Lipid analysis Week 3: GAS LIQUID CHROMATOGRAPHY

Duringthisweek,youwillanalyzethefattyacidcompositionoftheindividuallipidfractionsrecoveredfromtheTLCplate.Gaschromatographyisaverysensitivemethodfortheseparationandquantificationofchemicals,anditisperfectfortheanalysisoffattyacidcomponents.Likeinanyotherchromatographictechnique,separationofcompoundsdependsontheirpartitionbetweenastationaryandamobilephase.Ingaschromatography,themobilephaseisagasthatismovedthroughthecolumn,whilethestationaryphaseisaliquidfilmthatcoatsthecolumnfilling(inpackedcolumns)orthecolumnwall(incapillarycolumns).Hence,thecorrectnameforgaschromatographyis"GasLiquidChromatography",abbreviatedGLC.Compoundsareinjectedontothecolumnandcarriedthroughitbythemobilephase;dependingontheirpartitionintothestationaryphase,theymoveslowerorfaster.Asensitivedetectorisrequiredattheendofthecolumntodetectandquantifythecompoundsastheyleavethecolumn.

Whiletheseparationprincipleissimple,practicalconsiderationsnecessitateratheracomplexexperimentalset-up.CompoundsmustbepresentinthegasphasesothatpartitionbetweenthegaseousmobilephaseandtheliquidstationaryphaseispossIBLe.Thus,GLCmustbecarriedoutattemperaturesabovetheboilingpointofthecompoundstobeseparated.Inpractice,theboilingpointofmanycompounds,includingglyceridesandfreefattyacids,istoohighforGLCanalysis.Therefore,compoundsarefrequentlyderivatized,i.e.chemicallytransformedintoanalogsthataremorevolatile.Inthecaseoflipids,thisisachievedbytransformingfattyacidsintotheirmethylesters.

Ifwewanttoanalyzelipidsotherthanfreefattyacids,wecouldreleasethefreefattyacidsbyhydrolyzingtheglyceridesandthentransformthefreefattyacidsintotheirmethylesters.However,frequentlyitismoreconvenientto"methanolyze"theacylglycerides,i.e.toimmediatelytransformthefattyacidglycerolestersintofattyacidmethylesters.ThiswasachievedinthetransmethylationreactionwithBF₃andmethanol.

ThemobilephaseinGLCisaninertgas(nitrogen);fattyacidmethylestersareretainedmoreorlessbydissolvinginthestationaryphase.Thevolatilityoffattyacidmethylestersandtheirinteractionwiththestationaryphasedependsonthechainlengthanddegreeofdesaturation.Increasedchainlengthleadstolowervolatilityandincreasedretention;hence,methylesterswithshortfattyacylchainscomeoutfirst,whilelongeroneshavehigherretentiontimes.Inordertoavoidextremelylongretentiontimes,onecanincreasethecolumntemperature,thusincreasingthevolatilityofthelongerfattyacidmethylesters.However,shortfattyacidswillthennotseparate.Ifoneisinterestedinachievinggoodseparationofboth,shortandlongfattyacidmethylesters,isisadvantageoustouseatemperatureprogram:Forthefirstpartoftherun,thecolumntemperatureislow;aftertheshortfattyacidshavepassedthecolumn,thetemperatureisgraduallyincreaseduntilallcomponentshaveleft.

Theinteractionofthemobilephasedependsonitschemicalnature.Non-polarphases(asSP-1)justseparateaccordingtochainlength,whilepolarphasesbindpolarcomponentsmorestronglythannon-polarones.Forfattyacids,thenon-polarphaseusedhereseparatesallfattyacidsonlyaccordingtotheirchainlength.Unsaturatedfattyacidsareelutedbeforethesaturatedmoleculewiththesamenumberofcarbonatoms.Atypicalelutionprofilewouldlooklikethis:

Variouspossibilitiesexistforthedetectionoftheelutedcompounds.ThemostcommonlyuseddeviceforfattyacidanalysisisaFlameionizationdetector.WhentheelutedcompoundsentertheFIDdetector,theyarecombustedbyanintenseflameandbrokenupintoionizedfragments,whichcanbequantitativelydetected.

