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ÄKTApurifier User Guide - GE Healthcare Life Sciences

1. B1 B2 44 e AKTApurifier UPC Monitor UPC 900 a high precision on line combined monitor for measuring UV absorption conductivity and pH optional Components such as the mixer column and injection valve are mounted to the right Columns are snapped in place in column holders The separation unit is controlled from UNICORN software Pump P 900 Monitor UV 900 and Monitor UPC 900 can also be controlled individually from the modules without UNICORN software This is described in the respective component manuals 3 1 1 Liquid flow path The flow path between the different components in the system is shown and described below Mixer Sample Column Monitoring Fractionation Conductivity monitor optional for AKTApurifier Injection valve UV monitor Flow Outlet valve restrictor optional On line filter AKTApurifier pH monitor optional Outlet fraction Waste To fraction collector optional 8 1 The pump has four pump heads two for pump module A and two for pump module B Pump module A is the one closest to the front 2 Pump inlets A and B are immersed in buffer A and B respectively The buffer solutions are pumped to a mixer by the pump Inlet A1 and B1 are placed in buffer A and B respectively Inlet A2 and B2 are used when buffers are prepared automatically by BufferPrep They can also be used for changing buffer during the me
2. Standard mode Prep mode Frac 950 can be used for both small scale standard mode and preparative scale prep mode purification with AKTApurifier Up to 392 fractions can be collected in time or volume base as fixed volume and or peak fractionation The fractionation order can be selected as serpentine row row by row serpentine column or column by column For standard mode four sizes of tube racks for tube diameters of 12 18 and 30 mm tubes and 96 well microplates are available For prep mode two sizes of racks for tube diameter of 30 mm tubes and 250 ml bottles are available A Funnel to flask kit for transporting the fractions to other vessels is also available Using Frac 950 in prep mode requires a dispenser arm specially made for the prep mode racks Sample loss during tube change can be eliminated by using drop synchronisation or by using a built in accumulator for intermediate liquid storage between tube change Installation 1 Unpack and assemble Frac 950 according to the instructions given in the Fraction collector Frac 950 User Manual 2 Put Frac 950 on the workbench to the right of AKTApurifier 3 Locate the capillary kit included with Frac 950 and select if you want to include the accumulator in Frac 950 or not AKTApurifier User Guide 28 4079 53 Edition AC Installation 2 4 Connect the tubing from the flow restrictor to Frac 950 as described in the Frac 950 User Manual Injection
3. UV flow cell Valve INV 907 specifications Start protoco Superloop connect descripti filling system description System preparation changing tubing US 124 168 169 T technical Specifications eebe e na troubleshooting Bufferprep conductivity curve Mixer M 925 Monitor UPC 900 pressure curve 112 12 176 AKTApurifier User Guide 28 4079 53 Edition AC Index Pump P 900 UV curve valve Tubing kit CANNES SE Re es Anna Le tubings changing overview TE COM MEN GOON nee ee EE e 135 U UniNet 1 19 UniNet 2 19 UV cell 167 UV flow cell 167 V Valve INV 907 wees troubleshooting W Wieler disposal deh SA TAN al 12 Wetted MOTEL Scene unnes nasa NAN ANN NN Lan 162 AKTApurifier User Guide 28 4079 53 Edition AC 177 Index 178 AKTApurifier User Guide 28 4079 53 Edition AC For contact information for your local office please visit www gelifesciences com contact GE Healthcare Bio Sciences AB Bjorkgatan 30 751 84 Uppsala Sweden www gelifesciences com imagination at work GE imagination at work and GE monogram are trademarks of General Electric Company Drop Design Capto GSTPrep GSTrap HiLoad HiPrep HiTrap HisPrep HisTrap MabSelect MabSelect SuRe MabSelect Xtra ini O Mini S Mono Q Mono S Mono P RESOURCE SOURCE S
4. WARNING Superloop 10 ml and Superloop 50 ml must not be used at pressures above 4 MPa 40bar 580 psi Superloop 150 ml must not be used above 2 MPa 20 bar 290 psi At higher pressures the glass tube might shatter gt AKTApurifier User Guide 28 4079 53 Edition AC 11 1 Introduction 1 4 Typographical conventions 12 gt gt gt WARNING Never use i d 0 25 mm tubing or smaller with columns that can only withstand a low maximum pressure and that allow high flow rates as the columns might rupture resulting in injury WARNING In KTApurifier 100 never use i d 0 50 or 0 75 mm tubing or smaller with columns that can only withstand a low maximum pressure and that allow high flow rates as the columns might rupture resulting in injury WARNING Never attempt to remove the mains fuse while mains voltage is applied to the system For continued protection against risk of fire replace only with fuse of the specified type and rating Please refer to the Technical specifications for fuse data LA Typographical conventions Menu commands field names and dialog box prompts are identified in the text by bold italic text A colon separates menu levels thus File Open refers to the Open command in the File menu Hardware items for example keyboard key names key combinations key sequences hard key names on the equipment and connector port names located on the equipment are id
5. Eng Bar 101 AOL Ba SEM EL i 250 Page lofi Preview Mode Adding 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 e Free text e Picture e Text method e Chromatogram e Documentation e Evaluation log e Quantitate and molsize optional e Frac 950 optional 84 AkTApurifier User Guide 28 4079 53 Edition AC 4 Move the mouse pointer into the page area of the window You will notice Operation 4 that the mouse pointer has an additional symbol according to the item type you selected to insert 5 Click and drag to create a box of the desired size Release the mouse button 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 for example select page size and items to be included in the header and in the footer The information selected here will be printed in the report Click OK Page Setup xi Page Setup First Header Header Footer The selected items will be included in the report m Select Items Run user Current date amp time MRun date amp time ViReport title MResult file name Method file name
6. Inlet or outlet check valves not functioning correctly Piston seal leaking Clean the valves according to Chapter 4 of Pump P 900 User Manual Replace the piston seal according to the instructions in Chapter 4 of Pump P 900 User Manual Blockage or partial blockage of flow path Flush through to clear the blockage If necessary replace tubing Check the inlet tubing filter It can become clogged if unfiltered buffers or samples are applied See instructions for flushing through at the end of the run in Chapter 3 of Pump P 900 User Manual 7 4 Error symptom Conductivity measurement with the same buffer appears to change over time Possible cause Action Conductivity curve Clean the flow cell according to Monitor UPC 900 User Manual or Monitor pH C 900 User Manual The ambient temperature might have decreased Use a temperature compensation factor see Reference information in Monitor UPC 900 User Manual or Monitor pH C 900 User Manual Ghost peaks appear in the gradient profile A charged sample has been detected e g protein Air bubbles are passing through the flow cell Check for loose tubing connections Use a flow restrictor AKTApurifier User Guide 28 4079 53 Edition AC 109 7 Troubleshooting 7 4 Conductivity curve Error symptom Baseline drift or noisy signal Possible cause Action There may be air in the flow cell Check for leaking tubing conn
7. Pump P 900 e Replace piston seals Refer to Chapter A in the Pump P 900 User Manual e Replace piston Refer to Chapter 4 in the Pump P 900 User Manual e Clean or replace the inlet our outlet check valves Refer to the Chapter 4 in Pump P 900 User Manual e Calibrate the pressure sensor Refer to Section 6 4 in Calibration procedures Monitor UPC 900 e Clean the flow cell according to Chapter 4 in Monitor UPC 900 User Manual e Calibrate the temperature sensor when changing conductivity cell Refer to Section 6 4 in Calibration procedures e Enter a new cell constant when changing conductivity cell Refer to Section 6 4 in Calibration procedures e Calibrate the cell constant if specific conductivity with high accuracy is measured Refer to Section 6 4 in Calibration procedures On line filter e Replace the on line filter 6 2 Cleaning the system surface The system surface should be kept dry and clean to maintain proper safety and function of the system Chemical stains and dust should be removed To clean the system surface 1 Wipe the surface regularly with a damp cloth 2 To remove stains spray the system with 20 ethanol and wipe off the excess Note Be careful not to bend any tubing 3 Let the system dry completely before using it 104 AKTApurifier User Guide 28 4079 53 Edition AC Maintenance 6 6 3 Cleaning the system flow path The procedures below describe how to clean the syste
8. 2 Start Pump P 900 and let it run until the movable seal has reached the bottom of Superloop 3 Stop Pump P 900 and set the injection valve to position LOAD A Load a large volume syringe with sample 5 Gently load the syringe contents into Superloop through port 3 6 Leave the syringe in position The loaded sample can be injected all at once or in several smaller volumes down to 1 ml portions 7 The volume to inject is set by the variable Empty_loop_with in the Sample_Injection block 8 The sample is applied to the column when the injection valve is set to position INJECT When the required volume has been injected set the valve to LOAD WARNING Make sure that the upper pressure limit of the system is lower than the maximum allowed pressure of the Superloop 443 Mixing gradients There are two different techniques available for mixing gradients The standard technique using two separate buffers one to each pump module and the BufferPrep technique using four solutions two to each pump module generating the buffer on line with a switch valve before each pump module The minimum flow rate for BufferPrep is 1 0 ml min The outputs of the pump modules are routed to a mixer The BufferPrep method is described in section 5 2 BufferPrep Note To be able to use the BufferPrep function the BufferPrep kit including two switch valves must be installed on the pump see Section 5 2 2 Installin
9. System rack Serial Number ixer M 925 onitor UV 900 onitor UPC 900 Pump P 900 NV 907 Computer onitor AKTApurifier User Guide 28 4079 53 Edition AC 41 2 Installation 2 6 Registration form 42 AKTApurifier User Guide 28 4079 53 Edition AC System overview 3 3 System overview This chapter gives a brief overview of AKTApurifier with the separation unit and UNICORN software Note Two versions are available AKTApurifier with monitor UV 900 for multiple wavelengths detection and AKTApurifier UPC with monitor UPC 900 for combined measurement of UV absorption pH and conductivity When instructions or specifications are similar for both the models only AkTApurifier is mentioned Fig 3 1 AKTApurifier to the left and AKTApurifier UPC to the right 3 1 Separation unit AKTApurifier is an automated liquid chromatography system designed for method development and research applications The separation unit of the chromatography system has two main modules which are stacked on each other The modules are e Pump P 900 a binary high performance gradient pump and e AKTApurifier Monitor UV 900 a multi wavelength UV Vis monitor for measuring up to three wavelengths simultaneously AKTApurifier User Guide 28 4079 53 Edition AC 43 3 System overview 3 1 Separation unit System pump O SS Switch valves optional
10. e Increase the concentration of all buffers in the broad range recipe Increase the acid concentration by the same factor as the buffer concentration Note that a new recipe has to be created The ionic strength of the start eluent will also be increased lonic strength Buffer components with several pKa values will give a higher ionic strength at the start when the pH is set above the second pKa and even higher above the third pKa for acidic buffer components This may cause problems if you have peaks that elute early in the gradient To reduce this use low concentrations or if possible change to buffer components that only have one pKa Example A recipe with 0 1 M Citrate pKa 3 13 4 76 and 6 40 will have an ionic strength of approximately 0 22 at pH 6 but only 0 05 M at pH 4 Use a 0 03 M solution instead or use 0 1 M MES at pH 6 Choice of eluent To avoid local disturbances in pH caused by buffering ions participating in the ion exchange process select an eluent with buffering ions of the same charge as the substituting groups on the ion exchanger Choose the start buffer pH so that substances to be bound to the ion exchanger are charged e g at least 1 pH unit below the isoelectric point for cation exchangers Example A recipe with Na HPO pKa 2 15 7 20 and 12 33 will not give accurate pH above approximately pH 7 6 when titrated with HCl AKTApurifier User Guide 28 4079 53 Edition AC Reference information 8
11. 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 4 8 1 Viewing 1 After a run you can view the result Click the UNICORN Manager icon Double click a result file icon in the list to the right 2 The Chromatogram window is opened automatically in the Evaluation workspace when you open a result file The Chromatogram window contains all the curves Note that the term chromatogram is used here when talking about the whole window containing all the different curves Evaluation Example Result002 1 vAUNICORNI LocalNfil folder result Example Result002 Example Result002 res sk File Edit View Integrate Operations Procedures Window Help Recent Runs Files Find Name Li Example Result001 4 Example Result002 Lil Example Result003 li Example Result004 585 A Example Result002 1_UV 01 PEAK1 Area mAU min Height mAU No Peak name Retention min Internal st 5 85 Peak A 0 2 6 53 Peak B 0 2 7 06 Peak C 0 2 7 76 60 5051 14 6082 20 4377 36 9911 336 828 95 761 125 560 147 623 Pomme a The result file from a run holds a complete record of the run including method system settings curve data and run log Note Original raw data cur
12. 156 AKTApurifier User Guide 28 4079 53 Edition AC Reference information 8 8 6 Technical specifications For the complete specifications for each component refer to the individual User Manuals and Instructions The relevant system specifications are listed below 8 6 1 Operating data Pump P 901 Flow rate range 0 01 100 ml min Flow rate increments 0 01 ml min Pressure range 0 10 MPa 100 bar 1450 psi pH stability range 1 13 1 14 lt 1 day exposure Viscosity Max 5 cP Flow rate accuracy 2 or 20 pl min whichever is greater Flow rate reproducibility rsd lt 0 5 at flow rate 0 5 ml min Gradient composition Accuracy Reproducibility Internal volume 1 at 0 5 100 ml min rsd lt 0 25 at flow rate lt 0 5 100 ml min Stroke volume 286 ul Total volume 1850 ul Pump P 903 Flow rate range Flow rate increments 0 001 10 ml min 0 001 ml min Pressure range 0 25 MPa 250 bar 3625 psi pH stability range 1 13 1 14 lt 1 day exposure lt iscosity Max 5 cP n ow rate accuracy 2 or 2 l min whichever is greater with compression compensation activated Flow rate reproducibility rsd lt 0 5 at flow rate 0 05 ml min Gradient composition Accuracy Reproducibility 1 at 0 05 10 ml min rsd lt 0 25 at flow rate lt 0 05 10 ml min Internal volume Stroke volume Total volume 35 6 ul 5
13. Q 1 ml x 11 0013 03 HiTrap Capto Q 5 ml RF 28 9165 37 HiTrap Capto DEAE 1 ml X 28 9165 40 HiTrap Capto DEAE 5 ml RF 17 5441 22 HiTrap Capto S 1 ml X 17 5441 23 HiTrap Capto S 5 ml RF 11 0032 73 HiTrap Capto MMC 1 ml X KTApurifier User Guide 28 4079 53 Edition AC 135 8 Reference information 8 4 Recommended tubing and columns Code no Column name 0 25 0 50 11 0032 75 HiTrap Capto MMC 5 ml RF 28 4058 44 HiTrap Capto adhere 1 ml x 28 4058 46 HiTrap Capto adhere 5 ml RF 17 1153 01 HiTrap Q HP 1 ml X 17 1154 01 HiTrap Q HP 5 ml RF 17 1151 01 HiTrap SP HP 1 ml X 17 1152 01 HiTrap SP HP 5 ml RF 17 6002 33 HiTrap IEX Selection Kit 7x1 ml X 17 5053 01 HiTrap Q FF 1 ml X 17 5156 01 HiTrap Q FF 5 ml RF 17 5054 01 HiTrap SP FF 1 ml X 17 5157 01 HiTrap SP FF 5 ml RF 17 5055 01 HiTrap DEAE FF 1 ml X 17 5154 01 HiTrap DEAE FF 5 ml RF 17 5056 01 HiTrap CM FF 1 ml X 17 5155 01 HiTrap CM FF 5 ml RF 17 5162 01 HiTrap ANX FF high sub 1 ml X 17 5163 01 HiTrap ANX FF high sub 5 ml RF 17 5158 01 HiTrap Q XL 1 ml X 17 5159 01 HiTrap Q XL 5 ml RF 17 5160 01 HiTrap SP XL 1 ml X 17 5161 01 HiTrap SP XL 5 ml RF 17 5092 01 HiPrep 16 10 Q XL RF 17 5093 01 HiPrep 16 10 SP XL RF 17 5090 01 HiPrep 16 10 DEAE FF RF 17 5091 01 HiPrep 16 10 CM FF RF 17 5190 01 HiPrep 16 10 Q FF RF 17 5191 01 HiPrep 16 10 A
14. The filter should be replaced every week When changing the filter use a tool if necessary to unscrew the filter body When assembling the on line filter tighten the filter body by hand only Never use a tool 8 2 9 Monitor pH C 900 optional Monitor pH C 900 is an option for AKTApurifier It is acombined monitor for accurate on line monitoring of pH conductivity and temperature in a wide range of liquid chromatography applications Its accurate response coupled with A high precision over a wide measuring range makes it ideal for use in all chromatography techniques from reversed phase with very low conductivity eluents to hydrophobic interaction chromatography in high salt solutions 8 2 10 Fraction collector optional A fraction collector Frac 920 or Frac 950 can be used for both small scale and preparative scale purifications with AKTApurifier A number of racks for different tubes sizes are supplied with the fraction collector In AKTApurifier the fraction collector allows fixed volume fractionation or automatic peak fractionation The latter function is based on UV peak detection using slopes or level sensing Fraction marks and fraction numbers make it easy to identify fractions and peaks AKTApurifier User Guide 28 4079 53 Edition AC Reference information 8 8 3 Optional configurations AKTApurifier standard system configuration can easily be changed to optional configurations This built in flexibility in the
15. Page number AR snort format name WM Font Select All Clear All m Logo T Logo m Layout M Line over IP Line under Free text Poo Font 2 Click Print to print the report 4 8 3 Printing all method instructions with explanations Report title UNICORN Report Font 1 Select Method Editor File and click Print This opens a window containing instructions that are printable 2 Make sure that the Instruction Set box is checked and clear any unwanted items 3 Click Print to print the instructions AKTApurifier User Guide 28 4079 53 Edition AC 85 4 Operation 4 9 Completion of a run and storage 86 4 9 Completion of a run and storage All valves return to default positions i e position 1 after a run 49 1 Storage Overnight Let the system run isocratically at a low flow rate approximately 10 of the used flow rate Weekend and long term storage Flush the system with water and then fill it with 20 ethanol not the pH electrode if used see separate instructions in Optional Configurations AKTApurifier User manual 4 10 Cold room operation Cold room operation is sometimes necessary to keep the biomoleculels of interest stable 410 1 Preparation 1 Place the separation unit in the cold room 2 Place the computer outside the cold room A 15 m UniNet cable is available as an accessory and should be used to con
16. take all suitable protective measures such as wearing protective glasses and gloves resistant to the chemicals used Follow local regulations and instructions for safe operation and maintenance of the system WARNING Only use spare parts that are approved or supplied by GE Healthcare for maintaining or servicing the system WARNING Only use tubing supplied by GE Healthcare to make sure that the pressure specifications of the tubing are fulfilled WARNING When using hazardous chemicals make sure that the entire system has been flushed thoroughly with bacteriostatic solution e g NaOH and distilled water before service and maintenance WARNING For continued protection against risk of fire replace only with a fuse of the specified type and rating Refer to Reference information for fuse data WARNING If the system is turned or the fraction collector removed the external capillaries and other tubing may become entangled in nearby objects and be pulled from their connections causing leakage WARNING There must always be a sample loop or Superloop connected to ports 2 and 6 of the injection valve This is to prevent liquid spraying out of the ports when switching the valve This is especially dangerous if hazardous chemicals are used gt Bb Pbb EE When using a sample pump for sample application directly onto the column a tubing must be connected between ports 3 and 6 on the Injection valve
17. 0 50 UV cell to conductivity cell H7 80 0 50 Conductivity cell to flow restrictor H8 140 0 50 Flow restrictor to outlet valve centre port H9 600 0 50 Outlet valve port 2 to fraction collector optional 1 G General tubing H High flow tubing W Waste tubing A Inlet tubing A B Inlet tubing B F Fraction tubing AKTApurifier User Guide 28 4079 53 Edition AC 125 8 Reference information 8 1 System description AKTApurifier UPC 10 The column is installed either using the tubing supplied with the column or with PEEK tubing cut by the user to suitable lengths i d 0 5 mm orange PEEK tubing is supplied with AKTApurifier UPC 10 A male male union connects the conductivity cell to the flow restrictor Tubing Length Tubing i d Tubing o d Material Color Volume of design 10 cm A1 B1 170 cm 1 6 MM 1 8 Teflon Clear 201 1 ul G1 G2 33 cm 0 50 mm 1 16 PEEK Orange 19 6 ul G3 25 cm 0 50 mm 1 16 PEEK Orange 19 6 ul G4 30cm 0 25 mm 1 16 PEEK Blue 4 9 ul G5 17 cm 0 25 mm 1 16 PEEK Blue 4 9 ul W1 W3 100 cm 0 75 mm 1 16 Tefzel Clear 44 2 ul AKTApurifier UPC 100 The column is installed either using the tubing supplied with the column or with PEEK tubing cut by the user to suitable lengths i d 0 75 mm green PEEK tubing is supplied with AKTApurifier UPC 100 A male male union connects the conductivity cell to the flow
18. 10 300 GL X 17 5172 01 Superose 6 10 300 GL X 17 5173 01 Superose 12 10 300 GL X 17 1408 01 HiTrap Desalting 5 ml x 17 5087 01 HiPrep 26 10 Desalting x 17 1139 01 HiLoad 16 60 Superdex 30 prep x grade 17 1140 01 HiLoad 26 60 Superdex 30 prep x grade 17 1068 01 HiLoad 16 60 Superdex 75 prep x grade 17 1070 01 HiLoad 26 60 Superdex 75 prep x grade 17 1069 01 HiLoad 16 60 Superdex 200 prep x grade 17 1071 01 HiLoad 26 60 Superdex 200 prep x grade 17 1165 01 HiPrep 16 60 Sephacry x S 100 HR 17 1194 01 HiPrep 26 60 Sephacryl S 100 HR x 17 1166 01 HiPrep 16 60 Sephacryl S 200 HR x 17 1195 01 HiPrep 26 60 Sephacryl S 200 HR x 17 1167 01 HiPrep 16 60 Sephacryl S 300 HR x 17 1196 01 HiPrep 26 60 Sephacryl S 300 HR x AkTApurifier User Guide 28 4079 53 Edition AC 143 8 Reference information 8 4 Recommended tubing and columns Hydrophobic Interaction Columns Code no Column name 0 50 0 75 1 0 17 1184 01 RESOURCE ETH 1 ml X 17 1185 01 RESOURCE ISO 1m X 17 1186 01 RESOURCE PHE 1 ml X 17 1187 01 RESOURCE HIC Test kit 3 x 1 ml X 28 4110 07 HiTrap HIC Selection Kit 7 x 1 ml x 17 1085 01 HiLoad 16 10 Phenyl Sepharose x x HP 17 1086 01 HiLoad 26 10 Phenyl Sepharose x x HP 17 5095 01 HiPrep 16 10 Phenyl FF high sub x 17 5094 01 HiPrep 16 10 Phenyl FF low sub x 17 5096 01 HiPrep 16 10 Butyl FF X 17 5097 01 HiPrep 16 10 Oc
19. 10 ml min with water Note the back pressure Bp1 on the pump display or in the Run Data pane A Connect flow restrictor to the open end of the tubing observe he IN marking Put the flow restrictor in the waste container 5 Run the pump at 10 ml min with water Note the back pressure Bp2 on the pump display or in the Run Data pane 6 Calculate the back pressure Bp2 Bp1 of the flow restrictor If is not within the limit replace or contact GE Healthcare Every 3 months Monitor UV 900 Monitor UPC 900 Check the cell and optical fibre connectors according to Chapter 4 in Monitor UV 900 User Manual Check the instrument according to Chapter 4 in Monitor UPC 900 User Manual Every 6 months Monitor UV 900 Monitor UPC 900 Check the instrument according to Chapter 4 in Monitor UV 900 User Manual Clean the flow cell according to Chapter 4 in Monitor UPC 900 User Manual Mixer M 925 Check that the mixer chamber is clean and without damage Check the tubing connectors Replace if reguired Yearly Valve INV 907 Check for external and or internal leakage Replace the distribution plate yearly or when reguired Refer to Chapter 4 in the valve s Instructions AKTApurifier User Guide 28 4079 53 Edition AC 103 6 Maintenance 6 2 Cleaning the system surface Interval Every 2 years Action Mixer M 925 e Replace the complete mixer chamber When required
20. 2 3 4 5 6 7 8 10 11 42 5 0 9 5 pH AIEX 5 0 9 5 6 0 9 0 pH CIEX 6 0 9 1 methyl 5 0 5 5 piperazine Bis Tris 6 0 7 7 Piperazine 6 6 7 Tris 7 5 8 5 1 methyl 8 6 10 3 piperazine Piperazine 9 4 10 5 2 3 amp 5 6 7 8 9 10 11 12 AkTApurifier User Guide 28 4079 53 Edition AC 155 8 Reference information 8 5 BufferPrep details Data for pre programmed CIEX recipes Gradient 0 0 1 0 M NaCl Buffer Titrate pH Default Start with range correction conductivity at0 B 100 B low high pH 3 0 7 5 pH CIEX 0 1 M HCl 3 0 7 5 0 0 0 2 4 2 5 7 mS cm 0 03 M Phosphate Na HPO 0 03 M Formate Na 0 06 M Acetate Na 0 1 M Acetate Na 0 1 M HCI 4 0 5 7 0 0 0 1 3 2 3 8mS cm 0 1 M Bicine 0 1M NaOH 7 0 9 0 0 1 0 0 0 4 1 7 mS cm 0 03 M Citrate Na3 0 1 M HCI 2 5 6 2 0 0 0 2 4 2 3 0mS cm 0 1 M Formate Na 0 1 M HCI 2 5 4 5 0 0 0 2 4 4 3 5 mS cm 0 1 M HEPES 0 1MNaOH 6 8 8 0 0 2 0 3 0 6 1 6 mS cm 0 1MMES 0 1M NaOH 5 5 6 7 0 1 0 1 0 7 1 6 mS cm 0 03 M Phosphate Na HPO 0 1 M HCI 2 2 3 0 0 0 0 3 2 4 2 6 mS cm 6 4 7 4 0 0 0 3 5 0 2 6 MS cm 3 0 7 5 pH CIEX 3 0 25 Phosphate 2 2 3 0 Formate 2 5 45 Citrate 2 5 6 2 Acetate 4 0 SF MES 5 5 6 7 Phosphate 6 4 7 4 HEPES 6 8 8 0 Bicine 7 0 9 0
