ÄKTAbasic 10 XT upgrade kit - GE Healthcare Life Sciences
Contents
1. SyringeEnd aspirates a syringe volume of wash solvent from the wash solvent bottle and fills the wash solvent tubing SyringeHome dispenses the syringe contents to the syringe waste Repeat this action a number of times until the wash solvent tubing and the syringe are completely filled 13 After the wash solvent tubing and the syringe are filled use the InitialWash instruction to perform a standard wash All tubing connected to the syringe valve will be filled and flushed with wash solvent Purging the syringe in A 900 1 Manually run the SyringeEnd and SyringeHome instructions alternately 2 Repeat until no more air is left in the syringe more than 5 repeats can be necessary 3 Finish by performing the InitialWash instruction Checking the needle height in A 900 1 Remove the tray segment next to on the left side the one directly below the needle arm 2 Puta vial with a flat bottom filled with e g water in the tray segment directly below the needle arm The bottom of the vial must be clearly visible 3 Carry out a manual injection by issuing the System Control Manual Flowpath InjectionFlushed instruction 4 Check visually that the needle stops approximately 2 mm above the vial bottom 5 If not go to System Settings Special to adjust the needle height The default setting is 2 mm Note Check the needle height with 2 mm as default setting whenever the needle is replaced The 2 mm setting is only a relative2. Description 28 A 1 UNICORN version 3 0 UNICORN is a control system for real time control of chromatographic separation systems from a personal computer UNICORN operates together with the AKTAbasic 10 chromatography system It runs under the operating system Windows NT UNICORN version 3 0 software enables the use of UNICORN Analysis module software A 2 UNICORN Analysis module UNICORN Analysis module software extends the UNICORN Evaluation module with functions for quantification and molecular size calculation It allows the user to determine the absolute quantity or concentration of a sample component and determine its molecular size The Analysis module is accessed in the Evaluation window under two new menus Quantitate and Mol size The Analysis module uses many of the facilities within the Evaluation module The Quantitate function uses peak data from standards to produce calibration curves which can then be used to evaluate the amount and concentration of components in a sample The Mol size function uses a molecular size curve prepared from one or more standards to determine the molecular size of components in a sample AKTAbasic 10 XT upgrade kit User Manual 18 1120 23 Edition AB Reference information A A 3 Autosampler A 900 sequence of valve switching and syringe dispenser controlled sample withdrawal enables the A 900 to inject sample volumes in a range from a few microliters to 1 ml in a simple
3. 1 Introduction 1 1 General AKTA basic 10 XT upgrade kit is used for upgrading AKTAbasic 10 chromatography system with Autosampler A 900 Strategy templates and adviser to support automatic sample application performed under UNICORN 3 0 command UNICORN 3 0 Analysis module that extends the UNICORN 3 0 Evaluation module functions After installation and verification your AKTAbasic 10 chromatography system is extended to perform the following functions Automated multiple sample injections Quantify amount concentration and molecular size Strategy templates and adviser The following strategy and templates are new e Strategy B10X Allows UNICORN to control the Autosampler A 900 e Template Aut y zz Allows autosampler functions to be utilised in an advanced method In the column adviser the column recommendations are modified to suit the columns when autosampling with A 900 is used In the method adviser support for programming standard protocols for the B10X strategy is included zversion number AKTAbasic 10 XT upgrade kit User Manual 18 1120 21 Edition AB 1 1 Introduction 1 2 Safety UNICORN 3 0 features Upgrading to UNICORN version 3 0 offers the possibility to utilise the UNICORN Analysis module which allows you to perform External standard quantification nternal standard quantification Standard addition Recovery calculations Molecule size calculatio
4. KTA desi AKTAbasic 10 XT upgrade kit User Manual Amersham 18 1120 28 Y Biosciences Important user information AN Meaning Consult the instruction manual to avoid personal injury or damage to the product or other equipment WARNING The Warning sign is used to call attention to the necessity to follow an instruction in detail 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 shall be followed to avoid damage to the product or other equipment 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 or optimal use of the product Should you have any comments on this instruction we will be pleased to receive them at Amersham Biosciences AB SE 751 84 Uppsala Sweden Trademarks AKTA and UNICORN are trademarks of Amersham Amersham Biosciences Limited or its subsidiaries Amersham and Amersham Biosciences is a trademark of Amersham plc Windows is a trademark of MIcrosoft Corp Terms and Conditions of Sale 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 of sale
