biosystems engineering hplc standard operating procedure sop... · the purity of the buffer and...

UniversityofKentucky,BiosystemsandAgriculturalEngineeringDept

HPLCSTANDARDOPERATINGPROCEDURE

Aminex87Hand87Panalyticalcolumns

BAE1/28/2015

BAEHPLCStandardOperatingProcedure:87HcolumnLastUpdated:12December2014READ&UNDERSTANDTHEENTIRESOPBEFOREBEGINNINGNote:AnSOPcannotcoveralltheneededinformationforproperinstrumentunderstandingandoperation,orcoverallscenarios;norcananSOPeliminatethenecessityofawareness,attentiontodetailsandmostimportantlyactivethinking!Pleaserefertotheinstrumentmanualsfortheneededbackgroundinformation,reviewliterature,andalwayskeepthemostpowerfulinstrument,thebrain,turnedon.Whenindoubtaboutsomething,ask!

HPLCSystemInformation

1. Hardware:DionexThermoFisherUltimate3000w/autosampler(HPLC#2),P680pump,TCC100ColumnCompartment,VariableWavelengthDetector(UV);ShodexR01RefractiveIndexdetector

2. Consumables:AnalyticalcolumnBioRadAminexHPX87H(order#1250140)(ionexclusion);GuardcolumnisaBioRadMicroGuardCationHcartridge(order#1250129);columntypicallyoperatesat50C

3. Thecolumntypeisspecifictotheanalytes.The87Hcolumnisahydrogenformcolumnusedforanalysisofcarbohydratesinsolutionwithcarboxylicacids,volatilefattyacids,shortchainfattyacids,alcohols,ketones,andmanyneutralmetabolicbyproducts.Mostoftenusedfororganicacidanalysis,thiscolumnisalsousefulforfermentationmonitoring,biologicalfluidanalysis,andacetylatedaminosugarseparations.Seehttp://www.biorad.com/fordetailedinformationonotheranalyticalcolumnchemistryandpurpose.

HPLCOperatingProcedure

A. MobilePhase(alsoknownasworkingbuffer,buffer,orsolvent)

1. Flowrateistypically0.4mL/min,constantflowrate(isocratic),typicallya2070minuterunruntimewillbeafunctionoftheelutiontimeoftheanalytesbeingquantified.

2. Mobilephaseis5mMsulfuricacidforthe87Hcolumn.(Fisher#AC124645001)

B. MobilePhase&FlushWaterPreparationforthe87HColumn

1. Themobilephaseisdefinedbythecolumntypedifferentcolumnsmayhaveadifferentmobilephasechemistry.

2. Thevolumeofmobilephase/bufferneededwillbecalculatedbyChromeleonafteryouvesetuptherun.SeeD.9below.Confirmtheamountneeded,checktheexistingvolumeinthebufferrackontheHPLC,andmakemoreifneeded.

3. Tomakebuffer,preparea500mMstocksolutionofsulfuricacidusingthecertificateofanalysistodefinethepurityoftheconcentratedacid.Yourcalculationsshouldyieldamixtureofabout2.8mLofconcentratedsulfuricacidinto97.2mLofdoubledeionizedwater(DIwater);notethattheDIwaterunithasa0.2micronfilterinline.

4. Add10mLofthe550mMstocksolutionto990mLof(DIwater)usingaclean,DIwaterrinsed,glass1Lgraduatedcylindertoyielda5mMfinalconcentration.

5. Vacuumfilterthebufferfromthegraduatedcylinderthrougha0.2mx47mmnylonfilter(PallCorporation,order#66602)todegasandcleanbuffer;useaglassfunnel/supportsystem(Pall

BAEHPLCStandardOperatingProcedure:87HcolumnLastUpdated:12December2014

Corporation,order#4013or#4012).Storefilteredbufferintoanappropriatecontainer(1,2or4Lglassbottlewith33mmthreadedtop)

6. Thepurityofthebufferandflushwateriscriticaldonotskipthefiltrationstep.Poorlyprepared,contaminatedbuffercandestroyaguardcolumn,and/ortheanalyticalcolumn.

