Download hplc standard operating procedure

University of Kentucky, Biosystems and Agricultural Engineering Dept HPLC STANDARD OPERATING PROCEDURE Aminex 87‐H and 87‐P analytical columns BAE
1/28/2015
BAEHPLCStandardOperatingProcedure:87Hcolumn
LastUpdated:12December2014
READ & UNDERSTAND THE ENTIRE SOP BEFORE BEGINNING Note:AnSOPcannotcoveralltheneededinformationforproperinstrumentunderstandingandoperation,
orcoverallscenarios;norcananSOPeliminatethenecessityofawareness,attentiontodetailsandmost
importantlyactivethinking!Pleaserefertotheinstrumentmanualsfortheneededbackground
information,reviewliterature,andalwayskeepthemostpowerfulinstrument,thebrain,turnedon.When
indoubtaboutsomething,ask!
HPLC System Information 1. Hardware:Dionex‐ThermoFisherUltimate3000w/auto‐sampler(HPLC#2),P‐680pump,TCC‐100
ColumnCompartment,VariableWavelengthDetector(UV);ShodexR01RefractiveIndexdetector
2. Consumables:AnalyticalcolumnBio‐RadAminexHPX‐87H(order#125‐0140)(ionexclusion);Guard
columnisaBio‐RadMicro‐GuardCation‐Hcartridge(order#1250129);columntypicallyoperatesat
50°C
3. Thecolumntypeisspecifictotheanalytes.The87Hcolumnisahydrogen‐formcolumnusedfor
analysisofcarbohydratesinsolutionwithcarboxylicacids,volatilefattyacids,shortchainfattyacids,
alcohols,ketones,andmanyneutralmetabolicby‐products.Mostoftenusedfororganicacidanalysis,
thiscolumnisalsousefulforfermentationmonitoring,biologicalfluidanalysis,andacetylatedamino
sugarseparations.Seehttp://www.bio‐rad.com/fordetailedinformationonotheranalyticalcolumn
chemistryandpurpose.
HPLC Operating Procedure A. Mobile Phase (also known as working buffer, buffer, or solvent) 1. Flowrateistypically0.4mL/min,constantflowrate(isocratic),typicallya20‐70minuterun–run
timewillbeafunctionoftheelutiontimeoftheanalytesbeingquantified.
2. Mobilephaseis5mMsulfuricacidforthe87Hcolumn.(Fisher#AC124645001)
B. Mobile Phase & Flush Water Preparation for the 87H Column 1. Themobilephaseisdefinedbythecolumntype–differentcolumnsmayhaveadifferentmobile
phasechemistry.
2. Thevolumeofmobilephase/bufferneededwillbecalculatedbyChromeleonafteryou’vesetupthe
run.SeeD.9below.Confirmtheamountneeded,checktheexistingvolumeinthebufferrackon
theHPLC,andmakemoreifneeded.
3. Tomakebuffer,preparea500mMstocksolutionofsulfuricacidusingthecertificateofanalysisto
definethepurityoftheconcentratedacid.Yourcalculationsshouldyieldamixtureofabout2.8mL
ofconcentratedsulfuricacidinto97.2mLofdoubledeionizedwater(DIwater);notethattheDI
waterunithasa0.2micronfilterin‐line.
4. Add10mLofthe550mMstocksolutionto990mLof(DIwater)usingaclean,DIwaterrinsed,glass
1Lgraduatedcylindertoyielda5mMfinalconcentration.
5. Vacuumfilterthebufferfromthegraduatedcylinderthrougha0.2µmx47mmnylonfilter(Pall
Corporation,order#66602)todegasandcleanbuffer;useaglassfunnel/supportsystem(Pall
BAEHPLCStandardOperatingProcedure:87Hcolumn
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Corporation,order#4013or#4012).Storefilteredbufferintoanappropriatecontainer(1,2or4L
glassbottlewith33mmthreadedtop)
6. Thepurityofthebufferandflushwateriscritical–donotskipthefiltrationstep.Poorlyprepared,
contaminatedbuffercandestroyaguardcolumn,and/ortheanalyticalcolumn.