5. 1120 23 Edition AB 21 3 Operation 3 4 InjectionPick up 3 5 InitialWash The InjectionPickup instruction selects the ul pick up injection mode for A 900 The syringe dispenser aspirates transport liquid from the transport vial into the sample line The A 900 injection valve is turned to LOAD position The needle moves from the transport vial to a sample vial and aspirates the selected injection volume The needle moves back to the transport vial and the sample is quantitatively transported into the sample loop with transport liquid After each injection the needle is rinsed e f WashExtzNO is selected in the method the needle is only washed internally If WashExt YES is selected in the method the needle is also washed on the outside In both cases the buffering tubing is also washed An air segment is not recommended for this injection mode Air segmentzNoAir because the air segment at the front of the sample plug is injected More details of pl pick up are found in the Autosampler A 900 User Manual Reference information A 3 6 RotateTray The InitialWash instruction performs a complete wash of the needle and the syringe The syringe is first washed using one syringe volume Then the needle is moved to its home position and washed internally Finally the needle moves to its front position and washed both internally and externally 22 The RotateTray instruction is used to rotate the tray e
6. Disconnect the UniNet 1 cable running between Frac 900 and the computer in the computer end Note If no fraction collector Frac 900 is used disconnect the UniNet 1 cable running between the Pump P 900 and the computer Termination Frac 901 PUG AKTAbasic 10 rear panel UniNet 1 chain Disconnect at the computer end and route to the A 900 UniNet 1 m di connector To computer AKTAbasic 10 XT upgrade kit User Manual 18 1120 23 Edition AB 2 Installation 2 Connect this UniNet 1 cable to any of the two UniNet 1 connectors in A 900 Autosampler A 900 TOTTI UniNet 1 chain To computer 3 Connect a new UniNet 1 cable supplied with the A 900 from the other UniNet 1 connector on the A 900 to the computer The UniNet 1 connection to the computer must be made to the board with four green LEDs Four green LEDs AKTAbasic 10 XT upgrade kit 8 User Manual 18 1120 23 Edition AB Installation 2 Connecting A 900 to mains voltage WARNING A 900 must be connected to a grounded mains socket WARNING For continued protection against risk of fire replace only with fuses of specified type and rating See Technical specifications in the A 900 User Manual for fuse data Before connecting the mains cord to the AKTAbasic 10 base platform check that the voltage set
7. and reproducible manner Three different injection methods can be selected e Flushed loop The sample loop is completely quantitatively filled with sample resulting in extremely good reproducibility better than 0 3 e Partial loopfill The sample loop is partially filled with sample giving low sample loss and allowing programmable injection volumes pl pick up After aspiration from the vial the sample volume is transported into the loop with transport liquid mobile phase from another vial This eliminates sample loss The A 900 uses a syringe to aspirate the sample from a vial into the sample loop To prevent contamination of the syringe the A 900 is equipped with a buffer tubing between the syringe and the injection valve An integrated washing mechanism removes the sample from the buffer tubing and sample needle and also rinses both components UniNet 1 computer network Ventilation Injection valve holes Tubing guide P Needle arm Tray Segment Syringe waste ue Mains switch Syringe dispenser Mains input Wash solvent ottle Fuses and Wash position voltage selector Drain wash position Condensed water and leakage drain The A 900 contains no
8. g when new vials are loaded into the tray segments AKTAbasic 10 XT upgrade kit User Manual 18 1120 23 Edition AB Operation 3 3 7 SyringeHome End The SyringeHome End instructions are used when the syringe is replaced They are also used for purging the syringe i e to remove air SyringeHome empties the syringe contents to the syringe waste container SyringeEnd aspirates wash solvent into the syringe from the wash bottle Note When the performed action is completed make sure the syringe is run to the Home position before leaving the manual function 3 8 NeedleHome Front The NeedleHome Front instructions are used when the sample needle is replaced NeedleHome moves the needle to its normal position NeedleFront moves the needle closer to the front of A 900 3 9 AutosamplerSyringeSpeed The AutosamplerSyringeSpeed parameters are used to manually preset the speed of the syringe The syringe speed needs adjusting when changing syringe size using highly viscous samples or using volatile eluents Three different speed settings are available Low Normal default setting High The actual speed is dependent on the speed setting and a scaling factor and on the syringe volume The scaling factor range of 0 1 1 0 is used to reduce the basic speed setting Speed setting low and scaling factor 0 2 means that low speed is reduced to 1 5 of original low speed Syringe volume Viscosity Spee
