learning about plant colors and pigmentation developed by
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LEARNINGABOUTPLANTCOLORSANDPIGMENTATION DevelopedbyJuliaDupin,2017 Thisactivitywasdesignedtoshowcasethediversityofpigmenttypesinplantsandshowhowtheycanbe‘extracted’fromplanttissuesusingsimplesolvents.Plantscolorscomefromdifferenttypesofchemicalcompounds,calledpigments.Themostabundantplantpigmentischlorophyll,thecompoundthatmakesplantleavesappeargreentooureyes.However,thisisjustoneofmanypigmentsthatplantsnaturallymanufacture.Otherphotosyntheticpigmentsincludecarotenoids,yellow-orangecompoundsthataresimilarinchemicalstructureandstoredwithchlorophyllinorganellescalledplastids.Additionalcolorsarisefromanthocyaninandbetalainpigments.Thesewatersolublepigmentsareredtopurple,dependingonpH,andarefoundinthevacuole,thelargestoragepoolatthecenterofplantcells.Anthocyaninsaremorecommonthanbetalains,whichareonlyfoundinsmallgroupofplantspeciesthatincludesbeets.Bothanthocyaninsandbetalainsareprincipallyimportantforcoloration,althoughanthocyaninsareinvolvedinresponsetoUVlight,actinglikeasortofplantsunscreen.Theyalsoserveasantioxidantsinourbodies,andhavemajorhealthbenefitswheneaten.Thisactivityisfocusedonthechemicalextractionofanthocyanins.Thesuggestedsamples,redapple,blackberry,eggplant,andradish,havedifferenttypesofanthocyanins.
EXTRACTIONOFANTHOCYANINPIGMENTSFROMREDAPPLESKIN,EGGPLANTSKIN,RADISHSKIN,AND
BLACKBERRIES,ANDSEPARATIONWITHPAPERCHROMATOGRAPHYMaterials/stepsmarkedwitha★shouldbehandledonlybytheinstructor,orbyastudentinclosesupervisionMaterials(seeFig.1):-mortarandpestle(thekitchenoneworksjustfine)-fruit/rootsamples-1/2teaspoonmeasuringspoon-disposableplasticpipette(tomeasure0.5and1mLofisopropanol)-dropperbottle-containerswithlidforsamplestorage-smallglassvial(forblackberrysolution)-coffeefilters-funnel-plasticpipettetips(volumeupto200µL)-glassjars(e.g.Masonjarsofpintsize)-chromatographypaper(therearemultipletypesofchromatographypaper,wehavetestedthismethodusingthe‘Whatman™Grade3MMChr’ChromatographyPaper.Othertypesofpaperofsimilargraderecommendedforchromatographyuseshouldyieldthesamequalityresults)
-lemonjuice-★isopropanol(rubbingalcohol),~91%(thisseemedtoworkslightlybetterthanthe70%)-★acetone100%(somebrandsofnailpolishremoverhavethisconcentration)-★singleedgeblade-★vegetablepeeler-★regulartableknifeForeachstudent/smallgroupworkingwithonesampleyouwillneed:-onemortarandpestleset-onepipettetip(minimum)-masonjarwithacetone
-ifthesampleisblackberry,theywillalsoneedonecoffeefilter,onefunnel,andonesmallglassvialAllothermaterialscanbepreparedinlargeamountsandsharedinalargeclassroom.Foranestimateoftheamountofplantsampletopreparegivenaclasssizeseetheinformationunder"Preparingtheskinsamples"andthedetailsundertheextractionofpigmentsfromblackberry.PreparationsforPigmentextraction------------------------------------------------------Beforeclass------------------------------------------------------Selectingthefruits/roots-whenbuyingthevegetablesgoforthosethathavethedarkestcolors,especiallyinthecaseofapplesPreparingtheskinsamples-beforegoingintothedetails,hereissomeimportantinformation
-first,asaheadsup,knowthattheeasiestsampletoworkwithistheradishskinbecauseitlacksthewaxylayerpresentbothontheappleandeggplantskins
-second,youwillneedabout1/2teaspoonofsampleskinperpersonforthisexperiment-third,ifyouneedtopreparesamplesforalargenumberofpeopledosoacoupleofdaysinadvance,thesecanbestoredinthefridgeinsealedcontainersandstilllookfreshafter3to5days.Thiswayyoucanavoidhavingtopeelalargeamountofthingsonthedaybefore,yourfingertipswillbethankful.
