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ColumnChromatographyofPlantPigments
fromSpinachExtract
Description:Adeep,yellowgreenextractderivedfromrawspinachiselutedthrougha
chromatographycolumncontainingsilica.Theresultisaseparationofthecarotene
andchlorophyllpigmentsinthespinach.Whenthedemohasbeencompleted,the
studentsshouldbeabletoseedistinctyellowandgreenbandsinthecolumn.Ifdesired,
thepigmentscanbecollectedasfractionsandanabsorbancespectrumofthetwocanbe
showntotheclass.
Concept:Moleculescontainedwithinamixturecanbeseparatedbasedontheir
differingaffinitiesforvarioussolvents.Thecarotene/chlorophyllmixturecanbe
extractedfromspinachusingpetroleumetherandacetone.Thecarotenehasanaffinity
forpetroleumether,whilethechlorophyllhasanaffinityforacetone.Afterthemixture
hasbeendeliveredtoasilicapackedcolumn,theadditionofpetroleumethercan
successfullyelutetheyellowcarotene.Thelateradditionofacetonecanelutethe
chlorophyllthatwasunaffectedbytheadditionofpetroleumether.
Materials:
LargeMortarandPestle
FreshSpinach
PetroleumEther
Acetone
125mLErlenmeyerflaskwithstopper
Large,glasschromatographycolumn(fromundergraduatelabs)
StoppertofitcolumnorParafilm
Shortlengthoftubingtofitcolumn
Pinchclamp
Glasswool
Sand
Silica
2plasticweighingboats
Plasticfunnel
Large,glassstirringrod
Largecrystallizingdish
Ringstand
2fingerclamps
Greenpipetpump
3,5mLMohrpipets

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Procedure:
PreparingtheExtract:
Tear1015gofspinachleavesintosmallpieces,andplacetheminthelarge
mortar.Addabout50mLofan80:20volumetovolumemixtureofpetroleumetherand
acetonetothespinach.Grindthemixtureuntilthesolventtakesonadeep,greencolor.
Decanttheliquidandretainitina125mLErlenmeyerflaskfittedwithastopper.Ifmore
extractisdesired,continuetoaddsolventtothemixturealongwithafewsmallpiecesof
spinachbeforecontinuingtogrind.
PreparingtheColumn:(seethediagramonthenextpage)
Mountthecolumnatthetopandbottomtoaringstandusingtwofingerclamps.
Soakasmallamount(~0.1g)ofglasswoolinpetroleumetherandpushittothebottom
ofthecolumnusingalarge,glassstirringrod.Makingsurethatthetubeatthebottomof
thecolumnissecurelyclampedshut,addenoughpetroleumethertothecolumn(usinga
funnel)toreachaheightofabout15cm.Pouringfromaweighingboat,addenoughsand
tothecolumntoreachaheightof1cmabovetheglasswool.Tapthesideofthecolumn
gentlywiththestirrodtoensurethatthesandhassettledevenly.Slowly,usinga
weighingboat,addenoughsilicatothecolumntoobtainaheightofabout10cm.You
willneedtoaddthisinsmallportionswhiletappingthecolumnwiththestirrodto
ensureevensettling.Addanother1cmofsandtothetopofthecolumninasimilar
manner.Thereshouldbeenoughpetroleumetherinthecolumntoreachabovetheheight
ofthislastadditionofsand.Openthepinchclampanddraintheexcesspetroleumether
intothecrystallizingdishuntiltheleveloftheetherisjustabovethetoplayerofsand.
StopperthecolumnoruseParafilmtokeepthepetroleumetherfromevaporatingbefore
thedemoistobeperformed.
InClass:(seethediagramonthenextpage)
Carefullypipetabout5mLofthespinachextractontothetopofthecolumn.
Withthecrystallizingdishrestingbelowthecolumn,openthepinchclamptoallow
elutingtobegin.Ayellowbandofcarotenewillbegintomovedownthecolumn.
UsingafunnelorMohrpipet,continuetoaddpetroleumethercarefullytothecolumn
untiltheyellowbandhasreachedthedesireddistanceoftravel.Donotletthelevelof
thepetroleumetherfallbelowthetoplayerofsandatanypoint.Thecarotenecanbe
elutedthroughthebottomofthecolumntobecollected,oritcanremaininthecolumnso
thattwodistinctbandsofcolorcanbelaterpointedout.Tobegintheelutionof
chlorophyll,carefullyaddabout5mLofacetonetothetopofthecolumn.Thiswill
produceagreenbandinthecolumn.Thischlorophyllcanbecollectedorretainedinthe
columnwiththecarotene.Formostpurposes,itissufficienttoleavethetwopigments
inthecolumnsothatthestudentscanseethattheextracthasseparatedintotwo
components.Ifrequested,transparenciescontainingtheabsorbancespectraofthetwo
pigmentscanbeprovided.

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Safety:
Wear gloves and goggles at all times while preparing and performing this demonstration.
Do not place the acetone or petroleum ether near any source of heat, sparks or flame.
Clean-Up:
Retain the column and the contents of the evaporating dish for the demonstration
technician to dispose of

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