7. Theworkingbufferandflushwatercanbedegassedifdesired;however,theHPLChasade‐gassing
pumpsomanualde‐gassingisnotneeded.
8. ClearlyLABELallsolutionbottleswiththechemicalcompositionofthecontents,thedateprepared,
andthenameofthepreparer.
9. Dionexrecommendsreplacingaqueoussolventsatleasteverytwoweeks.Donot‘topoff’solvent
bottlesduetopotentialbuildupofunwantedcomponents.Donotletsolventbottlesrundry!Ifa
solventbottlelookslikeitwillbeemptybeforetheanalysisiscomplete,stoptherunbetween
samples;seesectionD.1fortherequirementstochangesolventbottles.
C. Standards & Standard Curves NOTE:Standardcurvesaredevelopedbytheoperatorusingcalibrationstandardsandareusedbythe
HPLCsoftwaretoquantifytheconcentrationofanalyteinthesample;thestandardsallowtheoperator
toestablishtheelutiontimeofanalytes,andaccountforcolumndriftorshiftingelutiontimesduring
analysis(anormaloccurrence).CalibrationstandardsMUSTbeusedwitheveryHPLCanalysisevent.
1. Calibrationstandards(preparedsampleswithknownconcentrationsusedtocalibratethe
instrument)shallbepreparedaheadoftimeforeachanalyteofinterest.Thecalibrationstandards
preparedforeachanalyteshallincludenotlessthanatwo(2)lowendconcentrations,two(2)
differentmid‐rangeconcentrations,andahighendconcentrationforatotaloffive;thisrangeof
concentrationvaluesshouldbrackettheexpectedrangeofconcentrationsinthesample.Typically,
theconcentrationsshouldbefrom70%ofthelowestto130%ofthehighestexpectedinthe
sample;notethatblanks(zeroes)donothaveapeak.Astartingpointtodeveloptheappropriate
standardconcentrationswouldbetouseliteraturevaluesfrommultiplesourcestoestablishthe
bracketingconcentrationswithmultiplepointsaroundthetypicalconcentrationsreported.Askthe
HPLCoperatorabouttheinstrumentdetectionlimit(s)foryouranalytes.Note:theconcentrations
neededforthecalibrationsampleswilllikelybedifferentforeachanalyte.
2. Thecalibrationisvalidonlywithintheconcentrationrangeofthestandardsusedandnotbeyondit;
thussamplesthatareoutsidetherangeofconcentrationsinthestandardswillproduceunreliable
results.Agoodunderstandingofthecalibrationprocess,linearregression,andstatisticsisofgreat
value‐seethe“TheoryofCalibration”inChromeleon6.80UserManual,startingonpage109.The
manualisfoundonthereferencelibraryCD(D:\library\manuals\software\CM_680_V30.pdf)
3. Combiningmultipleanalytesintoasinglestandardvialisacceptable,providedthatthereisgood
separationbetweenpeaks;ifindoubt,makeseparatecalibrationsamples.Ensureconcentration
calculationsarecorrect–usetheCertificateofAnalysis(CoA)concentrationsfortheactualsource
bottleratherthanthenominalconcentrationprintedonthelabel.IftheCoAconcentrationisnot
onthesourcebottle,itcanusuallybefoundonthesupplier’swebsite–usetheLotNumberand
catalognumberprintedonthelabelassearchparameters.
4. Allofthecalibrationstandardsshallberunatthebeginning,andthenunknownsamples;providea
blank,aspikedsample,andaduplicateunknownsampleforeachrun,orforevery15to17
unknownsamples.Thethreeextrasamplesareusedforqualitycontrol(QC);these18to20
BAEHPLCStandardOperatingProcedure:87Hcolumn
LastUpdated:12December2014
samplesrepresentagroupofsamples–theunknownsplusthequalitycontrolsamples.Theblank
willbeasamplethatincludesthebackgroundmatrix,i.e.,anyreagentsusedinsamplepreparation,
freshmicrobialmedia,etc.,butdoesnotcontaintheanalytesofinterest[theblankprovidesa
chromatogramofthebackgroundmatrix].Thespikedsampleshouldbepreparedbysplittingan
existingunknownsampleandthenaddingaknownquantityofeachanalytetothesample[the
spikewillshowthebackgroundmatrixeffectontheanalytes,ifanyexists].Theduplicatesampleis
asplitunknownsamplethatcancomefromanyunknown–itisoftentakenfromthesamesample
thatusedinthespikingprocess[theduplicatewillshowinstrumentvariability].Thequality
controlsamplesshallbeevenlyspacedamongtheunknownsamplessothattheQCcheckoccurs
throughoutthegrouping.