21. 2 Installation No visible damage Injection valve waste tubings port 4 and 5 marked W1 and W2 to waste reservoir Fraction collector installed optional Waste tubing marked W3 extended to waste reservoir Monitor pH C 900 installed optional pH electrode hold er installed optional Computer and pri nter installed UniNet 1 and Un Net 2 optional cabling installed Mains power cab ing installed Column holder installed Computer and pri 28 nter installed AKTApurifier User Guide 28 4079 53 Edition AC Installation 2 3 Installation test Solutions prepared Tubings to piston seal rinsing system in 20 ethanol AKTApurifier prepared nstallation Test method run nstallation Test results evaluated Test Record completed Registration Form completed Test Record and copy of Registration form stored in System Logbook Registration form posted to Service Administration nstallation Guide stored in User Manual box for future use Table 2 1 Installation Record 2 4 Installation test The installation test checks the function of the liquid delivery and the UV monitoring system of AKTApurifier The installation test can also be used at any time to check the condition of the system for example after a prolonged stop Correct gradient formation is tested by producing a linear gradie
22. 60 Superdex 75 prep grade x 17 1069 01 HiLoad 16 60 Superdex 200 prep grade x 17 1071 01 HiLoad 26 60 Superdex 200 prep grade x 17 1165 01 HiPrep 16 60 Sephacryl S 100 HR RF 17 1194 01 HiPrep 26 60 Sephacryl S 100 HR RF 17 1166 01 HiPrep 16 60 Sephacryl S 200 HR RF 17 1195 01 HiPrep 26 60 Sephacryl S 200 HR RF AkTApurifier User Guide 28 4079 53 Edition AC 137 8 Reference information 8 4 Recommended tubing and columns 138 Code no Column name 0 25 0 50 17 1167 01 HiPrep 16 60 Sephacryl S 300 HR RF 17 1196 01 HiPrep 26 60 Sephacryl S 300 HR RF Hydrophobic Interaction Columns Code no Column name 0 25 0 50 17 5186 01 SOURCE 15PHE 4 6 100 PE X 17 1184 01 RESOURCE ETH 1 ml X 17 1185 01 RESOURCE ISO 1 ml X 17 1186 01 RESOURCE PHE 1 ml X 17 1187 01 RESOURCE HIC Test kit 3x1 ml X 28 4110 07 HiTrap HIC Selection Kit 7x1 ml X 17 1085 01 HiLoad 16 10 Phenyl Sepharose HP x 17 1086 01 HiLoad 26 10 Phenyl Sepharose HP x 17 5095 01 HiPrep 16 10 Phenyl FF high sub RF 17 5094 01 HiPrep 16 10 Phenyl FF low sub RF 17 5096 01 HiPrep 16 10 Butyl FF RF 17 5097 01 HiPrep 16 10 Octyl FF RF 17 1355 01 HiTrap Phenyl FF high sub 1 ml x 17 5193 01 HiTrap Phenyl FF high sub 5 ml RF 17 1353 01 HiTrap Phenyl FF low sub 1 ml X 17 5194 01 HiTrap Phenyl FF low sub 5 ml RF 17 1351 01 HiTrap Phenyl HP 1 ml X 17 5195 01 HiTrap Phe
23. 8 11 The systems Fig 8 1 AKTApurifier to the left and AKTApurifier UPC to the right AKTApurifier consists of a compact separation unit and a personal computer running UNICORN software version 5 10 or higher The individual modules and components are briefly described in this chapter Detailed information on the components can be found in their respective User Manuals and Instructions UNICORN is described in the separate UNICORN user documentation supplied Communication between the computer and the various components of AKTApurifier is achieved via high speed data network connections UniNet 1 and UniNet 2 All of the fluid handling components of AKTApurifier are mounted on the same side of the main components This allows easy access to all components tubing and other fluid components located on Pump P 900 and Monitor UV 900 Monitor UPC 900 AKTApurifier User Guide 28 4079 53 Edition AC 115 8 Reference information 8 1 System description 8 12 Component locations The illustration below shows the location of the components of the separation unit Column holder f ZS Box 900 Injection valve INV 907 Monitor UV 900 UV Flow cell Pump P 900 P 901 P 903 Mixer chamber ON OFF button Mixer M 925 Fig 8 2 AKTApurifier component locations 116 AKTApurifier User Guide 28 4079 53 Edition AC Reference information 8 Conductivity cell Optical u
24. AkTApurifier is mentioned AKTApurifier features e Flow rates up to AKTApurifier 100 P 901 100 ml min at pressures up to 10 MPa AKTApurifier 10 P 903 10 ml min at pressures up to 25 MPa e One working platform for most liquid chromatography techniques from micro gram to gram scale Fig 1 1 AKTApurifier to the left and AKTApurifier UPC to the right AKTApurifier consists of a compact separation unit a personal computer running UNICORN control system version 5 10 or higher and as options e a fraction collector and ea monitor for pH and conductivity measurement included for AKTApurifier UPC AKTApurifier User Guide 28 4079 53 Edition AC 7 1 Introduction 11 General The chromatography system is described in detail in Section 8 1 in System description Brief descriptions of the individual components are given in Section 8 2 in Component description Detailed information on the components can be found in their respective User Manuals and Instructions The UNICORN control system is described in the separate UNICORN User Manuals The location of the modules and components in the separation units are shown in the following illustrations Column Column holder Injection valve INV 907 Box 900 Monitor UV 900 C IV Vi Pump P 900 a P 901 P 903 r UV Flow gell we me IC Mixer M 925 ON OFF button
25. Conversion of apparent pKa s to thermodynamic pKa s Only pKa values at thermodynamic conditions pKa at infinite dilution must be entered into BufferPrep When entering a new buffer substance with only apparent pKa known a conversion to thermodynamic pKa has to be performed The apparent pKa is measured at a non zero concentration Find the thermodynamic pKa by taking the apparent pKa and add the value found in the table below for the buffer concentration at which the apparent pKa has been measured Conc MJ Base pKa1 Base pKa2 Acid pKal Acid pKa2 0 02 0 05 0 26 0 05 0 26 0 05 0 07 0 37 0 07 0 37 0 1 0 09 0 47 0 09 0 47 0 2 0 12 0 60 0 12 0 60 Example Bis Tris has a listed pKa of 6 46 at a concentration of 0 1 M This is a base with only one pKa Using the table above we find that 6 37 should give more accurate results 6 46 0 09 6 37 Zwitter ions Using zwitter ions in a BufferPrep recipe can be difficult All zwitter ions have at least two pKa values Since BufferPrep has to know all pKa values for the buffer component do not use a zwitter ionic buffer component if the pKa values are not known The second important issue is to know in which form the component is i e if the molecule contains acidic protons Example These examples can be used as a template for zwitter ions Bicine pKal 1 84 dpKa1 dT value unknown insert 0 pKa2 8 33 dpKa2 dT 0 017 number of acidic
26. Flow rate 0 20 ml min Max pressure 4 0 MPa 40 bar 580 psi Max back pressure 0 02 MPa at 20 ml min Optical path length 5mm Cell volume 6 ul 10 pl detector volume Conductivity unit Conductivity range Deviation from theoretical conductivity 1 uS cm 999 9 mS cm Max 2 of full scale calibration range or 10 yS cm whichever is greater in the range 1 uS cm 300 mS cm Reproducibility Max 1 maximum or 5 yS cm whichever is greater in the range 1 uS cm 300 mS cm oise Conductivity flow cell Max 0 5 of full scale calibrated range Flow rate 0 100 ml min ax pressure 5 MPa 50 bar 725 psi ax back pressure 0 01 MPa at 100 ml min pH measurement optional pH range Oto 14 AKTApurifier User Guide 28 4079 53 Edition AC 159 8 Reference information 8 6 Technical specifications 8 6 2 Physical data Degree of protection IP 20 Power requirement 100 240 V 50 60 Hz Power consumption 600 VA Fuse specification KTApurifier T 6 3 AL 250 VAC Dimensions W x H x D 450 x 610 x 490mm Weight AKTApurifier 41 kg AKTApurifier UPC 38 kg Environment 4 to 40 C 10 95 relative humidity non condensing 84 106 kPa 840 1060 mbar atmospheric pressure 8 6 3 Compliance with safety standards The CE symbol and declaration The CE symbol and corresponding of conformity declaration of conform
27. Fraction marks and fraction numbers make it easy to identify fractions and peaks Fast tube change minimizes spill between tubes eliminating it entirely below flow rates of 5 ml min Drop synchronization eliminates sample loss during tube change Installation 1 Unpack and assemble Frac 920 according to the instructions in the Fraction Collector Frac 920 User Manual AKTApurifier User Guide 28 4079 53 Edition AC 2 Drain tubing Installation 2 Place Frac 920 to the right of the system Injection Valve V1 Connect a tubing from the outlet of the To deliver flow restrictor to the IN port on the flow ym INC y diversion valve hy Note Use tubing with the same inner diameter as G5 to connect to the Towoste IN and NC ports From flow NO restrictor IN Connect tubing from the NC normally closed port on the flow diversion valve to the delivery arm on Frac 920 Follow the instructions given in the Frac 920 User Manual Connect tubing W3 from the NO normally open port on the flow diversion valve to waste Place the valve in the hole behind the bowl Extend the drain tubing from Frac 920 34 to a suitable waste bottle Set the delay volume in UNCORN see Section 8 1 6 Setting the delay volume AKTApurifier User Guide 28 4079 53 Edition AC 23 2 2 3 Installation of AKTApurifier 24 2 3 7 Installing Fraction collector Frac 950 optional
28. Select Flow Select FlowRate Click Execute 2 Start a run manually at 0 B Make sure the buffer valve optional is set to the required inlet A11 A18 3 Check the pH reading when stable 4 Change to 100 B by setting the Gradient instruction in Manual Pump to 100 B for Target and 0 for Length Click Execute 5 Check the pH reading when stable at 100 B 6 If the reading is acceptable at both O and 100 B the correction factors do not need to be changed 7 Ifthe pH reading is not acceptable it is possible to correct the factors for the recipe Select Edit BufferPrep Recipes in the Method Editor Click New In the New Recipe dialog click Correction factors Enter the pH deviation at 0 and 100 B For example if the pH set is 7 0 and the actual pH is 7 1 enter 0 1 or if the actual pH is 6 9 enter 0 1 Note Some of the pre programmed recipes have default correction factors Add your deviation to these to obtain the correct value Example If your pre set correction factor is 0 2 and your reading at pH 7 is 7 1 enter 0 2 0 1 0 1 8 Click Save As to save the recipe AKTApurifier User Guide 28 4079 53 Edition AC 151 8 Reference information 8 5 BufferPrep details 152 8 5 5 Examples and tips Buffering capacity If the buffer capacity of the broad range BufferPrep recipes are too low there are two alternatives e Switch to a recipe with one buffer component with a pKa close to the required pH
29. WARNING The covers of the modules and components must not be removed by the user The modules and components contain high voltage circuits that can give a lethal electric shock WARNING The optical units of Monitor UPC 900 and Monitor UV 900 uses high intensity ultra violet light Do not disassemble the optical unit while the lamp is ON WARNING Incorrectly fitted tubing may loosen causing a jet of liquid to spray out This is especially dangerous if hazardous chemicals are used Connect the tubing by first inserting the tubing fully then tightening the connector finger tight PEEK tubing should be tightened a further 1 4 turn using the key supplied Do not tighten Teflon tubing further as this will damage the end of the tubing WARNING Never place waste containers on top of the system If they become full and overflow liquid might penetrate the system causing a short circuit WARNING Always disconnect the power supply before attempting to replace any item on the system during maintenance WARNING In case of an emergency situation the system mains switch must always be easy to access and the mains plug must always be easy to disconnect AKTApurifier User Guide 28 4079 53 Edition AC Introduction 1 WARNING The computer must be placed in a dry location and according to the environmental requirements stated in the manufacturer s instructions WARNING When using hazardous chemicals
30. WC i 600000000066 f f 0000000000 000000000600 a NN 866608808086 000000000000 IT Define Set default ae 0000806688 C8 L 60006 TANT Be no lt Back Cancel AKTApurifier User Guide 28 4079 53 Edition AC Operation 4 4 The Variables page is the same page as in the Method Editor Here you can verify and fine tune the method before you proceed This is very convenient when repeating runs with minor adjustments Block Variable Value Range Main Column HiLoad 16 10 D Sepharose FF e RE Flow Rate Fon Rate GoD E ban Column_Pressure_Limit Column_PressureLimit MPa 0 500 0 000 5 000 Corn ae toum Poston Fas es 2 1 000 S666 000 Peak_Frac_Size ml 0 000 0 000 99999 000 Length of Gradient Cv 20 00 0 00 9999 00 PF Show details T Show unused variables FN Display tooltip for extended variable cells Help lt F reg ende Avbryt Some variables are normally hidden Check the Show details box to view them 5 On the Evaluation Procedures page you select the automated operations you want the system to perform after the run Select Print_Chromatogram to have an automatic print out after the run 6 The Method Information page contains a summary of the 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 7 Onthe Result Name page you name the result file and defi
31. account 2 Room temperature and limited over pressure is assumed Note Chemical influences are time and pressure dependent Unless otherwise stated all concentrations are 100 Chemical Exposure Comments lt iday upto 2 months Acetaldehyde OK OK Acetic acid lt 5 OK OK Acetic acid 70 OK OK Acetonitrile OK OK FFKM PP and PE swell PEEK is affected by long term use Acetone 10 OK Avoid PVDF is affected by long term use Ammonia 30 OK OK Silicone is affected by long term use Ammonium chloride OK OK Ammonium bicarbonate OK OK Ammonium nitrate OK OK Ammonium sulphate OK OK 1 Butanol Ok OK 2 Butanol OK OK Citric acid OK OK Chloroform OK Avoid ECTFE PP and PE are affected by long term use Cyclohexane OK OK Detergents OK OK Dimethyl sulphoxide Avoid Avoid PVDF is affected by long term use 1 4 Dioxane Avoid Avoid ETFE PP PE and PVDF are affected by long term use Ethanol OK OK AkTApurifier User Guide 28 4079 53 Edition AC 163 8 Reference information 8 7 Chemical resistance guide and chemical compatibility Chemical Exposure Comments lt 1day upto 2 months Ethyl acetate OK Avoid Silicone not resistant Pressure limit for PEEK decreases Ethylene glycol OK OK Formic acid OK OK Silicone not resistant Glycerol OK OK Guanidinium hydrochloride OK OK Hexane OK Avoid Silic
32. acidic protons for the buffer substance in the form that is actually weighed in Example For NaH PO enter 2 for Na HPO enter 1 for Tris enter 0 6 Enter the Charge of the completely deprotonated ion This will be a negative value for an acid 0 for a base Example For NaH PO enter 3 for Tris enter 0 7 Click OK to add the new buffer substance to the list of available buffers 8 5 3 Defining a new salt When defining a new salt make sure that the new salt is inert i e a salt with no buffering properties 1 In the New Recipe dialog click Salt and click New in the Define Salt dialog 2 In the New Component dialog enter the name of the new salt Click OK AKTApurifier User Guide 28 4079 53 Edition AC Reference information 8 3 Enter the Charge of the Anion Example for Cl enter 1 for SO enter 2 4 Enter the Charge of the Cation Example For Nat enter 1 for Mg2 enter 2 5 Click OK to add the new salt to the list of available salts 8 5 4 Correction factors To obtain higher pH accuracy the recipe can be fine tuned around a specific pH at the flow rate to be used When scouting over a broad pH range fine tuning is less beneficial Run BufferPrep manually at 0 and 100 B as follows 1 From the System Control Manual menu select Other In the Other instructions dialog select BufferPrep Recipe Select the recipe in the Recipe Name list Click Execute Select Pump to switch to the Pump instructions dialog
33. cable 1 18 1108 40 fill port needle and syringe holder are not included Injection kit INV 907 including 1 18 1110 89 fill port 0 7 mm needle and syringe holder Valve kit INV 907 including channel plate and 1 18 1109 05 distribution plate Mounting bracket 1 18 1109 11 Sample loops 10 ul 1 18 1120 39 100 ul 1 18 1113 98 500 ul a 18 1113 99 1 ml 1 18 1114 01 2 mi ik 18 1114 02 Cables UniNet 0 18 m 18 1109 72 UniNet 0 3 m a 18 1109 73 UniNet 0 7 m 1 18 1109 74 UniNet 1 5 m 1 18 1117 75 UniNet 3 0m 1 18 1109 75 UniNet 15 0 m 1 18 1117 74 ains cable US standard 1 19 2447 01 ains cable EU standard 1 19 2448 01 Signal Cable 6 pin mini DIN open 1 18 1110 64 Inlet filter Inlet filter set 5 11 0004 14 Filter holder kit 1 11 0004 07 Screw lid GL45 kit 1 11 0004 10 Inlet filter kit 1 filter 1 net 1 lid GL45 1 18 1113 15 Tubing PEEK tubing i d 0 25 mm o d 1 16 2m 18 1120 95 PEEK tubing i d 0 50 mm o d 1 16 2m 18 1113 68 PEEK tubing i d 0 75 mm o d 1 16 2m 18 1112 53 PEEK tubing i d 1 0 mm o d 1 16 2m 18 1115 83 168 KTApurifier User Guide 28 4079 53 Edition AC Reference information 8 Item Quant Codeno pack Tubing Tefzel tubing i d 0 25 mm o d 1 16 2m 18 1121 36 Tefzel tubing i d 0 50 mm o d 1 16 2m 18 1120 96 Tefzel tubing i d 0 75 mm o d 1 16 2m 18 1119 74 Teflon tubing i d 0 75 mm o d 1 16 2m 18 1112 54 Teflon tu
34. displays the monitor signal values graphically To customize the Curves pane 1 Right click in the Curves pane and select Properties or select View Properties from the System Control menu to select the curves to be displayed All curves are always stored in the result file 2 Click the different tabs in the Curve Properties pane to set the properties for the different curves Normally the curves are scaled with auto scaling Le the scale is adjusted continually to the highest and lowest values for each curve Curve Style and Color Flow Scheme Logbook Run Data Groups Run Data Color Curves Axis Y Axis r Display curves MUY So Select All MCondz Clear All MConc os Flow MITemp ok Avbiyt 3 Tofixthe Y axis scale for a curve mark the curve click Y axis click Fixed and enter the max and min values You can repeat this for other curves Click OK 4 To maximize the Curve Data pane position the cursor in the Curve Data pane Click the right mouse button and select Maximize Go back to normal size by clicking Restore 5 To shift to a scale for another curve click the Y axis scale or click the curve name at the top of the Curve Data 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 Flow scheme The flow scheme is a graphical representation of the flow path in the chromatography system During a run the flow sche
35. filling is achieved as follows 1 Set the injection valve to position LOAD 2 Load the syringe with sample 2 5 times the loop volume 3 Gently load the syringe contents into the loop 4 Leave the syringe in position The sample will be injected onto the column when the valve is switched to INJECT in the method Note If the syringe is taken out before the sample is injected onto the column self drainage will occur and the loop will be emptied Emptying the sample loop When emptying the sample loop a buffer volume of at least 5 times the sample loop volume should be used to flush the loop and ensure that all sample is injected onto the column The volume to inject is set by the variable Empty_loop_with in the Sample_Injection block AKTApurifier User Guide 28 4079 53 Edition AC 65 4 Operation 4 4 Sample application 4 4 2 Applying large sample volumes using a Superloop WARNING Superloop 10 ml and Superloop 50 ml must not be used at pressures above 4 MPa 40 bar 580 psi Superloop 150 ml must not be used above 2 MPa 20 bar 290 psi At higher pressures the glass tube might shatter Superloop permits the introduction of larger volumes of sample 1 150 ml onto the column Superloop must not be used with columns generating a column pressure higher than that stated in the table below If the column pressure is higher the flow must be reduced in order not to exceed the pressure limit Set the pressure limit in the meth
36. intended for your system KTApurifier User guide 28 4079 53 Edition AC 5 Optional functions 5 Connect the two short tubings in position A3 and B3 using the connectors and ferrules supplied see the figure above Note the direction of the ferrule Ferrule Gs Connect the four long tubings in position A1 A2 B1 and B2 Setting up UNICORN for BufferPrep To enable the BufferPrep function in UNICORN the switch valves must be selected as components 1 2 In UNICORN Manager select Administration System Setup In the System Setup dialog select your system and click Edit In the Edit System dialog click Component In the Component dialog select Switch valves Click OK twice and finally Close 5 2 3 Strategy for using BufferPrep BufferPrep should be used to quickly find the optimal pH for your protein purification procedure Use it as follows 1 Start with a recipe covering a broad pH range and scout for pH in steps of for example 0 5 1 pH units Select the pH that gives the best result Either continue with the broad pH range recipe or change to a single buffer recipe within the pH range that gave the best result AKTApurifier User guide 28 4079 53 Edition AC 91 5 Optional functions 5 2 BufferPrep 92 5 2 4 Creating a method for pH scouting 1 Create a new method in the Method Wizard by selecting BufferPrep on the first page Also select which recipe to use and the desired pH Method Wizard for Syste
37. kit 0 25 installed Tubing kit 0 50 should be fitted when columns with a low max pressure are used at high flow rates When changing from to Tubing kit 0 25 to from Tubing kit 0 50 change the tubings designated G6 G9 with H6 H9 Refer to the flow diagram in section 8 1 5 for their location in the fluid handling path 124 AKTApurifier User Guide 28 4079 53 Edition AC Reference information 8 Tubing Length mm i d mm Location G1 330 0 50 Pump P 900A inner to mixer G2 330 0 50 Pump P 900B outer to mixer G3 200 0 50 Mixer to on line filter G4 180 0 50 On line filter to injection valve pos 7 G5 400 0 50 Bypass injection valve pos 1 to UV cell Tubing kit 0 25 mm G6 160 0 25 UV cell to conductivity cell G7 80 0 25 Conductivity cell to flow restrictor G8 140 0 25 Flow restrictor to outlet valve centre port G9 600 0 25 Outlet valve port 2 to fraction collector optional F3 1000 0 50 Outlet valve port 3 A3 150 1 6 SV 903A IN to pump P 900A B3 150 1 6 SV 903B IN to pump P 900B Al 1700 1 6 Buffer vessel A1 to SV 903A NO A2 1900 1 6 Buffer vessel A2 to SV 903A NC B1 1500 1 6 Buffer vessel B1 to SV 903B NO B2 1700 1 6 Buffer vessel B2 to SV 903B NC W1 1000 0 75 Injection valve port 4 to waste W2 1000 0 75 Injection valve port 5 to waste W3 1000 0 75 Outlet valve port 1 to waste Tubing kit 0 50 mm H6 160
38. method Method Wizard By default flow regulation is not included when creating a method using the Method Wizard To include flow regulation 1 Select Flow Regulation of the System Pump on the first page in the Method Wizard 2 Enter suitable pressure and flow rate limits see Section below Note Turning on the flow regulation means that the feedback control in UNICORN is activated It will only be activated during sample application and wash out of unbound sample Turning on flow regulation in an existing method 1 Select View Run Setup in Method Editor 2 Click the Variables tab and select Show details AKTApurifier User guide 28 4079 53 Edition AC Optional functions 5 3 Inthe Aut_PressureFlow_Regulation block select PressFlowControl mode for the System_Pump variable 4 Enterthe pressure level in System_PressLevel choose a value slightly below the column pressure limit and the minimum allowed flow in System_MinFlow These parameter values as well as the PID parameter values can later be changed separately by selecting System Settings in System Control Note If the flow rate falls below the System_MinFlow value an Alarm will be issued and the system set to Pause Therefore we recommend using a Watch instruction WatchPar_Flow that is activated above this value A suitable action is to continue to the next block To prevent pressure peaks when continuing use a lower flow rate in the block after the Watch inst
39. ml X 28 9075 47 StrepTrap HP 5 ml RF 28 4082 53 HiTrap MabSelect 1 ml x 28 4082 55 HiTrap MabSelect 5 ml RF 11 0034 93 HiTrap MabSelect SuRe 1 ml X 11 0034 94 HiTrap MabSelect SuRe 5 ml RF 28 4082 58 HiTrap MabSelect Xtra 1 ml x 28 4082 60 HiTrap MabSelect Xtra 5 ml RF 139 AKTApurifier User Guide 28 4079 53 Edition AC 8 Reference information 8 4 Recommended tubing and columns Code no Column name 0 25 0 50 17 5079 01 HiTrap rProtein FF 1 ml X 17 5080 01 HiTrap rProtein A FF 5 ml RF 17 0402 01 HiTrap Protein A HP 1 ml X 17 0403 01 HiTrap Protein A HP 5 ml RF 17 0404 01 HiTrap Protein G HP 1 ml X 17 0405 01 HiTrap Protein G HP 5 ml RF 17 0406 01 HiTrap Heparin HP 1 ml X 17 0407 03 HiTrap Heparin HP 5 ml RF 17 5189 01 HiPrep 16 10 Heparin FF RF 17 0412 01 HiTrap Blue HP 1 ml X 17 0413 01 HiTrap Blue HP 5 ml RF 17 5110 01 HiTrap IgM Purification HP 1 ml X 17 5111 01 HiTrap IgY Purification HP 5 ml RF 17 5112 01 HiTrap Streptavidin HP 1 ml X 17 5143 01 HiTrap Benzamidine FF high sub 1 ml X 17 5144 01 HiTrap Benzamidine FF high sub 5 ml RF Chromatofocusing Columns Code no Column name 0 25 0 50 17 5171 01 Mono P 5 200 GL X 17 5170 01 Mono P 5 50 GL X 140 KTApurifier User Guide 28 4079 53 Edition AC Reference information 8 8 4 2 AKTApurifier 100 Note When using low pressure columns such as Hi
40. ml mixer chamber Absolute conductivity value wrong Turn the flow cell so that the end with the screws is facing the flow restrictor FR 902 Recalibrate the conductivity cell Calibration solution 1 00 M NaCl not prepared correctly Prepare a new calibration solution and recalibrate the conductivity cell Unstable conductivity curve 7 8 Try using a larger mixer chamber instead of the standard 0 6 ml mixer chamber Mixer M 925 Error symptom Leakage Possible cause Action Check the tubing connections Retighten or replace if necessary Check the mixer chamber Replace if liquid has penetrated the mixer chamber walls and sealings Function test Test the mixer function by placing a stirrer bar on top of the mixer housing The stirrer bar should rotate when the system is in Run mode The mixer function can also be checked by running the installation test 112 AKTApurifier User Guide 28 4079 53 Edition AC 7 9 Error symptom The valve is not switching Possible cause Action Troubleshooting 7 Valve INV 907 Check the connection to the pump The valve should be connected to the UniNet 2 socket Check the ID switch on the valve The ID number should correspond to the number set in UNICORN Check the UniNet cable and replace if required The valve is switching to wrong position The valve parts might have been incorrectly reassembled after replacemen
