sop for use of the sx19 stopped-flow instrument · sop for use of the sx19 stopped-flow instrument...

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SOPforuseoftheSX19Stopped-FlowInstrument

Themaincomponentsofthestoppedflowsystemare:

Alwaysbookthesystembeforeuse.

StartingupandpreparingtheSX19foruse:

1. Turnonthecompressedairsupply.

An8bargassupplypowerthepneumaticramsandstopvalveofthesample-handlingunit.Openthelargelevervalueonthewallandcheckthepressureis8bar.

2. Turnonthewaterbath.

Turnonthewaterbathonthefloorundertheinstrument.Checkthereissufficientwatercoveringtheheatercoil,andtopupwithmQwaterasnecessary(thereisalargeplasticcontainerbythesinkin0.14forthispurpose).(LetKatherineorJoeknowifthereisairinthesamplehandlingunit,whichthewaterbathfillsandcontrols.)

3. Turnonthelamp.

Poweronthelamppowersupplyunitusingtherockerswitchattherear.Thegreenlightonthe“arclampignite”switchshouldilluminate.Waitforatleast30secondsbeforepressingthegreenbuttonfirmlytoignitethelamp.Ensuretheshutterbetweenthelamphousingandthemonochromatorisintheopenposition.

Youcancheckthelampisonsincelightcomesoutoftheslitinthelamphousing:

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Ifthelampfailstoignite,thenwaitfortwominutesbeforepressingthegreenbuttonagain.

4. Turnontheelectronicsunit.

Powerontheelectronicsunitviatheblackrockerswitchonthefrontpanel.Therearethreelightemittingdiodes,onegreenandtwored.Thegreendiodewillflashforashorttimeaftertheunitispoweredon,butshouldthenremainonconstantly.Wheneverthesoftwareisrunningthereddiodeswillflashcontinuously.Itisrecommendedthatiftheinstrumentistobeuseddailytheelectronicsunitisleftpoweredon.

5. PoweronthecomputerandloadthePro-Datasoftware.

Poweronthecomputerandlogon.Thedefaultaccountis“lab”whichhasthepasswordbcssxxxx(seethestickeronthesideofthePC).LoadPro-DataSXusingthedesktopicon:

6. LoadthePro-DataViewersoftwareandsetthefilesavelocationandseednames.

StartthePro-DataViewerprogramusingtheViewericononthePro-DataViewertoolbar:

NavigateinthePro-DataViewerfilebrowsertoyourpreferredsavedirectoryandrightclickonitselectingSetWorkingDirectoryHere.Newdatafileswillnowautomaticallybesavedinthisdirectory:

NowopentheFilesNamesdialogboxbyclickingontheicononthetoolbar:

Enteranameforthenewdatafilesandbaselinefile(whererelevant).Subsequentfilesofeachtypewillbenumberedstartingfromthevaluessetinthewindow:

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Preparingthesamplehandlingunit:

1. Configuretheflowlines.

Thefollowingdescriptionappliestoasingle-mixingstoppedflowexperimentwitha1:1mixingratio.(Seethemanualfordetailsofhowtoperformmorecomplicatedexperiments.)Thesingle-mixingexperimentalwaysutilisestheleft-handdriveramandthereagentsarealwaysloadedintheF(left)andC(right)drivesyringes:

Checkthattheinstrumentisintheconfigurationabovebeforeproceeding.(Thedrivevolumeispre-settoapproximately120μl.Donotadjustthisunderanycircumstances.)

2. Flushthesystemwithwater.

Thestopped-flowunitshouldhavebeenleftfromprevioususewithmQwater/ethanolintheflowcircuitandcell.InanyeventitisbesttoensurethatmQwater/bufferisintheflowcircuitandcellpriortoloadingsample.Todothis:

a)Setthebeigedrivevalvestothe“load”position:

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b)Emptyanycontentsofthedrivesyringesintowastereservoirsyringesbypushingthedrivesyringepistonupwardsintoreservoirsyringes.Alwaysbecarefulwhenhandlingthedrivesyringepistonsastheycanbreak.

c)ReplacethereservoirsyringeswithonescontainingcleanmQwater/buffer.

d)Loadeachdrivesyringewithabout1mlofwater/bufferbypushingdownoneachreservoirsyringepistonwhileatthesametimepullingonthedrivesyringepiston.Takecarenottointroduceairbubblesintotheflow-circuit:oneofthemostcommoncausesofartifactsinstopped-flowdataisthepresenceofairbubbles.Repeattheupanddownmotionafewtimestodevelopyourtechniqueforeliminatingairbubbles*.