5. Provideatleastonecalibrationverificationsamplepergrouportwoperrun.Acalibration
verificationsampleisapreviouslyinjectedcalibrationstandardthatisinjectedagaintocheckthe
accuracyandrepeatabilityofthecalibrationandanalyticalresults.Thesamplemustbedesignated
asa“validationsample”inthe“type”columnwhensettingupthesequencerun–seeD.1.8.
6. Runnotlessthanonestandardfromeachanalytegroupseveraltimesduringtheruninorderto
determineifthepeakisshiftingintimeandtoprovideadditionalcalibrationpointsorcalibration
verificationpoints.Itisgoodpracticetoincludemoreinjectionsofthelowendcalibration
standardstoprovideanaveragecalibrationpointandsome“weight”tobalancetheinfluenceofthe
higherconcentrations[seeitem2aboutthenecessityofunderstandingthecalibration(regression)
process].
7. TheFIRSTcalibrationstandardinjectionisa“waste”(thesystemusesthisfirstinjectionaspriming
forthecolumnsoitneverisasgoodassubsequentinjections);useanyofcalibrationstandards
becauseitwillnotbeincorporatedintothestandardcurve.ThesequencecouldbeS2,S1,S2,S3,
S4,andsoon,wherethefirstS2injectionisthe“waste”injection.Thenextthreeormoreinjections
shallbestandardsloadedinorderofincreasingconcentration.
8. Onestandardfromeachconcentrationgroupingshallberunafterthelastsampleattheendofthe
run.Thetotalnumberofcalibrationstandards,verificationsamples,QCsamples,andunknown
sampleshastobeconsideredtodeterminetheneededbuffervolume–Chromeleonwillcalculate
theneededvolumeafterthesequencehasbeensetup.
9. Foreachgroupofunknownsamplestobeanalyzed,randomlyselectnotlessthanthreebutnot
morethan10samplesandanalyzeusingthepreviouslyselectedstandards.Reviewthe
chromatogramstoensurethatthecorrectanalytes[allthepeaksfromtheunknownsamplesare
accountedfor]andcorrectcalibrationstandardconcentrationshavebeenchosen[alltheunknown
sampleconcentrationsliewithinthecalibrationstandardconcentrations];adjustasneededbefore
analyzingtheremainingsamples.
10. Thebestpracticeistoinjectthesmallestamountpossibleforbothstandardsandunknown
samples;itissuggestedtostartat20microlitersandincreasefromthereifthereisnotasufficient
response–usetheinjectionsdescribedinC.9toassesstheresponseofagivenconcentrationand
injectionvolume–adjustasrequired.
11. Theinjectionvolumewilldeterminehowmuchsample/standardvolumeshouldbeinthevial,e.g.a
100μlinjectionmusthave600μlinthevial.Typically,theHPLCsamplevialscontainatleastone
mL.Iftheavailablesamplevolumeislimited,thereareinserts(250ultotalvolume)thatfitinside
thestandardvials.Ifyou’reusinganinsert,adjusttheinjectionvolumeonthesamplesetuppagein
theHPLCsoftware,andchangeneedleheight.Failuretoadjustthesoftwaresettingforneedle
BAEHPLCStandardOperatingProcedure:87Hcolumn
LastUpdated:12December2014
heightforvialswithinsertswillresultinbrokeninsertsand/orneedlessincetheneedlegoestothe
bottomofthevialwhenextractingthesample.