9. if you intend to use the 0 25 tubing kit and the capillaries marked G A1 and H A2 if you intend to use the 0 50 tubing kit See AKTAbasic 10 User Manual Section 3 3 for general recommendations on selecting column and tubing kit 3 Both capillaries are routed in the space between the UV monitor UV 900 and the pH Conductivity monitor pH C 900 4 Connect the capillary marked G A1 12750 mm from the tubing kit between the AKTAbasic 10 Injection valve port 1 and the A 900 Injection valve port 1 marked pump 5 Connect the capillary marked G A2 H A2 1 800 mm from the tubing kit between the A 900 Injection valve port 6 marked column and the UV flow cell inlet Note This connection is only temporary When tbe installation test is successfully performed G A2 H A2 is connected to the top inlet of the column and the capillary connecting the column outlet and the UV flow cell inlet is refitted 6 Check additional internal and waste tubing connections on A 900 according to the A 900 User Manual instructions AKTAbasic 10 XT upgrade kit User Manual 18 1120 23 Edition AB Installation 2 Preparing for the installation test 1 Switch on A 900 at the rear panel Then switch on the AKTAbasic 10 chromatography system with the mains switch located at the rear 2 Switch on the computer and the printer Mains switch 3 Perform a login procedure see Chapter 2 in Making your first runs press the Control Syst
10. measure to ensure that the needle stops above the vial bottom regardless of which type of vial used Adjust the final setting in small steps until a needle height suitable for the actual use is obtained type of vial sample volume available etc AKTAbasic 10 XT upgrade kit 12 User Manual 18 1120 23 Edition AB Installation 2 2 5 Installation test General The installation test performs a total check of the liquid processing parts of the AKTAbasic 10 separation unit This is to establish that the integration of the additional parts in AKTAbasic 10 XT upgrade kit has not disturbed the total system function Preparation 1 Verify that the Pump P 900 is primed and purged according to the instructions in the AKTAbasic 10 User Maual 2 Prepare a flask containing 500 ml of 0 4 acetone in water solution 3 Fill two new standard vials with this solution and seal them properly with the correct type of cap or septum 4 Put one vial in position 1 and the other vial in position 2 in a standard tray 5 Immerse the inlet tubing B1 into the rest of the 0 496 acetone solution 6 Immerse the inlet tubing A1 into a flask containing distilled water ps menu 7 Click on the Main menu button in the Task bar utton Astart lt SUNICORN Main Menu Method Editor untitled 1 System Control 1 Bjorn d ig Evaluation 8 Select File Printer setup from the menu bar Select the appropriate printer from the list and click on Jo
11. reacts sete ere ees ese oe esso ee Pere Pete PEE H eR Se FUE SEEKS 23 3 8 NeedleHome BEront sies HS e E RC daria iter EE E celia 23 3 9 A tosamplerSyringe Speed sd even un ever e ere eaae cs eae e ers ER EPES 23 3410 Hardware related parameters eiie RH ERR RENEE ERSEN RENREN n 24 4 Maintenance MEE UU M 25 5 Trouble shooting 5 JFaults and actions aee ite eR Re E e e RE eee e eae 26 Reference information A Description 12 ced ete repite epe SHE OSEANE NEEE ENEA S ERE SEREESE ERES E ERE eves EXE PARE eges 28 A UNICORIVEISIOR 20 neret bte rri tree eret eene EEE ESNEA SEERE 28 A 2 UNICORN Analysis module eese eese eee esten ee ten nette setas etna 28 A3 Autosaimpler A 900 us ied mte mene ee e t PE E eR Re WR Yee eR ERROR RR U eO 29 B Technical specifications i ne ce erat eere ao ER SHEER EE HER EEELREE RE CEPR S E PERPE REPRE 30 C Accessories ANIC spare PANTS uc oseiro soo ea EEA Pee evhee eve re re ev eee eu Reve 31 Short instructions on back page AKTAbasic 10 XT upgrade kit User Manual 18 1120 23 Edition AB 1 Contents About this manual This manual comprises two parts a practical part sections 1 5 and a reference part sections A C Sections 1 5 contain the necessary information for installing operating and maintaining the XT upgrade equipment AKTAbasic 10 XT upgrade kit User Manual 18 1120 23 Edition AB Introduction 1
12. to 0 50 tubing kit Sample needle bent no injection performed 1 Wrong type of septa deformed septa or needle height set too low Replace needle and re adjust needle height if necessary Needle destroyed in injection using micro vial segments 1 Segments badly oriented or septa bent down Replace needle Check segments and replace septa Air bubbles 1 Wrong needle height Adjust the needle height 2 No wash solution or not properly purged syringe Check the wash solvent bottle and or purge the syringe 3 Syringe speed too high Lower the syringe speed Bad reproducibility 1 Injection valve switched dry Replace rotor seal 2 Syringe valve coupling leaking Check and tighten coupling Replace if necessary 3 Syringe leaking problem Replace syringe 4 Peaks of different height Connectors not tightened in the injection valve Tighten connectors 5 Airin the syringe See Air bubbles above AKTAbasic 10 XT upgrade kit 26 User Manual 18 1120 23 Edition AB Trouble shooting 5 Fault Possible cause and Action Error message during initial wash 1 Syringe valve not switched properly Check syringe installation Refit if necessary Error message during other commands 1 Syringe in End position Issue a SyringeHome command 2 A 900 is not ready to receive a command Wait a while and try again AKTAbasic 10 XT upgrade kit User Manual 18 1120 23 Edition AB 57 A Reference information Heference information A
