Good ÄKTA™ system practice Cue Cards
Contents
1. column positions to be washed NOTE Make sure no columns are attached All outlets including outlet to Fraction collector Injection valve Fraction collector if present Clean the syringe port of the injection valve with a syringe Whipe off the Dispenser head and Clean the its sensor diode pH electrode windows when if present Fraction collector is present Follow the Comprehensive Clean the instructions in System Wash pH valve s restrictor UNICORN and move and pH positions Use inlets to the next and clea aliale a flow rate so that the cleaning solution maximum pressure when needed does not exceed 0 8 MPa Method outline for Method outline for System Wash System Cleaning Use the predefined method for Use the predefined method for System Preparation System CIP Method settings Method settings System Preparation Water System CIP Water Start run of Place all System Preparation inlets in the first OR cleaning solution System Preparation Buffer System CIP 0 1 0 5 M NaOH System CIP method System CIP Buffer System CIP 20 ethanol Wash of Fraction collector accumulator is included If water is used instead of buffer this step will take longer time For further details see KTA avant User Manual and KTA pure User Manual References on page 14 ii Good KTA system practice Cue Cards 29109616 AA 5 5 UNICORN system notifications Autom2. Good AKTA system practice Cue Cards eS ee St rs 6 1 gja 6 2 6 3 6 4 6 5 6 6 6 7 6 8 6 9 Good KTA system practice Run Essentials Fraction collector F9 and F9 C operation Comprehensive System Wash and cleaning UNICORN system notifications Troubleshooting Conductivity signal a useful troubleshooting tool Pressure alarms Backpressure contributions Fluctuating pressure Fluctuating UV and conductivity signal examples and solutions Cleaning the pH electrode and UV flow cell Air bubble in a flow cell Broken connector tip stuck in valve Tubing guide 7 Abbreviations and References Good KTA system practice Cue Cards 29109616 AA Oo oo oo N N WD oO fF U N 10 11 12 13 13 14 1 Good AKTA system practice Keep the system and Fraction collector clean both on the outside and the inside Use inlet filters and filtered buffers preferably degassed buffers For optimal results use buffers system and columns at ambient temperature during a purification run Centrifuge or filter the samples prior sample loading unless columns that can handle crude samples are used Use the Comprehensive System Wash and cleaning regularly for optimal performance of the whole instrument including Fraction collector See p 5 To prevent cross contamination and bacterial growth in the instrument perform a System cleaning in place System CIP after each run See p 5 Limit system exposure to hars
3. TA system practice Cue Cards 29109616 AA 7 6 2 Pressure alarms Change online filter Dirty online filter Comprehensive System Wash or change tubing Replace clean column Partly blocked flow path Column Pressure alarm Worn pump piston seal De Cold room crease run flow rate Nicked folded or too narrow tubing Uncalibrated pressure sensors Calibrate pressure sensors Replace tubing For further details see KTA avant User Manual and KTA pure User Manual References on page 14 6 3 Backpressure contributions Source Tubing Inline filter Buffer solution Temperature Sample Column How to minimize the contribution Keep the tubing as short as possible and optimize the i d Change the filter regularly Decrease the flow rate when running high viscosity buffers solutions Decrease the flow rate when running at low temperature Dilute viscous samples or decrease the flow rate during sample application Remove the sample inlet filter and if using the system pump to apply sample bypass the mixer Clean the column Do not use smaller beads or column diameter than the application requires A larger i d will decrease the back pressure but will have a negative effect on resolution See p 13 The inline filter will prevent particles in the solutions from entering the flow path and column With time the filter will start to clog and the p
4. ated maintenance notifications for the system can remind you Fb Khon when your action is needed Follow the steps below in order to seta new Scheduled maint Change pump rinsing solution notification In this exemple an automatic notification for changing the srt F Enable Event inci i i DO Wet valve 8 pump rinsing solution is set gt aii S ma 6 1 Injection valve ae es 1 Inthe System Control module select System Maintenance Manager oa Recurs every 1 weeks to open the Maintenance Manager dialog no emcees Next notification at 2014 06 03 is 3 Quatemary valve 2 Inthe Maintenance Manager dialog click the New System Notification Result The NewNotification field appears 3 Inthe NewNotification field a Enter a name for the notification e g Change pump rinsing solution Please change the pump rinsing solution with a freshly made 20 ethanol solution Select a time interval after which the notification will be issued e g one week Write a message in the message Input field that will be shown for the maintenance notification e g Please change the pump Cancel rinsing solution with a freshly made 20 ethanol solution Click Apply to save the changes and apply the notification settings 4 Handle the maintenance notification when the set time interval has Maintenance Notification AKTA avant 25 been reached e Click Acknowledge to reset the counter for a new maintenan