*Ingeneral,tominimisetheintroductionofbubblesandtofacilitatetheirremoval,withthedrivevalvesinthe“load”position,shuntliquidsbackandforthgentlythroughthedrivevalveswhilstkeepingtheliquidcompressedfrombothendsoftheflow(byapplyingdownwardpressureonthepistononthereservoirsyringeandupwardpressureonthedrivesyringepiston).Whenloadingdrivesyringesavoidsimplypullingthesyringesdown,tosuckliquidfromthereservoirs,asthiswillencouragecavitationandbubbleformation.Fillthembybothpushingliquidinfromthereservoirsabove,whilepullingthedrivesyringepistonsdown,tokeepapositivepressureinthevalveandsyringecavity.

e)Withthedrivevalvesstillinthe“load”position,raisethedriveramuntilitisincontactwithbothdrivesyringepistons.

f)Nowsetthebeigedrivevalvestothe“drive”position:

g)SelectDriveontheSamplehandlingunitpanel(usingtheDrivefunctionratherthantheAcquirefunctionensuresthattherewillbenodatacollectionandgenerationofadatafile*).

*ItishowevermoreefficienttorunwashesusingAcquire:use<10datapointsand<1stimeframe,tickthepanellabelled“repeats”andenter“10”asthenumberofrepeats.Thefilesgeneratedcaneasilybedeletedafterwards.

h)Doatleast5repeatdrivestoensurethattheflowcircuitandcellcontentshavebeencompletelydisplacedbythecleanwater.

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Beforeexecutingadrivealwayscheckthatthedrivevalvesareinthe“drive”position(notthe“load”position)andthedriveramisincontactwiththesyringeplungers.Failuretodosomayresultindamagetotheflowcircuit.

Stopped-FlowAbsorbanceKinetics

1. SelectAbsorbancemode.

OntheSignalpanelofthePro-DatamainwindowselectAbsorbancefromthedropdownlist.EnsurethatAutoHVisticked:

2. Selectkineticsmode,settimebaseandpoints.

EnsurethatKineticsmodeisselectedontheSequencerpanel:

SelectExternalfromthedropdownlistintheTriggerpaneltoactivatestopped-flowoperation:

OntheTimebasepanelchooseanappropriateacquisitiontimebasedontheexpectedtimescaleofthereaction.ThePointsshouldnormallybesettoavaluebetween400and1000datapoints.

3. Selectmonochromatorwavelengthandslitwidth.

OntheMonochromatorpanelentertheselectedwavelength:

Usingthedialsonthefrontofthemonochromatorsettheslitwidthstogivetherequiredabsorbancesignal.

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4. Zerothedetector.

Filltheopticalcellwithasuitablereferencematerial(e.g.waterorothersolvent).ClicktheReferencebuttonintheSignalpaneltozerotheabsorbancedetector.

5. Loadthesamples.

Samplesmaynowbeloadedintothedrivesyringes.Atleast1.3mLisnecessaryforacompleteseriesofapprox.25drives.

Donottopush/pullthedrivesyringepistonswhiletheyareinthe“drive”positionafterloading.Equalizingthepistonlevelsshouldonlybedoneinthe“load”position,otherwisethesamplefromonesyringe(i.e.enzyme)iseasilypushedintotheothersyringe(i.e.substrate).Thereactionwillthenoccurprematurelyinthedrivesyringesandyouwillhavetodiscardthecontaminatedmaterial,andthenwashandloadthesyringesagainusingfreshsamples.

6. Acquiredata.

Ensurethatthedrivevalvesareinthe“drive”positionandclicktheAcquirebuttontoperformadriveandcollectthedata.Thefirstfewdriveswillconsistofbufferandthendilutedsample.Bythefourthorfifthdrivecleansampleshouldbemixedwitheachdriveandresultsshouldbeconsistent.