13. and the XT components General The A 900 needs approximately 28 cm of bench space and a mains connection of 220 240 V or 100 120 V 50 60 Hz preferably taken from the AKTAbasic 10 base platform The recommended position is to the left of the separation equipment of AKTAbasic 10 with approximately 3 cm spacing The A 900 has two transportation safety devices which must be removed Remove the locking screw holding the front cover It is located at the right side of the front cover Save the locking screw A red plastic cap is attached over the needle washing mechanism below the front cover Remove the red plastic cap and save it See also the installation instructions given in the Autosampler A 900 User Manual Chapter 2 Locking screw AKTAbasic 10 XT upgrade kit 6 User Manual 18 1120 23 Edition AB Installation 2 Connecting to the UniNet 1 communication network CAUTION The mains power to AKTAbasic 10 and the computer must be switched OFF before disconnecting or connecting the UniNet 1 cables A 900 is controlled from a PC running UNICORN version 3 0 or higher using UniNet 1 cables Autosampler A 900 rear panel UniNet 1 computer network Ventilation holes Mains switch Mains input Fuses and voltage selector 1
14. b properties Under Page Orientation select Landscape then click on Save Click on OK to acknowledge the printer chosen 9 Click on the Instant Run button 10 Select the method Insttest in the templates list and make sure that Any is selected for both Technique and Column 11 Click on Run 12 When the Method window is shown click on Next and then Next again 13 Click on Start to start the Installation test The run time is approximately 30 minutes AKTAbasic 10 XT upgrade kit User Manual 18 1120 23 Edition AB 13 2 Installation Evaluating the installation test result 1 Click on the Main menu button in the Task bar Double click on Gde in the results panel and then double click on the Instte01 icon to open the result file Maximise the chromatogram window by clicking on EL in the upper right corner 1 UV3 280nm903 SMTH 4 h h i i b i i Ret Width Area Area Peak area Height min min mAU min time mAU E 1 1 49 1 34 102 1047 56 61 194 971 2 4 31 1 13 51 1771 33 39 107 472 Total number of detected peaks 7 Total area 4331 4293 mAU min Area in evaluated peaks 153 2818 mAU min Ratio peak area total area 0 035388 Total peak width 2 47 min Calculated from insttell l UV1 265nm Baseline insttell 1 UV1 265nmGe01 BASE C E 14 4 Click in the chromatogram window with the right mouse button and select Properties De select the first t
15. cted value interval mAU value 100 95 94 96 70 69 71 30 29 31 5 4 6 0 UV response test result UV response table 1 2 3 4 5 Wavelength Value read Base line Absorbance Allowed nm corrected ratio interval 100 B 096 B value 254 265 254 1 14 1 24 265 265 280 1 32 1 42 280 Autosampler peak test result Autosampler peak table 1 2 3 4 Peak no Area Area Peak area Allowed mAU x min 96 interval 1 66 1 67 2 2 32 8 33 9 AKTAbasic 10 XT upgrade kit User Manual 18 1120 23 Edition AB 19 3 Operation 3 Operation 3 1 General 20 A 900 is controlled from a PC running UNICORN version 3 0 or higher It cannot be used as a stand alone module A 900 can be controlled using the B10X strategy either automatically from a template method or manually via commands The A 900 can be used for automatic sample injection from a few microlitres to one millilitre of sample solution To achieve optimal performance selection of sample loop volume syringe size and speed plus the washing method between injections must be considered The sample injection techniques which can be programmed in the method editor and then utilised in automatic runs are as follows InjectionFlushed The sample loop is completely quantitatively filled with sample resulting in extremely good reproducibility e InjectionPartial The sample loop is partially filled with sample giving low sample loss and allo
16. d setting Scaling factor 250 ul Normal Normal 1 0 250 ul High 5 10 cP Low 0 2 500 wl Normal Low 1 0 1000 ul Normal Low 1 0 Note If the sample is viscous the flush volume can be enlarged and the syringe speed reduced to enbance performance AKTAbasic 10 XT upgrade kit User Manual 18 1120 23 Edition AB 23 3 Operation 3 10 Hardware related parameters 24 All of the following parameters are found under System Settings Special AutosamplerParameters Parameter Value Comment LoopVol 10 1000 pl 100 ul is default Sample loop volume SyringeVol 100 250 500 or 1000 ul 250 ul is default Syringe volume FlushVol 30 100 wl 40 ul is default Flush volume The amount of sample or transport liquid taken from a vial before the loop is filled with sample WashVol 300 9999 ul 300 ul is default Wash volume Sets the volume for the external wash WashExt programmable in the different injection modes TrayType StandardVial_A A Standard vial O D 12 mm MicroVial_D D Micro vial O D 7 mm HeadPress No Yes Head space pressure Applied via the outer air needle Assures that no air or vapour bubbles are formed during sample aspiration NeedleHgt 0 40 mm 2 mm is default AutosamplerSyringeSpeed Scaling Low Normal High Normal is default 0 1 1 0 1 0 is default Needle height A relative measure used to adjust the space between the needle and the bottom of the vial used Syringe speed setting S