41. protons 1 charge of deprotonated ion 1 HEPES pKal 3 dpKa1 dT value unknown insert 0 pKa2 7 39 dpKa2 dT 0 014 number of acidic protons 1 charge of deprotonated ion 1 AKTApurifier User Guide 28 4079 53 Edition AC 153 8 Reference information 8 5 BufferPrep details 154 Cold room If the dpKa dT values are correct there is no problem using BufferPrep in a cold room To fine tune the AIEX and CIEX recipes use the following correction factors AIEX at 5 C 0 B 0 10 100 B 0 00 CIEX at 5 C 0 B 0 00 100 B 0 40 AKTApurifier User Guide 28 4079 53 Edition AC Reference information 8 8 5 6 Recipe overview Data for pre programmed AIEX recipes Gradient 0 0 1 0 M NaC Buffer Titrate pHrange Default Start conductivity with correction at low high pH 0 B 100 B 5 0 9 5 pH AIEX mixture 0 1 M HCI 5 0 9 5 0 0 0 0 3 2 0 8 mS cm 0 05 M 1 methyl piperazine 0 05 M Bis Tris 0 025 M Tris 6 0 9 0 pH AIEX mixture 0 1 M HC 6 0 9 0 0 0 0 0 2 5 0 4 mS cm 0 07 M Bis Tris 0 05 M Tris 0 1 M Bis Tris 0 1 M HC 6 0 7 7 0 2 0 0 2 2 0 4 mS cm 0 1 M 1 methyl piperazine 0 1 M HC 5 0 5 5 0 0 0 1 2 4 0 6 mS cm 8 6 10 3 0 1 0 2 3 3 2 8mS cm 0 1 M Piperazine 0 1 M HC 6 0 6 7 0 1 0 3 2 4 1 1 mS cm 9 4 10 5 0 2 0 4 3 4 2 9 mS cm 0 1 M Tris 0 1 M HCI 7 5 8 5 0 0 0 2 2 4 1 3 mS cm
42. restrictor Tubing Length Tubing i d Tubing o d Material Color Volume of design 10 cm A1 B1 170 cm 2 9 mm 1 16 Teflon Clear 660 ul G1 G2 33 cm 0 75 mm 1 16 PEEK Green 44 2 ul G3 25 cm 0 75 mm 1 16 PEEK Green 44 2 ul G4 30 cm 0 75 mm 1 16 PEEK Green 44 2 ul G5 17 cm 0 75 mm 1 16 PEEK Green 44 2 ul W1 W3 100 cm 0 75 mm 1 16 Tefzel Clear 44 2 ul 126 AKTApurifier User Guide 28 4079 53 Edition AC Reference information 8 8 16 Setting the delay volume The delay volume is the total volume of the tubing and components between the UV cell and the outlet of the fraction collector This volume is used in UNICORN for synchronizing the UV peak detection with the fraction collection Note The delay volume is not set at system delivery because it depends on the length of the outlet tubing When the installation is finished the delay volume should be calculated and then set in UNICORN When the system configuration after the UV cell has been changed for example by changing a component or tubing the delay volume might change as well The new delay volume must then be calculated and set in UNICORN Calculating the delay volume Calculate the contribution from each component and tubing from the UV cell to the fraction collector outlet The table below lists the theoretical volumes of a number of components and tubing Component capillary Volume ul Valve PV 908 7 Valve IV 908 27 Valv
43. system pump P 901 903 REMOTE connector e Connection of an AD converter 134 AKTApurifier User Guide 28 4079 53 Edition AC Reference information 8 8 4 Recommended tubing and columns The tables below shows which tubing kit should be used for each column It is important that the recommendations in these tables are followed The tubing to be changed is described in Section 8 1 2 in Component locations 8 4 1 AKTApurifier10 Note In order to use low pressure columns such as HiTrap HiPrep and HiLoad an extra system pressure measurement must be performed This is described later in this Section Xx recommended tubing kit P recommended for purity checks only RF can be used with indicated tubing if the optimal recommended flow rate in the column list is reduced in order not to exceed the column maximum pressure lon Exchange Columns Code no Column name 0 25 0 50 17 5177 01 ini Om 4 6 50 PE x P 17 5166 01 ono Q 5 50 GL X 17 5167 01 ono Q 10 100 GL RF x 17 0506 01 ono Q HR 16 10 RF x 17 5178 01 ini S 4 6 50 PE x P 17 5168 01 ono S 5 50 GL X 17 5169 01 ono S 10 100 GL RF x 17 0507 01 ono S HR 16 10 RF x 17 5181 01 SOURCE 15Q 4 6 100 PE RF x 17 5182 01 SOURCE 15S 4 6 100 PE RF x 17 1177 01 RESOURCE Q 1 ml x 17 1179 01 RESOURCE Q 6 ml x 17 1178 01 RESOURCES 1 ml x 17 1180 01 RESOURCE S 6 ml x 11 0013 02 HiTrap Capto
44. the mixer chamber thoroughly with distilled water before removing it At delivery the mixer is delivered with a 0 6 ml mixer chamber It can be used at gradient volumes down to 5 ml binary gradients The recommended minimum gradient volume for each mixing chamber is specified in the table below Recommended minimum gradient volume Mixing chamber yalume Binary gradient BufferPrep gradient 0 6 ml 5 ml 2ml 16 ml 60ml 5ml 38 ml 80 ml 12 ml 90 ml 113 ml When using eluents that are more difficult to mix such as isopropanol and water a larger mixer volume can be used to get optimum mixing Note Ifthe pH and conductivity curves indicate uneven mixing of your buffers unstable curves change to a larger mixer chamber KTApurifier User guide 28 4079 53 Edition AC 87 5 Optional functions 5 2 BufferPrep 88 Note Always place the buffer bottles lower than the mixer when changing chambers to prevent draining 5 2 BufferPrep 5 2 1 What is BufferPrep BufferPrep eliminates the time consuming manual buffer preparation and titration usually needed for every pH change in chromatography For any pH and salt concentration entered BufferPrep automatically calculates and prepares the composition of the buffer on line from four stock solutions Linear and step salt gradients can be run and pH can be used as a variable scouting parameter Note To be able to use the BufferPrep function the Bu
45. valve INV 907 in AKTApurifier Lift off the Mixer M 925 mixer to remove the LEE protective foam and then AKTApurifier UPC refit e Optical unit Lift the optical unit gently to remove the plastic foam packing e Capillary loops Remove all red tape holding capillaries and loops attached to valve INV 907 INV 907 e UVcell cover AKTApurifier located above the UV flow UV cell cover cell Remove the red tapes Mixer M 925 AKTApurifier AKTApurifier User Guide 28 4079 53 Edition AC 17 2 Installation 2 3 Installation of AKTApurifier 18 Should the cover over the UV cell compartment come loose it is refitted in the following way 1 The cover is a simple push fit onto the cell holder Two small lugs on the cover locate in holes at the front and rear of the cell holder 2 The cover is then lowered over the cell holder Waste tubings Place the waste tubings marked W1 W2 and W3 ina waste bottle and place the bottle in a convenient place Inlet tubing Unpack the inlet tubing contained in a plastic bag taped to the buffer tray on top of Box 900 Make sure all items are securely fitted and that no capillary connection has worked loose or been tangled Column holder AKTApurifier Attach the column holder enclosed in Box 900 above the UV cell Select a slot to suite the height of the column to be used AKTApurifier UPC Select a position that minimizes the
46. 00 Note If using Frac 920 with AKTApurifier 10 we recommend a maximum flow rate of 3 5 ml min with the d 0 25 mm tubing kit At higher flow rates the drops will turn to a continuous liquid stream 68 AKTApurifier User Guide 28 4079 53 Edition AC Operation 4 Note If tubing with too large inner diameter is used the peaks will become broader than necessary If tubing with too small inner diameter is used the backpressure from the tubing might become higher than the maximum pressure for the column and the run will stop immediately after it is started 2 Immerse inlet tubing A in buffer A and inlet tubing B in buffer B A2 and B2 respectively if you changed this in the method 3 Make sure that the waste tubing from the flow restrictor is put into an empty waste bottle 4 If there is air in the inlet tubing or if you suspect air in the pump purge the pump with a syringe as described in 2 4 1 Preparing AKTApurifier or in Chapter 2 in Pump P 900 User Manual 5 IfpH measurement is desired calibrate the pH monitor Refer to the UNICORN User Manuals or the Monitor pH C 900 User Manual Mount the pH electrode in the flow cell Checking the monitors 1 Make sure that the correct UV cell 2 or 5 mm and filter is installed and the correct wavelength set in the optical unit See Chapter 2 in Monitor UPC 900 User Manual 2 Make sure that pressure sensor conductivity cell and pH electrode optional are calibrated according to t
47. 01 SMTH Installation Test01 1_UV 01 SMTH Move the vertical bar to the constant Section of each plateau by dragging it Enter the absorbance values in MAU in column 2 in the Step response table of the Test record see page 39 leaving out the decimals 8 Click Print under File to print the chromatogram 9 AKTApurifier Read the absorbance for the plateaus corresponding to 0 B and 100 B for the curve Installation Test01 1_UV1_215nm 01 SMTH and enter the values in column 2 in the UV response table of the Test record Evaluating the gradient Place a ruler along the gradient part of curve AKTApurifier AKTApurifier UPC Installation Test01 1_UV1_215nm 01 SMTH Installation Test01 1_UV 01 SMTH in the printed report The curve should be linear between 10 B and 90 B and void of discontinuities AKTApurifier User Guide 28 4079 53 Edition AC Installation 2 Evaluating the step response Calculate the relative adsorption plateau heights for curve AKTApurifier AKTApurifier UPC Installation Test01 1_UV1_215nm 01 SMTH Installation Test01 1_UV 01 SMTH as follows e Subtract the base line value 0 B from each of the values in column 2 in the Step response table of the Test record see page 39 and enter the results in column 3 e 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 The values of column 4 should all fall within the inte
48. 04 must be replaced with the supplied flow restrictor FR 902 Otherwise the long term stability and lifetime of the pH electrode will be reduced AKTApurifier User Guide 28 4079 53 Edition AC 21 2 Installation 2 3 Installation of AKTApurifier 22 2 3 5 Installing the computer and printer Unpack and install the computer and printer according to the manufacturer s instructions Place them to the left of the system Note Do not turn them on yet WARNING In case of an emergency situation the system mains switch must always be easy to access and the mains plug must always be easy to disconnect WARNING The computer must be placed in a dry location and according to the environmental requirements stated in the manufacturer s instructions WARNING The computer should be installed and used according to the instructions provided by the manufacturer of the computer 2 3 6 Installing Fraction collector Frac 920 optional Frac 920 can be used for both small scale and preparative scale purification with AKTApurifier It collects up to 175 fractions in 12 mm diameter tubes up to 95 fractions in 10 18 mm diameter tubes and up to 40 fractions in 30 mm diameter tubes An optional flow diversion valve FV 923 allows flow diversion to waste Frac 920 allows fixed volume fractionation and or automatic peak fractionation The latter function is based on peak detection using slope or level sensing
49. 10 ul 25 MPa 18 1120 39 Loop 100 ul 25 MPa 18 1113 98 Loop 500 ul 10 MPa 18 1113 99 Loop 1 ml 10 MPa 18 1114 01 Loop 2 ml 10 MPa 18 1114 02 Operation 4 When filling the loop with a sample volume equal to the loop volume about 15 to 25 of the sample will be lost to waste because the fluid velocity in the sample loop tubing varies from a maximum at the tube axis to almost zero at the tubing wall The exact amount of sample lost depends on the delivery flow rate Two techniques can be used for filling the sample loop partial or complete filling Type of filling Partial filling Volume to load Max 50 of the sample loop volume Complete filling 2 5 times the sample loop volume AKTApurifier User Guide 28 4079 53 Edition AC 63 4 Operation 4 4 Sample application Partial filling Partial filling is used when high recovery is required The sample volume loaded should be at maximum 50 of the loop volume The volumetric accuracy and precision is that of the syringe Partial filling allows the injected volume to be changed without changing the loop and does not waste sample The sample loop must be completely filled with buffer before the sample can be loaded Partial filling is achieved as follows Note The flow must be off before starting the filling procedure 1 64 Set the injection valve to position LOAD Load the syringe with a large volume of buffer 5 times
50. 112 01 HiTrap Streptavidin HP 1 ml x 17 5143 01 HiTrap Benzamidine FF x high sub 1 ml 17 5144 01 HiTrap Benzamidine FF RF high sub 5 ml Chromatofocusing Columns Code no Column name 0 25 0 75 1 0 17 5171 01 Mono P 5 200 GL X 17 5170 01 Mono P 5 50 GL X KTApurifier User Guide 28 4079 53 Edition AC 147 8 Reference information 8 4 Recommended tubing and columns 8 4 3 Selecting tubing kit for other columns For the columns not available in the tables in 8 4 in Recommended tubing and columns select the tubing kit as described below Note Before starting to perform the described method below make sure that the column itself is clean and does not generate too high back pressure 1 Select View Run Setup in the Method Editor for the method that is used 2 Note the maximum specified back pressure for the column at the variable Column_PressureLimit on the Variables page Note The maximum allowed back pressure on a self packed column should never exceed 75 of the back pressure used during the packing procedure 3 Install the column and test to run at the flow rate and with the eluents to be used with the i d 0 75 mm tubing kit 4 Select the appropriate tubing kit as follows e If the generated back pressure at the flow to be used is within the set column pressure limit use the tubing kit already installed e If the generated back pressure at the flow rate to be used is well below the c
51. 56 jul KTApurifier User Guide 28 4079 53 Edition AC 157 8 Reference information 8 6 Technical specifications 158 Monitor UV 900 Wavelength range 190 700 nm in step of 1 nm 3 wavelengths simultaneously Bandwidth 4nm Wavelength accuracy 2nm Wavelength reproducibility 0 01nm Wavelength switch time lt 500 ms one cycle from 214 nm to 254 nm and back to 214 Linearity lt 2 deviation up to 2 AU at 260 nm with Uracil at pH 2 Noise at 230 nm Drifti at 254 nm lt 6x107 AU with 10 mm cell H 0 at 1 ml min lt 2x10 AU h Flow cell Max flow rate Max pressure 100 ml min 2 MPa 20 bar 290 psi 1 Specified for 10 mm flow cell 2 Typical values at room temperature after warm up Monitor UPC 900 UV measurement Absorbance range 0 001 5 0 AU full scale Autozero range 0 2 2 0 AU Baseline adjust Adjustable 0 100 of full scale Wavelengths Hg lamp fixed 254 and 280 nm by changing filter 313 365 405 436 and 546 nm Zn lamp 214nm UV flow cell 2mm Flow rate 0 100 ml min Max pressure 4 0 MPa 40 bar 580 psi Max back pressure 0 05 MPa at 100 ml min Liquid temperature range 4 to 60 C Optical path length 2mm AKTApurifier User Guide 28 4079 53 Edition AC Reference information 8 Cell volume 2 pl 30 ul detector volume UV flow cell 5mm
52. 8 Reference information BL System descriptions ananda aan at 115 8 2 Componentide SG BLOM sise tnt 129 8 3 Optional CONIQUATIONS ns ainun ane h 133 8 4 Recommended tubing and COLUMNS nree oooooocoooooooooooo 135 8 5 BufferPrep details 8 6 Technical SPECIFICATIONS oooooocooooooooooco coco 157 8 7 Chemical resistance guide and chemical compatibility 163 BP Ordering Inf OF Matin sienne enam tu 166 e Ee 173 About this manual This manual describes the operation of AKTApurifier and AKTApurifier UPC 10 and 100 The description is common for all the systems unless otherwise clearly stated in the contents The differences are few and indicated with divergent typeface Installation description system description system maintenance and trouble shooting are also found in this manual AKTApurifier User Guide 28 4079 53 Edition AC Introduction 1 1 Introduction 1 1 General AKTApurifier is an automated liquid chromatography system designed for method development and research applications The system simplifies the transition from laboratory to full scale production Scale up to production is predictable and trouble free Two versions are available AKTApurifier with monitor UV 900 for multiple wavelengths detection and AKTApurifier UPC with monitor UPC 900 for combined measurement of UV absorption pH and conductivity Note When instructions or specifications are similar for both the models only
53. 9 53 Edition AC System overview 3 3 3 UNICORN software UNICORN is a software package for control and supervision of the AKTAdesign chromatography system It runs on an IBM compatible PC under Microsoft Windows operating system and includes hardware for interfacing the controlling PC to the chromatography liquid handling parts of AKTApurifier 3 3 1 Starting UNICORN To start UNICORN 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 Ab appears AZ 2 Start UNICORN by double clicking the UNICORN icon UNICORN i An information window appears during start up 3 Inthe Logon dialog select a user from the Users list and enter the password If you log on for the very first time select user default and enter the password default Click OK Note You should enter users and individual passwords before starting using KTApurifier on a regular basis See UNICORN User manuals Aw KE UNICORN logon User name default Password es Cancel Hep AKTApurifier User Guide 28 4079 53 Edition AC 47 3 System overview 3 3 UNICORN software 4 Eventually the UNICORN Manager module appears on the screen COR ages BER File View Administration Tools Window Help An EC se Methods x ID Results c Default Example files Example Result N Size 3 Prev Folder ES Prev Folder S BAS UPC Affi Segm BAS_UPCIOF 239K
54. 9 M etha CA Swste Say E shstio Additional buttons are provided in the toolbar These are e Instant run Opens a dialog window where you directly can create a method to run This is handy for starting routine runs instantly e Logon Logoff Opens a dialog window to control the logon logoff process e Method Queue Opens a dialog window for defining a new J Method Queue A e Running Method Queues Opens a dialog window for showing a Ma Method Queue that is running D Method Queues are used to link several methods together AKTApurifier User Guide 28 4079 53 Edition AC 49 3 System overview 3 3 UNICORN software 3 3 3 Help Comprehensive on line help is available To get help about an instruction or module e Place the cursor on the instruction module and press the F1 key or e 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 with using the current active dialog 50 AKTApurifier User Guide 28 4079 53 Edition AC Operation 4 4 Operation In this chapter you will learn how to operate AKTApurifier from UNICORN Note Two versions are available AKTApurifier with monitor UV 900 for multiple wavelengths detection and AKTApurifier UPC with monitor UPC 900 for combined measurement of UV absorption
55. Apurifier UPC the connections on AKTApurifier are similar 1 Connect the power converter cable to the Input port on CU 950 The CU 950 should be attached to the left side of the system 2 Connect a mains cable supplied between the power converter and the mains supply socket on the system 3 Ifa fraction collector is used connect a mains cable supplied between the fraction collector and the mains supply socket on the system AKTApurifier User Guide 28 4079 53 Edition AC 21 2 Installation 2 3 Installation of AKTApurifier 4 Connect a mains cable supplied between AKTApurifier and a properly grounded mains socket according to Figure 2 3 Do not switch on 5 All other mains cables are connected at delivery Complete the two first Sections of the Installation record The unpacking and installation phase of AKTApurifier is now completed 2 3 11 Settings and calibrations 1 If fraction collection should be used remember to change the delay volume This setting is found in System Control in System Settings Specials To calculate the new delay volume see Section 8 1 6 Setting the delay volume 2 Calibrate the pH electrode and set up the additional monitor functions desired for pH measurement These procedures are described in the Monitor UPC 900 Monitor pH C 900 User Manual and in UNICORN User Manuals Check 1 Unpacking Sign Remarks Contents according to packing lists All packing mater ial removed
56. B MethodFile 2004 09 29 2004 09 29 E Result001 Result File 2002 04 24 2004 08 31 gt AJ Example Result002 Result File 2002 04 24 2004 08 31 Li Example Result003 Result File 2002 04 24 2004 08 31 Lil Example Result004 Result File 2002 04 24 2004 08 31 I User default 5 UNICORN Manager module is the central part of the UNICORN displays It is mainly used for file handling and administration From this module you navigate through the control system In the Methods pane to the left 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 48 AkTApurifier User Guide 28 4079 53 Edition AC System overview 3 3 3 2 UNICORN modules and buttons In general UNICORN consists of 4 different modules of which UNICORN Manager is one The other modules are represented by icons in the toolbar These modules are e Method Editor Opens a dialog window for creating new methods e System control Opens 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 click their respective button in the task bar at the bottom of the screen Se ONICO J 4
57. Block 5 01 ml Block AutoZero_UV 5 01 ml Base SameAsMain 5 01 ml AutozeroUV 5 01 ml End Block 5 01 ml Block Aut PressureFlow Regulation 5 01 ml Base SameAsMain 5 01 ml SystemPumpControlMode Normal 0 50 MPa 0 100 ml min 5 01 ml End Block 5 01 ml Block Flowthrough_Fractionation Find Help Print Cancel 4 8 2 Printing and creating a report 1 To print the chromatogram select File Report The Generate Report dialog opens Format Contents Global BP Chromatogr Report Customized Report Format New Global BP Full Report New Global Chromatofocusing Global Chromatogram Si Global Chromatogram_Peaks Global Chromatogram_Quant Global Chromatogram Report Global Full Report Global Installation Test 2 Select for example format Global Chromatogram This will create a report containing the chromatogram and the guestions on one page AKTApurifier User Guide 28 4079 53 Edition AC 83 4 Operation 4 8 Viewing and printing the result 3 Click Preview to view the report on the screen Customize sa Chromatogram Modified Layout Help Ins ET ot Prev Page OnePage Zoomin aa Add Page a4 a SARs delia n UNICORN 5 01 Bi 318 Ze AHB 2002 04 11 15 2634 W Eom m Daylight Tina Bt 2 Diwtunph fk Eunph Tardfiomph Fern Usted Su S i Eramp Farah UNICORN Report Chromatogram Cheomatagesm Questions Ya intonation to petat TT Enh el DVI Ham
58. C HP 1 ml x 17 0920 05 HiTrap IMAC HP 5 ml x 17 0921 02 HiTrap IMAC FF 1 ml X 17 0921 04 HiTrap IMAC FF 5 ml X 17 0921 06 HiPrep IMAC FF 16 10 X 17 0408 01 HiTrap Chelating HP 1 ml X 17 0409 01 HiTrap Chelating HP 5 ml x 17 0716 01 HiTrap NHS activated HP 1 ml x 17 0717 01 HiTrap NHS activated HP 5 ml X 17 5281 01 GSTrap HP 1 ml X 17 5282 01 GSTrap HP 5 ml X 17 5130 01 GSTrap FF 1 ml X 17 5131 01 GSTrap FF 5 ml X 17 5234 01 GSTPrep FF 16 10 X 28 4082 53 HiTrap MabSelect 1 ml X 28 4082 55 HiTrap MabSelect 5 ml X 11 0034 93 HiTrap MabSelect SuRe 1 ml X 11 0034 94 HiTrap MabSelect SuRe 5 ml X 28 4082 58 HiTrap MabSelect Xtra 1 ml X 28 4082 60 HiTrap MabSelect Xtra 5 ml X 17 5079 01 HiTrap rProtein A FF 1 ml X 17 5080 01 HiTrap rProtein A FF 5 ml X 17 0402 01 HiTrap Protein A HP 1 ml X 17 0403 01 HiTrap Protein A HP 5 ml X 17 0404 01 HiTrap Protein G HP 1 ml X 17 0405 01 HiTrap Protein G HP 5 ml X KTApurifier User Guide 28 4079 53 Edition AC Reference information 8 Code no Column name 0 50 0 75 1 0 17 0406 01 HiTrap Heparin HP 1 ml x 17 0407 01 HiTrap Heparin HP 5 ml RF 17 5189 01 HiPrep 16 10 Heparin FF x 17 0412 01 HiTrap Blue HP 1 ml x 17 0413 01 HiTrap Blue HP 5 ml RF 17 5110 01 HiTrap IgM Purification HP x 1ml 17 5111 01 HiTrap IgY Purification HP X 5ml 17 5
59. GE Healthcare AKTApurifier User Guide O KTA Important user information CE Certification All users must read this entire manual to fully understand the safe use of AKTApurifier IMPORTANT AKTApurifier is intended for laboratory use only not for clinical or in vitro use or for diagnostic purposes WARNING 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 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 Recycling This symbol indicates that the waste of electrical and electronic equipment must not be disposed as unsorted municipal waste and must be collected separately Please contact an authorized representative of the manufacturer for information concerning the decommissioning of equipment WARNING This is a class A product In a domestic environment it may cause radio interference in which case the user may be required to take suitable measures WARNING All repairs should be done by personnel authorized b
60. Immediately repeat steps 3 and 4 with distilled water to rinse the system of NaOH AKTApurifier User Guide 28 4079 53 Edition AC 105 6 Maintenance 6 4 Calibration procedures 106 6 3 4 Cleaning in place After repeated separation cycles contaminating material might progressively build up in the system and on the column This material may not have been removed by the cleaning step described previously The nature and degree of contamination depends on the sample and the chromatographic conditions employed These should be considered when designing a cleaning process 6 4 Calibration procedures The table below lists the type and frequency of calibrations that can be done on AKTApurifier Refer to the UNICORN User Manuals and to the individual component User Manuals and Instructions for descriptions of how to perform these calibrations The calibration is performed in UNICORN by selecting System Calibrate in System Control Component How often Conductivity flow cell Cell constant Only necessary if specific conductivity with high accuracy is measured Cond_Calib Temperature Must be done when changing the conductivity cell Temp Entering a new cell Must be done when changing the conductivity cell constant Cond Cell Pressure reading Once a year or when required pH electrode optional Every day 6 5 Moving the system Two persons are required to lift the system CAUTION Never lift the system b
