How do levels of anthocyanins in blueberries change due … · How do levels of anthocyanins in blueberries ... How do levels of anthocyanins in blueberries change due to temperature

Howdolevelsofanthocyaninsinblueberrieschangeduetotemperatureandlocationoforigin?

GwennythCarroll

CandidateNumber:1203-0016

CentreNumber:1203

ChemistryExtendedEssay

InternationalBaccalaureate

WordCount:3992

InternationalSchoolofToulouse

GwennythCarroll 1203-0016

2

Abstract

Thisessayinvestigates:Howdolevelsofanthocyaninsinblueberrieschangeduetotemperatureandlocationoforigin?Blueberriesareagoodsourceofdietaryantioxidants,free-radicalscavengingchemicals.TheBriggs-Rauscherreactionwasusedtoanalyselevelsofanthocyanins(amainantioxidantinblueberries)insolutionsofblueberryjuiceandwater.Alongertimeforoneoscillationsignifieshigherlevelsofantioxidants,asittakeslongerforionstobuilduptoasuitableconcentrationforthereactiontooscillate.Arangeoftemperatures,from25°Cto75°C,ofablueberrysolutionsample,fromMorocco,wastestedaswereblueberrysolutionswithoriginsinCanada(wild),WashingtonState(organic),andSpain.

Theresultsofthetestusingblueberriesfromdifferentlocationsatthesametemperaturedemonstratesasizeabledifferenceinthetimeforoneoscillationcorrelatingwithasignificantdifferenceinlevelsofanthocyanins.TheCanadianwildblueberrieshavethehighestconcentrationofanthocyaninswithatimeforoneoscillationof60.1sfollowedbyWashingtonStateorganicblueberries(45.8s),Moroccanblueberries(10.2s)andfinallythelowestconcentrationofanthocyaninsintheSpanishblueberrieswithanoscillationtimeof9.0s.Thedifferenceinlevelsofanthocyaninscanbecontributedtothevarietyofblueberryandthecultivationpracticesusedtogrowthem.

Thetemperaturetestwasinconclusive.Howeverthegeneraltrend,whencombinedwithacademicresearch,indicatesthatlevelsofanthocyaninsinblueberriesdecreasewithtemperature.At25°Cthetimeforoneoscillationwas10.2swhileat75°Cthetimewas9.4s.Thismaybeduetotheslightincreaseintemperatureofthereactionsolutionsincreasingtherateofreaction,therebydecreasingthetimeforoneoscillation.Anthocyaninsarereportedtobethermo-sensitive,howeverdegradationhasbeenobservedabove70°Csoisoutoftherangeofthisexperiment.

WordCount:300


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TableofContents

Listoffigures 4

Introduction 5-7

Method 8-10

Assumptions 8

Variables 8-9

Preparationandreactionsummary 9-10

Problemsencounteredinpreliminary 10

DataCollection 11-17

Massofblueberryinblueberrysolution 11

Rawdata 12

ProcessingData 13,17

ProcessedData 13-14

Graphs 14-16

InterpretationandJustificationofData 17-20

Typeofblueberryaccordingtolocation 17-18

Temperature 19-20

Conclusion 20

UnansweredandAdditionalquestions 21

Evaluation 21-23

Justifyingrange 21

Limitations 21

RandomError 22

SystematicError 22-23

ErrorinDesignandData 23

Reliabilityofdata 23

Bibliography 24-25

Appendix 26-31

Method 26-29

Disposal 30

Safety 30

Preliminary 31


4

ListofFigures

Figure1–Skeletalstructureofanthocyanins

Figure2–Anthocyaninscommonlypresentinblueberries,theirstructures,sugarmoieties,andcolour

Figure3–SummaryoftheBriggs-Rauscherreaction

Figure4–DiscussingControlVariables

Figure5–Massofblueberryjuicein100mlofdistilledwaterinpreparationofblueberrysolution

Figure6–Calculatinguncertaintyofblueberryjuiceinsolution

Figure7–Testforanthocyaninlevelsindifferentblueberrysamples–Rawdata

Figure8–Testforanthocyaninlevelsatdifferenttemperatures–RawData

Figure9–Examplecalculationofprocessingoscillationtimeanduncertainty

Figure10–Testforanthocyaninlevelsindifferentblueberries–ProcessedData

Figure11–Testforanthocyaninlevelsatdifferenttemperatures–ProcessedData

Figure12–Howthetimeforoneoscillationchangeswiththetypeofblueberry

Figure13–Howthetemperatureoftheblueberrysolutionaffectsthetimeforoneoscillation



Figure16–Ratiotocomparetherelativetimeforoneoscillationinblueberriesfromdifferentlocations

Figure17–PoliticalMapofWorldshowingtwodifferentregionsoforiginofblueberrysamples

Figure18–Differentcoloursoftheblueberrysolutions

Figure19–Thermaldegradationoftwocommonanthocyanins


5

Introduction

Antioxidants,suchasanthocyanins,havebeen,‘ahottopicinrecentyears’accordingtoDr.MarizaSnyderandDr.LaurenClum(2011)inthescientificcommunityandthegeneralpublicduetotheirpotentialhealthbenefits.Theymayprotectagainstsomecancers,maculardegeneration,Alzheimer’sandatherosclerosis,amongstothers(WebMD,2014).

