21/07/2016 TipsandTricksfortheLab:ColumnChoices::Education::ChemistryViews

TipsandTricksfortheLab:ColumnChoices
Author:SarahMillar
PublishedDate:03Julho2012
Copyright:WileyVCHVerlagGmbH&Co.KGaA,Weinheim

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Magazine:Bestof2014

Columnchromatographyisacommonlyusedpurificationtechniqueinlabsacrosstheworld.Donerightitcan
simplyandquicklyisolatedesiredcompoundsfromamixture.Butlikemanyaspectsofpracticalchemistry,thequickand
efficientsettingupandrunningofacolumnissomethingthatcantakeyearstomaster.Herewepresentsomeofthetips
andtricksofthetradetohelpyouoptimizealltheexperimentalparameterstogetthebestseparationofyourcompounds.

Columnsaregenerallyusedinorganicandinorganiclabstoremoveunreactedstartingmaterialorisolateonedesired
productfromarangeofbyproductsafterareactionhasbeenperformed.Todothis,themixedsampleispasseddowna
verticalglasstubepackedwithsilicaoraluminaandcollectedinsmallportions,orfractions,attheend.Thevarious
componentsofthesampleareseparatedintotheirdifferentcompoundtypesthroughinteractionwiththesolventandthe
silica.Polarcompoundswillinteractwiththesilicamorestronglythannonpolaronessowillcomeoffthecolumn,orelute,
afternonpolarcompounds.Whenasamplecontainscompoundswithsimilarpolarity,theseparationbetweenthencanbe
smallandrecoveringallofyourcleansamplecanbecomeachallenge.

Whilecorrectlypackingyourcolumnisarguablythemostimportantexperimentalfactor,thereremainseveralotherfactors
thatcanbeoptimizedtohelpyouachievethebestseparationpossibleintheshortestamountoftime.Hereweguideyou
throughhowtochoosethebestsolventsystemforyoursample,andlookathowmuchsilicayouwillneedandinwhich
sizecolumn.

ChoosingaSolventSystem
Thisisthemostdifficultchoicetomakeandthemostimportant.Ineveryorganicchemistrylabtherearearangeof
solventstochoosefrom.Sowheredoyoubegin?

Columnchromatography,particularlyflashchromatography,isnormallycarriedoutwithamixtureoftwosolventsasthe
mobilephase:onepolar,onenonpolar.Occasionally,asinglesolventcanbeusedoramixtureofthreesolventsisneeded.

Manypeoplehavetheirfavoritesolventssystemsandwillautomaticallystartwiththeseandadjustthemixasneeded.
CommonsolventsandsolventsystemsaregiveninTab.1.

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Table1.Commonsolventsandsolventsystemsforcolumnandflashchromatography.

Togetagoodseparationbetweenyourproductandanyimpuritiesand,where
possible,touseaslittlesolventaspossible,thekeyistotestpotentialsolvent
systemsbeforesettingupyourcolumn.Todothis,youwillneedtorunthinlayer
chromatography(TLC)usingglassoraluminiumsheetscoatedwithsilica(the
aluminiumsheetsarecheaper,soaremorecommon).

ToRunaTestTLC

Youwillneed:

TLCsheet

Scissors/Stanleyknife/boxcutter

Pencil

Glasscontainersuchasasmallbeakerwithwatchglassorpetridishasalid

Solvent(s)tobetested

CapillaryTLCspotterormicropipette(see:HowtoMakeaCapillaryTLCSpotter)

Tweezers

UVlamporpotassiumpermanganatediptovisualize

Method:

CuttheTLCsheetintostripsapproximately2cmx7cm.
Drawapencillineontheshortside,approximately0.5cmfromthebottom.Donotusepenastheinkwilldissolveinthe
organicsolventandseparateout,obscuringorcontaminatingyourresults.
Pourthesolvent(s)tobetestedintotheglasscontainer.Thesolventshouldbe23mmdeepsothatthespottedsample
andthelinearenotsubmerged.ThisensuresthesampledoesnotdissolveintothesolventandtraveluptheTLCplate
withthesolvent.
Useacapillaryspotterormicropipettetospotsomeofyoursampleontotheline:Touchthespotterorpipettetothesilica
TLCplategentlyandallowcapillaryactiontodrawthesampledownontotheplate.Itisbettertotouchthespottertothe
TLC23timesforshortamountsoftime(>1s)thantoholditagainsttheplateforalongertime.Theresultingspotshould
be12mmindiameter.
StandtheTLCstripinthecontainerwiththesolvent(s).