D. Instrument Preparation & Sample Analysis NOTES:
a. Thefollowingscreenshotsareintheappropriateorderofoperations–NoShortcuts!Themobile
phasemustbeflowingbeforeheatingthecolumnorwarmingtheUVlamp.
b. TheUVdetectorisnotneededwhenanalyzingonlycarbohydrates(sugars)onthe87H
c. BoththeUV&RIdetectorsshallbeusedforfermentationanalyses
d. RefertotheChromeleonsoftwaremanualfordetailedinformationoncreatingnewinstrument
methodsandprocessingmethods
I.
Getting Started 1. Open
Chromeleon
usingthetoolbar
Icon.Itwillopen
tothisscreen:
2. Selectthe
instrumenttabin
thelowerleft
corner,select
UNIVERSITYOFKE
N_1intheleft
pane,andselect
theHometabfor
generalstatus.
BAEHPLCStandardOperatingProcedure:87Hcolumn
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3. Onthepump
moduletab,you
canensurethat
thepumpis
connected,setthe
flowrate,choose
themobilephase
bottle,turnthe
pumpon/off,and
startapurgeto
setupanew
solventbottle.
a. Topurgethelinesofairafterinstallinga
newbottleofmobilephase(airinthelines
isBAD),openthepumpmoduledoorand
turntheknurledbrassknobtwofullturns
(thisbypassesthecolumnandsendsthe
flowdirectlytothewastecontainer).
b. Thenputacheckinthepurgeboxandthiswindow
willpopup.Startthepurge–thepurgeprocess
hasabuiltintimer.Ifthereisstillvisibleairinthe
lines,purgeasecondtime.Oncethetimerhas
elapsed,thepurgebuttonwillgofromgreenback
togray.BesuretoCLOSETHEPURGEVALVE!
BAEHPLCStandardOperatingProcedure:87Hcolumn
LastUpdated:12December2014
c. Nowstartinitialflowthroughthecolumntopreparethesystem
i. Checkthemotoronbox–itwillturngreen
ii. Settheflowrateat0.1mL/minandletitflowfor30minutes–thesystempressure
shouldbestableafter30minutes–ifnot,continueflowat0.1mLuntilpressureisstable.
iii. Settheflowrateat0.2mL/minfornotlessthan15minutes(thisensuresthatthecolumn
isfullbeforeheatingbegins)
4. Gotothecolumntab,
adjustthedesired
temperaturesetpoint
andturnon
“Temperature”.After
thecolumnreaches
thetemperatureset
point,increasethe
flowin0.100mL
increments,wait15‐
20minutes,and
repeat(increaseflow
rate,wait15‐20min)
uptothefinaldesired
flowrate.
5. GototheRITaband
checkthepurgebox;
purgefornotlessthan
5minutestoremove
air,differentsolvents,
etc.UNCHECKTHE
PURGEBOXBEFORE
STARTINGTHE
SAMPLERUN
SEQUENCE!
BAEHPLCStandardOperatingProcedure:87Hcolumn
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6. IfusingtheUV
detector,gototheUV
tabandsetthedesired
wavelengthvaluein
oneofthe4channels
,i.e.,UV1toUV4(the
channelusedis
specifiedinthe
instrumentmethod
usedinasequence).
TurnontheUVlamp;
itwilltakeatleast30
minutesforthelamp
towarmup–system
willnotproceedwith
therununtilthelamphasreachedoperatingtemperature.
7. Gotothesampletab.
Usethedefaulttray
controlsettingsfor
eachcolor(thecolors
representsectionsof
thetray).Setthetray
temperaturetothe
appropriatesetting
foryoursamples,e.g.,
8°Cistypical,
however4°maybe
appropriatefor
sampleswith
microbes,orhigherif
needed.Usethe
“startup”and“inject”defaultvalues.Theinjectionvolumevalue(seeC.9above)willbeoverwritten
bythevalueusedinthesequencewrittenforyoursamples.
BAEHPLCStandardOperatingProcedure:87Hcolumn
LastUpdated:12December2014
8. Nowthatthe
instrumentisready,
youcancreateanew
sequenceoropenan
existingsequencefile
fromthedatapane
thathasthesametype
ofsamplesasyours
andexecutingasave
as“YYYYMMDD_file
descriptor”,e.g.