5. ce notification period N Name Change pump rinsing solution Click Ignore to close the dialog without action Please change the pump rinsing solution with a freshly made 20 ethanol solution NOTE The Maintenance Notification will be displayed each Notification limit 2014 06 03 time the System Control module is opened until the notification is acknowledged Click Acknowledge to restart the notification counter Good AKTA system practice Cue Cards 29109616 AA 6 6 Troubleshooting 6 1 Conductivity signal a useful troubleshooting tool a lt Conductivity e Gradient concentration Check the gradient performance Check that the conductivity signal is stable before sample injection 60 58 56 54 52 50 48 a6 1 44 42 40 38 36 34 32 30 28 26 24 22 20 Possible to identify when the buffer has been completely exchanged washed out oe d ot oN amp DOW WOH amp D Run 8 27 2013 4 23 09 PM 02 00 Method conductivity signal Resuk JD old 8127 2013 4 23 31 PM 0200 gt 962013 4 24 35 PM 0200 bd 8 27720134 2818PM40200 2 3 4 Note The conductivity signal becomes distorted at higher salt concentration since the ion activity decreases when the concentration increases i e a different curvature than the theoretical one is therefore normal at high salt concentrations For further details see AKTA avant User Manual and AKTA pure User Manual References on page 14 Good AK
6. e air bubble moves backwards For 150 ml min systems buffers can also be placed next to the system if preferred before stopping After test Purge the pump and make sure that the air bubble is not stuck in the pump For further details see chapter on Troubleshooting in KTA avant User Manual and KTA pure User Manual References on page 14 Good KTA system practice Cue Cards 29109616 AA 9 6 5 Fluctuating UV and conductivity signal oReessseSSESSES ESET ESES ERSTE SE Gaa an a a m 8 a 2 ai 3 m 37 d gt 34 33 32 3 2 28 2 7 z EREET TEN LELLI ELT examples and solutions Air spikes in UV signal Small air bubbles passing UV and cond flow cell Large air segments passing UV and cond flow cell Small air bubbles passing UV and cond flow cell w Conductivity w System flow inline Large air segments Air bubble flushed passing UV and out with increased cond flow cell flow rate Make sure the flow restrictor is inline Air in UV flow cell Flow restrictor is offline Dirt in the UV flow cell Clean the UV flow cell Temperate or de gas buffers Cold buffers runat RT Effects arr due to air De gas in buffers TEER Cold sample run at RT Reduce the flow rate Good KTA system practice Cue Cards 29109616 AA 10 6 6 Cleaning the pH electrode and UV flow cell Contaminant pH electrode Salt deposits Lipid deposits P
7. ent is in state READY 2 Open and close the Fraction collector door 3 The Fraction collector performs a Full scan NOTE The Fraction collector content does not reset after the method has ended unless the door has been opened and closed Pause the run Open the Fraction collector door Remove the desired tubes Close the the Fraction collector door The Fraction collector performs a Quick scan Press Continue os a a a a NOTE If deep well plates have been removed these need to be replaced with deep well plates of the same type before continuing the run Pause the run Open the Fraction collector door Remove the desired cassette s Replace any removed cassette s with the same type filled or empty Close the Fraction collector door The Fraction collector performs a Quick scan Press Continue ee ee Open the Fraction collector door Remove all cassette s that has been fully utilized Replace any removed cassette s with the same type Close the Fraction collector door The Fraction collector performs a Quick scan Press Continue nM ge A ie The Fraction collector content is updated and all cassettes are ready to be used To view the content of the Fraction collector in the System control module select View Fraction Collector Content Fractionation continues in the next tube well Fractionation continues into the next tube well Fractionation continues in the first tube well of the replaced cass
