InvestigatingtheTechniquesofSolid-LiquidExtractionbyisolating
lycopenefromtomatopasteandColumnChromatography&Thin-Layer
Chromatography(TLC)bypurifyinglycopene
MengyingLi
DepartmentofChemicalEngineering,ThePennsylvaniaStateUniversity,PA16802
[email protected]
Abstract
Theseparationtechniqueofsolid-liquidextractionwasusedtoisolatelycopene
fromtomatopaste.Columnchromatographywasalsousedtopurifylycopene
fromtheothercarotenecompoundssuchasGrbeta-caroteneinthecrude
product.TLCtestwasperformedafterwardstoshowtheeffectivenessof
purification.AnUV/Visspectrumofthepurifiedlycopenewasalsoperformed
andanalyzedintheendofthelab.
Introduction
Lycopene[1]isabrightredC40–carotenoidpigmentfoundintomatoes.Therich
concentrationoflycopeneintomatoisprovedtobehelpfulinbonehealthdueto
itsoutstandingantioxidantproperties.(1)
Figure1.Structureoflycopene
Lycopenecanbeisolatedfromtomatopaste(babyfood)throughsolid-liquid
extraction.Insolid-liquidextraction,asolventthathasdistinctpropertiessuch
ashydrophilicorhydrophobicisaddedtoasolidcompound.Thesoluble
materialisextractedintothesolventfromthesolidcompoundandinsoluble
materialisleftbehindwiththesolidcompoundthuscompletestheseparation.
Lycopene,asanorganichydrophobiccompound,willbeextractedintothe
hydrophobicorganiclayerandseparatedfromotherwater-solublecompounds
andsolidresidue.Otherhydrophilicsubstances,suchasacetone,willbeeasily
extractedintowaterandsodiumchloridethusitpurifieslycopenetosome
extent.
Columnchromatographyisagreatwaytoseparateandpurifycompoundfrom
theimpuritiesthathavesimilarcharacteristicwiththecompoundinterested.
Whilesolid-liquidextractioncanextractlycopenefromtomatopaste,italso
extractssomeimpuritiesthatarecloselyrelatedinstructurewithlycopene.In
thislab,wewillusecolumnchromatographytoseparatelycopenefromother
carotenoidcompoundssuchascarotenesandxanthophylls.Column
chromatographyseparatescompoundsbasedontheirpolarity.Inthislab,
aluminawillbeusedasthepolarstationaryphaseandhexaneasthenonpolar
mobilephase.Whentheanalytesareelutedwiththemobilephaseinthecolumn,
themorepolarcompoundwillbeelutedslowlysinceitwillhaveastronger
interactionwiththestationaryphase.Thelesspolarcompoundwillbeeluted
fastersinceittendstostickwiththenonpolarsolventdueto‘likedissolveslike’
principle.Differentpolarityanddifferentretentiontimeisthekeyofseparation.
Thin-layer-chromatography,orTLC,isoftenusedtodeterminetheeffectiveness
ofthepurification.(3)TLChasthesameseparationprinciplesasthecolumn
chromatography,butithasadifferentsetup.Thestationaryphaseisusuallya
silicaplate.Theanalytesaredippedontotheplateandmobilephaseisgradually
climbingupontheplateandcausesthemovementofthespots.Thespotscanbe
visualizedbyUVlightoriodinechamber.TheretentionfactorRfcanbe
calculateddependsondistancesthatthespotstraveledandthedistancesolvent
travels.ThebestseparationareusuallyachievedwhentheRfvaluesfallbetween
0.3and0.7(3)
ResultandDiscussion
Solid-liquidextractionoflycopene
Thesolid-liquidextractionwasprovedtobeaneffectivemethodforisolating
lycopenefromtomatopaste.Tomatopastewasfirstextractedwith10mlacetone
andthenextracted3timeswith10mldichloromethane.Thecombinedfiltration
wastreatedwithwaterandsodiumchloridetobreaktheemulsionsandremove
acetoneandotherwater-solublecomponent.(3)Thewater-insoluble,orange
hydrocarboncarotenoidwasthenseparatedintotheorganiclayeranddried
overanhydrouscalciumchloride.
DriedcarotenoidwasdissolvedinafewdropsofdichloromethaneandaTLCtest
wasthenperformedonthemixture.ThemobilephaseofTLCconsisted8ml
hexaneand2mlacetone.Onespotwasmadeofhighconcentrationmixtureby
repeatedlytouchingtheplateinthesamelocation,andtheotherspotwasmade
ofdilutedmixturebytouchingtheplateonlyonce.Astheelutionwascompleted,
thespotswerevisualizedbyputtingtheplateintotheiodinechambersinceno
spotsweredetectedbyeyesandUVlights.Alignwiththeconcentratedspot,
thereweretwospotswithRfvalueof0.418and0.945.Alignwiththediluted
spot,onlyonespotwasseenwithRfvalueof0.945.Itcanbeconcludedthatthere
existedtwocomponentsintheorangecarotenoidsolution.Thereasonthatthere
wasonlyonespotinthedilutedonecouldbeassumedthattheamountofoneof
thecomponentwastoolittleinthedilutedsolutionthusitwashardtodetectits
existence.AftertheTLCtest,therestofthedichloromethanesolutionwasdried
overasteamofnitrogengasandstoredinthedarkplace.