“20140613_JohnDoe_F
ermentation”.Fora
newsequence,you
Changethevaluesin
“name,type,and
position”columnstomatchyoursamples.Usetheappropriateinjectionvolume(C.9above).Usethe
existinginstrumentandprocessingmethodsunlessyou’vecreatednewones.WhenusingtheUV,
opentheinstrumentmethodandconfirmthatthechannel&wavelengtharewhatyouwant.Select
the“start”dropdownmenuandselect“addsequencetothequeue”.
9. Gotothequeuetaband
selectthe“readycheck”
intherightpane.The
readycheckwill
calculatetheneeded
mobilephase(a.k.a
buffer,solvent)volume
neededanddisplayat
thebottomofthe
windowontheright
side.Ensurethatthere
ismorebufferinthe
bottlethanthe
calculatedvolume;ifnot
seeB.2–B.9;oncethe
buffervolumesupplyisconfirmedthenclickthestartbutton.Therunwillstoponitsownonce
completeandusethe“Smartshutdown”thatisalreadyprogrammed.Removethesamplesand
disposeofthevialsintheproperlocation.
10. Ifthereareerrorsduringyourrun,consultwiththeHPLCoperator,astheactionsrequireddepend
onthenatureoftheproblem.Aftertheerrorisresolved,youcanopenthesequence,changethelast
samplefrom“interrupted”to“idle”andthensavethesequence.Beforerestartingtherun:
a. Gototheconsole,samplertab,selectthewashbufferloopbutton.Thiswillcleantheflowpath
toensurethereisnocrosssamplecontamination.Oncethewashiscomplete(thewashbuffer
BAEHPLCStandardOperatingProcedure:87Hcolumn
LastUpdated:12December2014
loopbuttonwillchangebacktogray),restarttheflowrateat100uLandfollowtheflowrate
incrementalincreaseprocessdiscussedaboveinD.1.4.Oncetheflowrateisbacktothedesired
value,restartthesequence.
II.
Results Quality Control & Data Archival NOTE:Theareaunderthecurveofeachpeakisusedwiththestandardcurvetogeneratea
concentrationvalue.Thus,anyerrorinareadelineationonthechromatogramand/orcalculation
directlyandsignificantlyimpactstheresults.
1. StandardCurves(calibrationcurves)shallbedevelopedfromthestandardsforeachindividualrun
–donotre‐usestandardcurves.ThedefaultregressionoptionsinChromeleonMUSTbereviewed
foreachanalysiseventtoensurethechosenregressionoptionsmakesense.Thetypicalsettings
wouldbe“linearwithoffset”,“averagingallresponsevaluesforeachcalibrationlevelbeforecurve
fitting”,and“noweighting”.IfanyregressioncurvehasanR‐squaredvalueoflessthan0.999,
investigatetheproblemandrepeattheprocesstoachieveanacceptableregression.Seethe
Chromeleon6.80UserManual,startingonpage109.Themanualisfoundonthereferencelibrary
CD(D:\library\manuals\software\CM_680_V30.pdf)itprovidesexcellentbackgroundinformation
oncalibration.
2. Chromeleon’sautomatedtoolcanbeusedtoestablishbaselinesforeachpeak,identifythepeak,
andthedroplinesforadjacentpeaks.However,thesoftwareisonlyastartingpoint–the
chromatogramforeachsampleshallbereviewedmanuallyandcorrectedtoensuretheproper
placementofthelinesdefiningthepeak’sarealboundary–theareadefinitioniscriticaland
requiresintensefocus.
3. Thearealboundarylines(baselinesanddroplines)maybedefinedmanuallyusingtheHPLC
software,theareavaluesexportedtoexcel,regressed,andconcentrationscalculatedwithoutusing
theChromeleonalgorithms.
4. Thedata(chromatogramfile)fromeverysampleanalysiseventMUSTbearchivedinMSexcel
formatonastoragedeviceotherthanthecomputeroperatingtheHPLC.Thedatashallbeexported
onaweeklybasisorasneeded(suchaspriortoaHPLCsoftwareupdate).Thedatainthearchive
MUSTbemaintainedforaperiodof10yearsfromtheanalysisdate.Thedatashallbearchivedin
primaryfoldersbyexperimenter’sname,then(years)andsecondaryfolders(months).TheMS
Excelfilesshallincludeaprimaryidentifierinthename,sotheanalysistypecanbereadily
identified.Thedocumentpropertiesdialogboxisaconvenientplacetoutilizekeywords,etc.,to
facilitateasearchfunctionwithinthearchive.