61. NX FF high sub RF 17 5192 01 HiPrep 16 10 SP FF RF 17 1064 01 HiLoad 16 10 Q Sepharose HP X 17 1066 01 HiLoad 26 10 Q Sepharose HP X 17 1137 01 HiLoad 16 10 SP Sepharose HP X 17 1138 01 HiLoad 26 10 SP Sepharose HP x 136 AkTApurifier User Guide 28 4079 53 Edition AC Reference information 8 Reversed Phase Columns Code no Column name 0 25 0 50 17 5068 01 SOURCE 15RPC ST 4 6 100 X 17 5116 01 SOURCE 5RPC ST 4 6 150 X 17 1181 01 RESOURCE RPC 1 ml RF X 17 1182 01 RESOURCE RPC 3 ml RF X 17 0704 01 uRPC C2 C18 SC 2 1 10 xX P 17 5057 01 URPC C2 C18 ST 4 6 100 xX P Size Exclusion Columns Code no Column name 0 25 0 50 17 1458 01 Superdex Peptide PC 3 2 30 x P 17 5176 01 Superdex Peptide 10 300 GL X 17 0771 01 Superdex 75 PC 3 2 30 xX P 28 9205 04 Superdex 75 5 50 GL x P 17 5174 01 Superdex 75 10 300 GL X 17 1089 01 Superdex 200 PC 3 2 30 x P 17 5175 01 Superdex 200 10 300 GL X 28 9065 61 Superdex 200 5 150 GL xX P 17 5172 01 Superose 6 10 300 GL X 17 0673 01 Superose 6 PC 3 2 30 xX P 17 5173 01 Superose 12 10 300 GL X 17 0674 01 Superose 12 PC 3 2 30 xX P 17 1408 01 HiTrap Desalting 5 ml RF 17 5087 01 HiPrep 26 10 Desalting RF 17 1139 01 HiLoad 16 60 Superdex 30 prep grade x 17 1140 01 HiLoad 26 60 Superdex 30 prep grade x 17 1068 01 HiLoad 16 60 Superdex 75 prep grade x 17 1070 01 HiLoad 26
62. Notes Gradient BufferPrep Columns Reference Curves Gradient Block name 0 00 Main The length of each block is marked at the bottom of the graph Click the x axis to view the method in time volume or column volumes 56 AKTApurifier User Guide 28 4079 53 Edition AC Operation 4 9 Click the Start Protocol tab to decide which of the Run Setup pages is to be displayed at the start of a method run Frac 350 Variables Scouting Notes Gradient BufferPrep Columns Reference Curves Evaluation Procedures Method Information Start Protocol Questions Result Name Checked items are displayed before method is started i eee VVariables Scouting Text Method VINotes Gradient BufferPrep C Columns _IReference Curves Evaluation Procedures Method Information Settings Calibration Questions MResult Name AS r Scouting start protocol First run only Alliruns 10 To save the method select File Save In the Save As dialog enter a name Store the method in the directory of your choice by double clicking on a directory Click OK In UNICORN Manager module the method name appears in the Methods pane 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 Section 4 6 Starting a run For more information on e
63. The computer communicates with the CU 950 controller unit through a USB cable The UniNet 1 data communication cable is routed from the CU 950 to the rear of Pump P 900 UniNet 1 is terminated at Monitor UV 900 Monitor UPC 900 with a termination plug or if used at the fraction collector UniNet 2 KTApurifier KTApurifier UPC From UniNet 2 connector on rear of Pump P 900 The UniNet 2 data communication cable which supplies the valve and mixer with control signals and power is routed from the rear of Pump P 900 and links components on the right side of the system The cable is terminated at the injection valve V1 with a termination plug KTApurifier User Guide 28 4079 53 Edition AC 119 8 Reference information 8 1 System description Mains cables Fraction collector optional Fuseholder with Fuse ae To mains socket CU 950 7 2 grounded Fig 8 5 Mains cables in AKTApurifier system Mains cables in AKTApurifier UPC is similar WARNING Never attempt to remove the mains fuse while mains voltage is applied to the system For continued protection against risk of fire replace only with fuse of the specified type and rating Please ref
64. Trap and HiLoad change to the Flow restrictor FR 902 If the pressure exceeds the pressure limit value given for these columns an extra system pressure measurement must be performed This is described later in this Section X recommended tubing kit may improve resolution x can be used if flow lt 5 ml min o should be used if flow gt 30 ml min lon Exchange Columns Code no Column name 0 50 0 75 1 0 17 5166 01 ono Q 5 50 GL x 17 5167 01 ono Q 10 100 GL x 17 5168 01 ono S 5 50 GL x 17 5169 01 ono S 10 100 GL x 17 1177 01 RESOURCE Q 1 ml x 17 1179 01 RESOURCE Q 6 ml x o 17 1178 01 RESOURCES 1 ml X 17 1180 01 RESOURCES 6 ml x o 11 0013 02 HiTrap Capto Q 1 ml x 11 0013 03 HiTrap Capto Q 5 ml X 11 0032 73 HiTrap Capto MMC 1 ml x 11 0032 75 HiTrap Capto MMC 5 ml X 17 1153 01 HiTrap Q HP 1m X 17 1154 01 HiTrap Q HP 5 m X 28 9165 37 HiTrap Capto DEAE 1 ml X 28 9165 40 HiTrap Capto DEAE 5 ml X 17 5441 22 HiTrap Capto S 1 ml x 17 5441 23 HiTrap Capto S 5 ml X 17 1151 01 HiTrap SP HP 1 ml X 17 1152 01 HiTrap SP HP 5 ml X 28 4058 44 HiTrap Capto adhere 1 ml x 28 4058 46 HiTrap Capto adhere 5 ml x 17 6002 33 HiTrap IEX Selection Kit 7 x 1 ml x 17 5053 01 HiTrap Q FF 1 ml x AkTApurifier User Guide 28 4079 53 Edition AC 141 8 Reference informa
65. Valve V1 Frac 950 5 Connect the included waste tubing W3 to the waste outlet on Frac 950 and route it to a suitable waste container 6 Set the delay volume in UNCORN see Section 8 1 6 Setting the delay volume 2 3 8 Installing Controller unit CU 950 POWER pc SYSTEM 1 Unpack CU 950 2 Hang the CU 950 on the left side of the system by inserting the hooks on the front of CU 950 into the channel on the side of the UV 900 and rotating into position AKTApurifier User Guide 28 4079 53 Edition AC 25 2 Installation 2 3 Installation of AKTApurifier 26 2 3 9 Connecting the UniNet cables CAUTION The mains power to AKTApurifier must be turned OFF before installing the UniNet 1 cables Monitor UPC 900 Pump P 900 Fraction collector UniNet 1 Termination plug Only in Frac 950 Frac 920 has only one UniNet 1 UniNet 1 socket To computer USB cable Fig 2 2 Illustration of AKTApurifier UPC connections AKTApurifier connections are similar 1 Connect a UniNet 1 cable between the UniNet 1 port on CU 950 and Pump P 900 in the system 2 Connect a UniNet 1 cable between Pump P 900 and Monitor UPC 900 Monitor UV 900 3 When using a fraction collector e connect a UniNet 1 cable between Monitor UPC 900 Moni
66. air in the flow cell Check that the flow restrictor generates the following back pressure FR 904 0 4 0 05 MPa FR 902 0 2 0 05 MPa Check the back pressure as follows 1 Disconnect the flow restrictor 2 Connect a piece of tubing approx 1 m i d 1 0 mm to a free port in injection valve V2 Set the valve manually to this port Put the open end in the waste container 3 Run the pump manually at 10 ml min with water Note the back pressure Bp1 on the pump display or in the Run Data pane A Connect the flow restrictor to the open end of the capillary note the IN marking Put the flow restrictor in the waste container 5 Run the pump at 10 ml min with water Note the back pressure Bp2 on the pump display or in the Run Data pane 6 Calculate the back pressure Bp2 Bp1 of the flow restrictor If it is not within limit replace or contact GE Healthcare Degas the buffer before use Clean the UV flow cell see Chapter 4 of Monitor UPC 900 User Manual or UV 900 User Manual 108 AKTApurifier User Guide 28 4079 53 Edition AC 7 3 Error symptom Erratic flow noisy baseline signal irregular pressure trace Possible causes are Possible cause Action Troubleshooting 7 Pressure curve Air bubbles passing through or trapped in the pump Check that there is sufficient eluent present in the reservoirs Check all connections for leaks Follow the instructions in Chapter 5 of Pump P 900 User Manual
67. an zoom further on the enlarged part To return to the complete chromatogram right click and select Undo or Reset zoom 7 To change to a scale for another curve click on the Y axis scale The style and color of a curve its Y scale and its X scale can all be changed KTApurifier User Guide 28 4079 53 Edition AC 81 4 Operation 4 8 Viewing and printing the result 82 10 11 12 13 Open the Chromatogram Layout window again Click on 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 To fix the Y axis scale mark a curve click Fixed and enter the max and min values for that curve You can repeat this for other curves To fix the X axis scale click Fixed in the X axis field and enter the min and max values for the X axis Click OK Click OK at the bottom of the Chromatogram Layout window to execute all the changes When you have made the necessary changes in the Chromatogram layout window they can be saved as a new layout e Click the Layout Library tab at the top of the Chromatogram layout window e Click Save current layout as and type a name Click OK Layouts can be selected in Save current layout and all your saved selections will apply Saved layouts can be applied to any result file Minimize the chromatogram window by clicking on the smaller squares in the upp
68. at an angle of 30 from the vertical with the outlet placed higher than the inlet to prevent air bubbles being trapped in the cell The flow direction is marked on the flow cell 3 Unpack the pH electrode Ensure that it is not broken or dry 4 Prior to first using the electrode remove the electrode end cover and immerse the glass bulb in buffer for 30 minutes AKTApurifier User Guide 28 4079 53 Edition AC Installation 2 5 Remove the dummy electrode from the To instrument flow cell and store it in the flow cell holder rear pH Probe Ce 6 Carefully insert the electrode in the flow cell Tighten the nut by hand to secure the electrode Electrode Note If the electrode is not fully inserted the system will leak and a dead volume will occur in the holder 7 Cut a new capillary of 90 mm i d 0 50 mm and connect the outlet the screw head end from the conductivity flow cell to the inlet on the pH flow cell lower connection marked IN WT Flow cell 8 Connect the outlet from the pH flow cell marked OUT to the inlet on the FR 902 marked IN using the male male union 9 Connect the pH electrode BNC connector to the pH Probe connector on the rear of Monitor p HC 900 UPC 900 Should interference from static fields occur connect the pH flow cell to the pH Ground connection as well using a standard laboratory 4 mm banana plug cable Note When using the pH electrode the flow restrictor FR 9
69. ays be easy to access and the mains plug must always be easy to disconnect e To maintain correct ventilation the system requires an appropriate amount of free space Do not block the ventilation inlet on the front panels and the outlets on the rear of the instrument e AKTApurifier requires 100 120 220 240 V 50 60 Hz electrical supply with protective grounding e Pliers are recommended for cutting plastic straps e Awaste flask e The installation test requires the following solutions 1000 ml of distilled water for priming and purging the pump 200 ml of distilled water 200 ml of 0 4 acetone in distilled water 100 ml of 20 ethanol in distilled water AKTApurifier User Guide 28 4079 53 Edition AC 15 2 Installation 2 3 Installation of AKTApurifier 2 3 Installation of AKTApurifier Create a clean and dry working area of 200 x 80 cm that allows easy access Follow the step by step instructions below and check the Installation Record as you go along see page 29 Note Some items are packed in separate boxes delivered with the system Note Some packing lists are included in the boxes 2 3 1 Unpacking the system 1 After removing the cardboard hood and other packing material GENEE check the contents against the zy enclosed packing list Also check all enclosed boxes Store all the boxes and plastic bags in a convenient nearby place 2 Check the system for obvious damage which migh
70. bing i d 1 6 mm o d 1 8 3m 18 1121 16 Teflon tubing i d 2 9 mm o d 3 16 3m 18 1112 47 Tubing kit AKTApurifier 10 i d 0 25 mm 1 18 1123 34 Tubing kit AKTApurifier 10 i d 0 5 mm 1 18 1123 33 Connectors and unions Fingertight connector 1 16 10 18 1112 55 for 1 16 o d tubing PEEK Tubing connector 10 18 1112 49 Loo for 3 16 o d tubing PEEK Ferrules for 3 16 o d tubing PEEK 10 18 1112 48 TF E ER Tubing connector 10 18 1121 17 for 1 8 o d tubing PEEK Ferrules for 1 8 o d tubing PEEK 10 18 1121 18 Tubing connector 10 18 1127 07 for 1 16 o d tubing PEEK Ferrules for 1 16 o d tubing PEEK 10 18 1127 06 Union 1 16 female M6 male PEEK 6 18 1112 57 Union luer female 1 16 male PEEK 2 18 1112 51 Union M6 female 1 16 male PEEK 8 18 1112 58 Union 5 16 32 female HPLC male PEEK 8 18 1142 08 Union 1 16 male 1 16 male 10 18 1120 92 for 1 16 o d tubing PEEK AKTApurifier User Guide 28 4079 53 Edition AC 169 8 Reference information 8 8 Ordering information Item Quant Code no pack Connectors and unions Union 1 16 female 1 16 female 1 18 3855 01 for 1 16 o d tubing titanium Stop plug 1 16 PEEK 5 18 1112 52 Stop plug 5 16 PEEK 5 18 1112 50 Tubi
71. by turning the dial clockwise It is also possible to view all three wavelengths simultaneously by turning the dial one step further only three digits AKTApurifier UPC The main operating menu 1 of Monitor UPC 900 shows the absorbance value with 6 digits for the selected wavelength the conductivity as a percentage of full scale and the pH value optional By turning the dial one click an alternative display of the conductivity is shown main menu 2 This display shows pH temperature and the actual conductivity value in mS cm or US cm together with the percentage value 77 4 Operation 4 7 During a run 78 4 7 2 Changing parameters From UNICORN AKTApurifier can be controlled with manual instructions issued from the Manual menu in System Control in UNICORN These instructions can be used during a run to alter system conditions in response to the results observed The Manual menu in System Control opens a dialog box similar to the text instruction box in the Method editor For example to change the flow rate 1 Select Manual Pump The Instructions dialog is opened Instructions Parameters neat Pump Pump C Flowpath SystemPumpControlMode OFF Con Delete PN BER D aama SC Frac e OFF Con Tea Other T Auto update If this is checked the parameter fields will be updated during method run 2 Select Pump and then Flow 3 Typeanew flow rate under Para
72. cedures Block Variable Value Range Main Column ml num 0 100 993999 000 Flow Rate Flow Fate ml min 1 000 0 000 10 000 Column_Pressure_Limit Column_PressureLimit MPa 4 00 nes Groden Mage Cone 8 ker eer Length_of_Gradient CV 20 000 T Show details T Show unused variables IV Display tooltip for extended variable cells Edit Variable Run setup consists of a number of pages You select a page by clicking the respective tab at the top of the window Some of the more important pages are described below 7 Onthe Variables page the method is presented by a number of blocks The blocks represent the typical steps in a chromatographic run e Start instructions e Column equilibration AKTApurifier User Guide 28 4079 53 Edition AC 55 4 Operation 4 2 Creating a method e Sample injection see also section 4 4 Sample application e Wash out unbound sample e Fractionation see also section 4 3 Fraction collection e Gradient e Clean after elution e Re equilibration Some of the blocks contain a number of variables with suitable default values The values can be changed to suit your application Some of the variables are normally hidden but can be shown by checking the Show details box 8 Click the Gradient tab to view the method graphically Evaluation Procedures Method Information Start Protocol Questions Result Name Frac 950 Variables Scouting
73. change without disturbing the column Sample volumes up to 150 ml can be applied via loops connected to the injection valve e Using a range of fixed volume loops for applying samples from 10 ul to 2 ml with accuracy and precision e Using Superloop 10 ml Superloop 50 ml or Superloop 150 ml for applying samples in the range 1 10 ml 1 50 ml and 1 150ml respectively All three are loaded by a syringe AKTApurifier User Guide 28 4079 53 Edition AC 131 8 Reference information 8 2 Component description 132 8 2 7 Flow restrictor FR 902 904 The flow restrictor generates a steady back pressure to prevent air bubbles being formed in the flow cells FR 902 is set at the factory to 0 2 MPa and is used when a pH cell optional is fitted in the system or when using low pressure columns FR 902 is mounted in the system at delivery FR 904 is set at the factory to 0 4 MPa This flow restrictor should be used when working with organic solvents such as in RPC applications to avoid air bubble formation in the flow cell 8 2 8 On line filter The on line filter is fitted between the output of Mixer M 925 and position 7 of the injection valve Arrows on the on line filter indicates the flow direction It generates a back pressure of 0 5 MPa maximum The filter is a depth type filter and has a pore size of 2 um The filter used in AKTApurifier 10 systems is made of titanium In AKTApurifier 100 systems it is made of polypropene
74. ct loop or Superloop 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 73 Apply the sample manually with a syringe More information on filling the loops is found in Section 4 4 Sample application When using the fraction collector 1 Check that the tubing from the flow restrictor is connected to the fraction collector 2 Put the waste tubing from the fraction collector into a waste bottle 3 Insert a sufficient number of tubes into the fraction collector Note The delay volume UV cell to fraction collector outlet is not set at system delivery because it depends on the length of your outlet tubing To set the delay volume in UNICORN see Section 8 1 6 Setting the delay volume 70 AKTApurifier User Guide 28 4079 53 Edition AC Operation 4 4 6 Starting a run 4 6 1 Final checks Before starting any method we recommend a number of checks to make sure that no problems occur once the run has been started 1 Check that the inlet tubings are immersed in the correct bottles for the method selected 2 Check that there is sufficient eluent available 3 Check that the waste bottle is not full and will accept the volume diverted to it during the run 4 Check that the pump has been purged i e no air in the inlet tubing If not purge the pump with a syringe as described in 2 4 1 Preparing AKTApurifier or in Chapt
75. de 1 28 4079 53 AKTApurifier Getting started 1 28 4079 54 Optional Configurations AKTApurifier User manual 1 28 4079 56 Pump P 900 User Manual d 18 1120 04 Monitor UPC 900 User Manual 1 18 1125 55 Monitor UV 900 User Manual 1 18 1120 05 AkTApurifier User Guide 28 4079 53 Edition AC 171 8 Reference information 8 8 Ordering information 172 AKTApurifier User Guide 28 4079 53 Edition AC Index Index A OCCESSONES pesanan ana add a column Scouting page B buffer recipes buffer substances BufferPrep creating your own recipes details examples and tips fine tuning in cold room mixing gradients ordering information preparing the system for recipe overview strategy for using what is it BufferPrep kit C calculate th delay Volumes nn E ie 127 calibrations CE symbol change a variable value changing parameters during a run changing tubing Changing tubing kit chemical resistance guide cleaning the system flow path cleaning the system surface ell geingt Oe ler ten ne rte EA NAN NIAT collecting fractions fixed volume fractionation flowthrough fractionation peak fractionation column holders columns and tubing complete filling of sample loops compliance with safety standards components description location computer requirements Conductivity cell conductivity cell connecting the colu
76. distance between the column and the optical unit 2 3 3 Installing pH C 900 optional Monitor pH C 900 is a high precision on line monitor for measurement of pH and conductivity in liquid chromatography Monitor pH C 900 is an option for AKTApurifier for detailed descriptions refer to Monitor pH C 900 User Manual Fig 2 1 Monitor pH C 900 AKTApurifier User Guide 28 4079 53 Edition AC Installation 2 Unpacking pH C 900 1 Unpack and assemble pH C 900 according to the instructions given in the Monitor pH C 900 User Manual 2 Place pH C 900 in AKTApurifier Installing the conductivity cell 1 Place the conductivity cell in its cell holder or in a suitable location as close to the optical unit column as possible The cell can be placed up to 1 5m from the monitor housing 2 Connect the conductivity cell to the socket Conductivity Flow Cell on the rear panel of the monitor si 3 Connect the tubing with the Fingertight connectors The flow cell itself does not have a recommended flow direction In conjunction with the pH electrode place the conductivity flow cell and select its flow direction so that the screw head end of the flow cell faces the pH flow cell in AKTApurifier systems the pH electrode is optional AKTApurifier User Guide 28 4079 53 Edition AC 19 2 Installation 2 3 Installation of AKTApurifier Flow direction 20 2 3 4 Installing a pH electrode optional The
77. e A To achieve aset pH the buffering stock solution and the acid base stock solution are mixed via the switch valve in a ratio that depends on the buffering substance characteristics at the set pH An example of the mixing ratios between the two stock solutions is shown below Mixing ratios in Pump module A at different pH Acid Buffer 0 50 25 25 50 0 4 7 10 Note that the shape of the mixing ratio curve will differ for different buffering substances AKTApurifier User guide 28 4079 53 Edition AC 89 5 Optional functions 5 2 BufferPrep 90 Pump module B To achieve the set eluent the eluent stock solution and water are mixed via the switch valve in a ratio that depends on the programmed eluent concentration variation during elution Mixing ratios in Pump module B during an elution gradient H 0 Eluent 0 50 25 25 50 0 0 50 100 B 5 2 2 Installing the BufferPrep kit The BufferPrep kit contains e two valves with valve holders e tubing and connectors for AKTApurifier 10 i d 1 6 mm yellow ferrules e tubing and connectors for AKTApurifier 100 i d 2 9 mm blue ferrules Installing the kit 1 Install the two switch valves between the respective pump heads by inserting the hooks into the channel in the pump Valve A Valve B A2 A3 Al B2 B3 B1 2 Connect the valve A cable to socket Valve A at the rear of the pump and the valve B cable to Valve B 3 Select the tubing and connectors
78. e FV 903 113 Conductivity cell 24 DH flow cell 100 Accumulator Frac 950 40 Union 1 16 male 1 16 male 0 25 mm i d 1 8 Union 1 16 male 1 16 male 0 5 mm i d 7 Flow restrictor FR 902 5 Capillary i d 0 25 x 100 mm 49 Capillary i d 0 50 x 100 mm 19 6 Capillary i d 0 75 x 100 mm 44 2 Capillary i d 1 0 x 100 mm 78 5 AkTApurifier User Guide 28 4079 53 Edition AC 127 8 Reference information 8 1 System description Example This example shows how to calculate the delay volume in AKTApurifier UPC 10 including fraction collector Frac 920 based on the tubing lengths on the previous page and the values in the table above The tubing length from the flow restrictor to the outlet on the delivery arm is assumed to be 1000 mm The tubing volume is calculated according to the following example A 300 mm long i d 0 25 mm tubing has the volume 3 x 4 9 14 7 ul Calculation of the delay volume Component Tubing Length mm Volume ul Tubing UV cell conductivity cell 170 1 7 x 4 9 8 3 i d 0 25 mm Conductivity cell 24 0 Union 1 16 male 1 16 male 18 i d 0 25 mm Flow restrictor FR 902 5 0 Tubing FR 902 flow diversion valve 700 7 x 4 9 41 0 on Frac 920 i d 0 25 mm Flow diversion valve FV 903 113 Tubing FV 903 delivery arm on 300 3 x 4 9 14 7 Frac 920 i d 0 25 mm Delay volume 207 8 Setting the delay volume in UNICORN To change the delay vol
79. ections Check that the column is equilibrated If necessary clean the column Check the operation of the mixer and the system pump The mixer function is checked by placing a stirrer bar on top of the mixer housing The stirrer bar should rotate when the system is in Run mode The pump and mixer functions can also be checked by running the installation test Clean the flow cell according to Monitor UPC 900 User Manual Waves on the gradient Check that the pump and the valves are operating properly and are programmed correctly Change to a larger mixing volume if necessary Check the operation of the mixer The mixer function is checked by placing a stirrer bar on top of the mixer housing The stirrer bar should rotate when the system is in Run mode The mixer function can also be checked by running the installation test Non linear gradients or slow response to B changes Check that the tubing has been washed properly and that the pump is operating If a larger mixer than the standard 0 6 ml chamber is used try changing to a smaller mixer volume Incorrect or unstable reading Check that the conductivity flow cell cable is connected properly to the rear of the monitor Check that the pump operates correctly If temperature compensation is used check that the temperature sensor is calibrated and that the correct temperature compensation factor is used Check that the column is equilibrated I
80. ed If so replace the connector e Tighten the connector properly with your fingers Irregular pump pressure To check the pump function record the pressure or check it in UNICORN By observing the piston status indicator in the Check menu together with the pressure trace the pump cylinder that is functioning abnormally can be identified see Reference information in the Pump P 900 User Manual There can be several causes of an abnormal pressure recording for example e air trapped in the pump cylinders e partially blocked solvent filters e leaking connections e piston seal leakage e pump valve malfunction e piston damaged Some examples of normal and abnormal pressure traces together with comments are shown in Chapter 5 of Pump P 900 User Manual AKTApurifier User Guide 28 4079 53 Edition AC 111 7 Troubleshooting 7 6 Monitor UV 900 7 6 Error symptom No text on the front display Possible cause Action Monitor UV 900 Check that the mains cable is connected and the power switch is in ON position Unstable UV baseline Try using a larger mixer chamber instead of the standard 0 6 ml mixer chamber 7 7 Monitor UPC 900 Error symptom No text on the front display Possible cause Action Check that the mains cable is connected and the power switch is in ON position Unstable UV baseline Try using a larger mixer chamber instead of the standard 0 6
