 method queue Shott CUt DUEEOI s cette teatri iai 12 method wizard eee estttisttorisertesteettestee inanin ni s 15 Shott cutDUEEO cbe entrent eee te pe teet 15 abc 7 TOW UUM cisterna totetedustiside needed ER 9 modules Shotrt cut DUEEORS uctus ei estote sebo 12 monitor PC O00 cct tre teet Ree teneam as 7 N Motes GC m 25 0 ONOFF DUNOMA E E REOOO 8 on line filter Ow paten seii neen aae EEEE 9 KTArPLc Making Your First Runs 18 1140 80 Edition AF 41 42 P ejecit io ra RR 10 pump A TOW PILI ae 9 pump B Ow patireia ett eee et te ER ee te dd 9 PUMP rA D ei enei 7 Q questions DOGSS sicsassessjcatsccsscsasdesecscseousiooastidccnsaisoeassdoscnsasatezandeisbvesaetandsasaaesedeeopdiiynapetdenned 25 R restore all settings e eeeeeeeennttttttttttttttt ttt 15 result file Mone R 27 SCGUI Osna nannaa edite trt ree eiie iate iH 40 result files displayed in the Results pane tts 11 Result Natme page accettate tectis treibt deesse iisi 27 R b Set paVidOoW ssun acce tete binc diei eem re dtes 16 S sample loop Vill ofo Pise MITES 21 NOW P Otisni ee cme ces 9 scouting qp
22. on AF Viewing a run 6 7 Toshiftto 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 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 instruction is reached in the method Close the box by clicking Close All manual interactions are recorded in the Logbook 10 If you want to stop the run before it is finished click the End button at the top p o Run Gontin End Elola TEE AKTAreic Making Your First Runs 18 1140 80 Edition AF 29 7 Viewing and printing the result 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 71 Viewing 1 After
23. ponents in the separation unit is shown and described below It is not necessary to go through this in detail to make your first runs Look at the right hand side of the system if you want to follow the description System pump Mixer Sample Column Monitoring Fractionation Injection On line valve UV conductivity pH optional Flow restrictor To fraction Waste collection Waste 1 The pump has 4 pump cylinders two for pump A and two for pump B Pump A is the upper pair of cylinders and the pump valve closest to the front 2 Pump inlets A and B are placed in buffer A and B respectively and the buffer solutions are pumped to a mixer 3 The flow path continues from the mixer via an on line filter to the injection valve 4 Asample loop is connected to the injection valve The sample loop is filled manually using a syringe To perform this connect a fill port to the injection valve 5 From the injection valve the flow is directed to the column and then to the UV flow cell in the optical unit and the conductivity flow cell located below the optical unit Note In the standard configuration the pH flow cell is not included In optional configurations when the pH flow cell is mounted in the flow path it is connected between the conductivity flow cell and the flow restrictor 6 The flow path continues to the fraction collection or waste AKTAreic Making Your First Runs 18 1140
24. st 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 this product may cause radio interference in witch case the user may be required to take adequate measures Contents Contents 1 Aboutthis guide 11 Pre requisites 12 Typographical conventions 2 The system and the software 2l Generalaren ane a ae e L 2 2 UNICORN overview 235 Poler 3 Creating a method nn 12 4 Preparing the system for a run 4 1 SYSTEM connection s s 4 2 General system preparation 4 2 1 Filling the inlet tubing 422 Filling the Sample loop s S Starting A FUN n 21 6 Viewing d run e hh 27 7 Viewing and printing the result fd MEWN cse 7 2 Printing and making a report B SCOUDIWEO aic ide aci ee ee E 36 9 Going further 10 Short instructions mmm 39 Index omnia 40 AKTArpe c Making Your First Runs 18 1140 80 Edition AF 3 About this guide 1 1 Aboutthis guide This guide is written for users who are not familiar with UNICORN software and AKTAretc Here you will learn the basics of UNICORN and how to operate AKTAretc from UNICORN UNICORN is a software package for control and supervision of the AKTAretc chromatography system It runs on an IBM compatible PC under Windows and includes hardware for interfacing the controlling PC to