7. Theworkingbufferandflushwatercanbedegassedifdesired;however,theHPLChasadegassingpumpsomanualdegassingisnotneeded.

8. ClearlyLABELallsolutionbottleswiththechemicalcompositionofthecontents,thedateprepared,andthenameofthepreparer.

9. Dionexrecommendsreplacingaqueoussolventsatleasteverytwoweeks.Donottopoffsolventbottlesduetopotentialbuildupofunwantedcomponents.Donotletsolventbottlesrundry!Ifasolventbottlelookslikeitwillbeemptybeforetheanalysisiscomplete,stoptherunbetweensamples;seesectionD.1fortherequirementstochangesolventbottles.

C. Standards&StandardCurvesNOTE:StandardcurvesaredevelopedbytheoperatorusingcalibrationstandardsandareusedbytheHPLCsoftwaretoquantifytheconcentrationofanalyteinthesample;thestandardsallowtheoperatortoestablishtheelutiontimeofanalytes,andaccountforcolumndriftorshiftingelutiontimesduringanalysis(anormaloccurrence).CalibrationstandardsMUSTbeusedwitheveryHPLCanalysisevent.1. Calibrationstandards(preparedsampleswithknownconcentrationsusedtocalibratethe

instrument)shallbepreparedaheadoftimeforeachanalyteofinterest.Thecalibrationstandardspreparedforeachanalyteshallincludenotlessthanatwo(2)lowendconcentrations,two(2)differentmidrangeconcentrations,andahighendconcentrationforatotaloffive;thisrangeofconcentrationvaluesshouldbrackettheexpectedrangeofconcentrationsinthesample.Typically,theconcentrationsshouldbefrom70%ofthelowestto130%ofthehighestexpectedinthesample;notethatblanks(zeroes)donothaveapeak.Astartingpointtodeveloptheappropriatestandardconcentrationswouldbetouseliteraturevaluesfrommultiplesourcestoestablishthebracketingconcentrationswithmultiplepointsaroundthetypicalconcentrationsreported.AsktheHPLCoperatorabouttheinstrumentdetectionlimit(s)foryouranalytes.Note:theconcentrationsneededforthecalibrationsampleswilllikelybedifferentforeachanalyte.

2. Thecalibrationisvalidonlywithintheconcentrationrangeofthestandardsusedandnotbeyondit;thussamplesthatareoutsidetherangeofconcentrationsinthestandardswillproduceunreliableresults.Agoodunderstandingofthecalibrationprocess,linearregression,andstatisticsisofgreatvalueseetheTheoryofCalibrationinChromeleon6.80UserManual,startingonpage109.ThemanualisfoundonthereferencelibraryCD(D:\library\manuals\software\CM_680_V30.pdf)

3. Combiningmultipleanalytesintoasinglestandardvialisacceptable,providedthatthereisgoodseparationbetweenpeaks;ifindoubt,makeseparatecalibrationsamples.EnsureconcentrationcalculationsarecorrectusetheCertificateofAnalysis(CoA)concentrationsfortheactualsourcebottleratherthanthenominalconcentrationprintedonthelabel.IftheCoAconcentrationisnotonthesourcebottle,itcanusuallybefoundonthesupplierswebsiteusetheLotNumberandcatalognumberprintedonthelabelassearchparameters.