81. ed in the method Note Ifyou cannot find your column in the list you can add one Refer to the UNICORN User Manual and to section 8 4 3 Selecting tubing kit for other columns Note If you manually alter the default values of the column and thereby exceed the recommended values for the selected column you will get a warning when you save your method Note Ifyou want to perform a test run without a column you should still select a column a small one is recommended to get suitable default parameters in the method Then when running the method use a piece of tubing to replace the column 54 AkTApurifier User Guide 28 4079 53 Edition AC Operation 4 4 Ifrequired select Flexible Flow Rates and or Flow Regulation of the System Pump 5 Click Next to go through the subsequent dialogs In each dialog select the appropriate parameter values Note The options available in the Method Wizard depend on the current system configuration For example if a fraction collector is installed options for setting up the fractionation will appear in the wizard Note Click Help in the dialog for more information on the options in the Method Wizard 6 Click Finish in the last dialog The Run Setup window appears If not select View Run Setup Click here to select page Method Information Start Protocol Questions Result Name Variables Scouting Notes Gradient BufferPrep Columns Reference Curves Evaluation Pro
82. elumnSand tu bing tnt art asa 46 3 3 JUNICORN SORWATE se ane nn re Rand 47 4 Operation AY Starting the SW SEEM anatara anna ANN NG 52 4 2 Creating a Methods na na NE 53 1135 KEPACtON CONECUON eege EE 58 AAs Sampe appa O nn nn et hs 61 Ap Preparing the system for A run urmena 68 4 6 Starting A run TL 4 7 During a run 48 Viewing and printing the result 80 49 Completion of a run and storage oceeeccecsscsssssssssssssnssseseesssseccsssesssnssseesses 86 4 10 Cold room operation in 86 5 Optional functions Sch UMIRINGSGRACIENTS Ha ARE eiaa na 87 52 CBR ea mann ANN anna 88 5 3 SEMU NG eta ANE NN Ba NE 95 5 4 Extra system pressure measurement o oo oooooooooooooocooocoo oo 97 5155 MEOW FEQUIGHON sien en An RAA MATE 98 6 Maintenance 6 1 Penodi maintenance Hen la NAN anakan 101 6 2 Cleaning the system surface w oooocooooooooooooooooooooooo o 104 6 3 Cleaning the system flow path weervxeeeooooocoocooooocoocoooooo 105 6 4 Calibration procedures xeeeeeeroooocoooooooooooccoo coco eunamaan 106 AKTApurifier User Guide 28 4079 53 Edition AC V Contents 65 Moving the sSysteM when amankan 106 7 Troubleshooting 7 1 Faults and actions 7 2 UV CUTIVE rana nenennnnennnannn 7 3 Pressure CURVE mme FAL ConduCtiVIt CUVE nian anna 109 7 5 Elle E 111 7 6 Monitor RE 112 7 7 Monitor UPE 900 henna balasan 112 7 8 Mixer MEI ae 112 7 9 Valve INVE90 7er res tte te 113 FLO BU PROP rs aa na 113
83. entified in the text by bold text AKTApurifier User Guide 28 4079 53 Edition AC Installation 2 2 Installation 2 1 General AKTApurifier is assembled and fully tested before delivery For safe transportation however some components have been secured and thus need to be detached before the system can be tested and used Cables capillaries accessories column holder etc are enclosed in paper boxes This chapter describes how to install AKTApurifier It is divided into two parts one describing the installation and one describing how to run the installation test After the installation procedure has been performed your AKTApurifier is ready for use Note Two versions are available AKTApurifier with monitor UV 900 for multiple wavelength detection and AKTApurifier UPC with monitor UPC 900 for combined measurement of UV absorption pH and conductivity When instructions or specifications are common to both models only AKTApurifier is mentioned The installation test can be performed at regular intervals for example to verify the solvent delivery and UV monitoring of your AKTApurifier chromatography system For full details of specifications methods maintenance etc refer to the User Manuals and Instructions for the components AKTApurifier User Guide 28 4079 53 Edition AC 13 2 Installation 2 1 General 14 211 Installation procedure overview Unpack AKTAU EE asser donntnee obs meta page 16 Detach secured items a
84. ephacryl Sepharose Superdex Superose Superloop UNICORN AKTA and AKTApurifier ar companies All third party trademarks are the property of t e trademarks of GE Healthcare heir respective owners 1992 2008 General Electric Company All rights reserved First published Nov 1992 All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supp ies them A copy of these terms and conditions is available on request Contact your local GE Healthcare representative for the most current information UNICORN Any use of this software is subject to GE Healthcare Standard Software End User License Agreement for Life GE Healthcare Europe GmbH Munzinger Strasse 5 D 79111 Freiburg Germany GE Healthcare UK Ltd 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 28 4079 53 AC 06 2008 Sciences Software Products
85. er 2 in Pump P 900 User Manual 5 Check that the correct column has been fitted and equilibrated if not included in the method 6 Check that the correct max pressure is set for the column selected 7 Check that the correct mixer chamber and tubing are installed for the method selected 8 Check that the fraction collector optional has sufficient tubes fitted and is connected to the flow restrictor AKTApurifier User Guide 28 4079 53 Edition AC 71 4 Operation 4 6 Starting a run 72 4 6 2 Setting up and starting the method Note AKTApurifier UPC screen shots are displayed in the example below 1 2 Click the System Control icon Select File Run Select the method to start Click OK the method will not start yet The method run is initiated in a series of pages in Run Setup in System Control Note Only the pages set in the Start Protocol for the method will appear during the initialization Some of the pages are briefly described in the steps below When a page is done click Next to proceed If using Frac 950 the Frac 950 page will appear if it has been selected in the Start Protocol On the Frac 950 page you can define the order of fractionation and set up the last tube used The system will be paused when the last tube is reached and the fractionation will stop Frac 950 x Hack ie amp 30 mmTubes Ge 2 Fr 7 di aa M lt 00000000000 000000000000 c Rowby
86. er right corner AKTApurifier User Guide 28 4079 53 Edition AC Operation 4 14 Click the View Documentation button A number of pages appear as in the Run Setup in the Method Editor All documentation about the run is stored EA here e g the method answers to guestions variables logbook etc For example click the Notes and Logbook pages to check the contents Close the Documentation window by clicking the X in the upper right corner Frac 350 Variables Scouting Text Method Notes Columns Reference Curves Evaluation Procedures Method Information Result Information Start Protocol Settings Calibration Logbook Evaluation Log Questions Result Name 1 00 mi Method Run 10 7 2004 30852 PM US Eastem Standard Time Method 1D10 Resula et 7 0 00 ml Batch ID B27E207B ESCF 41C6 A0C0 3568951F743F F Alarm Warning 0 00 ml Base CV 1 00 mi E Method 0 00 ml Block Stat wai PumpWerh Basic 0 00 ml Base SameAsMain IR Manual 0 00 ml PumpWashBasic Off Off FE 0 00 ml End Block nors 0 00 ml Block Start Conditions E System 0 00 ml Base SameAsMain 0 00 ml Alarm_Pressure Enabled O 80MPa 0 00 MPa 0 00 ml Flow 5 000 ml min 0 00 ml AveragingTimeUV 1 30 ESS 0 00 ml End Block j 0 00 ml Block Start Cone B8 C Time 0 00 ml Base SamedsMain ee 0 00 ml Gradient 0028 0 000 CV me TE com mm ration Adjust retention zero 0 00 mi Base SameAsMain I to injection number E 5 01 ml End
87. er to the Technical specifications for fuse data Only one mains inlet is reguired The modules CU 950 and fraction collector optional are connected to the mains supply socket at the rear of the system A WARNING Only use mains cables supplied or approved by GE Healthcare The mains input fuse is located to the right of the mains input To open the fuse holder after first removing the power to the system use a small bladed screwdriver to lever the holder outwards 120 AKTApurifier User Guide 28 4079 53 Edition AC Reference information 8 8 1 5 Fluid handling path The following illustration shows the positions of the components and tubing in AKTApurifier Refer to the flow diagram for their positions in the fluid handling path AkTApurifier AKTApurifier UPC f A lr AS Column i b Sa E T d A L Injection 7 JIN valve INV fe 907 i Li Al 1 inn wi O Fe 2 gg S D SCH S Q sue amr Ca I en 1 0 SAR WEN NL OP SEE Mixer M 925 L we ent f LS 5 Mixer chamber Mixer M 925 Pump A Pump B Pump A Pump B Flow restrictor Mixer chamber Cond optional Connected after the UV cell Si 477 pH optional On line Connected after filter Gs Bypass the conductivity cell G3 i H Flow res
88. ers are e start and end points of the fractions to be collected the slope or level of the UV curve e minimum peak width to be collected e peak fractionation volume Refer to UNICORN User manuals for more information on the peak slopes and levels properties Fixed volume and peak fractionation Fixed volume fractionation can also be combined with peak fractionation during the elution Fixed volumes Fraction Volume will then be collected until a peak is detected The peak is then collected in either one fraction volume or B in several using the Peak Fractionation In 1 2 3 45 678 9 101 Volume parameter When the peakendis detected the fixed volume fractionation Fraction Volume Peak Fractionation continues Volume AKTApurifier User Guide 28 4079 53 Edition AC 59 4 Operation 4 3 Fraction collection 4 3 2 Outlet valve optional An outlet valve can be used for directing the liquid flow to either waste or to fractionation To perform outlet fractionation select Outlet Valve in the Elution Fractionation dialog in the wizard e Port 1 valve default position should always be connected to a waste flask of suitable size W3 e A fraction collector can be connected to the outlet valve with tubing G9 H9 e Ports 2 8 of the outlet valve can also be used for collecting larger fractionations 60 AKTApurifier User Guide 28 4079 53 Edition AC Operation 4 4 4 Sample application With AKTApurifier the samp
89. es dependent on the amount of acid base that has to be used to reach the desired pH We recommend that the total concentration for all buffer substances selected for the recipe should be between 0 03 M and 0 2 M typically 0 1 M for ion exchange chromatography 5 Select either Acid or Base HCI or NaOH from the pull down list and set the inlet concentration typically 0 1 M 6 Select a salt from the pull down list If the required salt is not listed a new salt can be defined see Section 8 5 3 in Defining a new salt below 7 Set the maximum outlet concentration of the salt to 100 B usually 1 0 M The maximum outlet salt concentration is half the concentration of the inlet salt stock solution 8 Set the pH range required for the buffer Note To set a useful pH range the pKa must be known Click on the Buffer substance button and select the buffer component The pKa values are shown in the list Typically a pH range 0 5 units around the pKa is useful For exact ranges check the buffer tables 9 To add notes about the recipe click Notes and enter the required text in the box displayed Click OK to close the Recip Notes dialog 10 When you have created your recipe click Save As Give the recipe a name The recipe is checked and if the pH range selected is not feasible a warning is displayed giving the error condition and making suggestions for its correction Correct the recipe and click Save As again AKTApurifier Use
90. f necessary clean the column Check the operation of the mixer The mixer function is checked by placing a stirrer bar on top of the mixer housing The stirrer bar should rotate when the system is in RUN mode The mixer function can also be checked by running the installation test 110 AKTApurifier User Guide 28 4079 53 Edition AC Troubleshooting 7 7 5 Pump P 900 Error symptom No text on the front display Possible cause Action e Check that the mains cable is connected and the power switch is in ON position I Liquid leaking between the pump head and the side panel e Piston seal or rinsing membrane incorrectly fitted or worn 1 Replace or re install the seal or membrane 2 Run in carefully see Chapter 4 of Pump P 900 User Manual Low eluent flow and noise as the piston moves e Disassemble pump cylinder and examine the piston spring according to Chapter 4 of Pump P 900 User Manual Replace if necessary e If the spring is corroded check piston seal and rinse membrane Make sure that piston rinsing system is always used when working with aqueous buffers with high salt concentration e Check the piston for damage If damaged replace the piston according to Chapter 4 of Pump P 900 User Manual e Remember to replace the piston seal with new parts Leaking connection and or crystallized material around a connector e Unscrew the connector and check if it is worn or incorrectly fitt
91. fferPrep kit including two switch valves must be installed on the pump see Section 5 2 2 Installing the BufferPrep kit and Section 8 8 Ordering information BufferPrep is optimized for use in ion exchange chromatography method development but can also be used for size exclusion chromatography and for affinity chromatography The four stock solutions consist of 1 A mix of buffering components Up to five different buffering components with up to three pKa s each enabling a broad pH range to be covered 2 An acid HCl or base NaOH for on line titration of pH 3 Distilled water 4 An inert salt e g NaCl or KCl for elution gradient formation BufferPrep compensates for the pKa drift with changed ionic strength and temperature Anumber of pre defined recipes are available New recipes can also be created see Section 8 5 The pH is generated from pump module A and the salt concentration from pump module B The stock solutions containing the buffering component mix and the HCl or NaOH are connected to pump module A The stock solutions containing salt and water are connected to pump module B A switch valve on each pump module together with the BufferPrep algorithms generates the correct mixing ratios for the set pH Each pump module delivers 50 of the set flow AKTApurifier User guide 28 4079 53 Edition AC Optional functions 5 A2 Acid Base A1 Buffer stock B2 Salt stock Pump modul
92. fraction collection see Section 4 3 Fraction collection e using Scouting in a method see Section 5 3 Scouting e using BufferPrep in a method see Section 5 2 BufferPrep AKTApurifier User Guide 28 4079 53 Edition AC 57 4 Operation 4 3 Fraction collection 58 4 3 Fraction collection The fractions can be collected before and or during the elution by using a fraction collector The software provides the following methods for collecting fractions e Flowthrough fractionation e Fixed volume fractionation e Peak fractionation Refer to the respective user manual for more information on using your fraction collector Note The delay volume UV cell to fraction collector outlet is not set at system delivery because it depends on the length of your outlet tubing To set the delay volume in UNICORN see Section 8 1 6 Setting the delay volume 4 3 1 Fraction collection methods in the Method Wizard The Method Wizard will automatically contain the fraction collection methods when the fraction collector is selected in UNICORN By default a fraction collector is automatically selected when the correct strategies are used To check that the fraction collector is selected in UNICORN 1 In UNICORN Manager select Administration System Setup 2 In System Setup dialog select the correct system symbol and click Edit 3 In Edit System dialog click Component 4 Check that the fraction collector is selected Flowthrough frac
93. g the BufferPrep kit AKTApurifier User Guide 28 4079 53 Edition AC 67 4 Operation 4 5 Preparing the system for a run 4 5 Preparing the system for a run 4 5 1 System connection 1 Click the 1 System Control button in the Task bar at the bottom of the monitor TS 2 If YES is displayed in the Connection panel in the Run Data pane UNICORN EE is connected to a system Check that it is the correct system The system name is displayed in the title bar of the window fitis the correct system go to 4 5 2 Preparing the flow path components 3 If wrong system is connected or if the Connection panel says NO select Connection d S NO 3 System Disconnect and then click OK A Select System Connect The System Connect dialog window appears System Connect Eg Gk Cancel Help 5 Select your system from the list If you are not connected to a network only one system will be shown Click OK 6 When connected YES is displayed 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 login to UNICORN 4 5 2 Preparing the flow path components Preparing the tubing 1 Use the correct tubing kit for the column you intend to use See Section 8 4 Recommended tubing and columns For most columns the i d 0 50 tubing kit can be used in AKTApurifier 10 and the i d 0 75 tubing kit in AKTApurifier 1
94. global box However we recommend to clear the Save as global box in this situation Click OK For further information refer to UNICORN User Manuals KTApurifier User guide 28 4079 53 Edition AC 97 5 Optional functions 5 5 Flow regulation 98 5 5 Flow regulation Flow regulation of the system pump is used for e Maintaining the requested pump flow rate e Making sure that the maximum pressure limit is not exceeded Flow regulation is useful in applications where high back pressure can be expected or when the back pressure fluctuates for example when using samples with high viscosity 5 5 1 Regulation principle There are two regulators involved in tuning the system pump The first one is active as long as the pressure is below the set point This regulator tunes the actual pump flow rate to the set flow rate If the pressure exceeds the set point another regulator decreases the flow rate in order to reduce the pressure When the pressure falls below the set point the first regulator again tunes the actual flow rate to the set flow rate and so on The regulators use so called PID flow regulation where P and D are parameters that determine the tuning characteristics The default PID values in UNICORN provide a robust flow regulation that suits most purposes However the parameters can be further optimized to suit a specific application see 5 5 4 Optimizing the PID parameters 5 5 2 Including flow regulation in a
95. h 10 mm internal volume 8 ul is available as an accessory For higher detection sensitivity the 10 mm flow cell should be used 2 Monitor UPC 900 Check on the optical unit that the correct UV wavelength is set If not set the wavelength by selecting lamp Optical unit position indicated by a dot on the lamp housing in combination with the appropriate filter the dot on the lamp housing should be adjacent to the symbol Dot of Ge on the filter housing corresponding to the housing symbol on the filter wheel for the desired o bech filter A click will indicate that the filter is in labels position The illustration to the right shows Symbols on setting of the wavelength 254 nm ilter housing Priming the piston seal rinsing system of Pump P 900 1 Immerse the rinsing tubing in a flask 1 containing 20 ethanol in distilled water 2 Connect a syringe to the rinsing tubing that is connected underneath the left pump head 2 on pump A Slowly draw rinsing solution into the syringe 30 AKTApurifier User Guide 28 4079 53 Edition AC 1 6 Installation 2 When rinsing solution starts to enter the syringe continue to draw a few milliliters Loosen the syringe and immerse the tubing in the rinsing solution 1 Both ends should now be in the flask 1 Note For more detailed information refer to Pump P 900 User Manual Purging Pump P 900 Immerse the inlet tubing of all pump modules with filters in
96. he 2 mm cell fitted A 10 mm cell optical pathlength 10 mm internal volume 8 jl is available as an accessory For higher detection sensitivity the 10 mm flow cell should be used AKTApurifier UPC is delivered with the 2 mm e Conductivity flow cell fitted A 5 mm cell is also included in the CO cell with Tee JA integrated package If a lower detection sensitivity is temperature desired due to output signal limitation the sensor 2 mm cell should be used UV flow cells with 2 and 5 mm path lengths 130 KTApurifier User Guide 28 4079 53 Edition AC Reference information 8 8 2 5 Mixer M 925 Mixer M 925 is a dynamic single chamber mixer powered and controlled from Pump P 900 All eluents commonly used in ion exchange hydrophobic interaction affinity and reversed phase chromatography can be mixed with a high degree of accuracy and reproducibility The mixer is positioned directly after the Pump P 900 Mixer M 925 is supplied with a 0 6 ml mixing chamber which can be exchanged for larger chambers 2 5 and 12 ml mixer chambers are available as options to obtain optimal mixing over the entire flow rate range of AKTApurifier 8 2 6 Valve INV 907 A seven port motorized valve is used as a sample injection valve Three different operating positions to e Load a sample loop without disturbing column equilibration e Wash the sample loop while the column is in operation e Wash the pump for quick eluent ex
97. he intervals listed in Section 6 4 Calibration procedures Connecting the column Monitor UV 900 Connect the column between the injection valve port 1 and the inlet port of the UV cell Monitor UPC 900 The inlet port of the 5 mm a a sh UV cell is on top of the optical unit The inlet A d port of the 2 mm UV cell is underneath the TN Til Le unit view illustration 5 mm UV cell 2mm UV cell AKTApurifier User Guide 28 4079 53 Edition AC 69 4 Operation 4 5 Preparing the system for a run Filling the inlet tubing 1 Click the System Control button in the Task bar at the bottom of the monitor 2 Fill the inlet tubing with the correct solutions by selecting Manual Pump Select instruction PumpWash and set PumpA and PumpB to ON Purifier10 Pump Instructions x Instructions p Parameters E Pump ee Pump4 ne radient Flowpath SystemPumpControlMode OFF C ON Delete c SystemPIDParameters Alarms amp Mon PumpWashE xplorer PumpB C Frac OFF CON Close C Other _ Help wee IT Auto update If this is checked the parameter fields will be updated during method run 3 Click Execute to fill the inlet tubing The injection valve will automatically switch to waste during the pump wash 4 When the pump wash is finished click End in the System Control toolbar 5 Close the dialog Filling the sample loop or Superloop 1 Make sure that the corre
98. hromatogram and an evaluation of the test result e Ifthe gradient test result is OK the print out says Gradient linearity accepted e Ifthe step response test result is OK the print out says Step response accepted e Ifthe UV response test result is OK the print out says UV response accepted If any of the evaluated values falls outside the specified range go to Section 2 4 5 Correcting faulty evaluation results AKTApurifier User Guide 28 4079 53 Edition AC 35 2 2 4 36 Installation Installation test 2 4 4 Manual evaluation of the installation test results If the chromatography system deviates from the standard configuration for example if optional components have been installed the automatic evaluation will not give a reliable result A manual evaluation is required 1 Select the UNICORN Manager module 2 Clickon in the Results window and then double click on the Installation Test01 icon to open the result file 3 Right click in the Curves pane and select Properties 4 Click the Curves tab and select the following curves to be displayed AKTApurifier AKTApurifier UPC Installation Test01 1_UV1_215nm 01 SMTH Installation Test01 1_UV 01 SMTH Installation Test01 1 Conc Installation Test01 1_Conc 5 Click OK 6 Right click in the chromatogram window and select Marker 7 Read the absorbance for the steps corresponding to AKTApurifier AKTApurifier UPC Installation Test01 1_UV1_215nm
99. ipal waste and must be collected separately Please contact an authorized representative of the manufacturer for information concerning the decommissioning of your equipment Lx AKTApurifier User Guide 28 4079 53 Edition AC 101 6 Maintenance 6 1 Periodic maintenance 6 1 3 Maintenance schedule Interval Every day System Action Inspect the complete system for eluent leakage The system can be left filled with buffer overnight If you are not using the separation unit for a few days wash the flow path with distilled water Remove the column and the pH electrode optional Replace the column by a bypass capillary and fit the pH dummy electrode if applicable Then wash the system with 20 ethanol and store it in 20 ethanol Make sure that all tubing and all flow paths used are rinsed Pump P 900 Check for leakage If there are signs of liquid leaking between the pump head and the housing side panel or increased or decreased volume of rinsing solution replace the piston seals Refer to Chapter 4 in Pump P 900 User Manual When changing eluent it is important to remove trapped air Purge the pump according to Chapter 2 in the Pump P 900 User Manual If there is still air in the inlet tubing stop and purge the pump according to Chapter 5 in the Pump P 900 User Manual Note If air is allowed to enter the columns their performance can be adversely affected or destroyed pH electrode optional Every
100. is mounted from the factory from pump P 901 to the outlet The table below shows the tubing available for AKTApurifier 100 and where it is located in the system Tubing Length Tubing i d Tubing o d Material Color Volume of 10 design cm G1 G2 33 cm 0 75 mm 1 16 PEEK Green 44 2 u G3 9cm 0 75 mm 1 16 PEEK Green 44 2 u G4 16cm 0 75 mm 1 16 PEEK Green 44 2 u G5 30 cm 0 75 mm 1 16 PEEK Green 44 2 u G6 H6 16cm 0 75 mm 1 16 PEEK Green 44 2 u G7 H7 8 cm 0 75 mm 1 16 PEEK Green 44 2 u G8 H8 14 cm 0 75 mm 1 16 PEEK Green 44 2 u G9 H9 60 cm 0 75 mm 1 16 PEEK Green 44 2 u W1 W3 100 cm 0 75 mm 1 16 Tefzel Clear 44 2 u W1 W3 100 cm 1 00mm 1 16 Tefzel Clear 78 5 u Al B2 170 cm 2 9mm 3 16 Teflon Clear 660 ul A2 190 cm 2 9mm 3 16 Teflon Clear 660 ul B1 150 cm 2 9 mm 3 16 Teflon Clear 660 ul A3 B3 15cm 2 9mm 3 16 Teflon Clear 660 ul Changing tubing kits AKTApurifier 10 Two different tubing kits with different internal diameters are available for use from the injection valve to the outlet valve or the fraction collector if applicable in AKTApurifier 10 e Tubing kit 0 25 G6 G9 PEEK tubing blue marked G Installed from factory at delivery Used for most columns e Tubing kit 0 50 H6 H9 Tefzel tubing clear marked H For low pressure columns at high flow rates Tubing kit 0 25 and Tubing kit 0 50 The system is delivered with Tubing
