VanillinAssay

AnnE.Hagerman2002

Contents
Introduction..................................................................................................................................................1
Reagents........................................................................................................................................................3
PreparationofWorkingReagents.................................................................................................................3
Extraction......................................................................................................................................................3
AnalysisofStandards....................................................................................................................................3
AnalysisofSorghumextracts........................................................................................................................3

Introduction
FromPrice,vanScoyoc,andButler,J.Agric.FoodChem.26,12141218(1978)
Thisfunctionalgroupmethodforcondensedtanninsisespeciallywidelyusedbyagronomists.Thereare
significantdifficultiesininterpretationofthemethod.Werarelyusethismethodinmylab,sinceIfind
theacidbutanolmethodsimplerandmorereliable.
Understandingthestructuralchemistryofcondensedtanninsandflavanoidsisessentialtoproperuseof
thevanillinassay.Thevanillinreactioninvolvesreactionofanaromaticaldehyde,vanillin,withthe
metasubstitutedringofflavanolstoyieldaredadduct.Althoughthevanillinreactionhasbeenwidely
usedtoestimatecondensedtannin(proanthocyanidin),thereactionisnotspecificforcondensed
tannins.Anyappropriatelysubstitutedflavanolreactsintheassay.Thustheformal"monomer"ofthe
condensedtannins,catechin,alsoreactstoyieldaredcoloredadduct.Furthermore,becausethevanillin
reactsonlywithmetasubstitutedflavanoids,the5deoxyproanthocyanidins(e.g.quebracho)donot
producemuchcolorwithvanillin.Reactivitywithvanillinisnotsufficientevidenceforthepresenceof
condensedtannins.
Catechiniscommonlyusedtostandardizethevanillinreaction,butthereareproblemswithinterpreting
themeaningof"catechinequivalents".Underthenormalconditionsforthevanillinassay(methanol
solvent),tannins(proanthocyanidins)andcatechinbothreactwithvanillin,buttheratesofreactionof
thepolymerandthemonomerarequitedifferent.Ingeneral,thecoloryieldislowerforthemonomer
thanforthepolymer.Althoughtheabsorbancesobtainedfromrunningthevanillinreactioninmethanol
onanunknowntannincontainingsamplecanbeconvertedto"catechinequivalents"thecomplexities
ofthesystemmakeitdifficulttointerpretthemeaningofthoseequivalentsonthemolecularlevel.

Copyright 2002 by Ann E. Hagerman. All rights reserved.

Themodifiedvanillinmethodwasdevelopedtoovercomethoseproblems,butprovestobemoreuseful
forestimatingmolecularweightofcondensedtanninthanforquantitativeanalysis.Thevanillinmethod
describedherewasdevelopedforanalysisofcondensedtannininSorghumgrain;modificationofthe
O
OH

O
OH

OH

HO

OH

HO

H+
OH

OH

OH

OH
O

catechin
vanillin adduct
max 500 nm
O
OH
vanillin

OH
OH
reacts with acid butanol to yield colored product

HO

OH

OH
OH

OH
O

HO

reacts with vanillin, to yield colored adduct

OH
OH

proanthocyanidin

methodsforsamplepreparationwouldprobablybenecessarytousethemethodwithothertissues.
Themethoddescribedhereuses0.5%vanillinratherthanthe2%originallyrecommendedbyBurns.By
decreasingthevanillinconcentration,thedependenceofthereactionontemperatureisminimized.The
vanillinreactionisverysensitivetothepresenceofwater.Evenasmallamountofwaterinthereaction
mixturewillsubstantiallyquenchcoloryield.Allstandardsshouldbepreparedinanhydrousorganic
solvents(usuallymethanol).Ifwatermustbepresentinthesamplestobeanalyzed,thesameamount
ofwatershouldbeaddedtothestandards.Vanillinreactsonlywithfreeflavan3ols,orwiththe
terminalunitoftheproanthocyanidin.Thevanillinmethodcombinedwiththeacidbutanolmethod
providesandestimateofdegreeofpolymerization.

