Stopped-flow-technique

Stopped-FlowTechniquePerhapsthemostfrequentlyusedrapidkineticstechniquesisstopped-flow.Smallvolumesofsolutionsarerapidlydrivenfromsyringesintoahighefficiencymixertoinitiateafastreaction.Theresultantreactionvolumethendisplacesthecontentsofanobservationcellthusfillingitwithfreshlymixedreagents.Thevolumeinjectedislimitedbythestopsyringewhichprovidesthe“stopped-flow”.Justpriortostopping,asteadystateflowisachieved.Thesolutionenteringtheflowcellisonlymillisecondsold.Theageofthisreactionvolumeisalsoknownasthedeadtimeofthestopped-flowsystem.Asthesolutionfillsthestoppingsyringe,theplungerhitsablock,causingtheflowtobestoppedinstantaneously.Usingappropriatetechniques,thekineticsofthereactioncanbemeasuredinthecell.Traditionallystopped-flowhaslentitselftoUV/Visdetectionmethods–bothbyabsorptionandfluorescencespectroscopyandthesearestillthemostcommonmethodsemployed.TgKScientificofferacomprehensiverangeofstopped-flowequipmentfromsimplestopped-flowaccessoriesthatcanbeusedwithexistingabsorbanceandfluorescencespectrometerstocomplete,self-containedcomputercontrolledstopped-flowsystemswithdedicatedacquisitionandanalysissoftware.Mixingoptionsforstopped-flowincludemulti-mixing(alsoknownasdoublemixingorsequentialmixing)wheremorethantwosolutionsaremixedtogether,variableratiomixingusingdifferentsizesyringesandmicrovolumemixingforapplicationswhereonlyverysmallquantitiesofreactantsareavailable.Theenvironmentofthereagentsisalsoextremelyimportantandsoequipmentisavailableforanaerobicapplications,sub-zerotemperatureandhighpressurestopped-flowapplications.Anextensiverangeofsoftwarepackagesisalsoavailablefortheinterpretationandanalysisofthekineticdata.Inadditiontoequipmentforabsorbanceandfluorescencemeasurements,Hi-Techoffersdedicatedstopped-flowsystemsforcirculardichroism,conductivityandFT-IRmeasurement.Stopped-Flow-AtechniqueforstudyingrapidchemicalkineticsStopped-flowisoneofanumberoftechniquesusedtostudythekineticsofreactionsinsolution.Inthesimplestformofthetechnique,tworeactantsolutionsarerapidlymixedbybeingforcedintoamixingchamber,andthenthroughanobservationcell.Atsomepointintime,theflowissuddenlystopped,andthereactionmonitoredusingasuitablespectroscopicprobe,suchasabsorbance,fluorescenceorcirculardichroism.Thechangeinthespectroscopicsignalasafunctionoftimeisrecorded.Figure1showstheschematicofatypicalstopped-flowapparatus.Thestoppingmechanisminthisexampleisastop-syringe.Theflowfillsthestop-syringe,untiltheplungerhitsthetrigger-switch.Thissimultaneouslystopstheflowandstartsthedataacquisition.Therateconstantswhichdefinethereactionkinetics,canbemeasuredbyfittingthedatausingasuitablemodel.Theperformanceofastopped-flowinstrumentisdeterminedtoalargeextentbythedead-time.Thisisdefinedastheminimumtimeafterthereactanthavemixedthattheobservationstarts.Thedead-timeisessentiallytheageofthereactionasitenterstheobservationcell.Thelimitingfactorinthedead-timeofaparticularstopped-flowapparatusisdeterminedbythedistancebetweenthemixerandthecell,andthefinalvelocityoftheflowattheinstanttheflowisstopped.Anotherfactorwhichcanaffectthedead-timeistheefficiencyofthemixer.Typically,dedicatedstopped-flowinstrumentscanachievedead-timesintheregionofamillisecond.Usingultra-smallobservationcells,deadtimesoflessthan0.5mscanbeachieved.Typicallyhowever,thetrueexperimentallimitationistherateofmixingofsolutionsofthetworeactants.Sequentialmixing(ordoublemixing)isavariationofstopped-flowwheretworeactants(AandB)aremixedinapre-mixerandthenflowintoanageingloop.Afteraspecifiedperiodoftime,thatmixtureisforcedtomixwithathirdreactant(C)andthereactionstudiedaspreviously.Seefigure2foraschematicofthestopped-flowapparatususedinsequentialmixingmode.Syringesofdifferentsizescanbeusedtogethertoobtainmixingratiosotherthansimple1:1mixing.So-calledasymmetricmixingorratiomixingisacommonrequirementinstopped-flowwork.ApplicationsOneareaofparticularinterestatthemomentisthestudyofproteinfolding.Acircular-dichroismorfluorescencestopped-flowspectrophotometersuchastheAppliedPhotophysicsπ*-180ortheChirascanwithSF.3stopped-flowaccessorycanbeusedtostudytherapidfolding/refoldingofproteins.Anapplicationnotewhichdescribessuchastudycanbedownloadedhere.LinkstopageslistingpaperscitingtheuseofAppliedPhotophysics�stopped-flowinstrumentsarelistedbelow.Stopped-FlowatAppliedPhotophysicsAppliedPhotophysicshasacomprehensiverangeofstopped-flowinstruments.Theentry-levelistheRX.2000rapidmixingstopped-flowaccessorywhichcanbeusedwithanyexistingspectrophotometer.ThehighlysuccessfulSX-seriesstopped-flowistheworld�sbestsellingstopped-flow.Recently,stopped-flowhasbeenaddedtotheChirascancircular-dichroism(CD)spectrometer.Stopped-flowisaparticularlyflexibletechniqueandAppliedPhotophysicscansupplystopped-flowsystemsinmanydifferentconfigurations.Ofprimaryimportanceistheabilitytostabilizethetemperature.AllAppliedPhotophysics�stopped-flowinstrumentshavetheabilitytothermostaticallycontrolthereagentsbeforetheyaremixedandtheobservationcell.Additionally,theneedtoworkinananaerobicenvironmentisoftenrequired.AppliedPhotophysicscansupply