4. Allofthecalibrationstandardsshallberunatthebeginning,andthenunknownsamples;provideablank,aspikedsample,andaduplicateunknownsampleforeachrun,orforevery15to17unknownsamples.Thethreeextrasamplesareusedforqualitycontrol(QC);these18to20


samplesrepresentagroupofsamplestheunknownsplusthequalitycontrolsamples.Theblankwillbeasamplethatincludesthebackgroundmatrix,i.e.,anyreagentsusedinsamplepreparation,freshmicrobialmedia,etc.,butdoesnotcontaintheanalytesofinterest[theblankprovidesachromatogramofthebackgroundmatrix].Thespikedsampleshouldbepreparedbysplittinganexistingunknownsampleandthenaddingaknownquantityofeachanalytetothesample[thespikewillshowthebackgroundmatrixeffectontheanalytes,ifanyexists].Theduplicatesampleisasplitunknownsamplethatcancomefromanyunknownitisoftentakenfromthesamesamplethatusedinthespikingprocess[theduplicatewillshowinstrumentvariability].ThequalitycontrolsamplesshallbeevenlyspacedamongtheunknownsamplessothattheQCcheckoccursthroughoutthegrouping.

5. Provideatleastonecalibrationverificationsamplepergrouportwoperrun.Acalibrationverificationsampleisapreviouslyinjectedcalibrationstandardthatisinjectedagaintochecktheaccuracyandrepeatabilityofthecalibrationandanalyticalresults.ThesamplemustbedesignatedasavalidationsampleinthetypecolumnwhensettingupthesequencerunseeD.1.8.

6. Runnotlessthanonestandardfromeachanalytegroupseveraltimesduringtheruninordertodetermineifthepeakisshiftingintimeandtoprovideadditionalcalibrationpointsorcalibrationverificationpoints.Itisgoodpracticetoincludemoreinjectionsofthelowendcalibrationstandardstoprovideanaveragecalibrationpointandsomeweighttobalancetheinfluenceofthehigherconcentrations[seeitem2aboutthenecessityofunderstandingthecalibration(regression)process].

7. TheFIRSTcalibrationstandardinjectionisawaste(thesystemusesthisfirstinjectionasprimingforthecolumnsoitneverisasgoodassubsequentinjections);useanyofcalibrationstandardsbecauseitwillnotbeincorporatedintothestandardcurve.ThesequencecouldbeS2,S1,S2,S3,S4,andsoon,wherethefirstS2injectionisthewasteinjection.Thenextthreeormoreinjectionsshallbestandardsloadedinorderofincreasingconcentration.

8. Onestandardfromeachconcentrationgroupingshallberunafterthelastsampleattheendoftherun.Thetotalnumberofcalibrationstandards,verificationsamples,QCsamples,andunknownsampleshastobeconsideredtodeterminetheneededbuffervolumeChromeleonwillcalculatetheneededvolumeafterthesequencehasbeensetup.

9. Foreachgroupofunknownsamplestobeanalyzed,randomlyselectnotlessthanthreebutnotmorethan10samplesandanalyzeusingthepreviouslyselectedstandards.Reviewthechromatogramstoensurethatthecorrectanalytes[allthepeaksfromtheunknownsamplesareaccountedfor]andcorrectcalibrationstandardconcentrationshavebeenchosen[alltheunknownsampleconcentrationsliewithinthecalibrationstandardconcentrations];adjustasneededbeforeanalyzingtheremainingsamples.

10. Thebestpracticeistoinjectthesmallestamountpossibleforbothstandardsandunknownsamples;itissuggestedtostartat20microlitersandincreasefromthereifthereisnotasufficientresponseusetheinjectionsdescribedinC.9toassesstheresponseofagivenconcentrationandinjectionvolumeadjustasrequired.

11. Theinjectionvolumewilldeterminehowmuchsample/standardvolumeshouldbeinthevial,e.g.a100linjectionmusthave600linthevial.Typically,theHPLCsamplevialscontainatleastonemL.Iftheavailablesamplevolumeislimited,thereareinserts(250ultotalvolume)thatfitinsidethestandardvials.Ifyoureusinganinsert,adjusttheinjectionvolumeonthesamplesetuppageintheHPLCsoftware,andchangeneedleheight.Failuretoadjustthesoftwaresettingforneedle


heightforvialswithinsertswillresultinbrokeninsertsand/orneedlessincetheneedlegoestothebottomofthevialwhenextractingthesample.

