LEARNING ABOUT PLANT COLORS AND PIGMENTATION Developed by ...
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LEARNING ABOUT PLANT COLORS AND PIGMENTATION Developed by Julia Dupin, 2017 This activity was designed to showcase the diversity of pigment types in plants and show how they can be ‘extracted’ from plant tissues using simple solvents. Plants colors come from different types of chemical compounds, called pigments. The most abundant plant pigment is chlorophyll, the compound that makes plant leaves appear green to our eyes. However, this is just one of many pigments that plants naturally manufacture. Other photosynthetic pigments include carotenoids, yellow-orange compounds that are similar in chemical structure and stored with chlorophyll in organelles called plastids. Additional colors arise from anthocyanin and betalain pigments. These water soluble pigments are red to purple, depending on pH, and are found in the vacuole, the large storage pool at the center of plant cells. Anthocyanins are more common than betalains, which are only found in small group of plant species that includes beets. Both anthocyanins and betalains are principally important for coloration, although anthocyanins are involved in response to UV light, acting like a sort of plant sunscreen. They also serve as antioxidants in our bodies, and have major health benefits when eaten. This activity is focused on the chemical extraction of anthocyanins. The suggested samples, red apple, blackberry, eggplant, and radish, have different types of anthocyanins. EXTRACTION OF ANTHOCYANIN PIGMENTS FROM RED APPLE SKIN, EGGPLANT SKIN, RADISH SKIN, AND BLACKBERRIES, AND SEPARATION WITH PAPER CHROMATOGRAPHY Materials/steps marked with a ★ should be handled only by the instructor, or by a student in close supervision Materials (see Fig. 1): - mortar and pestle (the kitchen one works just fine) - fruit/root samples - 1/2 teaspoon measuring spoon - disposable plastic pipette (to measure 0.5 and 1 mL of isopropanol) - dropper bottle - containers with lid for sample storage - small glass vial (for blackberry solution) - coffee filters - funnel - plastic pipette tips (volume up to 200µL) - glass jars (e.g. Mason jars of pint size) - chromatography paper (there are multiple types of chromatography paper, we have tested this method using the ‘Whatman™ Grade 3MM Chr’ Chromatography Paper. Other types of paper of similar grade recommended for chromatography use should yield the same quality results) - lemon juice - ★ isopropanol (rubbing alcohol), ~91% (this seemed to work slightly better than the 70%) - ★ acetone 100% (some brands of nail polish remover have this concentration) - ★ single edge blade - ★ vegetable peeler - ★ regular table knife For each student/small group working with one sample you will need: - one mortar and pestle set - one pipette tip (minimum) - mason jar with acetone
Transcript of LEARNING ABOUT PLANT COLORS AND PIGMENTATION Developed by ...
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.
Figure4.Hereweseetheresultsforallthesamples,fromlefttoright:apple,blackberry,eggplant,andradish.ThelinesidentifiedasFrepresentthelengthcoveredbythesolutionwhilethepaperwasinthechamber.