WildblueberriesfreshlypickedinNorthernCanadacannotcompare,inmyopinion,withcultivatedblueberriesboughtinFrance.This,combinedwithanarticleIreadaboutthehealthbenefitsofblueberries,sparkedmyinterestinwhetherlevelsofantioxidantsinblueberrieschangedwiththelocationoforigin.Asanavidbaker,Ienjoyusingblueberriesasatoppingorbakedinmuffinsorcrumbles,leadingtomycuriosityoftheimpactoftemperatureonlevelsofantioxidants.Theseinterestsledmetotheresearchquestion:

Howdotemperatureandlocationoforiginimpactthelevelsofanthocyanininblueberries?

Freeradicals(atomsormoleculeswithunpairedelectronssotheyareextremelyreactive)causedamageinthebodybyinhibitingthefunctionofcells(HusneyandRomito,2013)andbydamagingkeymoleculessuchasfats,proteinsandDNA(Smythies,1998).Antioxidantsstabilisefreeradicals,therebyprotectingthebody(HusneyandRomito,2013),by‘scavenging’freeradicalsandremovingthemorbyreactingwiththem(Smythies,1998).

Antioxidantsareagroupofchemicalsthatinhibitoxidation.Flavonoidsandphenols,togethercalledphytochemicals,areaclassofantioxidantstowhichthemajorantioxidantsinblueberries,anthocyanins,belong(Smythies,1998).TheyhavethebasicskeletalstructureofC6C3C6.(RoutrayandOrsat,2011).

AnthocyaninsoccurnaturallyinplantsasaprotectivemechanismagainstenvironmentalstressessuchasUVlightanddrought,intheformofglycosidesandacyloglycosides(RoutrayandOrsat,2011),ananthocyanidinmoleculepairedwithasugar(Webb,2014).

Figure1:Skeletalstructureofanthocyanins(RoutrayandOrsat,2011)


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Variationsinthechemicalstructureofanthocyaninsaremainlyduetodifferencesinthenumberofhydroxylgroupsandthenatureandnumberofthesugarmoiety(Nijveldtandothers,2001).

ThemostpredominateanthocyanininblueberriesisMalvidin-3-galactosidewhilewildblueberriesarereportedlythebestsourceofpeturidin-andmalvidin-basedanthocyanins(RoutrayandOrsat,2011).

On-goingresearchisexpandingknowledgeofthebenefitsofantioxidantsinblueberries.OnearticleintroducestheresearchofDavidLipsettandDr.BrianStaveley,whoarelookingintotheeffectsofblueberryextractonParkinsonDisease.Theresults,‘suggeststhatadietsupplementedwithblueberryextractmayindeedhaveapositiveimpactonafruitflymodelofParkinsonDisease.’(Foss,2014)

Figure2:Anthocyaninscommonlypresentinblueberries,theirstructures,sugarmoieties,andcolour(RoutrayandOrsat,2011)


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TheBriggs-Rauscherreactioncanbeusedtocomparerelativeamountsofantioxidantsinasampleofblueberrysolution.Developedbytwohighschoolteachersin1973,itisanoscillatingreactionwherethreecolourlesssolutionsarecombinedtoformaresultingsolutionwherethecolouralternatesbetweencolourless,amberanddeepblue.

Thecolourchangeswiththeoscillatingnatureofthereaction.WhentheconcentrationofI2ishigh,thesolutionappearsamber.WhentheconcentrationofI-ishigh,thesolutioniscolourless.WhentheI-andI2ionsareinequalconcentration,theybondwiththestarchresultingintheblue-blackcolour(UCSB,dateunknown).

Thecomplexreactionhasmanysub-reactions,howeverthesimplifiedreactionmechanismis:

IO3-+2H2O2+CH2(CO2H)2+H+->ICH(CO2H)2+2O2+3H2O

Asimplifiedoverallschemeis:

Figure3:SummaryoftheBriggs-Rauscherreaction(USCB,dateunknown)


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Method

[SeeAppendixI,pg26,forfullmethodandpreliminary]

TheBriggs-Rauscherreactionisthemostsuitablemethodinahigh-schoollabbecause,althoughnoabsolutevaluecanbecalculated,theresultsallowforacomparison.Inaddition,theBriggs-Rauscherreactionisrelativelysimpletoexecute,eventhoughthechemistrybehindthereactioniscomplex.

ThemethodhasbeenadaptedfromMIT(2012)andFarusi(2009),changingtheproportionofreactantsandalteringtheapparatusaswellasclarifyingtheprocedure.ThemethodformakingblueberryjuiceandintegratingitintothereactionwasadaptedfromFarusi(2009).

Assumptions

- Acomparisonbetweenthesamplescanbemadebecausethepreparationoftheblueberrysolutionwasconsistentsoanylossesofantioxidantswouldbeequivalent.