25. t 3 on the injection valve and apply the sample manually with a syringe 20 Ettan Pump P 905 User Manual 18 1145 36 Edition AC Starting a run 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 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 last tube used in the fractionation The system will be paused when the last tube is reached and the fractionation will stop AKTAre c Making Your First Runs 18 1140 80 Edition AF 21 5 Starting a run 4 Click Next For example the next page can be Variables This is the same page you were working on in the Method Editor Here you can verify and fine tune the method before proceeding This is very convenient when repeating runs with minor adjustments f Variables Mn coms E FF RE t low Rate ml min 5 00 10 00 20 00 jue E ETI NC ee 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 Change the flow rate on the Variables page Con
26. te volume of buffer used A B is shown as well as how long time the method will take Method Information x Information Signatures Method Duration Duration Total volume approximateh 872 il Total time approximates 872 mn The time and volume calculations do NOT include watch blocks Wash commands or programmed Hold Furthermore the calculations do NOT compensate for splitter flow 24 AKTArPLc Making Your First Runs 18 1140 80 Edition AF Starting a run 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 AKTArptc Making Your First Runs 18 1140 80 Edition AF 25 5 Starting a run 10 Click START The run starts You will view the run in the System Control module The Curves pane shows curves during the run Q System Control 1 WINCORNPILOT Method HT op 06 05 D4 03 02 01 Default test m12 The Run Data pane shows current values for running parameters Result c Default test001 res 07 08 09 10 14 14 15 16 17 18 19 0 m min iecore ict The Flow scheme is a graphical representation of the chromatography system 26 The Logbook pane shows when the instructions in the method are executed during the run AKTAre c Making Your First Runs 18 1140 80 Edi
27. the method is presented by a number of blocks The blocks represent typical steps in a chromatographic run such as Start instructions Column equilibration Sample injection Wash out unbound sample Fractionation Gradient Clean after elution Re equilibration Each block contains 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 B 100 9o 80 70 60 50 40 3 20 10 0 The length of each block is marked at the bottom of the graph 16 Ettan Pump P 905 User Manual 18 1145 36 Edition AC Creating a method 3 Click the x axis to view the method in time volume or column volumes 9 Click the Start Protocol tab to decide which of the Run Setup pages to be displayed at the start of a method run 10 To save 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 Comment 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 8 to learn how to alter
28. tinue 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 22 AKTAre c Making Your First Runs 18 1140 80 Edition AF Starting a run 5 5 Click Next For example the Notes page appears You can write your own comments in the Starts Note tab Method Notes Statt Notes Run Notes Evaluation Notes a Find Help r Back e 6 Click Next For example the Questions page appears Type the answers on the questions The answers will be saved in the result file x Wavelength UV Cell Length Chrom Sample Volume and Type Chrom Column Chrom Eluent A Chrom Elen B Chrom Remarks Chrom AKTAreic Making Your First Runs 18 1140 80 Edition AF 25 5 Starting a run 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 Evaluation Procedures Selected E valuation Procedures will run at the end ol the method Integiale Full Report 8 Click Next For example the Method Information page appears Here you see information about the run Under the Method Duration tab the approxima
29. tion AF Viewing a run 6 6 Viewing a run When the system pump is running the text Run is shown in the Run Status Run Status qun panel in the Run Data pane 1 To choose which panes to display select View Windows In the Customise 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 AKTAreic Making Your First Runs 18 1140 80 Edition AF 27 6 Viewing a run 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 5 Normally the curves are scaled with auto scaling i e the scale is adjusted continually to the highest and lowest values for each curve 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 6 To maximize the Curves pane right click in the Curve Data pane and select Maximise Go back to normal size by clicking Restore 28 AKTAre c Making Your First Runs 18 1140 80 Editi
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