Copyright 2002 by Ann E. Hagerman. All rights reserved.

Reagents

1%vanillininmethanol(1.0gvanillinupto100mLwithabsolutemethanol).Storeinadark
bottleat4C
8%concentratedHClinmethanol(8.0mLconcentratedHClbroughtto100mLwithabsolute
methanol).
4%concentratedHClinmethanol(4.0mLconcentratedHClbroughtto100mLwithabsolute
methanol).
Constanttemperaturewaterbathsetat30C.(Ifthisisnotavailable,therewillbetemperature
dependentvariationinthedata).
0.3mg/mLcatechin(3.0mgcatechinbroughtto10.0mLwithabsolutemethanol).Storeina
darkbottleat4Cforuptothreedays.

PreparationofWorkingReagents
Theworkingvanillinreagentmustbeprepareddailyfromthesolutionsdescribedabove.Onepartofthe
1%vanillinsolutionismixedwithonepartofthe8%HClsolution.Theworkingvanillinreagentandthe
4%HClsolutionarebroughtto30Cinthewaterbathbeforestartingtheanalysiseachday.

Extraction
Forbestresults,theextractionandanalysisshouldbecarriedoutonasingleday.Thegrainshouldbe
groundnomorethanonedayinadvanceofanalysis.About200mggroundSorghumgrainisweighed
exactly,andthenextractedwith10.0mLabsolutemethanolfor20mininrotating(Labquakerotator)
screwcapculturetubes(13x100mm).Themixtureiscentrifugedfor10minat3000xg,andthe
supernatantisusedintheanalysis.

AnalysisofStandards
1. 0to1.0mLaliquotsofthecatechinstandardaredispensedintotwosetsofculturetubesand
eachsampleisbroughtto1.0mLbytheadditionofabsolutemethanol.Tubesareincubatedin
thewaterbath.
2.
5.0mLoftheworkingvanillinreagentisaddedat1.0minintervalstoonesetofstandards,and
5.0mLofthe4%HClsolutionisaddedat1.0minintervalstothesecondsetofstandards.
3.
Thesamplesareleftinthewaterbathforexactly20.0min,andarethenremovedandthe
absorbanceat500nmisread.
4.
Becausethecolorcontinuestodevelopastimepasses,youcannotgobackandrereadany
sample.Youmustmaintainthestrict1.0minintervalsforreadingthatyouusedintheaddition
ofreagents.
Theabsorbanceoftheblank(vanillinreagentwithnocatechin)issubtractedfromtheabsorbanceofthe
correspondingvanillincontainingsample.Astandardcurveisconstructed(Absvs.mgcatechin)
andthelinearportionofthecurveisextrapolatedtoproducethestandardcurve.

AnalysisofSorghumextracts
1. 1.0mLaliquotsofeachextractaredispensedintoculturetubes.Duplicatesofeachsample
arerequiredsothatthesampleandablankcanberunforeachsample.

Copyright 2002 by Ann E. Hagerman. All rights reserved.

2. Tubesareincubatedinthewaterbath.
3. 5.0mLofthevanillinreagentisaddedat1.0minintervalstoonesetofsamplesand5.0mL
ofthe4%HClsolutionisaddedat1.0minintervalstothesecondsetofsamples(the
blanks).
4. Thesamplesareleftinthewaterbathforexactly20.0min,
5. Theabsorbanceat500nmisread.
Becausethecolorcontinuestodevelopastimepasses,youcannotgobackandrereadanysample.You
mustmaintainthestrict1.0minintervalsforreadingthatyouusedintheadditionofreagents.
Theabsorbanceoftheblankissubtractedfromtheabsorbanceofthecorrespondingvanillincontaining
sample.Theblankcanbesubstantialfortissuesthatcontainlargeamountsofpigments.
Thevalueobtainediscomparedtothestandardcurvetoobtain"catechinequivalents".

Copyright 2002 by Ann E. Hagerman. All rights reserved.