Making Your First Run
Contents
1. KTA design Making your first runs Begin here with AKTA FPLC Amersham e Biosciences 18 1140 80 Important user information All users must read this entire manual to fully understand the safe use of AKTArrrc WARNING N The Warning sign highlights an instruction that must be strictly followed in order to avoid personal injury Be sure not to proceed until the instructions are clearly understood and all stated conditions are met Caution The Caution sign is used to call attention to instructions or conditions that must be followed to avoid damage to the product or other equipment in order to avoid personal injury Be sure not to proceed until the instructions are clearly understood and all stated conditions are met Note The Note sign is used to indicate information important for trouble free and optimal use of the product CE Certification This product meets all 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 connected to other CE marked Amersham Biosciences instruments or connected to other products recommended or described in this manual and used in the same state as it was delivered from Amersham Biosciences except for alterations described in this manual WARNING This is a Class A product I2. and includes hardware for interfacing the controlling PC to the separation equipment of AKTAFPLC 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 AKTAFPLC Making Your First Runs 18 1140 80 Edition AD 7 Ny About this guide 1 1 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 AKTAFPLC read all the safety information in AKTAFPLC System Manual 1 2 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 AKTAFPLC Making Your First Runs 18 1140 80 Edition AD The system and the software 2 2 The system and the software 2 1 General AKTAFPLC is a fully automated liquid chromatography system designed for method development and research applications The system has two main instrument components stacked on the base
3. 3 7 To shift 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 x WINCORNPILOT Pump Instructions E 5 Instructions m Parameters Pump Flowrate 0 00 20 00 C Flowpath Pumpwash 0 00 m min Delete Insert C Alatms amp Mon SampleFlow_960 C Frac DirectLoad_960 MethodBase_960 Close C Other SystemPumpControlMode tilk Help 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 Ifyou want to stop the run before it is finished click the End button at the top c Run Cont nu End jajaj el Ll AKTAFPLC Making Your First Runs 18 1140 80 Edition AD 31 7 m and printing the result 7 Viewing and printing the result If you are sati
4. platform They are e Pump P 920 a high performance laboratory pump for constant flow delivery e Monitor UV 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 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 AKTAFPLC Making Your First Runs 18 1140 80 Edition AD 9 ED The system and the software an e Switch on the chromatography system with the ON OFF button located on the front of the base platform to the bottom left Buffer tray Injection valve INV 907 Column holder with column On line filter Monitor UPC 900 ll Pump P 920 EA 7 OF Mixer M 925 Be with mixer ur chamber Sample loo dl E Optical unit EN m Tol UV lamp Mains switch PN 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 AKTAFPLC Making Your First Runs 18 1140 80 Edition AD The system and the software ez Co
5. Click the Gradient tab to view the method graphically Evaluation Procedures Method Information Start Protocol Result Name Frac 950 Variables Scouting Notes Questions Gradient Columns Reference Curves Gradient Block name 0 00 Main The length of each block is marked at the bottom of the graph 18 AKTAFPLC Making Your First Runs 18 1140 80 Edition AD Creating a method 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 Frac 950 Variables Scouting Notes Questions Gradient Columns Reference Curves Evaluation Procedures Method Information Start Protocol Result Name Checked items are displayed before method is started W Frac 950 vlVariables Scouting Text Method Gradient Columns Reference Curves Evaluation Procedures Method Information Settings Calibration Result Name Sl Scouting start protocol First run only Alltuns 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 double clicking 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 met