101. ition AC Reference information 8 Item Quant Codeno pack Monitor UPC 900 Monitor UPC 900 1 18 1128 95 HG lamp amp housing complete 1 18 1128 22 Zn lamp amp housing complete 1 18 1128 23 UV flow cell 5 mm 1 18 1128 24 UV flow cell 2 mm 1 18 1128 25 Filter 214 nm 1 18 0622 01 Filter 254 nm 1 18 0620 01 Filter 280 nm 1 18 0621 01 Filter 313 nm 1 18 0623 01 Filter 365 nm 1 18 0624 01 Filter 405 nm J 18 0625 01 Filter 436 nm al 18 0626 01 Filter 546 nm 1 18 0627 01 Filter wheel complete 1 18 0647 01 Monitor UV 900 onitor UV 900 complete but without flow cells 1 18 1108 35 Flow cell UV 900 2 mm 1 18 1111 10 Flow cell UV 900 10 mm 1 18 1111 11 Fibre detachment tool 1 18 1111 16 pH and conductivity monitoring pH electrode with cell and holder round tip 1 18 1134 84 pH electrode round tip 1 18 1111 26 Dummy electrode round tip 1 18 1111 03 Conductivity cell d 18 1111 05 Mixer M 925 Mixer M 925 including one UniNet cable 1 18 1118 89 Mixing chambers 0 6 ml 1 18 1118 90 2 mi 1 18 1118 91 5ml 1 18 1118 92 12 ml 1 18 1118 93 AKTApurifier User Guide 28 4079 53 Edition AC 167 8 Reference information 8 8 Ordering information Item Quant Code no pack Valve INV 907 Valve INV 907 including one UniNet
102. ity is valid for the instrument only if it is e used for laboratory purposes not for clinical or in vitro purposes e used as a stand alone unit e used in the same state as it was delivered from GE Healthcare except for alterations described in this User Manual e connected to other CE labelled GE Healthcare modules or other products as recommended Safety standards This product meets the requirement of the Low Voltage Directive LVD 73 23 EEC through the following harmonized standards e EN 61010 1 e IEC 61010 1 e CAN CSA C22 2 No 61010 1 e UL61010 1 160 KTApurifier User Guide 28 4079 53 Edition AC Reference information 8 EMC standards This device meets the requirements of the EMC Directive 89 336 EEC through the following harmonized standards e EN 61326 emission and immunity e EN 55011 GR 1 Class A emission This is a Class A product In a domestic environment it may cause radio interference in which case the user may be required to take suitable measures This device complies with part 15 of the FCC rules emission Operation is subject to the following two conditions e This device may not cause harmful interference e This device must accept any interference received including interference that may cause undesired operation 8 6 4 Hardware requirements Refer to Section System requirements in the UNICORN User Manuals supplied 8 6 5 Software requiremen
103. l help to keep your AKTApurifier running smoothly Follow the recommendations in this chapter to keep the system in good working order Do not allow spilt liquid to dry on the instrument Wipe the surface regularly with a damp cloth Let the system dry completely before using it For details on how to perform the actions and for replacing components please refer to the individual User Manuals and Instructions 6 1 1 General precautions WARNING Always disconnect the power supply before attempting to replace any item on the system during maintenance WARNING If there is a risk that large volumes of spilt liquid may penetrate the casing of the instruments and come into contact with the electrical components immediately disconnect the mains cable and contact an authorized service technician WARNING When using hazardous chemicals make sure that the entire system has been flushed thoroughly with bacteriostatic solution e g NaOH and distilled water before service and maintenance WARNING Only spare parts that are approved or supplied by GE Healthcare may be used for maintaining or servicing the system gt Bb EE CAUTION When servicing and performing maintenance on the system always place the buffer bottles on the laboratory bench to prevent draining 6 1 2 Recycling and disposal This symbol indicates that the waste of electrical and electronic equipment must not be disposed as unsorted munic
104. le 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 5 Values for variables selected for scouting are dimmed on the Variables page and cannot be changed there 6 To change a variable value position the he oo Rum cursor in the Run value field and double Iron Paie Flow_Rate ml min II click with the left mouse button Type the new value 7 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 KTApurifier User guide 28 4079 53 Edition AC 95 5 Optional functions 5 3 Scouting 96 10 11 12 If you want to insert a new run column after a specific column in the scouting scheme position the cursor in the column and click Insert A new column with identical values appears directly after the selected column Repeat step 6 until you have defined all the runs you require If necessary use the horizontal scroll bar to see more runs 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 To save the scouting method select File Save When the method is started all the runs in the scheme will be performed automatically and the set flow for each r
105. le can be applied in different ways to suit the application and the sample volume e Using sample loops manually filled with a syringe or a Superloop As an option the sample can also be applied using a sample pump for example Pump P 960 More information is provided in the Pump P 960 package e AKTApurifier Directly onto the column using Pump P 960 or the system pump e optional Using an autosampler A 900 or A 905 The following table shows which technique is recommended for different sample volumes Sample application technique Volume to inject Sample loop manual filling 0 21 ml automated filling optional 0 1 21 Directly onto the column using the system gt 1ml pump or Pump P 960 optional Superloop optional 1 150 ml Autosampler 1ul 500 ul 1 For partial filling of the sample loop the recommended volume is up to 1 ml 2 Depending on autosampler model and tubing selection Refer to the A 900 or A 905 User Manual For scouting runs If the sample volume is to be varied automatically in a series of scouting runs one of the following technigues can be used e Automated partial filling of the sample loop using Pump P 960 0 1 2 ml e Applying the sample directly onto the column with the Pump P 960 gt 1 ml e Using a Superloop 1 150 ml The subsequent Sections describe e applying small sample volumes using a sample loop e applying large sample volumes using a S
106. lected eluent on a separate pH monitor When using standard recipes use the following mixture to measure the pH separately Mix using a 25 ml pipette 25 ml of the buffer stock solution 25 ml of the acid and 50 ml of water Check the pH with a known good quality pH electrode The reading should be close to 5 0 9 5 pHAIEX pH65 6 0 9 0pHAIEX pH 6 1 3 0 7 5pHCIEX pH 3 7 If not prepare new buffers If mixture pH is correct Check for a dirty or broken pH electrode Check the valves are operating correctly If a non standard recipe is used Check that the pH is not set too far from the pKa of the buffer components Check if there is too low concentration of buffer components causing low buffering capacity Check that the correct pKa values are used and that all pKa values have been entered Drifting pH Set the injection valve to position WASTE Pump through at least 30 ml of buffer at the set pH to stabilize the pH Switch the valve back to position LOAD Equilibrate the column 114 AKTApurifier User Guide 28 4079 53 Edition AC Reference information 8 8 Reference information 8 1 System description Note Two versions are available AKTApurifier with monitor UV 900 for multiple wavelengths detection and AKTApurifier UPC with monitor UPC 900 for combined measurement of UV absorption pH and conductivity When instructions or specifications are similar for both the models only AkTApurifier is mentioned
107. m UPC_10 x Main Selection Column and BufferPrep Main Selection Anion Exchange L Column Any 7 T Flexible Flow Rates NOTE Different Flow Rates are Set on Variable Page Flow Regulation of the System P IV BufferPrep BufferPrep Recipe 5 0 9 5 pH AIEX Global x BufferPrep pH 0 14 7 Beck Next gt Finish Cancel Help Set Defaut 5 2 5 Preparing the system for a BufferPrep run 1 Calibrate the pH monitor Refer to AKTApurifier Monitor pH C 900 User Manual AKTApurifier UPC Monitor UPC 900 User Manual For high accuracy measurements calibration should be performed with the pH electrode fitted in the flow cell at the flow rate to be used in the scouting run Prepare the required solutions For details about the preparation and solutions select View Run Setup in Method Editor and click the BufferPrep tab Inlet Al Buffer stock Inlet A2 HCI or NaOH Inlet B1 H 0 InletB2 Eluent stock AKTApurifier User guide 28 4079 53 Edition AC Optional functions 5 3 Manually fill the inlet tubing with the stock solution by using the PumpWash instruction in UNICORN 4 If equilibration is not programmed into the method equilibrate the system manually without a column with the set pH before starting the run e In System Control select Manual Other and click BufferPrep Recipe e Select the same recipe as you have in your method Click Execute e Select Pump and select BufferPrep_pH and enter the same
108. m UPC_10 x Main Selection and Column Main Selection 1 6 In the Evaluation Procedures window select the procedure for your system for example e InstPurifier UPC for AKTApurifier e InstBasic UPC for AKTApurifier UPC e Click Next 7 Click Next in the Method Information window 8 Click START in the Result Name window to start the installation test The progress of the test is monitored in the System Control module The installation test run time is approximately 30 min When the test is finished the printer automatically prints the chromatogram and the test results Installation Test01 1 result Step response result a gt lt gt Customizing the Curves pane To customize the Curves pane 1 Right click in the Curves pane and select Properties 34 AkTApurifier User Guide 28 4079 53 Edition AC Installation 2 2 Click the Curves tab Curve Style and Color Flow Scheme Logbook Run Data Groups Run Data Color Cuves z vais r Display curves Cond ETS Cond Clear All MConc Pressure Flow _ITemp 3 Select the following curves to be displayed AKTApurifier AKTApurifier UPC UV1 215nm UV Conc Conc Clear all other curves Click OK 2 4 3 Automatic evaluation of the installation test results The system automatically prints the test result when the test is finished The print out consists of a c
109. m flow path To bypass the column use a capillary tubing supplied with your system If the column is to be left in the flow path make sure that the maximum allowed flow and pressure for the column are not exceeded For column cleaning procedures and column storage instructions please refer to the instruction supplied with the column 6 3 1 At the end of the day If the system will be used with the same buffers the next day let the system run isocratic at a low flow rate approximately 0 02 ml min If the system will be used with other buffers the next day rinse the pump and the system with distilled water using the PumpWash instruction as follows 1 Submerge the inlet tubings in distilled water 2 Run the instruction AKTApurifier AKTApurifier UPC PumpWashPurifier PumpWashBasic 6 3 2 Leaving the system for a few days Perform a PumpWashPurifier PumpWashBasic with distilled water Repeat with a bacteriostatic solution i e 20 ethanol 6 3 3 Monthly cleaning Clean the system every month or when problems such as ghost peaks occur Also use this procedure to clean the flow path before service or maintenance 1 Disconnect the column and replace it with bypass tubing 2 If using a pH electrode replace it with a dummy electrode 3 Place all the inlet tubings in 1 M NaOH 4 Manually perform PumpWashPurifier PumpWashBasic for both inlet tubings 5 Flush the whole system with 1 M NaOH for 20 minutes 1 ml min 6
110. me shows open flow paths and monitor signals with numerical displays KTApurifier User Guide 28 4079 53 Edition AC Run 13 40 ml min 2 00MPa 45 5 B 411215 1 123 AU 111254 0 02345 AU 111280 0 1234 AU AU Cond Tc pH 0 00002 015 0 12 50 pH12 50 Tc 22 4 C 735 8mS cm Tc 78 8 KTApurifier User Guide 28 4079 53 Edition AC Operation 4 Logbook All actions and unexpected conditions such as warnings are logged for every run with date time and current username The logbook provides a complete history of any given run The log is saved in the result file Front panel display The front panel displays of Monitor UV 900 Monitor UPC 900 and Pump P 900 can be set to show their current status In each case the main operating menu display shows the most important parameters The main operating menu of Pump P 900 shows the current flow rate together with a mode indication pressure and B if used The available modes are Run The pump is running with the set flow rate End The system is not running Pause The pump is stopped but the set flow rate and the gradient values are retained Hold The gradient is held at the value displayed and the pump continues to run The method is held in its current status KTApurifier The main operating menu of the Monitor UV 900 shows the absorbance values with 4 digits for up to 3 active wavelengths The display for the third wavelength is reached
111. meters and then click Execute The new flow rate will be used until the end of the run or until a new flow rate instruction is reached in the method A Close the box by clicking Close All manual interactions are recorded in the Logbook Some instructions for example gradient or fraction instructions might take time to complete To stop the run before it is finished click the End button at the top Run Continde End ajale KTApurifier User Guide 28 4079 53 Edition AC Operation 4 From the modules Manual changes can also be performed on the pump and UV monitors using the selection dial Manual changes in UNICORN or on the modules are equivalent Manual changes are normally recorded in the log book The selection dial on the modules can be set in one of three different access modes e Open the dial on the module can be used for manual changes e KeyLocked the dial on the module can be used to select different menus but cannot be used to change any parameters e KeyAndDialLocked Neither menu selection nor parameter changes can be performed To select access mode 1 Select System settings in System Control and then Specials Keyboard 2 Select Open KeyLocked or KeyAndDialLocked AKTApurifier User Guide 28 4079 53 Edition AC 79 4 Operation 4 8 Viewing and printing the result 4 8 Viewing and printing the result If you are satisfied with the automated printout obtained after the run if selected
112. mn connections AKTApurifier User Guide 28 4079 53 Edition AC 173 Index 174 UniNet 2 consumables control keys creating a method CU 950 D d fining anew Selten RAN 150 E el ctricaliconnections ss msn dant nat EES 118 valugtion procedures zidane une BNN 73 F aults and actions inal checks ixed volume fractionation 59 ow path ow rate scouting 95 ow regulation 98 Flow restrictor FR 902 904 170 Flow schematics 2023 ow schematics 122 owthrough fractionation et D I uid handling path 121 Frac 920 PSTN e Essen Ra AN Ra Bi Ren us tenu S 22 Frac 950 instal prep mode standard mode fraction collection fraction collector installing Frac 920 installing Frac 950 fractionation order Frac 950 Fuse G gradi H hardware Ee EE 161 injection valve inlet filter Installation test installing CU 950 installing fraction collectors atelier EE ifting the system 106 AKTApurifier User Guide 28 4079 53 Edition AC Index locking the selection didle ea enam slama ennemies 79 M mains connections 120 MAINTENANCE ex 101 manual filling of sample loops materials wetted methods creating mixer chamber Mixer M 925 ce
113. nd install items enclosed 16 Unpack and install the computer 22 Install PL C2900 BEE 18 Install Frac 920 6r Frac 9 50s en ANN RE tele PEER RES 22 Connect UniNet 1 data communication chain cabling 26 Connect mains power cabling 27 Complete the two first Sections of the Installation record 28 Prepare AKTApurifier for the installation test 30 Run the installation test Method 35 Evaluate the gradient 22 osc0c2s0e25i ae E saG ass denses 36 Evaludte the step response 22 ras eee ied bie 37 Evaluate the UV response 39 Completethe Test record wis rss ester AN ABE 39 Complete the Registration form 40 Complete the last Section of the Installation record 28 Store photocopies of all records and forms in the System Logbook AKTApurifier User Guide 28 4079 53 Edition AC Installation 2 2 2 Pre requisites WARNING AKTApurifier must be connected to a grounded mains socket A outlet e Two people are required to lift AKTApurifier onto a workbench e The system should be installed on a stable laboratory bench providing a suitable working area To install AKTApurifier a working area of about 200 x 80 cm is required The mains power switch must alw
114. nd printer 3 start UNICORN See section 3 3 1 Starting UNICORN AKTApurifier User Guide 28 4079 53 Edition AC Operation 4 4 2 Creating a method UNICORN control software is supplied with a Method Wizard used for creating new methods The Method Wizard consists of a number of dialogs in which you answer questions and receive instructions on how to create your method The options in subsequent dialogs depend on the choices you have made in the previous dialogs The Method Wizard covers the most commonly used chromatographic techniques Note AKTApurifier UPC screen shots are shown in the example To create a method 1 Click the Ki icon in the Method Editor module The Method Wizard is started or Select File New in the Method Editor module The New Method dialog appears Select Wizard New Method Select system UPC_10 Click OK To get detailed help texts click Help e Select Wizard from the Use options e Select which system you want to use AkTApurifier User Guide 28 4079 53 Edition AC 53 4 Operation 4 2 Creating a method e Click OK The Method Wizard is started 2 Select a chromatographic technique for example Anion Exchange from the Main Selection list Method Wizard for System UPC_10 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 implement
115. ne where it should be stored By default the result file name will either be the same as the method name the date of the run or a pre defined name The name is followed by a three digit sequence number starting with 001 You can change this name and select a new directory by clicking Browse AKTApurifier User Guide 28 4079 53 Edition AC 73 4 Operation 4 6 Starting a run 74 8 Click START The run will start You can view the run in the System Control module The Curves pane shows curves during the run lt gt DEMO System Control 1 systemi Method File View Manual Adviser Help Result The Run Data pane shows current values for running parameters R BPauses 300 Hod Continue 200 100 End Curves x3 Ei x5 xB x7 xB Wast i 1 ji Dm PumpB Injection Vawe D 00 min Base CV 0 96 ml RESOURCE_Q_1_ml 0 05 min Block Start Conditions 0 05 min Base SameAsMain 0 05 min ColumnPosition Position2 0 05 min Wavelength 280 nm OFF OFF 0 05 min AveragingTimeUV 1 28 sec 1 05 min Alarm Pressure Fnahled For Help press F1 1 50 MPa ANN MPa The Flow scheme is a graphical representation of the system flow path Qutiet K 0 00 min Run 1999 11 04 09 16 53 Method Manual Injection FP Segmented Result c DefaulManual Injection FP Segmented01 res 10 D 12 13 14 15 16 17 18 19 20 a e End Block Controlled By
116. nect the computer to the separation unit 3 Allow the separation unit to stabilize at the new temperature for at least 12 hours 4 Tighten all connections and pump water through the system to check for leaks 5 Tighten any leaking connector 4 10 2 Running in cold room Before starting a run check that the temperature of the buffers has reached the ambient temperature 4 10 3 Removal from cold room 1 Loosen all connections to prevent them sticking when the system returns to room temperature 2 Allow the separation unit to stabilize at room temperature for at least 12 hours 3 Tighten all connections and pump water through the system to check for leaks 4 Tighten any leaking connectors AKTApurifier User Guide 28 4079 53 Edition AC Optional functions 5 5 Optional functions This chapter describes how to use optional functions when operating AKTApurifier 51 Mixing gradients 5 11 Gradients There are two different technigues available for mixing gradients The standard technigue using two separate buffers one to each pump module and the BufferPrep technigue using four solutions two to each pump module generating the buffer on line with a switch valve before each pump module The minimum flow rate for BufferPrep is 1 0 ml min The outputs of the pump modules are routed to a mixer The BufferPrep method is described in Section 5 2 BufferPrep 5 1 2 Mixer WARNING When using hazardous chemicals flush
117. ng connector M6 male 1 18 1017 98 Accessories KTApurifier BufferPrep kit including two switch 1 28 4044 73 valves and holders Tubing and connectors for 10 systems and 100 systems Flow restrictor FR 902 0 2 MPa 1 18 1121 35 Flow restrictor FR 904 0 4 MPa 1 18 1119 63 Short column holder 1 18 1113 17 Long column holder 1 18 1126 32 Small column clamp 1 18 1149 98 Lab rod holder 1 18 1113 19 Tubing cutter 1 18 1112 46 On line filter 10 ml systems 1 18 1118 01 On line filter kit 10 ml systems 2 18 1120 94 2 filters On line filter 100 ml systems 1 18 1112 44 On line filter kit 100 ml systems 10 18 1027 11 10 filters and 2 nets On line filters 100 ml systems 25 18 1130 23 25 filters 170 AKTApurifier User Guide 28 4079 53 Edition AC Reference information 8 Item Quant Codeno pack User Manuals AKTApurifier Manual Box complete containing all User Manuals and Instructions for all components in AKTApurifier 10 100 1 11 0014 51 AKTApurifier UPC Manual Box complete containing all User Manuals and Instructions for all components in AKTApurifier UPC 10 100 1 11 0014 51 UNICORN 5 10 Manual kit including 1 28 4010 25 Getting Started User Reference Manual Chapter part 1 User Reference Manual Chapter part 2 Administration and Technical Manual AKTApurifier User Gui
118. nit UV cell Injection valve Column INV 907 Column holder Monitor UPC 900 Pump P 900 Mixer M 925 Mixer chamber Flow restrictor Fig 8 3 AKTApurifier UPC component locations AKTApurifier User Guide 28 4079 53 Edition AC 117 118 8 Reference information 8 1 System description 8 1 3 Purging parts The figure below shows the purging parts for Pump P 900 Purge valve Outlet manifold Pump module Purge tubing 8 1 4 Electrical connections WARNING Never attempt to remove the mains fuse while mains voltage is applied to the system For continued protection against risk of fire replace only with a fuse of the specified type and rating Refer to the Technical specifications for fuse data All electrical connections for AKTApurifier are located at the rear of the system Only one mains input is required for the complete system The supply voltage for the components in the system and the fraction collector is distributed from the rear of the system AKTApurifier User Guide 28 4079 53 Edition AC Reference information 8 UniNet 1 Fraction collector UniNet 1 Termination plug Only in Frac 950 Frac 920 has only one UniNet 1 CU 950 UniNet 1 socketj To computer USB cable Fig 8 4 UniNet 1 connections on AKTApurifier UPC The connections on AKTApurifier is sim ilar
119. nt and a series of concentration steps of acetone Correct UV monitoring is tested by monitoring the acetone concentration at e 254 nm Monitor UPC 900 e 265 254 and 280 nm and calculating the absorbance ratios 265 nm 254 nm and 265 nm 280 nm Monitor UV 900 WARNING When using hazardous chemicals take all suitable protective A measures such as wearing protective glasses and gloves resistant to the chemicals used Follow local regulations and instructions for safe operation and maintenance of the system AKTApurifier User Guide 28 4079 53 Edition AC 29 2 Installation 2 4 Installation test 2 4 1 Preparing AKTApurifier Starting the separation unit Turn on the separation unit using the mains switch located to the left on the base platform Starting the computer Turn on the computer the display and the printer according to the instructions in the manufacturer manuals Starting and logging on to UNICORN control software Start UNICORN as described in Section 3 3 UNICORN software Checking the optical unit 1 Check that the correct UV cell is fitted Monitor UV 900 AKTApurifier 10 is delivered with the 10 mm cell fitted A 2 mm cell optical path length 2 mm internal volume 2 ul is available as an accessory If a lower detection sensitivity is desired due to output signal limitation the 2 mm flow cell should be used AKTApurifier 100 is delivered with the 2 mm cell fitted A 10 mm cell optical path lengt
120. nyl HP 5 ml RF 17 1359 01 HiTrap Octyl FF 1 ml X 17 5196 01 HiTrap Octyl FF 5 ml RF 28 4110 01 HiTrap Butyl HP 1 ml X 28 4110 05 HiTrap Butyl HP 5 ml RF 17 1357 01 HiTrap Butyl FF 1 ml X 17 5197 01 HiTrap Butyl FF 5 ml RF 17 0978 13 HiTrap Butyl S FF 1 ml X 17 0978 14 HiTrap Butyl S FF 5 ml RF KTApurifier User Guide 28 4079 53 Edition AC Reference information 8 Affinity Columns Code no Column name 0 25 0 50 17 5247 01 HisTrap HP 1 ml x 17 5248 01 HisTrap HP 5 ml RF 17 5319 01 HisTrap FF 1 ml X 17 5255 01 HisTrap FF 5 ml RF 17 5256 01 HisPrep FF 16 10 RF 11 0004 58 HisTrap FF crude 1 ml x 17 5286 01 HisTrap FF crude 5 ml RF 17 0920 03 HiTrap IMAC HP 1 ml X 17 0920 05 HiTrap IMAC HP 5 ml RF 17 0921 02 HiTrap IMAC FF 1 ml X 17 0921 04 HiTrap IMAC FF 5 ml RF 17 0921 06 HiPrep IMAC FF 16 10 RF 17 0408 01 HiTrap Chelating HP 1 ml x 17 0409 01 HiTrap Chelating HP 5 ml RF 17 0716 01 HiTrap NHS activated HP 1 ml x 17 0717 01 HiTrap NHS activated HP 5 ml RF 17 5281 01 GSTrap HP 1 ml x 17 5282 01 GSTrap HP 5 ml RF 17 5130 01 GSTrap FF 1 ml 5 pcs X 17 5131 01 GSTrap FF 5 ml RF 17 5234 01 GSTPrep FF 16 10 RF 28 4017 45 GSTrap 4B 1 ml X 28 4017 46 GSTrap 4B 5 ml RF 28 9187 78 MBPTrap HP 1 ml X 28 9187 79 MBPTrap HP 5 ml RF 28 9075 46 StrepTrap HP 1
