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


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.


3. Removethedesiredcassette(s).

4. Replaceanyremovedcassette(s)withthesametype(filledorempty).

5. Close the Fraction collector door.


7. PressContinue.

Fractionation continues into the next tube/well.

Change tubes/plates after Last tube filled message


2. Removeallcassette(s)thathasbeenfullyutilized.

3. Replaceanyremovedcassette(s)withthesametype.

4. Close the Fraction collector door.


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).



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.


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.



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.



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



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.


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



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



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™



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.)


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


7. AbbreviationsandReferences

AbbreviationsCIP CleaningInPlace

i.d. inner diameter

RT Room Temperature

ReferencesÄKTALaboratory-scaleChromatographySystemsInstrumentManagementHandbook,29010831

ÄKTApureUserManual,29119969

ÄKTAavantUserManual,29035184


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Good ÄKTA system practice - Cytiva