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Waitandcheckregularly.DonotmoveordisturbtheplateortheTLCcontaineras
thiswillcauseripplesinthesolventandirregularprogressofthesolventfrontupthe
plate.
Whenthesolventfrontis0.51cmfromthetopoftheplate,carefullyremovethe
platefromthecontainerwithtweezers.
Allowthesolventontheplatetoevaporate,andthenvisualizetheplatebyusing
eitheraUVlamporpotassiumpermanganatedip.
Markinpencilwherethespotsareastheinitiallyintensecolorsfromthe
permanganatedipcanfadeovertimeanditishardtomanveuvertomeasureaRf
value(seebelow)underaUVlamp.

Youcannowdeterminewhetheryoursamplecomponentswillseparateinthe
selectedsolvent.Ifyougetpoorseparation,chooseanothersolventsystemandtry
again.

WhatisGoodSeparation?/WhatAmILookingForonmyTLC?

Theendresultshouldbeasolventsystemthatmovesthetargetcompoundabout1/3ofthetotalsolventpath(Rfvalue
between0.25and0.35,Fig.1).

Figure1.TypicalTLCplatelayoutanddefinitionofRfvalue.

AsolventsystemthatgivesanRfbetween0.25and0.35isrecommendedasitgivesthesampletimetoequilibrateonthe
columnandtherebybefullyresolvedintoallitscomponentparts.Don'tbetemptedtouseasolventsystemwithahigher
Rfespeciallyifthisistheonlysolventsystemyouhavetestedasafastelutioncanresultinanimpuritybeingmasked
byanotherspot(Fig.2).

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Figure2.Choicebetweenfastelution(left)andmissingahiddenimpurity(right).

WhileitispossibleforaspottohaveaRfthatistoohigh,theoppositeisalsotrueifthedesiredcompoundisoneofthe
laterelutingspots.Hereitispossibleforthespottobetooslowmoving(lowRf).Fig.3showsthedesiredspotbarely
movesoffthebaselinewhiletheimpuritiesraceahead.Youmaybetemptedtousethissolventsystemtoavoidthe
potentialcontaminationthatcomeswithaworseseparationbutahigherRfforyourproduct.Thiswillnormallyprovetobea
mistake.Thelongeryourcompoundspendsonthecolumn,themorechanceithastodegradeandthelessproductyouwill
recover.TryingtoisolateacompoundwithalowRfvaluewillalsotakealongtimeandalotofsolvent.Inthiscase,itis
bettertoattemptaslightlymessierseparationandusemoresilica(seebelow).

Figure3.Cleanseparation(left)versusfasterelution(right)whenproductissecondspot.

Whenyouhavefoundasolventsystemthatgivesreasonableseparation,youshouldtakeafewmomentstolookatthe
shapeofthespots.Ideally,thespotsshouldberoundanddistinctfromeachother(Fig.4,1).Spotswithlongtailsona
TLCplatecantranslatetowidebandsandpossiblytooverlappingcompoundsontheactualcolumn.Ifyouhavetried
severalsolventsystemsandcannotfindonethatgivesperfectlyroundspots,don'tworry.Aslongastheseparation
betweenthespotsisgood(Fig.4,2),youwillstillgetgoodseparationonthecolumn.Insituation3inFig.4,itisstill
possibletogetacleansampleofthetopcompound,forcharacterizationforexample,butyouwilllosesomeofyour
productasittailsintothefollowingbandonthecolumn.Thisisnotrecommendediftheyieldisimportant.

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Figure4.PossiblespotshapesonaTLCplate.

OtherFactorstoConsiderWhenChoosingaSolventSystem
Youwillsometimesfindthattwoormoresolventssystemsgiveadequateseparationofyourcompoundsoryouwillsee
peoplerunacolumnwithasolventsystemthatgivesadequate,butnotthebestseparationontheTLCplate.Sowhat
affectedtheirdecision?