17. e and the moving needle arm of the A 900 which if not handled with care could result in injury or damage or destruction of these parts AKTAbasic 10 XT upgrade kit User Manual 18 1120 23 Edition AB 3 2 Installation 2 2 1 Installation Unpacking Unpack the AKTAbasic 10 XT upgrade kit and check the items against the supplied packing list Inspect the items for obvious damage which may have occurred during transportation Unless already done unpack the Autosampler A 900 according to the installation description given in the Autosampler A 900 User Manual It is recommended to retain all packing material if onward transport of the Autosampler A 900 is expected CAUTION Before using the A 900 for the first time remove the safety screw marked with red tape from the bottom of the Autosampler and the red cap from below the front cover Allow the instrument to acclimatise for 1 hour CAUTION Always lift the A 900 by the base unit NEVER by the front cover CAUTION The following information should be read carefully to ensure that the AKTAbasic 10 XT upgrade kit is installed correctly AKTAbasic 10 XT upgrade kit User Manual 18 1120 23 Edition AB Installation 2 2 2 Pre requisites Hardware pre requisites The AKTAbasic 10 chromatography system must be installed and fully tested before the AKTAbasic 10 XT upgrade kit and the Autosampler A 900 are installed See the Installation Guid
18. e for AKTAbasic 10 Software pre requisites The computer connected to your AKTAbasic 10 chromatography system must be upgraded to run UNICORN 3 0 or higher together with new strategy templates and adviser The UNICORN Analysis module software should also be installed prior to the installation test Follow the instructions given in the supplied software upgrade information and manuals which are included in the AKTAbasic 10 XT upgrade kit General installation instructions for UNICORN software are described in the UNICORN User Manual Chapter 13 Specific upgrade information for UNICORN 3 0 or higher is given in the attached UNICORN Upgrade Instruction Remember to check the boxes for loading strategy templates and adviser Installing the UNICORN Analysis module software is described in the UNICORN Analysis module User Manual Section 1 4 Install and upgrade the software before installing the hardware CAUTION Make sure the AKTAbasic 10 chromatography system and the computer are switched off before installing the hardware 2 3 Installing Outlet valve and Fraction collector optional AKTAbasic 10 XT upgrade kit User Manual 18 1120 23 Edition AB General The Outlet valve PV 908 and Fraction collector Frac 901 are optional and not mounted at the factory The installation of these components is desc ribed in the AKTAbasic 10 User Manual Section A 5 2 Installation 2 4 Installing Autosampler A 900
19. em button and select System Settings Special 4 Check that the default sample loop volume 100 ul syringe volume 250 ul and needle height 2 mm are selected 5 Manually run the SyringeEnd instruction under System Control Manual Flowpath AutosamplerControl 6 Remove the syringe from the syringe valve Make sure that the Luer connector in the valve remains in place 7 Fill the syringe with with 2096 isopropanol in water from a vessel Note Methanol can be used instead of isopropanol 8 Replace the filled syringe Fully tighten the syringe so that a gap between the plunger tip and the syringe end is obtained after d having executed the SyringeHome instruction under System Control Manual Flowpath AutosamplerControl Note If the syringe holder is not fully tightened i e no gap is obtained the plunger tip moves forcefully into the syringe end which can damage the syringe 9 Fill the wash solvent bottle with 20 isopropanol in water If the sample contains large amounts of salt or if the sample is very sensitive to isopropanol use water as wash solvent instead degas and change the water every day 10 Screw the bottle into the wash solvent holder and place the holder on the A 900 11 Put the wash solvent tubing in the wash solvent bottle AKTAbasic 10 XT upgrade kit User Manual 18 1120 23 Edition AB 11 2 Installation 12 Fill the tubing using the SyringeEnd and SyringeHome instructions
