good Äkta system practice - cytiva
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Good ÄKTA system practiceCue Cards
1. Good ÄKTA system practice 22. Run-Essentials 33. Fraction collector F9 and F9-C operation 44. Comprehensive System Wash and cleaning 55. UNICORNsystemnotifications 66. Troubleshooting 7
6.1 Conductivitysignal–ausefultroubleshootingtool 76.2 Pressurealarms 86.3 Backpressurecontributions 86.4 Fluctuatingpressure 96.5 FluctuatingUVandconductivitysignal, examples and solutions 106.6 CleaningthepHelectrodeandUVflowcell 116.7 Airbubbleinaflowcell 126.8 Brokenconnectortipstuckinvalve 136.9 Tubingguide 13
7. AbbreviationsandReferences 14
Good ÄKTA system practice Cue Cards 29109616 AC2
1. Good ÄKTA system practice
• KeepthesystemandFractioncollectorclean,bothontheoutsideandtheinside.
• Useinletfiltersandfilteredbuffers,preferablydegassedbuffers.
• Foroptimalresults,usebuffers,systemandcolumnsatambienttemperatureduringa
purificationrun.
• Centrifugeorfilterthesamplespriorsampleloading,unlesscolumnsthatcanhandlecrude
samples are used.
• UsetheComprehensiveSystemWashandcleaningregularlyforoptimalperformanceofthe
wholeinstrument,includingFractioncollector.Seepage 5.
• Topreventcross-contaminationandbacterialgrowthintheinstrument,performaSystem
cleaninginplace(SystemCIP)aftereachrun.Seepage 5.
• Limit system exposure to harsh cleaning solutions to maximum 2 hours.
• Fillthesystemwith20%ethanoltominimizebacterialgrowthpriorstorage.
• SetupUNICORN™systemnotificationstoletthesystemremindyouwhenyouractionis
needed.Forexample,letamessageappearonceaweekstating:“Timetochangepump
rinsing solution”. See page6.
ForfurtherdetailsseeÄKTA™ avant User Manual and ÄKTA pure User Manual.
Good ÄKTA system practice Cue Cards 29109616 AC 3
For further details see ÄKTA avant User Manual and ÄKTA pure User Manual.
2. Run-Essentials
Before1. Checkthe20%ethanolpumprinsingsolution.Changeifitappearsopaqueorif
the solvent level in the container has decreased.
2. Checkalltubingtomakesurenotubingisnicked.
3. Makesureallbuffershavethesametemperatureasthesystemtominimizeairbubbleissues.
4. Primeinlets,i.e.fillallusedbufferinletswithliquid.
5. Purgepumpstoremoveairfromthepumpheads.
6. Startaflowandtightenconnectorsifleakageisobserved.
7. CalibratethepHelectrode(ifused).
8. PerformaSystemPreparationruntofilltheentireflowpathwiththerightbufferintothesystem(includingtheFractioncollector).
9. StartaflowtocheckifsignalsfrommonitorslookstableandrealisticIfnot,seep.7-12.
10. WipeofftheFractioncollectorsensors(codereader&dropsyncsensor)withatissueandfilltheFractioncollectorwiththetubes/platesneeded.
11. Attachneededcolumn(s)andsetcorrectcolumnpressure–ifrunningmanually.
DuringWatch out for:
• Overpressure,seepage8
• Fluctuatingpressure,seepage 9
• FluctuatingUVsignal,seepage 10
• Leakage
tighten connectors
• Last tube filled (uponfrac-tionation)
changetubes,seepage 4
Sources of sound during operation:
• Air ventilation
• UVmonitor–whenusing2-3wavelengths
• Pumpmovement
• Mixer
• Changing valve position
• Fraction collector movements
AfterFor short term storage:
1. Empty Fraction collector
2. PerformaComprehen-siveSystemWashand/orcleaning
3. Clean used columns in order to increase their lifetimeandprevent:
• sample contamination
• cross contamination
• protein precipitation
• column clogging
For long term storage:
Iftheinstrumentisnotgoingtobeusedforacoupleofdaysorlonger,cleanasaboveandthenfill
• instrument
• columns
• pHflowcellwithstorage
solution
Good ÄKTA system practice Cue Cards 29109616 AC4
3. Fraction collector F9 and F9-C operationHow to... Steps Outcome
Set Fraction collector content
1. MakesurethereisnorunongoingandtheinstrumentisinstateREADY.
2. Open and close the Fraction collector door.
3. TheFractioncollectorperformsaFull scan.
NOTE: TheFractioncollectorcontentdoesnotresetafterthemethodhasendedunlessthedoorhasbeenopened and closed.
The Fraction collector content is updated andallcassettesarereadytobeused.ToviewthecontentoftheFractioncollector,in the System control module select View:Fraction Collector Content.