Theintensityofthesignalsdependsofcourseontheamountinjectedontothecolumn.IncapillaryGLC,onlyverysmallvolumescanbecarriedthroughthecolumn.Sinceitisimpracticaltoinject1/20ofamicroliter,forexample,ourinstrumentusesasplitinjectionmode;withasplitratioof1:20,only5%ofthevolumeinjectedactuallyentersthecolumn,while95%arediverted.

FortheexactquantitationwiththeGLCprofileandseparatestandards,itwouldbenecessarytoknownwhatproportionofyourtotallipidfractionwasinjectedontothecolumn.This,however,isimpossible,giventhesmallinjectionvolumeandthevariousstepsinvolvedinthesamplepreparation.Therefore,weaddedpriortothelipidextractionstepamixtureofinternalstandards;thesewereequalamountsofphospholipids,mono-di-andtriacylglycerolandfreefattyacidswhichonlycontainedodd-numberedfattyacidchains;thesedonotnaturallyoccurinanimals,andthusanypeakintheGLCprofilebelongingtoanodd-numberedfattyacidmustcomefromtheinternalstandard.Sincelossesinhandlingetc.areidenticalfornaturallipidsandinternalstandards,wecandirectlydeterminehowmuchofeachlipidclasswaspresentinourlipidextractbysimplycomparingthesumofallevennumberedfattyacidpeakswiththepeakoftheinternalstandard.

Experimentalprotocol

1.Runablankwith1µlofhexane.

2.Run1µloffattyacidmethylesterstandardtocalibratethecolumn.

Name%Rt(example)Rtobserved14:0ME14.9min16:0ME47.5min18:3ME510.2min18:2ME1210.2min18:1ME6010.4min18:0ME310.8min20:0ME314.7min22:1ME517.9min22:0ME118.5min24:0ME322.3min

3.Runyoursamples:PL,MAG,DAG,FFA,TAG.Youmayhavetovarytheamountdependingontheamountoflipidyouhave.StartwithdissolvingthePLfractionin50µlofhexane,andinject1µl.Ifresponseistooweak,concentratedownto10µl.

NotethatPLandDAGcontainthemostlipid;dissolveMAG,FFA,andTAGinlesshexane.Youcanvarytheamountinjectedbetween0.5and2µl.

ExperimentalDetails:

A:Computersetup

1. Turnoncomputerifitisoff.
2. DoubleclickonGPCaliastolaunchtheLabviewdatacollectionprogram,thismustbedoneeachtimeasampleisrunthroughtheGC.
3. Afterashortperiodoftimeagridwillappearwiththefollowingsettings,Ymaxshouldread"100"andModeshouldread"Acquire".
4. Pressthewhitearrow"È",therunbutton,locatedintheupperleft.Agridshouldappearovertheplotandthescaleshouldchange.
5. Adialogueboxwillcomeupwiththedesktopfile/folderhierarchy,select"New".Anewdialogueboxwith"UntitledFolder"highlightedinblackwillappear.
6. ForyourfirstrunyoumustcreateafolderforyourgroupthatwillcontainthefilesofalltheGLCrunscarriedoutbyyourgroup.Typeanamerepresentativeofyourgroupoverthehighlighted"UntitledFolder".Select"Folder"afterthenameisentered.
7. Anotherdialogueboxwillappearwithyourfoldernameintheheader.Select"New".AdialogueboxwillnowappearaskingyoutonamethefileforyourGLCrun.Typeoverthe"UntitledFolder"withyourselectedname.Makethefilenamedescriptivefortherunyouarecarryingoutsoitwillbeeasytodiscernfromtherestlater.Select"File"afternamingtherun.
8. Theprogramwillnowreturntothegridtoallowyoutobegintherun.Itisprobablybesttokeepyourgroupfolderatthedesktoplevelofthehierarchysoitiseasytofind.Justmakesurethatallsubsequentfilesforyourgroupareplacedintothisfolder.

Foreachsubsequentrunyoumustquitthecurrentrun.Todothisclickthesmallboxintheupperleftofthegridandthegridwilldisappear.YoumustnowlaunchtheGPCaliasonthedesktopandgothroughtheaboveprocedureagainexceptforcreatingyourgroupfolder.Justmoveaboutthefolder/filehierarchytofindyourgroupfolder,openit,typeinanameforthenewruntobecarriedoutandselect"New".Theprogramwillmoveyoutothegridtobeginyournextrun.