Therearetwootherfactorstoconsider:priceandboilingpoint.

Price

Iftwosolventsystemsgivesimilarlygoodseparation,thecheapestofthenonhalogenatedsolventsshouldbeused.
Typically,thiswillresultinahexanes:ethylacetatemixture.Ifyouareunsureoftherelativecostofasolvent,agoodrule
ofthumbistolookatthesizeofthecontainer.Cheapersolventsaregenerallypurchasedinlargerquantitiesthantheir
moreexpensivecounterparts.

Itisworthnotingherethatsomecheaphexanesorpetroleumethers,especiallythoseinplasticcontainers,oftencontain
saturatedhydrocarbonsthatarehardtoremoveandcanmakeinterpretationofNMRspectradifficult,especiallyinthe1
1.5ppmrange.Inthesecases,thesolventcanbedistilledpriortouse(timeconsuming),oryoucanuseabettergrade
solvent(moreexpensive).

BoilingPoint

Theargumentforchoosingbetweentwosolventswithsimilarseparationanddifferentboilingpointsissimple:Always
chosethelowerboilingsolvent.Youwillsaveyourselfalotoftimeandeffortinthelongrunashigherboilingsolventswill
takelongertoevaporateandrequireheatingorreducedpressuretoremovewhenyoutrytorecoveryourcompoundfrom
solution.

ChoosingtheCorrectSizeColumn
Somewouldarguethatthisistheleastimportantfactorwhensettingupacolumn.Indeed,intheoryitispossibletousea
verythincolumnwithalonglengthofsilicaorathickcolumnwithshorterlengthofsilicatoequaleffect(Fig.5).

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Figure5.Choiceofcolumnsizeislessimportantthantheamountofsilicaused.

However,inpractice,mostpeoplewillchooseacolumnsizethatallowsthemtofillcolumnapproximatelyonethirdtoa
halffullofsilicanotincludingthesolventreservoir(Fig.6).This,ofcourse,dependsonhowmuchsilicayouwillbe
using.

Figure6.Roughguideforchoiceofcolumndiameter.

HowMuchSilicaDoINeed?
Howmuchsilicayouneedisdeterminedbyhowmuchofyoursampleyouhavetoseparate.Theratioofsilicatosample
doesnothavetobeexact,butshouldbeintherightareatopreventwasteofbothtimeandresources.Itisalsoadvisable
tomeasurethesilicabyweightratherthanvolumeasthedensityofsilicacanvarydependingontype.

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Theotherfactoraffectingtheamountofsilicarequiredishoweasyordifficulttheseparationisgoingtobe.Thisis
determinedbyrunningTLCaspartoftheprocessofchoosingasolvent(seeabove).
Thehardertheseparation,thelargerthesilicaweighttosampleweightratiowillbe.GoodguidelinesaregiveninTab.2.

Table2.Guidelinesforsilicatosampleratiodependingontheeaseoftheseparationtobeperformed.

WhatNext?
Onceyouhaveyoursolventsystempickedout,knowhowmuchsilicayouneed,andhavefoundacolumnofasuitable
size,youarereadytobeginpackingyourcolumn.Ofcourse,everycolumnisuniqueandnotallwillgiveTLCresultslike
theidealizedonesshownhere.Inpartthreewewilllookatsometroubleshootingtechniquesforcommonproblemsand
some"quickanddirty"alternativestorunningafullcolumn.

Furtherreading:

HighPerformanceGradientElution,
LloydSnyder,JohnDolan,
JohnWiley&Sons,NewYork,USA,2006.
ISBN:9780471706465
Alsoofinterest:

IntroductiontoModernLiquidChromatography,
LloydSnyder,JosephKirkland,JohnDolan,
JohnWiley&Sons,NewYork,USA,2010.
ISBN:9780470167540

Doyouhaveanytiportricksfortheperfectcolumn?Sharetheminthecommentssection...

ArticleInformation

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21/07/2016 TipsandTricksfortheLab:ColumnChoices::Education::ChemistryViews

DOI:10.1002/chemv.201200074

ArticleViews:150075

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