Take out fractions during a run or in the middle of scouting or method queue runs
1. Pausetherun.
2. Open the Fraction collector door.
3. Removethedesiredtubes.
4. Close the the Fraction collector door.
5. TheFractioncollectorperformsaQuick scan.
6. PressContinue.
NOTE:Ifdeepwellplateshavebeenremovedtheseneedtobereplacedwithdeepwellplatesofthesametypebeforecontinuingtherun.
Fractionationcontinuesinthenexttube/well.
Take out cassettes during a run or in the middle of scouting or method queue runs
1. Pausetherun.
2. Open the Fraction collector door.
3. Removethedesiredcassette(s).
4. Replaceanyremovedcassette(s)withthesametype(filledorempty).
5. Close the Fraction collector door.
6. TheFractioncollectorperformsaQuick scan.
7. PressContinue.
Fractionation continues into the next tube/well.
Change tubes/plates after Last tube filled message
1. Open the Fraction collector door.
2. Removeallcassette(s)thathasbeenfullyutilized.
3. Replaceanyremovedcassette(s)withthesametype.
4. Close the Fraction collector door.
5. TheFractioncollectorperformsaQuick scan.
6. PressContinue.
Fractionationcontinuesinthefirsttube/wellofthereplacedcassette.Fractionationalwaysstartsinthelowestcassettepositionnumber(1-6).
NOTE:Thefractioncollectorisspeciallydesignedtoautomaticallydetectthecassettespresentinthefractioncollectorbutalsotomaximizetheusageofthetubes/wells.i.e.everytubeinthefractioncollectorshouldbeavailableforfractionation.Thefractioncollectorkeepstrackofwhichtubes/wellshavebeenusedduringtherunsanditisthereforenotpossibletomanuallystartfractionationinaspecificcassettepositionorinaspecificrow.Itishoweverpossibletoselectnexttube/well,skiptwotubes/wells,nextline,ornextcassette.
NOTE: Thereisapossibilitytotryoutotherdeep-wellplatesthentheonesapprovedbyCytiva,providedthatthedeep-wellplatespecificationsarefulfilled.Insomecases,theautomaticcassettescanningmayneedtobedisabledinSystem Control/System Settings/Fraction collector/Cassette configurationtomakethemwork.Makesuretomanually update the Fraction collector content in System settingseverytimethecontentischanged.ForfurtherdetailsseeÄKTAavantUserManualandÄKTApureUserManual(“References”onpage14).
Good ÄKTA system practice Cue Cards 29109616 AC 5
For further details see ÄKTA avant User Manual and ÄKTA pure User Manual.
4. Comprehensive System Wash and clean-ing
Cleanthe syringe port
of the injection valvewith a syringe
ComprehensiveSystem Washand cleaning
Clean thepH electrode
(if present)
Place allinlets in the firstcleaning solution
Start run of:
System PreparationOR
System CIP
method
Follow theinstructions in
UNICORN and moveinlets to the nextcleaning solution
when needed
Whipe off theDispenser head and
its sensor diodewindows (when
Fraction collectoris present)
START
END
Clean thepH valve´s restrictor
and pH positions. Usea flow rate so that the
maximum pressure does not exceed
0.8 MPa
…containsboth manual (green)andautomated(white)procedures.Itisusedtowash/cleanthesystemflowpath,i.e.notanycolumn.
4.1 Create UNICORN method(s)
In all relevant method phases select modules and parts to cover the entireflowpath,forexample:
• AllA&Binlets
• All sample inlets
• Allcolumnpositionstobewashed
NOTE:Makesurenocolumnsareattached.
• Alloutlets(includingoutlettoFractioncollector)
• Injection valve
• Fractioncollector*(ifpresent)
4.2 Method outline forSystem Wash
(UsethepredefinedmethodforSystemPreparation)
4.3 Method outline forSystem Cleaning
(Usethepredefinedmethodfor
SystemCIP)
Method settings
SystemPreparation– Water
SystemPreparation– Buffer
Method settings
SystemCIP– Water
SystemCIP – 0.1-0.5 M NaOH
SystemCIP – Buffer**
SystemCIP – 20%ethanol
* WashofFractioncollectoraccumulatorisincluded.
**Ifwaterisusedinsteadofbuffer,thisstepwilltakelongertime.
Good ÄKTA system practice Cue Cards 29109616 AC6
5. UNICORNsystemnotifications
Automatedmaintenancenotificationsforthesystemcanremindyouwhenyouractionisneeded.Followthestepsbelowinordertosetanewnotification.Inthisexampleanautomaticnotificationforchangingthepumprinsingsolutionisset.