6. 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 waste 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 I port 1 of the injection valve and the UV flow cell Use a suitable length of 0 50 mm PEEK tubing orange da PE supplied with your system i 6 Insert a sufficient number of eee FREE d mm tlow ce mm tow Ce tubes into the fraction Connect from Connect from collector above below 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 JWINCORNPILOT Pump Instructions e rr Pee ad me Flowpath C OFF ON Delete Alarms amp Mon Sane 60 Rr EEE Bice irectLoa m C Oter Meine M 2 MEN gt Help 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 AKTAFPLC Making Your First Runs 18 1140 80 Edition AD
7. Setup window appears click the Scouting tab Evaluation Procedures Method Information Start Protocol Result Name Frac 950 Variables Scouting Notes Questions Gradient Columns Reference Curves Block Scouting Yariables olumn_PressureLimil e O Flowthrough_TubeType En O Flowthrough_FracSize O Flowthrough_Start t EBEN O Empty_loop_with O Eluate_Frac_Size O Peak_Frac_Size O Target_ConcB C Length_of_Gradient I Show details J Show unused variables cae _ He Display tooltip for extended scouting scheme cells Clear All Add Insert Delete Help Define other Scouting variables 3 A list 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 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 AKTAFPLC Making Your First Runs 18 1140 80 Edition AD 38 Scouting 2 E 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 af
8. 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 Select File Method Wizard in the Method Editor module or click LX 2 If necessary select a system and click OK 3 Go through the selections on the Method Wizard pages click Next to go to next page 4 Click Finish on the last page 5 Select File Save in the Method Editor module and give the method a name Click OK 6 Click the System Control button in the task bar 4 Syste 7 Select File Run Select the method and click Run 8 The start protocol will appear Check the method on the Variables page and change values as you require Click Next a few times 9 On the Evaluations procedures page select Print_Chromatogram to get a print out automatically after the run 10 Click the Start button on the last page the run starts Amersham e Biosciences TC information Uppsala Printed in Sweden by T K i Uppsala AB
9. operations that are performed after the run For instance select Print_Chromatogram and the chromatogram will automatically be printed after the run Evaluation Procedures x Selected Evaluation Procedures will run at the end of the method _JIntegrate_Full_Report MReport_Chromatogram M Integrate_and_Print W Print_Chromatogram DinstTest_FPLO Chromatofocusing Help lt Back Cancel 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 x Information Signatures Method Duration Duration Total volume approximately 872 mM 7 Total time approximately 872 mm The time and volume calculations do NOT include watch blocks Wash commands or programmed Hold Furthermore the calculations do NOT compensate for splitter flow 26 AKTAFPLC Making Your First Runs 18 1140 80 Edition AD Starting a run ea E o 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 AKTAFPLC Making Your First Runs 18 1140 80 Edition AD 27 ED Starting a run A Click START The run starts You will vie
10. 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 1 Layout Library Header CurveNames Y Axis XAxis Cuve Peak Table Select curves to display 01 Example Res hm l 02 Example Result001 1_UV2_280nm 03 Example Result001 1_UV3_Onm 104 Example Result001 1_Cond 05 Example Result001 1_Cond v06 Example Result001 1_Conc 307 Example Result001 1_pH 08 Example Result001 1_Pressure 09 Example Result001 1_Flow 10 Example Result001 1_Temp 11 Example Result001 1_Fractions 4112 Example Result001 1_Inject Highlight curves to view 17 Example Result001 1_UY1_215nm 01 BASEM 18 19 IT Apply to all chromatograms Cancel Hep 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 ina peak of interest left click and drag to create a rectangle When you release the mouse
11. the Run Setup in the Method Editor All documentation about E 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 Documentation x Frac 950 Variables Scouting Text Method Notes Questions BufferPrep Columns Reference Curves Evaluation Procedures Method Information Result Information Start Protocol Settings Calibration Logbook EvaluationLog ResultName 0 00 ml BufferPrep ON 1 Filter 00 ml Method Run 2002 04 11 14 25 32 Method Example Result Result v Niklas Exe M Alarm Warming ml Batch ID 20020411 1425 7303317114 Method ml Base Cv 0 98 A Mono_S_HR_5 5 Flow M Manual Alarm_Pressure ence 5 00MPa 0 00 MPa A ColumnPosition Position M Errors Wavelength 215 nm 280 nm OFF IM System BufferPrep_pH 5 400 Block eq Base SameAsMain L AutoZeroUV End Block pees Block inject C Time Base SameAsMain vane QuantitationData Sample Quantitate Info Injection Volume 0 050 ml Quantitate Info Type 0 Hold 2002 04 11 14 34 27 InjectionPartial 69 50 ul No NoAir Samplelnject O Continue 2002 04 11 14 35 17 End Block ml Block gradient 3 67 ml Base SameAsMain 3 67 ml Gradient 80 00 B 8 000 CV 12 61 ml Fractionation 18mm 0 250 ml FirstTube Volume 20 45 ml Gradient 0 00 8