ColumnChromatography
ColumnChromatographywasalsoprovedtobeasuccessfulmethodfor
separation.Inan8cmchromatographycolumn,3095mgaluminawasprepared
asthesolidphaseandhexanewaspreparedastheliquidphase.Thedry
carotenoidwasdissolvedinafewdropsofdichloromethane.ATLCtestwasfirst
performedonthecarotenoidsolutionbeforethecolumnchromatography.
Hexanewasfirstwasheddowntheinsideofthechromatographycolumnin
ordertoconsolidatethecarotenoidmixtureatthetopofthecolumn.(4)The
columnwasthenelutedwithhexane.Firsttwotubesofliquidthatcomingoutof
thecolumnwerecolorlessthuswerediscarded.Thecoloroffollowingtubesof
liquidwereyelloworange,orangeandyellow.Afterthis,thecoloroftheliquid
elutedwascolorless.TLCtestwasperformedontheliquidthathadcolor.For
eachtube,twospotsweremade,onewastheconcentratedspotandtheother
wasdilutedspot.SpotswerevisualizedbyUVlightandiodinechamber.The
resultoftheTLCplatewasshownintable1.
Table1RfValuesoftheTCLTestoncolumnchromatography.
Concentratedspot
Dilutedspot
CarotenoidSolution
0.54
0.54
Tube#1
0.36,0.54
0.36
Tube#2
0.843
0.843
Tube#3
0.145,0.843
0.145,0.843
Tube#4
0.145,0.54
0.145
TheTLCtestshowedthattheoriginalcarotenoidsolutionhadanRfvalueof0.54.
Tube#1mainlyhadonecompoundthathadanRfvaluesof0.36.Tube#2and
tube#3allhadthesamemajorcomponentthathadanRfvalueof0.843.Intube
#4themajorcompoundhadanRfvaluesof0.145.Thesolutionfromtube#2
andTube#3werecombinedsincetheyhadthesamemajorcompound.The
solutionfromTube#1,Tube#2&3,andTube#4wereconcentratedtosolidby
nitrogengasandstoredinthedarkplace
TheTLCtestshowedthatthereweremorethanonespotsinoneplate.This
situationcanbeassumedthattwoormoresubstancesmightco-eluteatthe
similartime.Puttingsmalleramountofthesolutionelutedinthetubesmight
helpwiththisproblem.TLCplatesalsoshowedthatthereweremorespotsin
oneplateafterthecolumnthanbefore.Beforethecolumnchromatographyit
hadbeenconfirmedthattherewereonlytwosubstancesinthemixture.(after
solid-liquidextraction)Afterthecolumnchromatography,4differentRfvalues
wereobserved.Thiscircumstancemightcausebythedifferentconcentrationof
thesamesubstanceintheoriginalmixture.Theoriginalcarotenoidmixturehad
notbeenmixedandshakenwellandthesamesubstancemighthavedifferent
concentrationindifferentplacesinthebeaker.OnlyoneRfvaluewasseenforthe
originalcarotenoidsolution.Thiscanbeassumedthattheanalyteanalyzedon
theTLCplatemightbeextractedfromthesameplacewherethesubstance’s
concentrationwasuniformed.
UV/Visspectrum
2-3dropsofethanolwasaddedtoeach3vialsofdriedcarotenoidpigmentand
UV/Visspectrumwasperformedonthese3samples.Theresultwasshowedin
table2.
Table2ResultofUV/VisSpectrum
Wavelength(nm)
Vial#1
464
Vial#2
467.4
Vial#3
472.2
Sinceinstandardsolution,𝛽-carotenehasthemaximaabsorbanceof448nm
andlycopenehasthemaximaabsorbanceof472nmand𝛾carotenehasthe
maximaabsorbanceof462nm.(TanandSoderstom,1989)Itcanbeassumed
thatinvial#1and#2thecarotenoidaremostly𝛾caroteneandinvial#3the
carotenoidismostlylycopene.Thisfurtherconfirmedthesuccessfulseparation
oflycopenefromotherimpurities.
Conclusion
Thesolid-liquidextractionwasprovedtobeeffectivewhenisolatinglycopene
fromtomatopastesincetheTLCtestshowedthattherewereonlytwo
componentsinthecoloredorganiclayer.Columnchromatographywasalsoa
greatwaytoseparatelycopenefromothercarotenoidcompoundsthathave
similarcharacteristics.TheTLCtestandtheUV/Visspectrumconfirmedthat
majorcomponentinthesolutioninthetube#4andvial#3werelycopene.Inthe
future,whencatchingthesolutioneludedfromthecolumn,puttingsmaller
amountofsolutionintoeachtubemighthelpinseparationbettersincetheTLC
platesshowedthatthereweredifferentcompoundsinthesametubes.Before
doingthecolumnchromatographyandtheTLCtestitisalsoimportanttoshake
andmixthesolutionwellinordertoachievetheuniformconcentrationforthe
samesubstanceinordertoavoidthesituationthatmorespotswereobservedin
theTLCplatesthanthespotsobservedbeforethecolumninthislab.
Experimental
GeneralMethods
AllcompoundswerepurchasedfromSigma-Aldrichandusedwithoutfurther
purification
Lycopene(1)
Tomatopaste(0.5310g)wasextractedwith10mlacetonefirstandthen10ml
dichloromethanefor3times.Theorganiclayerwasdriedoveranhydrous
calciumchloride.ThestationaryphaseforTLCwassilicaandforcolumn
chromatographywasalumina..UV(EtOH) 𝜆max472.2nm.
Acknowledgement
IwouldliketosincerelyandprofuselythankmychemistrylabTAStevenTaylor
forhisguidancetotheinstrumentusageandgreatsupportincompletingmy
project.
Reference
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