Step Action
1. In the System Controlmodule,selectSystem:Maintenance Manager to open the Maintenance Manager dialog.
2. In the Maintenance Managerdialog,clicktheNew System Notification.Result:TheNewNotificationfieldappears.
3. In the NewNotificationfield:a. Enteranameforthenotification,e.g.“Changepumprinsing
solution.”
b. Selectatimeintervalafterwhichthenotificationwillbeissued, e.g.oneweek.
c. Writeamessageinthemessageinputfieldthatwillbeshownforthemaintenancenotification,e.g.“Pleasechangethepumprinsingsolutionwithafreshlymade20%ethanolsolution.”
d. ClickApplytosavethechangesandapplythenotificationsettings.
4. Handlethemaintenancenotificationwhenthesettimeintervalhasbeenreached:• ClickAcknowledgetoresetthecounterforanewmaintenance
notificationperiod.• ClickIgnoretoclosethedialogwithoutaction.NOTE: TheMaintenanceNotificationwillbedisplayedeachtimetheSystemControlmoduleisopeneduntilthenotificationisacknowledged.
Good ÄKTA system practice Cue Cards 29109616 AC 7
For further details see ÄKTA avant User Manual and ÄKTA pure User Manual.
6. Troubleshooting
6.1 Conductivitysignal–ausefultroubleshootingtool
Conductivity
Gradient concentration
Possibletoidentifywhenthebufferhasbeencompletelyexchanged/washedout.
CheckthegradientperformanceCheckthatthe
conductivity signal is stablebeforesampleinjection
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.
Good ÄKTA system practice Cue Cards 29109616 AC8
For further details see ÄKTA avant User Manual and ÄKTA pure User Manual.
6.2 Pressurealarms 6.3 Backpressurecontributions
Source How to minimize the contribution
Note
TubingKeepthetubingasshortaspossibleandoptimizethei.d
Alargeri.d.willdecreasethebackpressurebutwillhaveanegativeeffectonresolution.See page 13
Inlinefilter Changethefilterregularly.
Theinlinefilterwillpreventparticlesinthesolutionsfromenteringtheflowpathandcol-umn.Withtime,thefilterwillstart to clog and the pressure willincrease.
Buffer/solutionDecreasetheflowratewhenrunninghigh-viscositybuffers/solutions.
Mixingdifferentliquids,e.g.,inagradient,canincreasetheviscosity and result in higher backpressure.
TemperatureDecreasetheflowratewhenrunningatlowtemperature.
Viscosityincreasesatlowertemperature.
Sample
Dilute viscous samples or decreasetheflowrateduringsample application. Remove thesampleinletfilterandifus-ing the system pump to apply sample,bypassthemixer.
Toavoidover-pressure,ÄKTApure and ÄKA avant systems have optional pressure-con-trolledsampleapplication,wheretheflowrateisdecreased as the pressure increases and vice versa.
Column
Clean the column. Seecolumninstructionsforcleaning.
Donotusesmallerbeadsorcolumn diameter than the applicationrequires.
Smallerbeadswillgivehigherresolutionbutalsohigherbackpressure.
Pressure alarm
Changeonline filter
Dirtyonlinefilter
Run aComprehensive System
Wash or change tubingPartly
blockedflow path
Changepumppiston
seal
Worn pumppiston seal
Replace tubing
Nicked,folded or
too narrowtubing
Calibrate pressuresensors
Uncalibrated pressure sensors
De-crease
flow rate
Cold roomrun
Replace/ clean
column
Column
Good ÄKTA system practice Cue Cards 29109616 AC 9
For further details see ÄKTA avant User Manual and ÄKTA pure User Manual.
6.4 Fluctuating pressure
Placebuffers on top
of system*
Buffersplaced below
system
Fluctuatingpressure
Airin pumps
Purge the
pump
Mal-functioning
check valves Clean
check valve
and use buffer inlet filters
Coldbuffers
run at RT
Temperatebuffers
Worn pump pistonseal (if rinsing
solutionincreases) Change
pump piston
seal
Leakage
Tighten connectors andreplaceanyleakingtubing
Air in pump, example
*For150ml/minsystems,bufferscanalsobeplacednexttothesystemifpreferred.
UV
System pressure
Conductivity
Conc. B
IncreasedpulsationonUVsignalduetohigherusageofpumpAinthebeginningofthegradient
AirstuckinpumpA
Diagnose malfunctioning check valves
Inlet check valve diagnostic test, ”air bubble test”:
• Runthepumpat0.5ml/min.
• Lifttheinlettubingafewsecondstoletasmallairbubblein.
• Normalfunction:airbubblemovesforwardandthenstops,andsoon,nobackwardmovement.
• Malfunctionininletcheckvalve:airbubblemovesbackwardsbeforestopping.