12. 0 000 Cv 20 45 ml End Block Adjust retention zero io a to injection number 1 tD to eD to eo oo 0 NASA 2222318388 88888 888888888 3333333333333333333 7 2 Printing and making a report 1 To print the chromatogram select File Report The Generate Report dialog opens Generate Report x Format Contents Global BP_Chromatogr Report Global BP_Full_Report Delete Edit Customised Report Format Global Chromatofocusing Global Chromatogram Global Chromatogram_Peaks Global Chromatogram_Quant Global Chromatogram_Report Global Full_ Report Global Installation_T est Preview Close Help 2 Select for example format Global BP Chromatogram This will create a report containing the chromatogram and the questions on one page AKTAFPLC Making Your First Runs 18 1140 80 Edition AD 35 Viewing and printing the result 36 3 Click Preview to view the report on the screen Customise Report Chromatogram _l Ix File Edt View Insert Layout Help Be Be One Page Zoom In ZH Jene je Exit Deg reli Tara dee S ate alar a Im 202 01 144524 DefadvEsemple le Es ple ReulvEseapleRouldo ln Expte Baule UNICORN Report Chromatogram Encamatogean Guertianz fia anramataon ta peamt Page 1of1 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
13. 21 ED Preparing the system for a run A 6 When finished click End in the System Control toolbar Run Continuel End aale 7 In the 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 port 3 on the injection valve and apply the sample manually with a syringe 22 AKTAFPLC Making Your First Runs 18 1140 80 Edition AD Starting a run ea 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 AKTAFPLC Making Your First Runs 18 1140 80 Edition AD 23 ED Starting a run 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 w
14. 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 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 AKTAFPLC Making Your First Runs 18 1140 80 Edition AD Viewing and printing the est 7 E 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 Page Setup Current date amp time WVRun date amp time VIReportt title VIResult file name meone e C ee 2 To print the report click Print AKTAFPLC Making Your First Runs 18 1140 80 Edition AD 37 Scouting 2 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
15. N 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 bi 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 FPLCFrac x 7 Main Selection and Column Main Selection Select technique _____ anion Exchange Column Select column MITER F Flexible Flow Rates NOTE Different Flow Rates are Set on Variable Page F Flow Regulation of the System Pump To get detailed help texts click Help Click Next age Nest Carcel Hao SetDeiaut 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 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 suitabl
16. OQ avatar a pease TES 9 N NOES Page wuss O 25 0 ON OFF BUTOR seen east diets Gee E da 10 on line filter HPA At otal sess chee geen 11 P PassWord ad 12 pump A DAA A a de He 11 pump B POWs pathi stat erteilen Aa 11 Bump P2920 ds Nua ae area 9 44 AKTAFPLC Making Your First Runs 18 1140 80 Edition AD Index E QRRQRK Q QUESTIONS PIES AE 25 R restoreall Settings wctncecescnnrcessacavsshisuds Won ridacenns et a A 16 result file A SM oe A en E E bee acces 27 SCOURING da 39 result files displayed in the Results pane ccccccccccncccncnnoninonananananonnnos 13 Result Name page oooocccccccococonoconoonnnncnononnnonnnononnnncnnnnnannannninnnns 27 RUM SSL B WINdOW wa ta ee harter 17 S sample loop A O 22 HOW Path atar na em 11 scouting POSUIETIISS Ar ar A dd 39 SCOUTING SCHEME italia ine 39 Scouting varna ples iia an atleti 38 ra AAA IA 19 23 Start The GUM sesso een 28 stat UNICORN rector rence dicto tai iria dans 12 switch on the chromatography system 10 system control Shorn cut DUON ia il aaa 14 Systermdeseti ti n smn nea tia ii it ains 9 U A debtt de devs a endbter binds cider uth a 12 UV flow cell TOW Paita hee Sie tera A be bed 11 V Valves anna ernannten EA Eaa aai Eae r ii 9 Variables page Tai ti n 18 KTAFPLC Making Your First Runs 18 1140 80 Edition AD 45 46 AKTAFPLC Making Your First Runs 18 1140 80 Edition AD Short instructions The following short instructions
