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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