After test:Purgethepumpandmakesurethattheairbubbleisnotstuckinthepump.
Good ÄKTA system practice Cue Cards 29109616 AC10
6.5 FluctuatingUVandconductivitysignal,examplesandsolutions
No air bubbles Small air bubles passing UV and cond flow cell
Small air bubles passing UV and cond flow cell
Small air bubles passing UV and cond flow cell
Small air bubles passing UV and cond flow cell
No air bubbles Air bubble flushed out with increased flow rate
Air bubble stuck in cond
UV
Conductivity
System flow
Airin buffers
De-gasbuffers
Temperate or de-gas
buffers
Coldbuffers
run at RT
Make sure the flow restrictor
is inline
Air inUV flow cell
Flow restrictoris offline
Set flowrestrictor
inline
Clean theUV flow cell
Dirt in theUV flow cell
Coldsample
run at RT
Reducethe flow rate
Effectsdue to air
Air spikes in UV signal
Large air segments passing the flow cells
Good ÄKTA system practice Cue Cards 29109616 AC 11
For further details see ÄKTA avant User Manual and ÄKTA pure User Manual.
6.6 CleaningthepHelectrodeandUVflowcell
Component Contaminant Cleaning solution
pHelectrode Salt deposits 0.1MNaOHand0.1MHCl
Lipid deposits Detergent or organic solvent
Proteindeposits 1%pepsinin0.1MHCl
UVflowcell Salt/lipid/proteindeposits
Detergent,suchasDeconex™
Good ÄKTA system practice Cue Cards 29109616 AC12
For further details see ÄKTA avant User Manual and ÄKTA pure User Manual.
6.7 Airbubbleinaflowcell
Large air segments assing UV and conductiv-ity flow cell
Air bubble stuck in cond flow cell
Air compressed int liquid and flushed out due to increased back pressure
UV
Conductivity
System flow
System pressure
YES
YES
YES
NO
NO
Is the flow restrictor
offline?
Set the flow restrictor inline and continue with the run.
Bypass the column and increase the flow rate to 5 ml/min.
Continuewith the run.
Connect an extra small i.d. tubing (see page 13) to the used outlet to increase the backpressure.
Remove the extra tubing and continue with the run.
Does the detector signal
go back to original level?
Does the detector signal
go back to original level?
Remove the extra tubing and flush the flow cells with 100% MeOH. (Make sure that the MeOH is not coming in contact with high salt concentration buffers.)
Good ÄKTA system practice Cue Cards 29109616 AC 13
6.8 Brokenconnectortipstuckinvalve
6.9 Tubingguide
Step Action
1. FindaTorx™T7screwdriver.
2. Firmlypressthetipofthescrewdriverintothebrokentip.
3. Unscrewthebrokenconnectortip.
• Therearemanydifferentsizesandtypesoftubingthatcanbeconnectedtoachromatographysystem.• Tubingwithasmalleri.d.holdslessdelayvolumeandwillthereforegeneratelessdilutionoftheproteinpeak.However,narrowtubingincreasesthesystem
pressure,especiallywhenrunningathighflowrates.• Thetubingandsystemusedshouldmatchtheapplicationneeds.
i.d. Color of tubing10 cm tubing corresponds to
100 cm tubing generates1
Standard tubing with/when
0.13 mm Red 1.3μl 24MPa Generatinghighbackpressure,whichisusefule.g.during
0.25 mm Blue 4.9 μl 1.7MPa troubleshootingandwhen“runningin”anewpumppistonseal
0.50 mm Orange 20μl 0.11MPa RunningatlowflowrateusingÄKTAavant25andÄKTApure25
0.75mm Green 44μl 0.02MPa ÄKTAavant25,ÄKTApure25andÄKTApure150
1.0 mm Beige 78μl 0.007MPa ÄKTA avant 150 and ÄKTA pure 150
1.0 mm Transparent 78μl 0.007MPa OutlettubingforÄKTAavantandÄKTApure
1.6mm Transparent 200μl –2 InlettubingforÄKTAavant25andÄKTApure25
2.9 mm Transparent 660μl –2 InlettubingforÄKTAavant150andÄKTApure150
1 Forwaterat10mL/minandroomtemperature2 Negligiblepressure
Good ÄKTA system practice Cue Cards 29109616 AC14
7. AbbreviationsandReferences
AbbreviationsCIP CleaningInPlace
i.d. inner diameter
RT Room Temperature
ReferencesÄKTALaboratory-scaleChromatographySystemsInstrumentManagementHandbook,29010831
ÄKTApureUserManual,29119969
ÄKTAavantUserManual,29035184
Good ÄKTA system practice Cue Cards 29109616 AC 15
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29109616AC01/2021