121. o Mixer chamber Fig 1 2 KTApurifier 8 KTApurifier User Guide 28 4079 53 Edition AC Box 900 Monitor UPC 900 Pump P 900 P 901 P 903 ON OFF button AKTApurifier User Guide 28 4079 53 Edition AC Introduction 1 Se Injection valve III INV 907 Optical unit Conductivity cell Mixer M 925 Mixer chamber Fig 1 3 AKTApurifier UPC 1 2 Labels The instrument has labels attached to the inside of the frame at the rear of the system The rating label is on the left side and identification labels are on the right viewed from the rear AKTApurifier Voltage Frequency Power max 100 240 V 50 60 Hz 600 VA Serial no Made in Sweden 12345678 X ce Code no XX XXXX XX Fig 1 4 Rating label and identification labels on KTApurifier 1 Introduction 1 3 Safety 10 gt gt Bb EE E 1 3 Safety e The system is designed for indoor use only e Do not use in a dusty atmosphere or close to spraying water e Operate in accordance with local safety instructions e Do not block the air inlet on the front panels or the outlets on the rear of the instrument WARNING This instrument is intended for laboratory use only not for clinical or in vitro purposes WARNING The system must be connected to a grounded mains socket to prevent system parts from becoming live
122. od with the variable Pressure_limit Superloop is an accessory available in three sizes 66 Volume Max allowed column pressure Code no 10 ml 4 MPa 18 1113 81 50 ml 4 MPa 18 1113 82 150 ml 2 MPa 18 1023 85 All the sample is applied which gives good reproducibility and high recovery The sample is not diluted as the buffer pushing the movable seal is kept separate The loaded sample can be injected all at once or in several smaller volumes down to 1 ml portions permitting automated repetition of sample injection Superloop should be manually filled before the method is started Preparation Prepare the injection valve and connect Superloop as follows 1 Connect the supplied Pos 1 LOAD Union Luer female 1 16 male to D 1 Tag 5 olumn port 3 of the injection valve 2 7 Pump 2 Ensure that tubing for the waste Sg 6 Top is connected to port 4 of the injection valve Superloop Bottom 3 Ensure that Superloop is filled D aste with liquid see separate Superloop Instruction A Mount Superloop in a column holder as close to the injection valve as possible 5 Connect the bottom tubing to injection valve port 2 AKTApurifier User Guide 28 4079 53 Edition AC Operation 4 6 Connect the top tubing to injection valve port 6 7 Make sure all connections are fingertight Filling Superloop Filling Superloop is achieved as follows 1 Set the injection valve to position INJECT
123. olumn pressure limit and the predicted peak volume is less than 1 ml it is preferable to change to a narrower tubing kit i d 0 5 mm If the demands on low band broadening are less critical use the tubing kit already installed e If the generated back pressure at the flow rate to be used exceeds the set column pressure limit change to a wider tubing kit i d 1 0 mm If the set column pressure limit is still exceeded with the i d 1 0 mm tubing kit installed change to Flow restrictor FR 902 and check the generated back pressure according to the description below Note The back pressure might increase during e g sample injection and gradient formation due to viscosity variations Make sure that these variations have been taken into consideration when selecting tubing i d 148 AKTApurifier User Guide 28 4079 53 Edition AC Reference information 8 8 5 BufferPrep details 8 5 1 Creating your own recipes 1 Select Edit BufferPrep Recipes in the Method Editor 2 Click New to open the New Recipe dialog 3 Select a buffer substance from the list If the required buffer substance is not listed a new buffer substance can be defined see Section 8 5 2 in Defining a new buffer substance below Up to 5 buffering components can be added 4 Set the stock concentration of the buffer Buffer concentrations of 2 4 times higher compared to normal preparation should be used When BufferPrep is used the buffer will be diluted between 2 10 tim
124. one not resistant Pressure limit for PEEK decreases Hydrochloric acid 0 1M OK OK Silicone not resistant Hydrochloric acid gt 0 1 M OK Avoid Silicone not resistant Titanium is affected by long term use isopropanol OK OK Methanol OK OK Nitric acid diluted OK Avoid Silicone not resistant Nitric acid 30 Avoid Avoid Elgiloy is affected by long term use Phosphoric acid 10 OK Avoid Titanium and aluminium oxide are affected by long term use Potassium carbonate OK OK Potassium chloride OK OK Pyridine Avoid Avoid ETFE PP and PE not resistant Sodium acetate OK OK Sodium bicarbonate OK OK Sodium bisulphate OK OK Sodium borate OK OK Sodium carbonate OK OK Sodium chloride OK OK Sodium hydroxide 1 2 M OK Avoid Titanium PVDF and borosilicate glass are affected by long term use Sodium sulphate OK OK Sulphuric acid diluted OK Avoid PEEK and titanium are affected by long term use Sulphuric acid medium Avoid Avoid concentration Tetrachloroethylene Avoid Avoid Silicone PP and PE are not resistant 164 AKTApurifier User Guide 28 4079 53 Edition AC Reference information 8 Chemical Exposure Comments lt iday upto 2 months Tetrahydrofuran Avoid Avoid Silicone ETFE CTFE PP and PE are not resistant Toluene OK Avoid Pressure limit for PEEK decreases Trichloroacetic acid 1 OK OK Trifluoroacetic acid 1 OK OK Urea OK OK o Xylene OK Avoid Silicone PP and PE are affected by l
125. ong p xylene term use AKTApurifier User Guide 28 4079 53 Edition AC 165 8 Reference information 8 8 Ordering information 8 8 Ordering information Item Quant Code no pack AKTApurifier 10 system 1 28 4062 64 AKTApurifier 100 system 1 28 4062 66 AKTApurifier UPC 10 system 1 28 4062 68 AKTApurifier UPC 100 system 1 28 4062 71 Monitor pH C 900 1 18 1107 76 Pump P 901 Pump P 901 1 18 1114 00 Sealing kit 100 ml including 1 18 1113 12 4 piston seals and 4 rinse membranes Piston kit 100 ml 1 18 1112 13 Inlet tubing kit 100 ml 1 18 1114 40 Pump head capillaries 100 ml two capillaries for 1 18 1117 52 one pump module Rinsing draining housing 100 ml 1 18 1112 03 Pump head 100 ml complete 1 18 1128 48 Pump P 903 Pump P 903 1 18 3000 00 Sealing kit 10 ml including 1 18 1120 77 2 piston seals 2 membranes and 2 O rings Piston kit 10 ml 1 18 1120 75 Inlet tubing kit 10 ml 1 18 1119 26 Pump head capillaries 10 ml two capillaries for one 1 18 1120 81 pump module Rinsing draining housing 10 ml 1 18 1120 76 Pump head 10 ml complete 1 18 1128 47 Pumps P 901 and P 903 Tubing kit for rinsing system 1 18 1113 32 Purge kit 2 18 1124 53 Purge valve 1 18 1128 87 O ring for purge valve 10 19 0036 01 Check valve kit including 1 18 1128 66 1 Inlet check valve 1 Outlet check valve 166 AkTApurifier User Guide 28 4079 53 Ed
126. or results in a stable stationary error between actual and requested flow The parameter eliminates the stationary error but results in a slight instability that might lead to oscillations in the actual flow The parameter can have values between 0 and infinity Smaller values have a greater effect and a value of infinity has no effect Note The value infinity is set as 9999 in UNICORN D In certain cases the D parameter can reduce the oscillations introduced by a Pl regulator D can have values between 0 and infinity where larger values have a greater effect and a value of 0 has no effect Note Most often a simple Pl regulator is preferable for control of flow rate and AKTApurifier is therefore configured by default with the D parameter set to zero Table 5 2 PID parameters Rules of thumb for optimizing the PID parameters e Use the default PID parameter values as start values To set the default values select System Settings in the System Control module and then Specials Select the PID parameter to be restored and click Set Selected Parameter To Strategy Default Value e Keep the D parameter set to zero i e use a simple Pl regulator e Start the pump before activating the regulator e Increasing P makes the regulator faster e Increasing reduces oscillations 100 AkTApurifier User guide 28 4079 53 Edition AC Maintenance 6 6 Maintenance 6 1 Periodic maintenance Regular maintenance wil
127. pH and conductivity When instructions or specifications are similar for both the models only AkTApurifier is mentioned The following topics are described e Starting the system e Creating methods e Preparing the system for a run e Performing and monitoring a run e Performing simple result evaluation e Viewing and printing results e Performing automatic method optimization Scouting e Preparing automatically buffers of any pH BufferPrep The chapter also contains information about fraction collection cold room operation mixing gradients and feed back tuning WARNING When using hazardous chemicals take all suitable protective A measures such as wearing protective glasses and gloves resistant to the chemicals used Follow local regulations and instructions for safe operation and maintenance of the system AKTApurifier User Guide 28 4079 53 Edition AC 51 4 Operation 4 1 Starting the system 52 41 Starting the system IMPORTANT Before using AKTApurifier read all the safety information in Section 1 3 Safety 4 1 1 Pre requisites Before starting a run make sure that e the system and software are installed and functioning e the monitors and pumps are tested This is described in Chapter 2 Installation 4 1 2 Starting the system To start the system 1 turn on the separation unit with the mains switch located to the left on the base platform 2 turn on the computer display a
128. pH as set in the method Click Execute e Seta flow rate and click Execute 5 Click the Variables page Use the equilibration volumes at minimum listed in he table below to obtain a steady pH reading The 0 6 ml mixer chamber is included in the systems at delivery the other mixer chambers are optional System Mixer chamber Equilibration volume ml volume ml AKTApurifier 10 0 6 9 2 14 AKTApurifier 100 0 6 20 2 30 5 50 12 100 6 When running pH scouting the sample should if possible have a pH close to he highest pH in the scouting run for AIEX and close to the lowest pH for CIEX The conductivity of the sample should be below 5 mS cm 7 Save the method 5 2 6 Fine tuning To obtain higher pH accuracy the recipe can be fine tuned around a specific pH When scouting over a broad pH range fine tuning is less beneficial Run BufferPrep manually at 0 and 100 B as follows 1 From the System Control Manual menu select the recipe under Other BufferPrep_pH and Flow under Pump Start a run manually at 0 B Make sure the switch valve is set correctly to the inlet A1 AKTApurifier User guide 28 4079 53 Edition AC 93 5 Optional functions 5 2 BufferPrep 94 2 Check the pH reading when stable 3 Change to 100 B by setting the Gradient instruction in Manual Pump to 100 B for Target and 0 for Length Click Execute A Check the pH reading when stable at 100 B 5 Ifthe reading is acceptable at bo
129. pH electrode is to be mounted in a separate pH flow cell housing the pH electrode itself as well as a dummy pH electrode The dummy pH electrode replaces the pH electrode whenever the pH electrode is to be removed from the system e g for storage The pH electrode contains a sealed Ag AgCl reference which cannot be refilled an internal electrolyte bridge of 4 M KCI saturated with Ag AgCl an outer electrolyte bridge of 1 M KNO3 an annular ceramic reference junction and a low profile pH membrane This is commonly known as a sealed combination double junction type The pH electrode is delivered with a transparent cover Note The flow cell should not be used with any other pH electrode Note Always use the 0 50 tubing kit and flow restrictor FR 902 when the flow cell is mounted Installation CAUTION Never use 0 25 mm i d tubing blue when the pH flow cell is mounted in the system The electrode might rupture due to high backpressure To install the pH flow cell and pH electrode between the conductivity cell and flow restrictor for more details refer to Monitor UPC 900 Monitor pH C 900 User Manual 1 Remove the flow restrictor and the male male union from the conductivity flow cell 2 Hook the flow cell holder directly to the right of the optical unit Secure it with the slide clamp The optical unit might have to be moved slightly to the left If the flow cell holder is not used the flow cell must still be installed
130. pump display that the pressure reading is stable variation less than 0 02 MPa AKTApurifier 100 Manually select pump gradient 100 B length 0 and run with 10 ml min distilled water Check on the pump display that the pressure reading is stable variation less than 0 2 MPa Proceed to step 5 if the pressure is stable If not consult the Pump P 900 User Manual for troubleshooting instructions Click END Check that the inlet filters are submerged in the corresponding buffers A and B according to the table below Installation Test Method Guide Buffer A Distilled water 200 ml Buffer B 1 0 acetone in distilled water 200 ml Test flow rate 5 ml min Test run time Approximately 30 minutes AKTApurifier User Guide 28 4079 53 Edition AC Installation 2 2 4 2 Running the installation test method 1 In UNICORN Manager module select File Printer Setup Select the appropriate printer from the list and select Landscape Then click OK to acknowledge the printer chosen Print Setup In System Control module select File Instant Run The Instant Run window opens InstantRun UPC_10 l P Eege Select the appropriate system from the For system list Click Run The Method Wizard is started AKTApurifier User Guide 28 4079 53 Edition AC 33 2 Installation 2 4 Installation test 5 Select Installation Test from the Main Selection list and click Run Method Wizard for Syste
131. r Guide 28 4079 53 Edition AC 149 8 Reference information 8 5 BufferPrep details 150 Note The check does not include whether the buffering capacity is large enough over the entire pH range 11 Click Close to close the BufferPrep Recipes dialog 8 5 2 Defining a new buffer substance Before defining a new buffer substance make sure that all pKa values for the substance are available The pKa values entered should be true pKa i e the pKa value at indefinite dilution as opposed to apparent pKa i e measured at a non zero concentration Refer to Section 8 5 5 in Examples and tips for information on true and apparent pKa The pKa values should be given at 25 C 1 Click New in the BufferPrep Recipes dialog to open the New Recipe dialog Click the Buffer substance button and click New in the Define Buffer Substance dialog that opens 2 In the New Component dialog enter the name of the new buffer substance Click OK 3 Inthe Define Buffer Substance table enter the pKa Value Up to 3 values pKa1 pKa3 can be entered for each buffering component The pKa values must be entered in order of increasing value starting with the lowest pKa value When the buffering component has less than three pKa the other pKa values should be set to zero 4 Enter the dpKa dT Value the change of pKa dependent on temperature for each pKa dpKa1 dT dpKa3 dT Zero means that the pKa does not change with temperature 5 Enter the Number of
132. rect tubing Consider the maximum allowed pressure for the column and the size of the column For low pressure columns such as HiTrap and HiLoad it is necessary to compensate for the pre column pressure as described in Section 5 4 in Extra system pressure measurement 3 2 1 AKTApurifier10 tubing WARNING Never use i d 0 25 mm tubing or smaller with columns that only withstand low maximum pressures and allow high flow rates as the columns might rupture resulting in injury On delivery the system is equipped with i d 0 50 mm tubing PEEK orange from the pump to the injection valve and i d 0 25 mm tubing PEEK blue from the injection valve to the conductivity cell I d 0 50 mm tubing can also be used from the injection valve to the conductivity cell This diameter should be used with columns that have a low maximum pressure limit and allow high flow rates If tubing with smaller inner diameter is used there is a risk that the back pressure will be too high and the columns might rupture The i d 0 25 mm tubing should also be changed to i d 0 50 mm tubing when a pH electrode optional is used Note Use only a maximum of 80 acetonitrile eluent at pressures above 10 MPa Otherwise the lifetime of the tubing will deteriorate 3 2 2 AKTApurifier 100 tubing On delivery the system is equipped with i d 0 75 mm tubing PEEK green from the pump to the conductivity cell AKTApurifier User Guide 28 407
133. ruction than used before the Watch instructions 5 5 3 Manual flow regulation instructions Flow regulation can also be used when running the system pump manually The instructions are found in the System Control module by selecting Manual Pump and are explained in Table 5 1 Instruction SystemPumpControlMode Parameter description To activate flow regulation select PressFlowControl PressLevel is the maximum allowed pressure MinFlow is the minimum allowed flow rate SystemPIDParameters Flow DG Flow I and Flow D are the parameters for tuning the actual flow rate to the set flow rate Active below the PressLevel value Press P Press Iand Press D are the parameters for reducing the flow rate and thereby decreasing the pressure to below the pressure set point Active above the PressLevel value Table 5 1 Flow regulation instructi AKTApurifier User guide 28 4079 53 Edition AC ons 99 5 Optional functions 5 5 Flow regulation 5 5 4 Optimizing the PID parameters The two regulators for the system pump have separate PID parameters The default PID parameters in UNICORN provide a robust flow regulation that is suitable for most purposes Note The PID parameters should only be changed by experienced users The table below describes the three PID parameters Parame Description ter P The P parameter reduces the effect of an error but does not completely eliminate it A simple P regulat
134. rvals given in column 5 AKTA purifier Evaluating the UV response Calculate the UV response ratios in the following way 1 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 see page 39 and enter the results in column 3 2 Calculate the absorbance ratios 265 nm 254 nm and 265 nm 280 nm using the values of column 3 and enter the results in column 4 The ratios obtained should all fall within the intervals given in column 5 AKTApurifier User Guide 28 4079 53 Edition AC 37 2 2 4 38 Installation Installation test 2 4 5 Correcting faulty evaluation results Should any of the evaluated values fall outside the specified range proceed as follows e If the system differs from the standard configuration evaluate the result manually e H the faulty evaluation result remains continue below Faulty gradient e The gradient is linear but the interval is too small the mixer chamber is too large or the mixer is faulty e Disturbances may arise from air in the pump pump valves or bad sealings in the pump Refer to the Pump P 900 User Manual Faulty step response e Ifall values are faulty air in the pump or a faulty pump e 5 and 95 faulty bad sealing in the pumps 5 faulty pump module B 95 faulty pump module A AKTApurifier User Guide 28 4079 53 Edition AC Installation 2 25 Test record 2 5 1 Gradient tes
135. sses troubleshooting E mixer Volumes ees modules and components location onitor pH C 900 calibration onitor UPC 900 troubleshooting onitor UV 900 moving the system N Ve e TEE 161 o Om Mete a sot itl ate Bui 170 changing the filter on line filter operating data Pump P 901 Pump P 903 P partial Time of SAM ple LOOPS EEN 64 peak fractionation periodic maintenance pH electrode mounting operation pH flow cell installation int BBB sms BN 20 pH scouting physical data PID floWredulation EEA LANANG une ee 98 optimizing parameters 100 pKa true and apparent 2153 power requirement preparing the flow path components preparing the system for a run printing method instructions Pump P 900 4 Pung eU bi NG EE AKTApurifier User Guide 28 4079 53 Edition AC 175 Index p rgima PUMP EE 31 R rating label EE 9 reguirements hardware network software result file SCOULING eeh Run Setup window running the installation test method S Safe NOM TE 9 Safety standards sample application overview using a sample loop sample loops compl t filigrane area 65 scouting result file scouting scheme scouting variables software requirements spare parts cables conductivity cell connectors Mixer M 925 Pump P 901 Pump P 903 tubing
136. standard AKTApurifier system allows the user to enhance already used purification methods and also to develop new more complex methods Optional configurations are selected installed and implemented by the user An optional configuration consists of both hardware components and software instructions To support the process of implementing optional configurations general guidelines regarding installation and operation are given in the separate manual AKTApurifier Optional Configurations User Manual included in AKTApurifier Manual Box Optional configurations are monitored and controlled via methods run by the UNICORN control system in the same way as the AKTApurifier standard configuration Optional configurations supported by AKTApurifier are e Connection of up to 9 motorised multi port valves These valves can be used to accomplish the following functions e Buffer selection e Sample selection e Column selection e Optional functions e On line pH measurement e Connection of a dedicated sample pump e Sample application using a Superloop e Connection of a fraction collector e Connection of an monitor for pH and conductivity measurement AKTApurifier e Connection of an autosampler e Connection of up to four air sensors AKTApurifier User Guide 28 4079 53 Edition AC 133 8 Reference information 8 3 Optional configurations e Connection of external equipment using digital input output signals through the
137. t Check that the distribution plate marking 3 is horizontal Refer to the relevant valve Instructions External leakage Internal leakage Check the tubing connections Tighten or replace if required Internal leakage can be detected at the small hole on the underside of the valve body Internal parts may be worn Change channel plate and distribution plate Refer to the relevant valve Instructions High back pressure Perform cleaning in place according Refer to the relevant valve Instructions Change channel plate and distribution plate Refer to the relevant valve Instructions 7 10 BufferPrep Error symptom Unstable pH Possible cause Action Check the system by running the method without a column or sample If still unstable Check that the correct mixer volume is used Check that the pump is operating correctly Check for a dirty or broken pH electrode Check that the valves are operating correctly Check that the pH is not set too far from the pKa of the buffer components If the pH is stable when running without column or sample check if there is too low concentration of buffer components causing low buffering capacity AKTApurifier User Guide 28 4079 53 Edition AC 113 7 Troubleshooting 7 10 BufferPrep Error symptom Incorrect pH Possible cause Action Collect some eluent Check the pH electrode calibration by measuring the pH of the col
138. t have occurred during transportation If unpacking AKTApurifier UPC continue with AKTApurifier UPC specific 3 Lift AKTApurifier onto the work area using the four strap handles 2 Two people are required to lift the system Do not raise to upright position yet Lay the system on the same side as on the pallet 4 Release the two red straps with the strap handles Allow the straps to remain taped to the plastic cover 5 Pull back the plastic cover 3 to uncover the swivel platform 4 and raise the system to an upright position 6 Pull off the plastic cover to uncover the system 7 Save all the original packing material If for any reason the equipment has to be repacked for transportation or otherwise it is important that the system can be safely packed using the original packing material 16 AKTApurifier User Guide 28 4079 53 Edition AC Installation 2 2 3 2 Unpacking the fluid handling components 1 Turn the system to access the fluid component side of the system 2 Detach the following items by cutting straps and removing red tape Also remove the desiccant bags Note Use pliers Be careful not to cut any capillaries by accident Do not lose any of the capillary marking tags e Valve INV 907 located at the top right side Lift off the valve to remove the protective foam Refit the valve into the top slot in the attachment bracket INV 907 e Mixer M 925 located below
139. t result Gradient linear from BHO B 10 90 2 5 2 Step response test result Step response table RP RIRE Si eg cor eines re Conc B read rected value value interval 100 95 94 96 70 69 71 30 29 31 5 4 6 0 2 5 3 UV response test result UV response table 1 2 3 4 5 Wavelength Value read Baseline Absorbance Allowed nm corrected ratio interval 100 B O0 B value 254 265 254 1 11 1 26 265 265 280 1 26 1 53 280 AkTApurifier User Guide 28 4079 53 Edition AC 39 2 Installation 2 6 Registration form 2 6 Registration form IMPORTANT WARRANTY REGISTRATION INFORMATION Please ensure that this form is completed and returned to Service Administration to register the users equipment under warranty N Co Lu EE Institute Compan is ss ANN BAN ANN d noncent Ades nine E TETEE EE Department loCationss iscssisssscescessescaschascesssscasscuesasessteavieicis icieecsasdccasausctatadesdzsctasezesdisecinactonaxacisaavesesnateescadiaceneasis POSE COG EE Phone Number Tax Number sis nine End U erer seess Lk UE Date of Installation ss Quote Noss hn an Ee Customer Order No InVoice No ss nn ENTER ei Note Fill in serial numbers overleaf 40 AKTApurifier User Guide 28 4079 53 Edition AC 2 6 1 Components AKTApurifier system serial numbers Installation 2 Qty Part Number Description