20. en curves and then check that the following curves are displayed 11 instte01 1_UV1_265nm 01 SMTH 13 instte01 1 UV2 254nm 202 SMTH 14 instte01 1 UV3 280nm 203 SMTH AKTAbasic 10 XT upgrade kit User Manual 18 1120 23 Edition AB Installation 2 AKTAbasic 10 XT upgrade kit User Manual 18 1120 23 Edition AB 5 Click on the Peak Table tag Check that Peak Table A is selected and select Percent of total peak area in the Peak Table Column Press OK Chromatogram layout 1 Ea Curve Style and Colour Edit Texts Save and Open Layouts l Header CuveNames Y amp s xs Cuve Peak Table Select peak table to display Select peak table columns Peak endpoint retentions Width at half height Percent of total area Percent of total peak area Type of peak limits Peak endpoint heights Filter Peaks T Height less than pooo T Width less than pooo I Arealess than p ti i S T Width more than fo M Peak must be one of fo largest Apply to all chromatograms Cancel Double click on 4I in the upper left corner of the chromatogram window and read the absorbance in mAU for the steps corresponding to instte01 1_UV1_265nm 01 SMTH by dragging the vertical bar to the constant section of each plateau Enter the absorbance values in mAU in column 2 in the Step response table of the Test record leaving out the decimals Read the absorbance for the plateaus corresponding to 0 and 100 B for the cur
21. internal user replaceable items KTAbasic 10 XT upgrade kit User Manual 18 1120 23 Edition AB 29 B Reference information B Technical specifications See Technical Specifications in the Autosampler A 900 User Manual and the AKTAbasic 10 User Manual AKTAbasic 10 XT upgrade kit 30 User Manual 18 1120 23 Edition AB Reference information C C Accessories and spare parts Item Quantity per pack Code no AKTAdesign 10 XT Tubing kit See also Accessories and spare parts in Autosampler A 900 User Manual 1 18 1122 65 AKTAbasic 10 XT upgrade kit User Manual 18 1120 23 Edition AB 31 9661 Jequianon Short instructions The following short instructions are intended as a guide to users who are fully familiar with the safety precautions and operating instructions described in this manual The instructions assume that the equipment is installed according to the installation instructions A 900 is controlled from UNICORN For method and manual control use the instructions and parameters given in the following Method Method editor Text instructions Flowpath Manual System Control Manual Flowpath 1 For Flushed loop injection select the instruction InjectionFlushed and enter the sample vial number 2 For Partial loopfill select the instruction InjectionPartial and enter the sample vial number and injection volume 3 For ul Pick up select the instruction InjectionPickup and enter the sam
22. is available on request Adresses Amersham Biosciences UK Limited Amersham Place Little Chalfont Buckinghamshire England HP7 9NA Amersham Biosciences AB SE 751 84 Uppsala Sweden Amersham Biosciences Inc 800 Centennial Avenue PO Box 1327 Piscataway NJ 08855 USA O Amersham Biosciences AB 1998 All rights reserved Contents Contents 1 Introduction L1 Generalized ene enr er ERE ERREUR ERES IER eds 1 1 2 3Salebyancecns ii ERU EH SURTURS URSI UREYEUNVENTTEXTETRERANEEN ERAN EEN EFEREEREERAT EUER ETEENEEPNI 2 2 Installation 2 npacking ue ieaonsai tec cites ee era ti erstes e rn T oa ere x 4 2 2 Pfesreguisitescssien ete rein nr err EE PEE rE EENES NESSES EESAN FEE Y Pe ERES 5 2 3 Installing Outlet valve and Fraction collector optional eesessss 5 2 4 Installing Autosampler A 900 and the XT components eese 6 20 dnstallatiQHn test ueetercieeter erre rere E eere Pi oin e epe been he prt Dd a ERE 13 2 6 Testecord icnic easet ir rimi ec rna e ere erii n erre ce eru 19 3 Operation EEG C M 20 3 2 InjectionElushed ii iiie t e e ERREUR GREEN EE E GEI RE alia E 21 3 9 JinjectionbParealienseieeire rere aN reden EEEE AE eee EEEE 21 3 4 TnjectionPicks poun aui ein min ERE e ER DER ER dn EE ETSI E E EEEa 22 S MEN ioc Fro 22 EMEN tocicilci me 22 Au 3Synnseblome Ernd uteceneteeseseveeeieve
23. n outlet to the UV flow cell inlet AKTAbasic 10 XT upgrade kit User Manual 18 1120 23 Edition AB 17 2 Installation 18 Correcting faulty evaluation results Faulty Gradient The gradient is linear but the interval is to small The mixer chamber is too large or the mixer is faulty Disturbances These may arise from air in the pump Purge again and repeat the test run If persistent faulty check valves or bad piston sealings in the pump may be the cause Refer to the Pump P 900 User Manual Faulty step response All values are faulty This may arise from air in the pump or a faulty pump Purge again and repeat the test run If persistent a faulty pump may be the cause Refer to the Pump P 900 User Manual 596 and 9596 faulty This may be due to bad sealings in the pump 5 faultyzpump module B 95 faultyzpump module A Refer to the Pump P 900 User Manual Faulty Autosampler peak values If values are faulty make sure that the A 900 is properly purged and that all tubing fittings are properly tightened AKTAbasic 10 XT upgrade kit User Manual 18 1120 23 Edition AB 2 6 Test record Installation 2 Gradient test result Gradient linear from 96 B to 96 B 10 90 96 Step response test result Step response table 1 2 3 4 5 Programmed Value Baseline Normalised Allowed Conc B read corre