E. ROUTINE INSTRUMENT MAINTANENCE Note:Refertotheequipmentmanualsforin‐depthmaintenancerequirementsincludingroutineand
periodicmaintenancefortheinstrumentsandanalyticalcolumns.SeetheattachedBio‐RadUse&Care
guideforbothanalyticalandguardcolumns(BRUC).
I.
HPLC Operation & Maintenance Logbook 1. AlogbookshallbemaintainedbytheoperatorforeachHPLCsystem.
2. Thelogbookwillbestoredateachinstrument’slocationandwillbeavailableforinspection
3. Thelogbookentriesshallincludetheinitialsofthepersonmakingtheentry,date,time,andnotes
regardingcolumn(guard&analytical)maintenance,replacement,andperformance,aswellas
BAEHPLCStandardOperatingProcedure:87Hcolumn
LastUpdated:12December2014
pertinentinformationaboutthesamplesbeinganalyzed,alongwithsystemperformance.Allcalls
toDionexandBio‐Radtechnicalsupportshallalsobedetailedinthelog.
II.
Guard & Analytical Columns Note:Theguardcolumnmaintenanceandreplacementfrequencydependsonthesamplesbeing
analyzedandthuscannotbepredicted.Ingeneral,replacementiswarrantedwhenstandardsamples
illustrateachangeinthemeasureddata(resolution)
1. Allcolumnprocedures(installation,start‐up,&maintenance)shallbedonepertheBRUC.
2. Anewguardcolumnshallbeinstalledwhenanewanalyticalcolumnisinstalled.
3. Thetotalsystem,analytical,andguardcolumnpressuresshallbedeterminedatnormalflowrates
whennewlyinstalledandrecordedinthelogbook.RefertotheBRUCfortheprocedureto
determinecomponentpressuresfromsystempressures.
4. Beforeanysamplesareanalyzed,allnewlyinstalledcolumnsshallbetestedandtheresults
comparedtothefactorysuppliedchromatogramtoensurepropercolumnandsystemperformance.
Oncethesuppliedandproducedchromatogramsmatch,samplesanalysiscanbegin.Boththe
supplier‐producedandinvestigator‐producedchromatographsshouldbedatedandtapedintothe
logbook.
5. TheHPLCpressurelimitcontrol(aoperatingsoftwaresetting)shallbesetsothatapressure
increaseof15%greaterthanthepressurefoundin#3(operatingpressure)shallcausethepumps
tostop.
6. Theguardcolumnshallbereplacedifitspressureisgreaterthan150%ofthepressurewhennew.
7. Withahighpressureshutdown,aflush&regenerationcyclewiththeproperregenerationsolvent
shallbecompletedontheanalyticalcolumn.SeetheBRUCtable#2forspecificsand
troubleshooting.
8. TheSystemwillneedtohavebufferrunthroughitfornotlessthan½houronceaweek(ifcolumn
isinplace)whenidlealongwithflushingpumplinewithddwater.
9. Agradualincreaseincolumnpressureisnormalasthecolumnagesandisproportionaltothe
numberofinjections.
10. Lessonslearnedregardingcolumnlife&performanceshallbeincorporatedtothisSOPbythe
operator.
F. SOP MAINTANENCE 1. TheHPLCoperatorshallreviewthisstandardoperatingprocedureevery6monthsandmakeall
neededchanges,andrevisethedateofupdate.
2. Dionex&Bio‐Radtechnicalsupportsolutionstospecificproblemsshallbeincorporatedintothe
SOP.
3. BestpracticeslearnedbytheHPLCoperatorforsamplepreparation,standards,standardranges,
etc.,shallbecommunicatedbytheoperatortothe“owner”oftheSOPforincorporationintothe
appropriateSOP.