D. InstrumentPreparation&SampleAnalysisNOTES:

a. ThefollowingscreenshotsareintheappropriateorderofoperationsNoShortcuts!ThemobilephasemustbeflowingbeforeheatingthecolumnorwarmingtheUVlamp.

b. TheUVdetectorisnotneededwhenanalyzingonlycarbohydrates(sugars)onthe87Hc. BoththeUV&RIdetectorsshallbeusedforfermentationanalysesd. RefertotheChromeleonsoftwaremanualfordetailedinformationoncreatingnewinstrument

methodsandprocessingmethods

I. GettingStarted

1. OpenChromeleonusingthetoolbarIcon.Itwillopentothisscreen:

2. Selectthe

instrumenttabinthelowerleftcorner,selectUNIVERSITYOFKEN_1intheleftpane,andselecttheHometabforgeneralstatus.


3. Onthepump

moduletab,youcanensurethatthepumpisconnected,settheflowrate,choosethemobilephasebottle,turnthepumpon/off,andstartapurgetosetupanewsolventbottle.

a. Topurgethelinesofairafterinstallinga

newbottleofmobilephase(airinthelinesisBAD),openthepumpmoduledoorandturntheknurledbrassknobtwofullturns(thisbypassesthecolumnandsendstheflowdirectlytothewastecontainer).

b. Thenputacheckinthepurgeboxandthiswindowwillpopup.Startthepurgethepurgeprocesshasabuiltintimer.Ifthereisstillvisibleairinthelines,purgeasecondtime.Oncethetimerhaselapsed,thepurgebuttonwillgofromgreenbacktogray.BesuretoCLOSETHEPURGEVALVE!


c. Nowstartinitialflowthroughthecolumntopreparethesystemi. Checkthemotoronboxitwillturngreenii. Settheflowrateat0.1mL/minandletitflowfor30minutesthesystempressure

shouldbestableafter30minutesifnot,continueflowat0.1mLuntilpressureisstable.iii. Settheflowrateat0.2mL/minfornotlessthan15minutes(thisensuresthatthecolumn

isfullbeforeheatingbegins)

4. Gotothecolumntab,adjustthedesiredtemperaturesetpointandturnonTemperature.Afterthecolumnreachesthetemperaturesetpoint,increasetheflowin0.100mLincrements,wait1520minutes,andrepeat(increaseflowrate,wait1520min)uptothefinaldesiredflowrate.

5. GototheRITabandcheckthepurgebox;purgefornotlessthan5minutestoremoveair,differentsolvents,etc.UNCHECKTHEPURGEBOXBEFORESTARTINGTHESAMPLERUNSEQUENCE!

BAEHPLCStandardOperatingProcedure:87HcolumnLastUpdated:12December20146. IfusingtheUV

detector,gototheUVtabandsetthedesiredwavelengthvalueinoneofthe4channels,i.e.,UV1toUV4(thechannelusedisspecifiedintheinstrumentmethodusedinasequence).TurnontheUVlamp;itwilltakeatleast30minutesforthelamptowarmupsystemwillnotproceedwiththerununtilthelamphasreachedoperatingtemperature.

7. Gotothesampletab.Usethedefaulttraycontrolsettingsforeachcolor(thecolorsrepresentsectionsofthetray).Setthetraytemperaturetotheappropriatesettingforyoursamples,e.g.,8Cistypical,however4maybeappropriateforsampleswithmicrobes,orhigherifneeded.Usethestartupandinjectdefaultvalues.Theinjectionvolumevalue(seeC.9above)willbeoverwrittenbythevalueusedinthesequencewrittenforyoursamples.