24. ns Quantification uses peak data from standards to produce calibration curves which can then be used to evaluate the amount and concentration of components in a sample The molecular size function determines the molecular size of components in a sample using a molecular size curve prepared from one or more standards The instrument is designed for indoor use only Do not use in a dusty atmosphere or close to spraying water Operate in accordance with local safety instructions WARNING The Autosampler A 900 must not be opened by the user It contains high voltage circuits which can deliver a lethal electric shock WARNING When using hazardous chemicals avoid spillage during autosampling and when the capillaries are removed WARNING When using hazardous chemicals all suitable protective measures such as protective glasses must be taken WARNING The Autosampler A 900 must be connected to a grounded mains socket AKTAbasic 10 XT upgrade kit User Manual 18 1120 23 Edition AB Introduction 1 WARNING Do not operate the A 900 with the front cover open WARNING For continued protection against risk of fire replace only with fuses of specified type and rating See Technical specifications for fuse data The A 900 contains the following safety symbol SHARP NEEDLE J AND MOVING PARTS KEEP HANDS CLEAR This sticker denotes the proximity of the sharp needl
25. nuities 3 Fill in the Test record Evaluating the step response 1 Calculate the relative adsorption plateau heights for curve instte01 1_UV1_265nm 01 SMTH in the following way Subtract the base line value 096 B from each of the values in column 2 in the Step response table of the Test record Enter the results in column 3 Divide each value in column 3 by the base line corrected value corresponding to 100 B Multiply by 100 and enter the results in column 4 2 The values of column 4 should all fall within the intervals given in column 5 Evaluating the UV response 1 Calculate the UV response ratios in the following way Subtract the base line values 0 B corresponding to each UV curve from the values in column 2 of the UV response table of the Test record Enter the results in column 3 Calculate the absorbance ratios 265 nm 254 nm and 265 nm 280 nm using the values from column 3 and enter the results in column 4 2 The ratios obtained should all fall within the intervals given in column 5 Evaluating the Autosampler peaks 1 In the printed report read the Area and Area Peak area 6 values for peaks No 1 and 2 2 Fill in the Test record 3 Check that the values fall within the given intervals The installation procedure is completed when all results fall inside the limits specified in the Test record Note Remember to connect the G A2 H A2 capillary to tbe top of the column and the capillary from the colum
26. ple vial and transport liquid vial numbers and the injection volume Manual System Control Manual Flowpath AutosamplerControl 4 To wash the needle and syringe use the instruction InitialWash 5 To rotate the tray segments use the instruction RotateTray 6 To purge the syringe use the instructions SyringeHome and SyringeEnd alternately to move the syringe plunger 7 To change the needle use the instructions NeedleHome and NeedleFront to move the needle Settings System Settings Special AutosamplerParameters 8 To set the relative needle height use the NeedleHgt parameter 9 To adjust the syringe speed use the AutosamplerSyringeSpeed and Scaling parameters Amersham Biosciences TC information ab Uppsala Printed in Sweden by T K i Uppsala AB
27. ting of the A 900 matches the local mains supply voltage and main fuses Use the supplied appliance with protective grounding If the indicated voltage is not correct select the proper voltage by removing inverting and then re inserting the voltage selector cartridge Check that the corect fuses are installed If not replace them with fuses as stated below For 110 120 VAC use two 5 AT fuses slow For 220 240 VAC use two 2 5 AT fuses slow When the voltage selection and fuses are correct for the local mains power source plug in the power cord in a free outlet in the base platform of the AKTAbasic 10 separation unit CAUTION DO NOT switch on any of the equipment yet AKTAbasic 10 XT upgrade kit User Manual 18 1120 23 Edition AB 9 2 Installation Pump B um Pump A 10 Connecting capillaries between A 900 and AKTAbasic 10 The tubings to be connected between A 900 and the AKTAbasic 10 separation unit are included in AKTAdesign 10XT Tubing kit 18 1122 65 The flow diagram below shows the location of A 900 in the AKTAbasic 10 flow path Autosampler Flow restrictor Injection valve V1 raction collector Sample loop NEA w2 eo Outlet valve V4 1 Remove the capillary connecting the Injection valve with the Column top inlet 2 Select the capillaries matching the tubing kit to be used in AKTAbasic 10 after the column Use the capillaries marked G A1 and G A2