17. S ccccccooconccoconononnnocononnnonononononcnanoninnnos 8 2 The system and the software 9 2i GOST AN 9 22 UNICORN overview isis gion ae ie els Reet ae 12 ES NACL ith tite Ah A wong tales Rese E eal TA anor 15 3 Creating a method eeenee 16 4 Preparing the system for a run 20 4 1 System Connection narra a 20 4 2 General system preparation occcccnnnncucuooooooooonononnnnonononcncnonononons 21 4 2 1 Filling Thesinlet tu DIN Eesti a nati 21 4 2 2 Filling the Sample LOOP ccccccccccceccccssescesessseeeeeseenenesnssasaaas 22 5 Starting a run aida 23 6 Viewing a run nenee 29 7 Viewing and printing the result 32 A crit ek deneen he hie belied elias die Ge creo ae ae 32 7 2 Printing and Making a report ooooooooooocnnccnnnnnncncnnnnnnnnnnanonononanananos 35 8 SCOUEING unten 38 9 Going further 40 Short instructions on back page KTAFPLC Making Your First Runs 18 1140 80 Edition AD 5 Contents 6 AKTAFPLC Making Your First Runs 18 1140 80 Edition AD About this guide er 1 About this guide This guide is written for users who are not familiar with UNICORN software and AKTAFPLC Here you will learn the basics of UNICORN and how to operate AKTAFPLC from UNICORN UNICORN is a software package for control and supervision of the AKTA FPLC chromatography system It runs on an IBM compatible PC under Windows
18. 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 AKTAFPLC Making Your First Runs 18 1140 80 Edition AD 33 Viewing and printing the result 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 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 e 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 To save 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 34 AKTAFPLC Making Your First Runs 18 1140 80 Edition AD Viewing and printing the est 7 11 Click the View Documentation button A number of pages appear as in
19. e default parameters in the method Then when running the method use a piece of tubing to replace the column 16 AKTAFPLC Making Your First Runs 18 1140 80 Edition AD Creating a method 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 Ss EEES 7 ne 7 a rs O ET a e a Ban stad IAN E A AS I EFE Cv ET ETA cz IIA 1 9D E A MAA HE A A a AA 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 AKTAFPLC Making Your First Runs 18 1140 80 Edition AD 17 Creating a method On the Variables page 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
20. hen repeating runs with minor adjustments Variables a A p a CA EI E 5000 AS Ft fee A LT td TOD PY fleet Gre Om an LL 80 a Ls a ay 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 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 24 AKTAFPLC Making Your First Runs 18 1140 80 Edition AD Eee 5 Method Notes Start Notes Run Notes Evaluation Notes Find Help Starting a run 5 Click Next For example the Notes page appears You can write your own comments in the Starts Note tab H ea 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 hhh Wavelength UY Cell Length Chrom Sample Yolume and Type Chrom Column Chrom Eluent A Chrom Eluent B Chrom Remarks Chrom Help lt Back Cancel AKTAFPLC Making Your First Runs 18 1140 80 Edition AD 25 ED Starting a run AA 7 Click Next For example the Evaluation Procedures page appears Evaluation procedures are automated evaluation
21. hnique select column select conductivity flow cell TOW Path mesura apta connect to the system connecting the column CONNECTION Panel sr an create a Metodista dada tata customize the pane s display ccccccnnnncccuonononononnnnnnononcncncncnnnons 29 E evaluation ShOnt Cut button ah aaa 14 evaluation procedures cccccccncnnnonononanananennnnonononcnnconananannnnnananos 26 existing method queue shortcut DUON u anne reden 14 F flow path dESErIptlION re Eh ds BE 11 flow fate SCOURING ur seen 38 flow restrictor flow path Frac 950 page fraction collector placement a o eas unas 9 G A A MAL de cohen OA 18 H Naci A lan lot dale du od at ae 15 AKTAFPLC Making Your First Runs 18 1140 80 Edition AD 43 njection till POMS di a ade 22 njection valve WD AAA AI errr ere 1 nlet A AA aa EN 1 nlet tubing THE rn retten 2 nlets B flow pathi dea eS AE 1 L ls to Windows ar raid 12 log on log off Shortcut DUON wasser 14 M Main Men WindOW sitari Ananas 13 method editor sh rt eut b tlon tata ia ae E 14 method files displayed in the Methods pane ccccccccccccnnnnononanananananonnnos 13 method queue SNO C T PUNO cta neueren USA PENILA LNA 14 methodiWizard Aura 16 short cut button oooooccccnconoccncnncononnnnnnnnnononcnnnnnononnnnnocinnnnnos 16 MXE e en dos 9 POW Bath aiii 11 modules SMOM CUT BUTONS vidal dictada pha dees 14 monitor UV J