8. ette Fractionation always starts in the lowest cassette position number 1 6 NOTE The fraction collector is specially designed to automatically detect the cassettes present in the fraction collector but also to maximize the usage of the tubes wells i e every tube in the fraction collector should be available for fractionation The fraction collector keeps track of which tubes wells have been used during the runs and it is therefore not possible to manually start fractionation in a specific cassette position or in a specific row NOTE There is a possibility to try out other deep well plates then the ones approved by GE Healthcare provided that the deep well plate specifications are fulfilled In some cases the automatic cassette scanning may need to be disabled in System Control System Settings Fraction collector Cassette configuration to make them work Make sure to manually update the Fraction collector content in System settings every time the content is changed For further details see AKTA avant User Manual and AKTA pure User Manual References on page 14 Good AKTA system practice Cue Cards 29109616 AA 4 4 Comprehensive System Wash and cleaning contains both manual blue and white procedures It is used to wash clean the system flowpath i e not any column Create UNICORN method s In all relevant method phases select modules and parts to cover the entire flowpath for example All A amp B inlets All sample inlets All
9. h cleaning solutions to maximum 2 hours Fill the system with 20 ethanol to minimize bacterial growth prior storage E Q Q Q E L Q L E Set up UNICORN system notifications to let the system remind you when your action is needed For example let a message appear once a week stating Time to change pump rinsing solution See p 6 For further details see KTA avant User Manual and KTA pure User Manual References on page 14 Good KTA system practice Cue Cards 29109616 AA 2 2 Run Essentials Before 1 Check the 20 ethanol pump rinsing solution Change if it appears opaque or if the solvent level in the container has decreased Check all tubing to make sure no tubing is nicked Make sure all buffers have the same temperature as the system to minimize air bubble issues Prime inlets i e fill all used buffer inlets with liquid Purge pumps to remove air from the pump heads Start a flow and tighten connectors if leakage is observed Calibrate the pH electrode if used Perform a System Preparation run to fill the entire flowpath with the During Watch out for Overpressure see p 8 Fluctuating pressure see p 9 Fluctuating UV signal see p 10 Leakage gt tighten connectors Last tube filled upon fractionation gt change tubes see p 4 After For short term storage Empty Fraction collector Perform a Comprehensive System Wash and or cleaning Clean used colum
10. ns in order to increase their lifetime and prevent e sample contamination cross contamination protein precipitation e column clogging Sources of sound during operation For long term storage If the instrument is not going to Air ventilation be used for a couple of days or UV monitor when using longer clean as above and then 2 3 wavelengths fill Pump movement Mixer Changing valve position Fraction collector movements right buffer into the system including the Fraction collector Start a flow to check if signals from monitors look stable and realistic If not see p 7 12 Wipe off the Fraction collector sensors code reader amp drop sync sensor with a tissue and fill the Fraction collector with the tubes plates needed e instrument e columns pH flow cell with storage solution 11 Attach needed column s and set correct column pressure if running manually For further details see KTA avant User Manual and KTA pure User Manual References on page 14 Good KTA system practice Cue Cards 29109616 AA 3 3 Fraction collector F9 and F9 C operation How to Outcome Set Fraction collector content Take out fractions during a run or in the middle of scouting or method queue runs Take out cassettes during a run or in the middle of scouting or method queue runs Change tubes plates after Last tube filled message 1 Make sure there is no run ongoing and the instrum
11. re representative for the most current information GE Healthcare Europe GmbH Munzinger Strasse 5 D 79111 Freiburg Germany GE Healthcare UK Limited Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK GE Healthcare Bio Sciences Corp 800 Centennial Avenue P O Box 1327 Piscataway NJ 08855 1327 USA GE Healthcare Japan Corporation Sanken Bldg 3 25 1 Hyakunincho Shinjuku ku Tokyo 169 0073 Japan 29109616 AA 06 2014 14