-Amountofsample-Ifoundthatifstudentsareworkingindividuallyoneradishhasenoughskinforabout4people,oneapplelikely10people,andoneeggplantmanypeople:),fortheblackberries,useoneperperson
-★peelingprocessforanyofthesamplesherethefinalproductshouldbesmallpiecesofskinthatareeasiertogrindthanlongstripofpeeledskin;thisisespeciallyimportantfortheappleandtheeggplant;thesepiecesshouldbeabout0.5cmx0.5cm
Radish-itisveryeasytoremoveskinsamplesjustbyusingaregularsingleedgebladeAppleandeggplant-itwillbetemptingtojustuseavegetablepeelertoremovetheskinsbuttheuseofitwillgeneratepeelsthathaveafairamountofwhitefruitfleshontheundersides.Soafollowupstepneededistousearegulartableknifetoscrapoffthefruitfleshandhaveafinalproductthatiscleaner(seeFig.2tochecktheidealfinallookofyoursamples)Blackberry-thereisnopeelingstepfortheblackberries,sojustkeepthemintact
-storageifthesamplesarenotgoingtobeusedimmediatelyyoucanplacetheseinasealedcontainerinthefridgeforabout3days(neverinthefreezer)
Lemonjuicestorage-tomakethingseasieryoucanstorethelemonjuiceinthedropperbottle★Chromatographychamberpreparation-givenhowvolatileacetoneis,itissafertohaveallmasonjarsthatwillbeusedalreadyfilledwithenoughacetonetofillup1cmatthebottomofeachjarChromatographypaperpreparation
-cutthepaperinto2cmwidestrips,thesestripsshouldnotbelongerthantheglassjarusedaschamber-usingapencil,measure2cmofheightandmakeahorizontallineononeofthesides(seeFig.3)Generalcommentaboutthepreparationofsamplesandsolutions:ifyouaredoingthisactivitywithmiddleandhighschoolstudents,andthereisnotimerush,thestudentscanbetheonescollectingthesamplesaslongasthereisenoughsupervisionforusingtheblades.Theycanalsodothemarkingofthechromatographypaperpreparationstepinclass.Pigmentextractionandpaperchromatography------------------------------------------------------Duringclass------------------------------------------------------Extractingthepigments
Forapple,eggplantandradish-inthemortaradd1/2teaspoonofachosensampleskin,0.5mLofrubbingalcoholand3dropsoflemonjuice,andgrindtheskinpieces
foryoungerkids,theradishisthebestchoicebecauseit'seasiertogrind appleandeggplantisrecommendedforolderkids
-youwillbedonegrindingonceyouhaveapigmented,likelythicksolution;youmayaddmoreofthelemonjuiceandalcoholifthetissuewasdryandnoliquidremainsaftergrinding.Stillitshouldbeconcentratedanddarklycolored.
Forblackberries-placeoneblackberryinthemortaralongwith1mLofrubbingalcoholand6dropsoflemonjuice,andgrindall-becausethereisnowtoomuchsolidmaterialinthisfinalmix,youwillneedtotransferafilteredsolutiontoasmallglassvial.Todothatuseafunnelandputacoffeefilterinthefunnel,placethosetwoitemsonyourvial,andgentlypourthesolutiononthecoffeefilter.Itmightatfirstjustsoakthefilterandnotreallygothroughit,soyoucanslowlysqueezethesolutiontoacceleratethefilteringprocess.