22. hod 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 variables systematically and automatically in repeated runs This is know as scouting and is a convenient easy to use function AKTAFPLC Making Your First Runs 18 1140 80 Edition AD 19 ED Preparing the system for a run 4 Preparing the system for a run Connection ces 4 Syste 20 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 To connect to a system Select System Connect The System connect dialog appears System Connect x 4 EXP100F FPLC 4 EXP100 E EXP10 KB EXP10F Cancel Help 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 KTAFPLC Making Your First Runs 18 1140 80 Edition AD Preparing the system for a run EN 4 2
23. inst001 ui instao2 bu Wizard Generated001 al Wizard Generated002 La Wizard Generated003 a Wizard Generated004 Prev Folder User Folder User Folder Result File Result File Result File Result File Result File Result File 2003 03 04 10 13 2003 04 30 14 23 2003 04 28 17 10 2003 04 28 17 15 2003 04 28 16 56 2003 04 28 17 04 2003 04 28 17 07 2003 04 28 17 12 2003 03 04 10 13 2003 04 30 14 23 2003 04 28 17 10 2003 04 28 17 15 2003 04 28 16 56 2003 04 28 17 04 2003 04 28 17 07 2003 04 28 17 12 User default ZA 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 KTAFPLC Making Your First Runs 18 1140 80 Edition AD 13 ED The system and the software 14 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 o
24. mment 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 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 filter dh UV conductivity pH optional 7 Flow restrictor To fraction 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 A sample 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 f
25. n a domestic environment this product may cause radio interference in which case the user may be required to take adequate measures Terms and Conditions of Sale Unless otherwise agreed in writing all goods and services are sold subject to the terms and conditions of sale of the company within the Amersham Biosciences group which supplies them A copy of these terms and conditions is available on request Should you have any comments on this product we will be pleased to receive them at Amersham Biosciences AB SE 751 84 Uppsala Sweden Trademarks Drop Design AKTA AKTAFPLC FPLC and UNICORN are trademarks of Amersham Biosciences Limited Amersham and Amersham Biosciences are trademarks of Amersham plc Windows is a registered trademark of Microsoft Corporation Office Addresses Amersham Biosciences AB SE 751 84 Uppsala Sweden Amersham Biosciences UK Limited Amersham Place Little Chalfont Buckinghamshire England HP7 9NA Amersham Biosciences Inc 800 Centennial Avenue P O Box 1327 Piscataway N J 08855 USA Amersham Biosciences Europe GmbH Munzinger Strasse 9 D 79111 Freiburg Germany Amersham Biosciences KK Sanken Building 3 25 1 Hyakunincho Shinjuku ku Tokyo 169 0073 Japan Copyright Amersham Biosciences 2003 All rights reserved Contents Contents 1 About this guide occacnnemmrmrmes 7 1 17 Presteq isites mii iia 8 1 2 Typographical CONVENTION
26. ograms 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 Read manual section The Method Handbook AKTAFPLC System Manual AKTAFPLC Optional Configurations User Manual AKTAFPLC System Manual UNICORN 4 12 User Manuals UNICORN 4 12 User Manuals UNICORN 4 12 User Manuals UNICORN 4 12 User Manuals UNICORN 4 12 User Manuals Click on Help button in the dialogue box that appears or look in the index in the UNICORN 4 12 User Manuals AKTAFPLc 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 AKTAFPLC Making Your First Runs 18 1140 80 Edition AD Going further 9 2 Security features UNICORN 4 12 User Manuals Controlling the system from a UNICORN 4 12 User Manuals remote computer AA AKTAFPLC Making Your First Runs 18 1140 80 Edition AD 41 ED Going further 42 AKTAFPLC Making Your First Runs 18 1140 80 Edition AD Index A add a column Scouting Page ooociocccnccnooccnonaconconnnonnncnnnonnnnos 39 air inthe inlet tubing inris sa aaa 21 MMS PUN DA add is See ty 21 c calibrate the pH monitor change a variable value chromatographic tec