28. ume After each injection the needle is rinsed e f WashExtzNO is selected in the method the needle is only washed internally e f WashExt YES is selected in the method the needle is also washed on the outside In both cases the buffer tubing is also washed An air segment Air segment Air is recommended to reduce the amount of the flush volume The air segment is not injected More details of flushed loop injection are found in the Autosampler A 900 User Manual Reference information A 3 3 InjectionPartial The InjectionPartial instruction selects the partial loopfill injection mode for A 900 The syringe dispenser aspirates a flush volume see also section 3 10 of sample into the sample line The A 900 injection valve is turned to LOAD position and the sample loop is filled by transporting the selected injection volume into the sample loop which is partially filled with sample After each injection the needle is rinsed e f WashExtzNO is selected in the method the needle is only washed internally e If WashExt YES is selected in the method the needle is also washed on the outside In both cases the buffer tubing is also washed An air segment Air segment Air is recommended to reduce the amount of the flush volume The air segment is not injected More details of partial loopfill are found in the Autosampler A 900 User Manual Reference information A AKTAbasic 10 XT upgrade kit User Manual 18
29. ves 11 instte01 1 UV1_265nm 01 SMTH 13 instte01 1 UV2 254nm 202 SMTH 14 instte01 1 UV3 280nm 203 SMTH and enter the values in column 2 in the UV response table of the Test record Change the curve to be measured by clicking on XH The colour of the value shown corresponds to the colour of the curve selected 15 2 Installation 8 Select File Report and select the boxes Header and Chromatogram Under Report contents click on the radio button to the right of the Chromatogram selection Report contents Select items Iv Header Current chromatogram All chromatograms Method C 1 12 Documentation Evaluation log C v Chromatogram CS LL r Chromatogram layout Print curves in landscape format Define Current Use thick curve lines Dee Curent 9 Under Select items highlight Current chromatograms 10 Click on Define and select only the following curves to be included in the report 06 instte01 1 Conc 11 instte01 1 UV1 265nm 201 SMTH 13 instte01 1 UV2 254nm 202 SMTH 14 instte01 1 UV3 280nm 203 SMTH Then click on OK 11 Click on Print to obtain a printed report of the results AKTAbasic 10 XT upgrade kit 16 User Manual 18 1120 23 Edition AB Installation 2 Evaluating the gradient 1 Place a ruler along the gradient part of curve instte01 1_UV1_265nm 01 SMTH in the printed report 2 Thecurve should be linear between 1096 B and 9096 B and void of disconti
30. wing programmable injection volumes InjectionPickup After aspiration from the vial the sample volume is transported into the loop with transport liquid mobile phase from another vial resulting in virtually no sample loss These sample injection techniques can also be ordered manually from System Control Manual Flowpath Manual functions used for setting up and maintaining the A 900 are found in System Control Manual Flowpath AutosamplerControl The available commands are as follows e InitialWash e RotateTray e SyringeHome End e NeedleHome Front Parameters for setting the syringe speed is found under System Settings Special AutosamplerParameters All of these functions are described in the following sections A table showing all the hardware related parameters for A 900 is given at the end of chapter 3 AKTAbasic 10 XT upgrade kit User Manual 18 1120 23 Edition AB Operation 3 3 2 InjectionFlushed The InjectionFlushed instruction selects the flushed loop injection mode for A 900 The syringe dispenser aspirates a flush volume see also section 3 10 of sample into the sample line The A 900 injection valve is turned to LOAD position and the sample loop is filled with 1 5 3 times the sample loop volume depending on the loop volume The injection volume equals the loop volume When the sample loop size is changed the loop volume setting must be changed in System Settings 100 ul is the default loop vol
31. yringe speed scaling factor AKTAbasic 10 XT upgrade kit User Manual 18 1120 23 Edition AB Maintenance 4 4 Maintenance The AKTAbasic 10 XT upgrade kit items require no periodic maintenance Autosampler A 900 specific maintenance requirements are described in the Autosampler A 900 User Manual chapter 4 4 1 Cleaning A 900 should be kept clean and spilled liquid should be wiped off before it dries The front cover should always be folded down when access to the internal parts is not necessary SHARP NEEDLE N AND MOVING PARTS KEEP HANDS CLEAR The module should be wiped regularly with a damp cloth Allow to dry completely before use AKTAbasic 10 XT upgrade kit User Manual 18 1120 23 Edition AB 25 5 Trouble shooting 5 Trouble shooting 5 1 Faults and actions If the suggested actions do not correct the fault call Amersham Biosciences WARNING Always disconnect the power supply before attempting to replace any item during maintenance WARNING The modules must not be opened by the user They contain high voltage circuits which can deliver a lethal electric shock CAUTION Only spare parts approved or supplied by Amersham Biosciences may be used for maintaining and servicing the modules Fault Possible cause and Action Ghost peaks at high pressure 1 Bad capillary connections Replace connectors if necessary Backpressure too high 1 Capillaries too narrow Change
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