B:GLCprocedure

TheGLCwillbepreparedforyouruse.PleasedonotattempttoturnontheGLConyourownifitisoff.Donotchangeanyofthesetupvaluesastheymustremainconsistentforallruns.

CheckoutthepaneloftheupperrightsideoftheGLC.Numerousbuttonsforcontrollingfunctionsarelocatedhere.Checkthefollowingsetupvaluesbydepressingthebuttononceandacknowledgingthereadout.

1. InitTemp-shouldread180°C,thisisthestartingoventemperature.
2. InjATemp-shouldread260°C,thisisthetemperatureoftheinjectorport(wherethesampleisinjectedintoandvaporized).
3. DetBTemp-shouldread280°C,thisisthetemperatureofthedetectorattheendofthecolumn.Itquantifiesmaterialspassingoutofthecolumnasvoltagethroughflameionization.
4. FinalTemp-shouldread260°C,thisistheupperoventemperature.
5. InitTime-shouldread1.0,thisistheamountoftimeinminutestheovenrunsattheinitialtemperature.Inthiscasetheovenrunsfor1minuteat180°Candthenstartsincreasingintemperature.
6. Rate-shouldread4°C,thisistherateofincreaseperminuteinoventemperature.
7. FinalTime-shouldread8minutes,thisisthetimetheovenwillholdatthefinaltemperature.Inthiscaseafter21minutestheovenwillreach260°Candrunfor8minutesatthattemperature.
8. Purge-shouldbesettoon.
9. Time-(locatedbesidepurge),shouldbeat0.0min(purgebeginsimmediately).
10. Sig1-outputsignalfordetectorB(FID)detectorweareusing).Oncetherunhasstartedleavethisontofollowthesignaloutputatthedetector.Theoutputvaluehereandonthecomputergridmaybeslightlyoffset,thisisnotaproblem.Avaluearound10.0orlesswillbeyourbaseline,thereADIngonthecomputergridwilllikelybeslightlyhigher.

Ifallthesetupparametershavebeenreachedthegreen"run"light,locatedabovethebuttons,shouldbelitup.Ifthesettingshavenotasyetbeenreachedthered"notready"lightwillbelitup.Duringasamplerunthethreeyellow"oven"lightswillbelitupinturnasthetrialprogresses.

Airandhydrogenareflowingoverthedetectorandhavebeenignitedwithanelectriccoil.Heliumisflowingthroughtheheatedcolumnactingasacarriergasforyoursampletobeinjected.

DonotadjustanyofthevalvesorcontrolstotheleftsideoftheGLC.Thesevalvescontroltheflowofgasesanddangerousconditionsmayoccuriftheflamegoesoutandthegaseskeepflowing.Ifthesignaldropswellbelowthebaselineforaperiodoftimewhilethetrialisrunningbringittotheattentionoftheinstructorortechnician.

C:Sampleinjection

1. 1.Usethe10.0µlsyringeprovidedandlabeledfortheGLConly,donotuseanyothersyringeforyoursamples.
2. Rinsethesyringeoutwithhexaneprovided.Aminimumof10timesimmediatelyafterinjectingasampleandbeforecollectingoneifthefirstrun.Todothisplacetheneedletipintothehexaneandfillthebarrelasfullaspossiblewithhexane.Ejectthehexaneintoawastebeakerprovidedtoevaporate.Repeatthisoverandover.Becarefulnottobendtheneedleplungerasitisdelicate.
3. Placethesyringeintothesampletoberun(hexaneforyourfirstrun).Removeairfromthesyringebyrunningthesampleinandoutofthesyringeuntilbubblesnolongerappearintheneedlebarrel.Loadthesyringetothe1.0µlmark.
4. Sandwichthesample.Afterloadingthesampletothe1.0µlmarkremovethesyringefromthesamplevialanddrawsomeairintotheneedle.Youshouldnowbeabletoseethetotalvolumesandwichedintheneedlebarrelwithaironeithersideofit.Thesamplevolumewillbe2.0µlastheneedleitselfholds1.0µlofsample.
5. Whenreadytorunthesample(donotletitsitintheneedleforlongasitisvolatile),computerissettogoandtheGLCisin"run"mode,placetheneedleintotheinjectorport.PortAlocatedontopofthemachinetothefrontleftofcenter.Theneedlecanbedifficulttoinjectthroughtherubberseptumasthereispressureontheotherside.Driveitdownasstraightaspossibleholdingtheglassbarrel.Pushitinuntilthebarrelbottomsout(gentlythough,becarefulnotthebendtheneedleastheyareexpensive.Thetechniquewillbedemonstratedtoyou.
6. Assoonastheneedleisfullyinsertedyoumustinjecttheentirecontentsinonequickmovement,carefulnottobendtheplunger.ImmediatelyhittherunbuttonontheGLCandusethemousetoclickthestartbuttononthecomputergrid.
7. Removetheneedlecarefullystraightupoutoftheinjectorport,ifleftinitcanaltergasflowpatterns.