Absorbancemeasurementswillnowbeinunitsofabsoluteabsorbancesincewaterwasusedwhenmeasuringthereference.Itispossibletomeasurethereferencewithsomethingotherthanwaterinthecell,inwhichcasethemeasurementswillnowberelativeabsorbance.Thismaybeconvenientwhenmeasuringsmallabsorbancechangesonsignificantbackgrounds.Collectingareferencewhenonlyonereagent(oftencolourless)ispushedintothecellisonetypicaloptionasisreferencecollectiontothechemicalendpointofthechemicalreactionunderinvestigation.

Whenyouhavefinishedyourexperiments:

1. QuitthesoftwareandsaveyourdatatoacleanUSBdrive.2. Poweroffthecomputer.3. Poweroffthelamp.4. Powerofftheelectronicsunit.5. Turnoffthewaterbath.

Cleaningtheobservationcellandflowcircuitafteruse:

1. Flushthedrivesyringes.

Withthebeigevalvesinthe“load”position,pushupthedrivesyringestoemptythemasfaraspossible(takingcareastheyarefragile).ReplacethereservoirsyringeswithonescontainingmQwaterandflushthedrivesyringeswithwater.Replacethereservoirsyringeswithonescontaining2%Hellmanex(providednexttotheinstrument;asupplyofneatHellmanexisunderthefumehoodandmQwaterisintheplasticcontainerbythesinkin0.14ifthissolutionrunsout).Flushthedrivesyringeswithatapprox.5mLofthiscleaningsolution.

2. Washthecellandstopsyringe.

Thecleaningsolutioncanbepushedthroughthecell,stopsyringeandassociatedcircuitryusingrepeateddrivesasdescribedonpages4-5(putthevalvesinthe“load”positionandthedriveramincontactwithbothdrivesyringepistons,thenputthevalvesinthe“drive”positionandperformrepeatedsingleDrives,orAcquirewithrepeats).However,thisisunlikelytofullyreplacetheliquidinthestopsyringe.Toflushthestopsyringe,cellandassociatedcircuitrymorecompletelyitisnecessarytoperformmanualdrives.Todothis,putthedrive

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valvesinthe“load”positionandmovethedriveramsoitisincontactwiththesyringeplungers,thenputthevalvesinthe“drive”position.

Next,turnoffthepressureusingthelargevalvehandleonthewall,andthenreleaseresidualpressurebydepressingthein-lineredbuttonbehindthesample-handlingunit:

Identifythestopvalve,whichwillbeinthe“inject”position(facingforwards):

Turnitbyhand180°clockwise(“waste”position;itwillonlyturnifthereisnopressureleftinthesystem)andemptythestopsyringemanuallybypushingitupwardsbeforereturningthestopvalvetoitsnormalposition.Whiledoingthis,takegreatcarenotto(a)movethestopassembly,whichisonlyweaklyfixedtothesamplehandlingunit,or(b)touchthecoppertriggerplate.FreshmQwater/buffercanthenbeintroducedintothedrivesyringes(“load”position)andflushedthroughtheflowcircuitmanually(byraisingthedriveramupwardsagainstthedrivesyringepistonsinthe“drive”position).Thestopsyringewillfill,andcanbeemptiedagainbyturningthestopvalveto“waste”.Thisprocedurecanberepeated(e.g.x5)untiltheuserisconfidentthatthecircuitiscleanandalsofreefromairbubbles.

Washthestopsyringeusingthemanualdriveproceduredescribedabove.Repeatapprox.5timesuntilyouareconfidentthatthecircuitisclean.Leaveitfilledwith80:20water:ethanolwhennotinuse.

3. Finalwash.

Finishbyreplacingthereservoirsyringeswithonescontainingcleanpurifiedwaterand20%ethanol.Flushthedrivesyringeswiththissolutionandleavethemfilledwith80:20water:ethanolwhennotinuse.

4. Turnofftheairsupply.

Turnoffthecompressedairsupplyonthewall,andreleasetheresidualpressurebydepressingtheredin-linebuttonbehindthesample-handlingunit.

5. Tidyup!

Pleasekeeptheareaaroundtheinstrumentcleanandclear.Re-fillemptybottlesandwipeupanyspillsimmediately.

KatherineStottandJoeMaman,January2019