12. ressure will increase Mixing different liquids e g in a gradient can increase the viscosity and result in higher back pressure Viscosity increases at lower temperature To avoid over pressure KTA pure and KA avant systems have optional pressure controlled sample application where the flow rate is decreased as the pressure increases and vice versa See column instructions for cleaning Smaller beads will give higher resolution but also higher back pressure Good AKTA system practice Cue Cards 29109616 AA 8 6 4 Fluctuating pressure Place buffers on top of system Air in pump example Tighten Buffers connectors and placed below replace system any leaking j Air tubing Leakage in pumps N System pressure Conductivity Conc B Air stuck in pump A HTM mny PATE r smn uiae due to highter usage of pump A in the beginning of the gradient Fluctuating Won pressure pump piston seal if rinsing solution increases Mal functioning check valves Cold buffers and use buffer run at RT inlet filters Diagnose malfunctioning check valves Inlet check valve diagnostic test air bubble test e Run the pump at 0 5 ml min bd e Lift the inlet tubing a few seconds to let a small air bubble in Temperate buffers e Normal function air bubble moves forward and then stops and so on no backward movement l l e Malfunction in inlet check valv
13. rotein deposits UV flow cell Salt lipid protein deposits Cleaning solution 0 1 M NaOH and 0 1 M HCI Detergent or organic solvent 1 pepsin in 0 1 M HCI Detergent such as Deconex For further details see AKTA avant User Manual and AKTA pure User Manual References on page 14 Good AKTA system practice Cue Cards 29109616 AA 11 6 7 Air bubble in a flow cell ahh Conductivity System flow 620 500 480 460 440 420 400 380 360 340 320 300 280 260 240 220 200 180 160 140 120 100 60 60 40 20 o Large air segments nee fe and Air bubble stuck in Air compressed into liquid and flushed out conductivity flow cell cond flow cell due to increased back pressure For further details see AKTA avant User Manual and AKTA pure User Manual References on page 14 System pressure Is the flow restrictor offline Set the flow restrictor Ne inline and continue with the run Bypass the column and increase the flow rate to 5 ml min Does the detector signal go back to original level YES Continue with the run Connect an extra small i d tubing see p 13 to the used outlet to increase the backpressure NO Does the detector signal go back to original level Remove the extra tubing and continue with the run NO Remove the extra tubing and flush the flow cells with 100 MeOH Make s
14. ure Negligible pressure Inlet tubing for AKTA avant 150 and AKTA pure 150 Good AKTA system practice Cue Cards 29109616 AA 13 7 Abbreviations and References Abbreviations CIP Cleaning In Place i d inner diameter RI Room Temperature References KTA Laboratory scale Chromatography Systems Instrument Management Handbook 29 0108 31 KTA pure User Manual 29 0206 55 KTA avant User Manual 29 0351 84 For local office contact information visit www gelifesciences com contact GE Healthcare Bio Sciences AB Bj rkgatan 30 751 84 Uppsala Sweden www gelifesciences com AKTA GE and GE monogram are trademarks of General Electric Company AKTA and UNICORN are trademarks of General Electric Company or one of its subsidiaries Deconex is a trademark of Borer Chemie AG All other third party trademarks are the property of their respective owner Any use of UNICORN is subject to GE Healthcare Standard Software End User License Agreement for Life Sciences Software Products A copy of this Standard Software End User License Agreement is available on request UNICORN 6 2009 2014 General Electric Company 2014 General Electric Company All rights reserved First published June 2014 All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them A copy of these terms and conditions is available on request Contact your local GE Healthca
15. ure that the MeOH is not coming in contact with high salt concentration buffers Good AKTA system practice Cue Cards 29109616 AA 12 6 8 Broken connector tip stuck in valve Step Action 1 Find a torx T7 screwdriver 2 Firmly press the tip of the screwdriver into the broken tip 3 Unscrew the broken connector tip There are many different sizes and types of tubing that can be connected to a chromatography system Tubing with a smaller i d holds less delay volume and will therefore generate less dilution of the protein peak However narrow tubing increases the system pressure especially when running at high flow rates The tubing and system used should match the application needs Color of 10 cm tubing tubing corresponds to 013mm GR 3 ul 100 cm tubing generates 24 MPa 0 25 mm 49 4 1 7 MPa Standard tubing with when Generating high back pressure which is useful e g during troubleshooting and when running in a new pump piston seal 0 50 mm 20 yi 0 11 MPa Running at low flow rate using KTA avant 25 and KTA pure 25 0 75 mm 44 yi 0 02 MPa KTA avant 25 KTA pure 25 and KTA pure 150 1 0 mm 78 0 007 MPa AKTA avant 150 and AKTA pure 150 1 0mm _ Transparent 78 ul 1 6mm Transparent 200 ul 0 007 MPa Outlet tubing for AKTA avant and AKTA pure Inlet tubing for AKTA avant 25 and AKTA pure 25 2 9mm Transparent 660 ul 1 For water at 10 ml min and room temperat
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