BAEHPLCStandardOperatingProcedure:87HcolumnLastUpdated:12December20148. Nowthatthe

instrumentisready,youcancreateanewsequenceoropenanexistingsequencefilefromthedatapanethathasthesametypeofsamplesasyoursandexecutingasaveasYYYYMMDD_filedescriptor,e.g.20140613_JohnDoe_Fermentation.Foranewsequence,youChangethevaluesinname,type,andpositioncolumnstomatchyoursamples.Usetheappropriateinjectionvolume(C.9above).Usetheexistinginstrumentandprocessingmethodsunlessyouvecreatednewones.WhenusingtheUV,opentheinstrumentmethodandconfirmthatthechannel&wavelengtharewhatyouwant.Selectthestartdropdownmenuandselectaddsequencetothequeue.

9. Gotothequeuetabandselectthereadycheckintherightpane.Thereadycheckwillcalculatetheneededmobilephase(a.k.abuffer,solvent)volumeneededanddisplayatthebottomofthewindowontherightside.Ensurethatthereismorebufferinthebottlethanthecalculatedvolume;ifnotseeB.2B.9;oncethebuffervolumesupplyisconfirmedthenclickthestartbutton.TherunwillstoponitsownoncecompleteandusetheSmartshutdownthatisalreadyprogrammed.Removethesamplesanddisposeofthevialsintheproperlocation.

10. Ifthereareerrorsduringyourrun,consultwiththeHPLCoperator,astheactionsrequireddependonthenatureoftheproblem.Aftertheerrorisresolved,youcanopenthesequence,changethelastsamplefrominterruptedtoidleandthensavethesequence.Beforerestartingtherun:a. Gototheconsole,samplertab,selectthewashbufferloopbutton.Thiswillcleantheflowpath

toensurethereisnocrosssamplecontamination.Oncethewashiscomplete(thewashbuffer


loopbuttonwillchangebacktogray),restarttheflowrateat100uLandfollowtheflowrateincrementalincreaseprocessdiscussedaboveinD.1.4.Oncetheflowrateisbacktothedesiredvalue,restartthesequence.

II. ResultsQualityControl&DataArchival

NOTE:Theareaunderthecurveofeachpeakisusedwiththestandardcurvetogenerateaconcentrationvalue.Thus,anyerrorinareadelineationonthechromatogramand/orcalculationdirectlyandsignificantlyimpactstheresults.1. StandardCurves(calibrationcurves)shallbedevelopedfromthestandardsforeachindividualrun

donotreusestandardcurves.ThedefaultregressionoptionsinChromeleonMUSTbereviewedforeachanalysiseventtoensurethechosenregressionoptionsmakesense.Thetypicalsettingswouldbelinearwithoffset,averagingallresponsevaluesforeachcalibrationlevelbeforecurvefitting,andnoweighting.IfanyregressioncurvehasanRsquaredvalueoflessthan0.999,investigatetheproblemandrepeattheprocesstoachieveanacceptableregression.SeetheChromeleon6.80UserManual,startingonpage109.ThemanualisfoundonthereferencelibraryCD(D:\library\manuals\software\CM_680_V30.pdf)itprovidesexcellentbackgroundinformationoncalibration.

2. Chromeleonsautomatedtoolcanbeusedtoestablishbaselinesforeachpeak,identifythepeak,andthedroplinesforadjacentpeaks.However,thesoftwareisonlyastartingpointthechromatogramforeachsampleshallbereviewedmanuallyandcorrectedtoensuretheproperplacementofthelinesdefiningthepeaksarealboundarytheareadefinitioniscriticalandrequiresintensefocus.

3. Thearealboundarylines(baselinesanddroplines)maybedefinedmanuallyusingtheHPLCsoftware,theareavaluesexportedtoexcel,regressed,andconcentrationscalculatedwithoutusingtheChromeleonalgorithms.

4. Thedata(chromatogramfile)fromeverysampleanalysiseventMUSTbearchivedinMSexcelformatonastoragedeviceotherthanthecomputeroperatingtheHPLC.Thedatashallbeexportedonaweeklybasisorasneeded(suchaspriortoaHPLCsoftwareupdate).ThedatainthearchiveMUSTbemaintainedforaperiodof10yearsfromtheanalysisdate.Thedatashallbearchivedinprimaryfoldersbyexperimentersname,then(years)andsecondaryfolders(months).TheMSExcelfilesshallincludeaprimaryidentifierinthename,sotheanalysistypecanbereadilyidentified.Thedocumentpropertiesdialogboxisaconvenientplacetoutilizekeywords,etc.,tofacilitateasearchfunctionwithinthearchive.