Paperchromatographytechnique-youwillnowneedtoastripofchromatographypaperandachromatographychamberalreadyfilledwith1cmofacetone
-makesureyourstripofpaperismarkedononesidewithahorizontalpencillineat2cm(donotusepen)(Fig.3).
-youwillnowapplysmallamountsofyoursolutiontothechromatographypaperusingapipettetip.Grabonepipettetipandtouchthepigmentsolutionwithitsverytipandholditthereforacoupleofseconds.Duetocapillaryaction,thesolutionwillnaturallystartmovingupthetip.Onceyouhavecollectedsomeofyoursolution(nottoomuch,seeFig.3forreference)gentlytapthechromatographypapertotransferittothepaper,youwanttotapexactlyonthemiddlepointofthatreferencepencilline(Fig.3).Youwillhavetorepeatthisstepthreetofourtimestoensurethatthereisenoughsample,alwaystappingthepaperonthesamemiddlelinespot.Itisimportanttowaitforthepapertodryabitinbetweenstepstokeepthesizeofthespotsmall.
-nowplaceyourstripofpaperinyourchromatographychamber,beingcarefultoopenandclosethechamberquickly.Thereisnoneedtoscrewthelidon,justplaceitontopofthejar.
-ifstudentsareworkingondifferentsamples,itisimportant,forcomparisonsake,tokeepthepaperstripsinthechambersforthesameamountoftime,sothesamplesaredevelopedequally(Fig.4).Anotherpointtokeepaneyeonisthatyouneedtoallowenoughtimeforthepigmenttomigratebutnottoomuchtimesothatitdoesn’trunsoffthepaper.Agooddistanceisabout2/3ofthepaper,andthisusuallytakesbetween5and10minutes.
-whatyoushouldseeisafaintcolorblobmovingupalongwiththeacetonethatisnowsoakingthepaper.
-oncethepigmentreachesabout2/3ofthepaperlengthyoucanremoveitfromthechamber(againquickly),beingcarefulnottotouchthewetpart.
-makeahorizontallinewithapenciltoindicatewherethesolutionstopped,thisiscalledthefrontofthesolution(i.e.,thepointbetweenthewetpartandthedrypart)(Fig.4)
-placeyourpaperstriponacountertopordeskandletitdryforatleast5minutes.Onceitstartsdryingyoushouldseethepigmentmoreclearlybeingabletotellhowmuchitmigratedonthepaper.
Howtointerprettheresults-Eachsamplepresentspigmentsthathavedifferentaffinityforthesolvent(acetone)sothepigmentswillthentraveldifferentdistancesonthechromatographypaper.-Differentpigmentshavedifferentcolorsonpaperliketheydointheplanttissues.Thepigmentinradishesskin(calledpelargonidin)isbrightredwhiletheoneineggplantskin(delphinidin)ispurple.-thisprotocolmimicstherealprotocolusedbyresearchersthatstudyplantcolors,herewehavethethreemaincomponentsofthepigmentextractionandseparationprotocol:analcohol(isopropanol),anacidsolution(lemonjuice)andthemediumforpigmentseparation(chromatographypaperandacetonesolution).AcknowledgementsThisworkwasfundedbytheNationalScienceFoundation(grantDEB1553114)
Figure1.Mostmaterialsusedinthisactivity.Notincludedinthispicture,coffeefilters,funnelandglasscontainersforstorage.
Figure2.Skinsamplesforapple,eggplantandradish,andextractionsolutionforblackberryafterfiltering.Noticethattheskinsampleshavelittletononefruitflesh(white).
Figure3.Stripsofchromatographypaperwithapencilmarkat2cm.Thestripontherightalreadyreceivedsomeoftheblackberrysolution.Werecommendcollectingroughlytheamountofsolutionindicatedonthepipettetipshownhere,foreachtimethesolutionistransferredtothepaper.Collectingtoomuchsolutioneachtimewillgeneratedotsthataretoobigandnotideal.