Thetrialhasnowstartedandwillrunfor30minutesintotal.Thehexanesolventspikewillappearinapproximately3minutes.TheoutputatfromtheFID(Sig1)willjumpoffscaleasthesolventfrontmovesacrossthedetector.Attheendofarunthecomputerwilldisplaytheentire30minutesofdataoutput,butonly20minutesduringdataacquisition.Theovenwillbeginresettingitselfforthenextrun,the"notready"lightwillbeonduringthistime.

D:Calibratingpeakareas

Youmayimmediatelycalculatetheareasunderthepeaksofyourchromatogramattheendofarunoryoucanwaitandcalluptherunforanalysislater,(seethenextsection).Onceacompletedchromatogramisdisplayedfollowthisproceduretocalculatetheareaundereachpeak.

1. Changethe"mode"from"acquire"to"measure",(modeislocatedtolowerright).
2. Toimprovepeakareameasurementresolutionyoucanselectaspecificareaofthegraphandmagnifyit.Youwillneedtodothisforeachpeak.Todothisclickthecursoronthemagnifyingglass(lowerleft)andholdit,movetotherightandselectthefirstofthethreegraphoptionsgiven,(dottedboxaroundpeaks).Youcannowencircleanyportionofthechromatogrambyclickingyourpositionedcursorandholdingthebuttonwhilemovingitaroundtheareatobemagnified.Trythisafewtimestogetthehangofit.Maketheareatobemeasuredaseasytodiscernaspossible.
3. Toreturntothefullchromatogramclickthe"X"and"Y"axisboxeslocatedtothelowerleft.
4. Whenyouhaveapeakisolatedandmagnifiedforareaintegrationlocatethetworedcursorcrosshairsinthelowercenterarea.Clickandholdoncursor1andselectbringtocenter.Dothesameforcursor2.
5. Nowyoumustchangethecursormodebyclickingtheplussign"+"locatedtothelowerleft.
6. a.Movecursor#1tothelowerandmostleftsideoftheselectedpeakandb.movecursor#2totheupperandmostrightsideoftheselectedpeak.Ifthebaselinechangesslightlyoverthewidthofthepeaksimplyaverageitbyplacingthecursorbaseinthemiddleregion.
7. Whenyouhavesatisfactorilyboxedintheentirepeakwiththe2cursorshitthe"run"button(whitearrowtothetopleft).Theareaunderthepeakwillbegiventoyouintheboxbesidethemodeselection.
8. Clickingonthe"X"and"Y"axisboxestothelowerleftwillreturnyoutofullchromatogramdisplayandyoucanstartoverwithanotherpeakarea.Ittakesalittletimetodisplaytheentireplot.Youmuststartatstep2inthislistforthenextareatobemeasured.

E:Recallingachromatogramthathasbeensaved

1. Ifachromatogramisalreadyopencloseit,clicktheboxlocatedtotheupperleft.
2. LaunchGPCaliasfromthedesktopbydoubleclickingit.
3. Underthemodebox(lowerright)changeto"reload".
4. Presstherunbutton(whitearrowtotopleft).Whenthedialogueboxcomesupmovethroughthehierarchytothefileyouwishtolaunchandselectit.
5. Itwilltakeawhiletoloadthefile(30secto1minute).Adjustthe"X"and"Y"axis(lowerleftcorner)afteritloadstogetthefullgraphinview.
6. Youcannowworkwiththechromatogramasdescribedintheprevioussection.

   (Shouldthedatafilebeunusuable,andyouonlyhaveahardcopyofthechromatogram,youcouldalsoanalyzethepeakareasintheNIHImagesoftware)