27. pens a dialog window for controlling the system and running your methods e 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 UNICO A Metho 4 Syste 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 e Method Queue opens a dialog window for defining a new Method Queue e Existing Method opens a dialog window for showing Queue the Method Queue that is running Method Queues are used to link several methods together AKTAFPLC Making Your First Runs 18 1140 80 Edition AD The system and the software ez 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 Index for a specific topic In any dialog click on the Help button to get help on how to use the current active dialog AKTAFPLC Making Your First Runs 18 1140 80 Edition AD 15 Creating a method 3 Creating a method The UNICOR
28. raction collection or waste AKTAFPLC Making Your First Runs 18 1140 80 Edition AD 11 ED The system and the software EZ 2 2 UNICORN overview Ak 1 Switch on the computer Log on to Windows by first pressing a7 Ctrl Alt Del and then clicking OK After a while the Windows ne desktop appears 2 Start UNICORN by double clicking on the UNICORN icon 3 An information window appears during start up Version 4 12 Copyright Amersham Biosciences 2003 4A Amersham e Y Biosciences 4 Inthe 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 AKTAFPLC on a regular basis Sr UNICORN logon User name defaut y Password Al 12 AKTAFPLC Making Your First Runs 18 1140 80 Edition AD The system and the software 2 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 UNICORN M M File View Administration Tools Window Help Bi 3 Methods Prev Folder Es inst WINCORNLAB 647KB Method File 2003 04 28 17 E Test WINCORNLAB 658KB Method File 2003 04 29 11 Ready 2003 04 28 17 2003 04 29 11 Results ex ADefault J Example files Manual runs System Contr EU
29. sfied 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 Evaluation Example Result001 1 v UNICORN Local fil Niklas result Example Result002 Example Result001 res uk File Edit View Wale Operations Procedures Quantitate Mol Size Window Help lal x Example Result001 1_UW1_215nm Example Result001 1_Cond Ex Example Result001 1_UV1_215nm 01 BASEM PAI 61 1651 44 0967 dre B 0 5 7 04 60 5619 351 079 Pa Peak c 0 5 7 74 115 9646 472 670 Ready FNMA 32 KTAFPLC Making Your First Runs 18 1140 80 Edition AD Viewing and printing the est 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
30. ter 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 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 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 AKTAFPLC Making Your First Runs 18 1140 80 Edition AD 39 ED Going further 9 Going further 40 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 AKTAFPLC manual package To learn about Protein purification strategies Different sample applications options Different fraction collection options Columns Calibrating monitors and pumps Comparing chromat
31. w the run in the System 28 Control module The Run Data pane shows current values for running parameters The Curves pane shows curves during the run Method c Default test m12 Result c Default test001 res Q System Control 1 WINCORNPILOT File View Manual System Help Continue End Pressure 0 03 min InjectionValve Inj 0 03 min Block Sample Injection 0 03 min Base Vojufne ml 0 04 min End 2603 05 20 11 04 39 Manual El O End No watch Controlled By default The Logbook pane shows when the instructions in the method are executed during the run The Flow scheme is a graphical representation of the chromatography system AKTAFPLC Making Your First Runs 18 1140 80 Edition AD 6 Viewing a run Viewing a run 3 rer When the system pump is running the text Run is shown in the Run Run Status 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 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 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 YAris Curve St
32. yle and Colour Flow Scheme Logbook Run Data Groups Run Data Colour Curves X Axis MlInstruments New Group Edit Group Delete Group Block Time Flow AKTAFPLC Making Your First Runs 18 1140 80 Edition AD E 29 6 Viewing a run 3 4 The Curves window shows the curves during the run All curves are 30 5 stored in the result file Under the Curves tab select which curves to show during the run Click OK xi Y Axis Curve Style and Colour Flow Scheme Logbook Run Data Groups Run Data Colour Curves xais Display curves P960_Press MP960_Flow Cones Help 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 xi Run Data Groups Run Data Colour Curves Axis YAris Curve Style and Colour FlowScheme Logbook r Y axis options r Scale C Auto 7000 0 7000 0 Fixed 1 118 0 882 mau 6 To maximize the Curves pane right click in the Curve Data pane and select Maximise Go back to normal size by clicking Restore KTAFPLC Making Your First Runs 18 1140 80 Edition AD Viewing a run
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