140. tem At delivery i d 0 5 mm PEEK tubing is installed from the pump to the UV cell and i d 0 25 mm Tefzel tubing from the outlet of the UV cell to the fraction collector if applicable The column is installed either by using the tubing supplied with the column or using pieces of 0 25 mm PEEK tubing cut by the user to suitab AKTApurifier 10 system e length 0 25 mm PEEK tubing blue is supplied with the Tubing Length Tubing i d Tubing o d Material Color Volume of 10 design cm INV 907 0 25 mm 1 16 PEEK Blue 49 ul column G5 40 cm 0 50 mm 1 16 PEEK Orange 19 6 ul UV Frac 0 25 mm 1 16 PEEK Blue 4 9 ul G1 33 cm 0 50 mm 1 16 PEEK Orange 19 6 ul G2 33 cm 0 50 mm 1 16 PEEK Orange 19 6 ul G3 20 cm 0 50 mm 1 16 PEEK Orange 19 6 ul G4 18cm 0 50 mm 1 16 PEEK Orange 19 6 ul H6 16 cm 0 50 mm 1 16 Tefzel Clear 19 6 ul H7 8 cm 0 50 mm 1 16 Tefzel Clear 19 6 ul H8 14cm 0 50 mm 1 16 Tefzel Clear 19 6 ul H9 60 cm 0 50 mm 1 16 Tefzel Clear 19 6 ul W1 W3 100 cm 0 75 mm 1 16 Tefzel Clear 44 2 ul Al B2 170 cm 1 6mm 1 8 Teflon Clear 201 1 ul A2 190 cm 1 6mm 1 8 Teflon Clear 201 1 ul B1 150 cm 1 6 mm 1 8 Teflon Clear 201 1 ul A3 B3 15 cm 1 6 mm 1 8 Teflon Clear 201 1 ul AKTApurifier User Guide 28 4079 53 Edition AC 123 8 Reference information 8 1 System description AKTApurifier 100 In AKTApurifier 100 i d 0 75 mm PEEK tubing green
141. template The Logbook pane shows when the instructions in the method are executed during the run AKTApurifier User Guide 28 4079 53 Edition AC Operation 4 4 7 During a run 4 7 1 Monitoring the run Viewing progress The progress of the method being used can be viewed in detail on UNICORN and the status of certain parameters of the instrument modules can be viewed directly on their front panel displays The System Control module in UNICORN displays the current status of AKTApurifier and can display up to four panes for monitoring different aspects of the run FF Click the Customize panes toolbar button or choose View Panes from the menu to select which panes to display Run Data The Run Data pane displays the current values for selected run parameters To customize the Run Data pane 1 Right click in the Run Data pane and select Properties or select View Properties from the System Control menu 2 Select the run data items to be displayed and click OK Curve Style and Color Flow Scheme Logbook Dun Data Groups RunDataColor Curves Keris Yaris Milnstruments New Group MIConnection Run Status Edit Group Scouting No Acc Volume Delete Group wi Block Volume WAcc Time wi Block Time Flow Pressure o TubeNumber xl Avbryt Help AKTApurifier User Guide 28 4079 53 Edition AC 75 4 Operation 4 7 During a run 76 Curves The curves pane
142. th O and 100 B the run can be started 6 If the pH reading is not acceptable select Correction Factors in the BufferPrep page of the Method Editor Enter the pH deviation at 0 and 100 B For example if the pH has been set to 7 and the actual pH is 7 1 enter 0 1 or if the actual pH is 6 9 enter 0 1 If default correction factors exists add the measured deviation to these factors 7 Save the method Note The new correction factors will only apply while this method is used When a new method is created and a recipe is selected default correction factors will apply To change default correction factors for a recipe see Section 8 5 4 5 2 7 Creating your own recipes Refer to Section 8 5 1 Creating your own recipes for details on how to create recipes or edit existing recipes AKTApurifier User guide 28 4079 53 Edition AC Optional functions 5 5 3 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 anew method as described in Section 4 2 Creating a method 2 When the Run Setup window appears click the Scouting tab Wavelength_1 Wavelength_2 Wavelength 3 BufferValve Al Inlet Pump A Inlet Pump B Inlet Target ConcB Length of Gradient Define other Scouting variables 3 Alist of all the variables will appear Select the variab
143. the distilled water Note Never place the reservoir flask below the level of the pump inlet Connect a male Luer syringe of at least 30 ml to the purge tubing Connect the other end of the purge tubing to the left purge valve at pump module A Turn the purge valve counter clockwise half a turn to open it and slowly draw eluent into the syringe Purge valve When fluid starts to enter the syringe continue to draw a few milliliters before closing the purge valve Check that there is no air left in the inlet tubing Repeat steps 3 to 5 for the other pump heads Testing pressure stability Perform a pressure test to establish that all air has disappeared from the pump heads 1 Connect a column bypass capillary between the injection valve port 1 and the top of the UV cell KTApurifier 10 Manually select pump flow 0 2 ml min and run at 0 B distilled water Check on the pump display that the pressure reading is stable variation less than 0 02 MPa AKTApurifier 100 Manually select pump flow 10 ml min and run at 096B distilled water Check on the pump display that the pressure reading is stable variation less than 0 2 MPa AKTApurifier User Guide 28 4079 53 Edition AC 31 2 2 4 32 Installation Installation test AKTApurifier 10 Manually select pump gradient 100 B length 0 and run with 0 2 ml min distilled water Check on the
144. the loop volume Fill the sample loop carefully with buffer Set the injection valve to position INJECT before taking out the A Column syringe Note Ifthe syringe is taken out Sample when the injection valveis TG in position LOAD self drainage will occur and air enter the sample loop Load the syringe with the required volume of sample Note No more than half 50 of a loop volume of sample should be loaded into the loop Insert the syringe into the injection fill port on the injection valve Set the injection valve to position LOAD Note Do not load the sample before the valve is in position LOAD AKTApurifier User Guide 28 4079 53 Edition AC Operation 4 7 Gently load the syringe contents into the sample loop 8 Leave the syringe in position The sample will be injected onto the column when the valve is switched to INJECT in the method Note If the syringe is taken out before the sample is injected onto the column self drainage can occur and the loop will be emptied Complete filling In this method an excess of sample is used to ensure that the sample loop is filled completely For analytical reproducibility use a sample volume that is 5 times the volume of the sample loop About 2 to 3 loop volumes of sample are required to achieve 95 of maximum loop volume Five loop volumes ensure better precision With complete filling the sample volume can only be changed by changing the loop size Complete
145. thod run 3 The flow path continues from the mixer to the injection valve For AKTApurifier via an online filter AKTApurifier User Guide 28 4079 53 Edition AC System overview 3 Asample loop is connected between ports 2 and 6 on the injection valve The sample loop is filled manually using a syringe anda fill port connected to port 3 in the injection valve After the injection valve the flow is directed through the column the UV cell in the optical unit and the conductivity cell located below the optical unit If a pH flow cell optional is included in the flow path it will be located after the conductivity cell The flow path then continues through the flow restrictor The flow restrictor generates a constant backpressure to eliminate the risk of air bubble formation The flow continues to the outlet valve optional which is used to switch the outlet flow to waste fraction collection or outlet fraction AKTApurifier User Guide 28 4079 53 Edition AC 45 3 System overview 3 2 Columns and tubing 46 3 2 Columns and tubing A wide range of pre packed columns for ion exchange size exclusion hydrophobic interaction reversed phase and affinity chromatography are suitable for use with AKTApurifier A comprehensive list of the recommended pre packed columns is given in Section 8 4 in Recommended tubing and columns together with information on the recommended tubing for each column It is very important to use the cor
146. tion 8 4 Recommended tubing and columns Code no Column name 0 50 0 75 1 0 17 5156 01 HiTrap Q FF 5 ml X 17 5054 01 HiTrap SP FF 1 ml x 17 5157 01 HiTrap SP FF 5 ml X 17 5055 01 HiTrap DEAE FF 1 ml X 17 5154 01 HiTrap DEAE FF 5 ml X 17 5056 01 HiTrap CM FF 1 ml X 17 5155 01 HiTrap CM FF 5 ml X 17 5162 01 HiTrap ANX FF high sub 1 ml x 17 5163 01 HiTrap ANX FF high sub 5 ml X 17 5158 01 HiTrap Q XL 1 ml x 17 5159 01 HiTrap Q XL 5 ml X 17 5160 01 HiTrap SP XL 1 ml X 17 5161 01 HiTrap SP XL 5 ml X 17 5092 01 HiPrep 16 10 Q XL X 17 5093 01 HiPrep 16 10 SP XL X 17 5090 01 HiPrep 16 10 DEAE FF X 17 5091 01 HiPrep 16 10 CM FF X 17 5190 01 HiPrep 16 10 Q FF X 17 5191 01 HiPrep 16 10 ANX FF high sub X 17 5192 01 HiPrep 16 10 SP FF X 17 5181 01 SOURCE 15Q 4 6 100 PE X 17 5182 01 SOURCE 15S 4 6 100 PE X 17 1064 01 HiLoad 16 10 Q Sepharose HP X 17 1066 01 HiLoad 26 10 Q Sepharose HP X 17 1137 01 HiLoad 16 10 SP Sepharose HP X 17 1138 01 HiLoad 26 10 SP Sepharose HP x 142 AKTApurifier User Guide 28 4079 53 Edition AC Size Exclusion Columns Reference information 8 Code no Column name 0 50 0 75 1 0 17 5176 01 Superdex Peptide 10 300 GL x 17 5174 01 Superdex 75 HR 10 300 GL X 17 5175 01 Superdex 200 HR
147. tionation Flowthrough fractionation means that fixed volumes are collected before the elution starts This Ae fractionation method is available in all purification techniques except for size exclusion The parameters are found on the Frac 920 950 Settings Flowthrough Fractionation page in the si i Noy FE Method Wizard Injaetisn 2 1234567 mark Fraction Volume AKTApurifier User Guide 28 4079 53 Edition AC Operation 4 Fixed volume fractionation op Fixed volume fractionation allows you to collect fixed volumes Fraction Volume during the elution The parameters are found on the Elution Injection ales 4567 Fractionation page in the Method Wizard SS Fraction Volume Peak fractionation Peak fractionation means that only the peaks are Peak Fractionation Volume UV collected during the elution There are two ways to bg collect peaks e Each peak is collected in one fraction The peak size is set to a volume Peak Fractionation ae Volume that is larger than the actual peak mark volume UV Start Slope UV End Slope 1 2 e Each peak is collected in several fractions The peak size is set to a volume Peak Fractionation Volume that is smaller than the actual peak volume Peak fractionation is selected on the Elution Fractionation page in the Method Wizard The parameters are found on the Peak Fractionation page Examples on peak fractionation paramet
148. tor UV 900 and the fraction collector e connect a termination plug to the empty UniNet 1 socket Frac 950 only 4 When not using a fraction collector connect a termination plug to the empty UniNet 1 socket in Monitor UPC 900 Monitor UV 900 5 Connect the USB cable to the USB port on CU 950 Note that the CU 950 should be attached to the left side of the system AKTApurifier User Guide 28 4079 53 Edition AC Installation 2 6 Connect the other end of the USB cable to the rear of the computer For connection to and installation on the computer please refer to UNICORN Administration and technical manual 7 All other UniNet 1 cables are connected at delivery 2 3 10 Connecting the mains cables WARNING AkTApurifier must always be used with the protective earth lead of the power cord correctly grounded to earth at the mains outlet WARNING In case of an emergency situation the system mains switch must always be easy to access and the mains plug must always be easy to gt E disconnect A WARNING Only use mains cables delivered or approved by GE Healthcare Q a wh i Jil o itl r 3 SER oo aa f Fraction EE collector 8 optional Ss Se Fuseholder E Ge with Fuse Power To mains socket CU 950 converter grounded LA w eg Fig 2 3 AKT
149. trictor Mixer D G1 G2 H Injection Valve V1 r Pump A Pump B H H H H Al B1 To fraction collector optional Fig 8 6 AKTApurifier flow diagram AKTApurifier User Guide 28 4079 53 Edition AC 121 8 Reference information 8 1 System description G3 4 pH optional Connected between Cond and Flow restrictor Bypass G1 Injection To fraction collector optional Fig 8 7 AKTApurifier UPC flow diagram The G5 capillary is mounted from the factory replacing the column It is used as a bypass when the column is removed from the separation unit e g at system wash Please note that there are two flow restrictors available for the system e FR 902 Mounted from the factory and pre adjusted to give a back pressure of 0 2 MPa over the nominal flow range This flow restrictor is used when a pH cell optional is fitted in the system or when a low pressure column is used s FR 904 Pre adjusted to give a back pressure of 0 4 MPa over the nominal flow range This flow restrictor should be used when working with organic solvents such as in RPC applications to avoid air bubble formation in the flow cell The table below shows the tubing dimensions installed at the factory and their location in the system 122 AKTApurifier User Guide 28 4079 53 Edition AC AKTApurifier 10 Reference information 8 The table below shows the tubing available for AKTApurifier 10 and where they are located in the sys
150. ts Refer to Section System requirements in the UNICORN User Manuals supplied 8 6 6 Network requirements Refer to Section System requirements in the UNICORN User Manuals supplied AKTApurifier User Guide 28 4079 53 Edition AC 161 8 Reference information 8 6 Technical specifications 8 6 7 AKTApurifier component materials The wetted materials of AKTApurifier are listed below v 2 N w 3 N ZS t route 2 lea Joe Ss 3 lz es hr Sole lu ju 2 a 5 6 5 Z2 N un lt x Pump P 900 x X X X X X X X X onitor x x x X UV 900 onitor x x x x X x X UPC 900 Flow X x x X restrictor ixer M 925 x X x INV 907 x Unions X x Connectors Online filter x x Inlet filters x x FFKM perfluororubber PEEK polyetheretherketone PTFE polytetrafluoroethylene ETFE ethylenetetrafluoroethylene CTFE chlorotrifluoroethylene ECTFE ethylenechlorotrifluoroethylene PP polypropylene PVDF polyvinylidenefluoride PE polyethylene 162 AKTApurifier User Guide 28 4079 53 Edition AC Reference information 8 8 7 Chemical resistance guide and chemical compati bility The chemical resistance of AKTApurifier to some of the most commonly used chemicals in liquid chromatography is indicated in the table below The ratings are based on the following assumptions 1 The synergistic effects of the chemical mixtures have not been taken into
151. tyl FF X 17 1355 01 HiTrap Phenyl FF high sub 1 ml x 17 5193 01 HiTrap Phenyl FF high sub 5 ml RF 17 1353 01 HiTrap Phenyl FF low sub 1 ml x 17 5194 01 HiTrap Phenyl FF low sub 5 ml RF 17 1351 01 HiTrap Phenyl HP 1 ml x 17 5195 01 HiTrap Phenyl HP 5 ml RF 17 1359 01 HiTrap Octyl FF 1 m x 17 5196 01 HiTrap Octyl FF 5 m RF 17 1357 01 HiTrap Butyl FF 1 m X 17 5197 01 HiTrap Butyl FF 5 m RF 17 0978 13 HiTrap Butyl S FF 1 ml X 17 0978 14 HiTrap Butyl S FF 5 ml RF 144 KTApurifier User Guide 28 4079 53 Edition AC Reference information 8 Reversed Phase Columns Code no Column name 0 50 0 75 1 0 17 5116 01 SOURCE 5RPC ST 4 6 150 X 17 5068 01 SOURCE 15RPC ST 4 6 100 X 17 1181 01 RESOURCE RPC 1 ml X 17 1182 01 RESOURCE RPC 3 ml X 17 0704 01 URPC C2 C18 SC 2 1 10 x P 17 5057 01 URPC C2 C18 ST 4 6 100 x P AkTApurifier User Guide 28 4079 53 Edition AC 145 8 Reference information 8 4 Recommended tubing and columns 146 Affinity Columns Code no Column name 0 50 0 75 1 0 17 5247 01 HisTrap HP 1 ml X 17 5248 01 HisTrap HP 5 ml X 17 5319 01 HisTrap FF 1 ml X 17 5255 01 HisTrap FF 5 ml X 17 5256 01 HisPrep FF 16 10 X 11 0004 58 HisTrap FF crude 1 ml x 17 5286 01 HisTrap FF crude 5 ml x 17 0920 03 HiTrap IMA
152. ue flow cell design ensures a high signal to noise ratio with a minimal drift and refractive index effects Monitor UV 900 consists of a main unit optical fibers and a choice of two flow cells AKTApurifier User Guide 28 4079 53 Edition AC 129 8 Reference information 8 2 Component description 8 2 3 Monitor UPC 900 Monitor UPC 900 is a high precision on line combined monitor for measurement of UV absorption pH and conductivity in liquid CH O gt chromatography The UPC 900 offers fixed wavelengths of 214 Zn lamp 254 or 280 nm EE Hg lamp fast response high accuracy and reproducibility and flow cells with low dead volumes Additional wavelengths are obtainable using optical filters accessories Monitor UPC 900 consists of a control unit an optical unit with lamp assembly and a choice of two flow cells optical path length 2 mm or 5 mm a conductivity flow cell with integrated temperature sensor and a pH flow cell with pH electrode optional 8 2 4 UV and conductivity flow cells The type of UV flow cell used depends on the sample amount applied and the size of the column AKTApurifier 10 is delivered with the 10 mm cell fitted 2 mm cell optical pathlength 2 S T g 5 mm mm internal volume 2 ul is available as an accessory Ifa A lower detection sensitivity is desired due to output signal limitation the 2 mm flow cell should be used AKTApurifier 100 is delivered with t
153. ume in UNICORN 1 Select System Settings in System Control 2 Select Specials and then FracParameters 3 Enter the delay volume and click OK 128 AKTApurifier User Guide 28 4079 53 Edition AC Reference information 8 8 2 Component description 8 2 1 Pump P 900 Pump P 900 is the collective name for a pump family P 900 is a high performance laboratory pump for use where accurately controlled liquid flow is required It is a low pulsation pump equipped with two pump modules A and B This allows for binary gradients with high pressure mixing A pressure sensor is connected to pump module A left hand pair of pump heads The model installed in AKTApurifier 100 has 100 ml pump heads and is referred to as Pump P 901 in the accessories list P 901 has an operating flow rate range of 0 01 100 ml min in both isocratic and gradient mode and a pressure range of 0 10 MPa 100 bar 1450 psi The model installed in AKTApurifier 10 has 10 ml pump heads and is referred to as Pump P 903 in the accessories list P 903 has an operating flow rate range of 0 001 10 ml min in both isocratic and gradient mode and a pressure range of 0 25 MPa 250 bar 3625 psi 8 2 2 Monitor UV 900 Monitor UV 900 is a multi wavelength UV Vis monitor that uses advanced fiber optic technology to monitor with high sensitivity at up to three wavelengths simultaneously in the wavelength range 190 700 nm The use of fiber optics together with a uniq
154. un 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 AKTApurifier User guide 28 4079 53 Edition AC Optional functions 5 5 4 Extra system pressure measurement For low pressure columns such as HiTrap and HiLoad it is necessary to compensate for the pre column pressure by measuring the pressure in the absence of the column This is achieved by using the following method 1 Set the injection valve INV 907 in position WASTE 2 Run the pump at the mandatory or intended flow rate 3 Make a note of the back pressure on the pump display or in the Run Data pane in UNICORN 4 Add this value to the pressure limit value for the column e g 0 5 MPa for HiLoad or 0 3 MPa for HiTrap The new total unit pressure value measured pressure max column pressure has to be introduced into the UNICORN column list and be defined as a personal column 1 nthe Method Editor select Edit Column list to open the Column List dialog Clicking a column in the list will display its parameters in the field to the right of the box N Q ick Edit to display the Edit Column dialog In the Parameter column enter n the field for Max pressure the new unit pressure limit 0 5 MPa the measured value 3 Click Save as and enter a new name of your column You can choose to save the column globally i e available to all users by checking the Save as
155. uperloop AKTApurifier User Guide 28 4079 53 Edition AC 61 4 Operation 4 4 Sample application 62 44 1 Applying small sample volumes using a sample loop The sample loop should be manually filled before the method is started Preparation Prepare the injection valve as follows 1 Loosely thread the supplied injection fill port screw into valve port 3 2 Insert the supplied injection needle o d 0 7 mm into the injection fill port 3 Tighten the fill port until the nozzle has formed a seal around the needle s tip i e when it feels as if you are penetrating a septum at the end of he injection fill port The seal should allow easy insertion and removal of the needle A Mount the syringe holder on the fill port 5 Ensure that waste tubing is connected to port 4 of the injection valve 6 Mount the sample loop between Pos 1 LOAD ports 2 and 6 of the injection valve 1 Column Note If the syringe is removed before the sample is injected Sample onto the column self syringe drainage can occur and the loop will be emptied A Union Luer female 1 16 male connector is supplied with AKTApurifier and is an alternative to the injection fill port If used the Union Luer connector replaces the injection fill port in port 3 of the injection valve Waste Waste AKTApurifier User Guide 28 4079 53 Edition AC Five sample loop sizes are available Sample loop Catalogue no Loop
156. ves can never be modified renamed or deleted from a result file 80 AKTApurifier User Guide 28 4079 53 Edition AC Operation 4 3 Maximize the Chromatogram window by clicking the square in the upper right corner A All changes regarding the presentation of the curves are done in the Chromatogram Layout window Position the cursor in the Chromatogram window Right click and select Properties or select Edit Chromatogram layout to activate this window Chromatogram Layout 1 xi Curve Style and Color Edit Texts Layout Library Header Curve Names Y Axis X Axis Curve Peak Table Select curves to display Highlight curves to view 703 04 Example Result002 1_Cond 105 Example Result002 1_Cond C106 Example Result002 1_Cone 107 708 Example Result002 1_Pressure 703 Example Result002 1_Flow 10 Example Result002 1_Temp 11 Example Result002 1_Fractions 12 Example Result002 1_Inject 013 Clear Select all IT Apply to all chromatograms Avbryt Help 5 Select the curves to view under Curves The curves are named as Result001 1_ curve where a curve can be for example UV_wavelength pressure etc Click OK at the bottom of the Chromatogram Layout window 6 Tozoom in on the curves click and drag in the chromatogram with the left mouse button A rectangle appears on the screen When you release the mouse button the part within the rectangle will be enlarged You c
157. week Calibrate the pH electrode optional Refer to Section 6 4 in Calibration procedures Inlet filters Check the inlet filters visually and replace them if necessary Pump rinsing solution Change rinsing solution Always use 2096 ethanol as rinsing solution f the volume of rinsing solution in the storage bottle has increased it can be an indication of internal pump leakage Replace the piston seals according to Chapter 4 in Pump P 900 User Manual f the volume of rinsing solution in the storage bottle has decreased significantly check if the rinsing system connectors are mounted properly f the rinsing system connectors are not leaking the rinsing membranes or piston seals may be leaking Replace the membranes and piston seals according to Chapter 4 in Pump P 900 User Manual Cleaning Clean the system see 6 2 Cleaning the system surface Fans Check that all fans are running 102 AKTApurifier User Guide 28 4079 53 Edition AC Maintenance 6 Interval Every month Action Flow restrictor Check that the flow restrictor generates the following back pressures FR 904 0 4 0 05 MPa FR 900 0 2 0 05 MPa Checking the back pressure 1 Disconnect the flow restrictor 2 Connect a piece of tubing approx 1 m i d 1 0 mm to a free port in injection valve V2 Set the valve manually to this port Put the open end in a waste container 3 Run the pump at
158. y by GE Healthcare do not open any covers or replace parts unless specifically stated in the instructions AKTApurifier User Guide 28407953 Edition AC This product complies with the European directives listed below by fulfilling corresponding standards A copy of the Declaration of Conformity is available on request e 2006 95 EC Low Voltage Directive e 2004 108 EC EMC Directive The CE logo and corresponding declaration of conformity is valid for the instrument when it is e used as a stand alone unit or e connected to other CE marked GE Healthcare instruments or e connected to other products recommended or described in this manual and e used in the same state as it was delivered from GE Healthcare except for alterations described in this manual Note The Declaration of conformity is valid only for systems that are marked with the CE logo CE AKTApurifier User Guide 28407953 Edition AC Contents Contents 1 Introduction ET G n ral ssl ESA FA LZ labels urines trente mt ne nf 9 13 Safety 10 LTA Typographicalconv ntiOns ssssss anna nuits 12 2 Installation 2 1 General ssh ont A NEK SN 2 2 Pre requisites ooooooooooooooo 2 3 Installation of AKTApurifier 2 4 Installation test ie Zb TESE TE CO Mentimun amankan amen anna 2 6 Registration forment entit ta 3 System overview 341 Separation LA naa antenne nantes dress 43 3 2 C
159. y the valves Before moving the system disconnect all cables and tubing connected to peripheral components and liquid containers Remove all items from the top of the system Grasp the system firmly by placing the fingers in the gap between the swivel platform and the base of the main unit and lift AKTApurifier User Guide 28 4079 53 Edition AC 7 Troubleshooting 7 1 Faults and actions Troubleshooting 7 This Section lists faults observed with specific monitor curves and specific modules The faults and actions are listed as follows Type Page UV curve 108 Pressure curve 109 Conductivity curve 109 Pump P 900 111 Monitor UV 900 112 Monitor UPC 900 112 Mixer M 925 112 Valve INV 907 113 BufferPrep 113 If the suggested actions do not correct the fault contact the local GE Healthcare service representative AKTApurifier User Guide 28 4079 53 Edition AC 107 7 Troubleshooting 7 2 UV curve 7 2 UV curve Error symptom Possible cause Action Ghost peaks Check that there is no air in the eluent Degas if necessary Clean the system in accordance with Chapter 4 Clean the column in accordance with the column instructions Check that the mixer is functioning properly and that the correct chamber volume is being used Noisy UV signal signal drift or instability The buffer may be impure Check if the signal is still noisy with water There may be

ÄKTApurifier User Guide - GE Healthcare Life Sciences

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&Auml;KTApurifier User Guide - GE Healthcare Life Sciences

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ÄKTApurifier User Guide - GE Healthcare Life Sciences