E. ROUTINEINSTRUMENTMAINTANENCENote:Refertotheequipmentmanualsforindepthmaintenancerequirementsincludingroutineandperiodicmaintenancefortheinstrumentsandanalyticalcolumns.SeetheattachedBioRadUse&Careguideforbothanalyticalandguardcolumns(BRUC).

I. HPLCOperation&MaintenanceLogbook1. AlogbookshallbemaintainedbytheoperatorforeachHPLCsystem.2. Thelogbookwillbestoredateachinstrumentslocationandwillbeavailableforinspection3. Thelogbookentriesshallincludetheinitialsofthepersonmakingtheentry,date,time,andnotes

regardingcolumn(guard&analytical)maintenance,replacement,andperformance,aswellas


pertinentinformationaboutthesamplesbeinganalyzed,alongwithsystemperformance.AllcallstoDionexandBioRadtechnicalsupportshallalsobedetailedinthelog.

II. Guard&AnalyticalColumnsNote:Theguardcolumnmaintenanceandreplacementfrequencydependsonthesamplesbeinganalyzedandthuscannotbepredicted.Ingeneral,replacementiswarrantedwhenstandardsamplesillustrateachangeinthemeasureddata(resolution)1. Allcolumnprocedures(installation,startup,&maintenance)shallbedonepertheBRUC.2. Anewguardcolumnshallbeinstalledwhenanewanalyticalcolumnisinstalled.3. Thetotalsystem,analytical,andguardcolumnpressuresshallbedeterminedatnormalflowrates

whennewlyinstalledandrecordedinthelogbook.RefertotheBRUCfortheproceduretodeterminecomponentpressuresfromsystempressures.

4. Beforeanysamplesareanalyzed,allnewlyinstalledcolumnsshallbetestedandtheresultscomparedtothefactorysuppliedchromatogramtoensurepropercolumnandsystemperformance.Oncethesuppliedandproducedchromatogramsmatch,samplesanalysiscanbegin.Boththesupplierproducedandinvestigatorproducedchromatographsshouldbedatedandtapedintothelogbook.

5. TheHPLCpressurelimitcontrol(aoperatingsoftwaresetting)shallbesetsothatapressureincreaseof15%greaterthanthepressurefoundin#3(operatingpressure)shallcausethepumpstostop.

6. Theguardcolumnshallbereplacedifitspressureisgreaterthan150%ofthepressurewhennew.7. Withahighpressureshutdown,aflush&regenerationcyclewiththeproperregenerationsolvent

shallbecompletedontheanalyticalcolumn.SeetheBRUCtable#2forspecificsandtroubleshooting.

8. TheSystemwillneedtohavebufferrunthroughitfornotlessthanhouronceaweek(ifcolumnisinplace)whenidlealongwithflushingpumplinewithddwater.

9. Agradualincreaseincolumnpressureisnormalasthecolumnagesandisproportionaltothenumberofinjections.

10. Lessonslearnedregardingcolumnlife&performanceshallbeincorporatedtothisSOPbytheoperator.

F. SOPMAINTANENCE1. TheHPLCoperatorshallreviewthisstandardoperatingprocedureevery6monthsandmakeall

neededchanges,andrevisethedateofupdate.2. Dionex&BioRadtechnicalsupportsolutionstospecificproblemsshallbeincorporatedintothe

SOP.3. BestpracticeslearnedbytheHPLCoperatorforsamplepreparation,standards,standardranges,

etc.,shallbecommunicatedbytheoperatortotheowneroftheSOPforincorporationintotheappropriateSOP.

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