- Thedensityoftheblueberryjuiceisthesameaswaterso1g=1ml.- Thereisnoheatlosswhentheblueberryjuiceisheated,orduringreaction.- Theheatedblueberryjuicedoesnottransferthermalenergytothereactionsolution.- HeathasnoimpactontheBriggs-Rauscherreaction(orontheoscillationtime).- Anthocyaninsaretheonlyantioxidants.[Blueberriescanalsocontainotherantioxidantssuchas

VitaminCorQuercetin(Nijveldtandothers,2001).]

Variables

Twoindependentvariablesweretested.Thefirstwastheblueberryspecies,accordingtowhereitwasgrown.Fourdifferentlocationswerechosen;Canada(wild),WashingtonState(organic),SpanishandMoroccan.Thesecondwasthetemperatureoftheblueberryjuice.Sixdifferenttemperaturewerechosen;25°C,35°C,40°C,50°C,65°Cand75°C.Whenoneoftheindependentvariableswastested,theotherwaskeptconstant.

ThedependentvariablewasthetimeforasingleoscillationoftheBriggs-Rauscherreaction,measuredusingastopwatch.


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Figure4:DiscussingControlVariablesVariable Effectonexperiment Howtocontrol

Temperature(whentestingdifferentblueberries)

LevelsofantioxidantsavailablemaybeimpactedbytemperatureasmaytherateofreactionoftheBriggs-Rauscherreaction,sotheoscillationtimewithchange.

Keepingallsolutionsatroomtemperature,controlledtemperaturewhentestingdifferentblueberrytypes.

Originofblueberry(whentemperatureistested).

Thetypeofblueberrywillimpacttheoscillationtimebecauseitmaycontaindifferentlevelsofantioxidants.

Usethesameblueberrysolution,ofasingleblueberrytype,forallthetemperaturetests.

VolumeofsolutionsA,BandCreacted

Thevolumeofeachsolutionwillimpacthowmanymolesofreactantsareavailable,thereforewillimpacttherateofreaction,andasaconsequence,oscillationtime.

Glassmeasuringcylinderswereusedtomeasure50mlofthesolutionsforeachtrial.

Concentrationofthesolutions

Itwillalsoimpactthenumberofmolesofreactantsandthereforetherateofreactionandtheoscillationtime.

ThiswascontrolledusingthesamesolutionA,BandC(fromapreparedreservoirsotheconcentrationofthesolutionswerethesame)foreachtrial.

Volumeofblueberrysolution

Affecttheamountofantioxidantsavailableandthereforetheoscillationtime.

Controlledusinga3mlglassmeasuringcylindertomeasure1mlofblueberrysolutionfromareservoir(sotheconcentrationofblueberrysolutionwasthesame)foreachtrial.

Methodoftimingoftheoscillations

Thisdirectlyimpactstheoscillationtime.Thetimesforconsecutiveoscillationswerenotthesamesothetimeforfouroscillationscouldbedifferent.

Fouroscillationsweretimedforeachtrial.Theblueberrysolutionwasaddedafterthesecondoscillationandthetimerstarted.

Preparationofreactionsolutions

- 1LofSolutionAinavolumetricflasko 400mlofdistilledwaterand410mlof30%H2O2.o Distilledwatertoppeduptothe1Lmark.

- 1LofSolutionBinavolumetricflasko 800mlofdistilledwaterand43gofpotassiumiodate,warmedgentlyo 4.3mlof18Msulphuricacido Distilledwatertothe1Lmark

- 1LofSolutionCinavolumetricflasko 500mlofdistilledwaterwith16gofmalonicacidand3.4gofmanganese(II)sulphate

monohydrate,stirredo 0.3gofstarchin5mlofdistilledwaterinabeakeraddedto50mlofboiling(distilled)

waterinaseparatebeakerthenaddedtothevolumetricflasko Distilledwatertothe1Lmark

- Allsolutionskeptinfumecupboard

Preparationofblueberrysolution

- Blueberrysamplemashedwithpestleandmortarthenstrainedo 100mlofdistilledwateraddedto2gofblueberryjuice

- Eachtypeofblueberrysolutionpreparedseparatelyafterwashinganddryingpestleandmortar.


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Briggs-RauscherReaction

- 50mlofSolutionA,BandCwerecombinedina400mlreactionbeaker.o Colourcycleoscillatedtwicethen1mlofblueberrysolutionwasaddedwhenthecolour

changedfromambertocolourlessandthestopwatchwasstarted.o Afterfourcycles,thestopwatchwasstoppedandthetimerecorded.

- Repeatedfortheremainingthreeblueberrysolutions- Moroccanblueberrysolutionsampleheatedtosixdifferenttemperaturesandprocess(above)

repeated.

Problemsencounteredinpreliminary

Inthepreliminary,thefirsttrialconsistingof10mleachofsolutionsA,BandCand3mlofblueberrysolutionwasunsuccessfulasnocolourchangewasobserved.Whensodiumthiosulfatewasaddedtoquenchthereaction,thereactionsolutionturnedreddish-brownwithatintoflilacandeventuallycolourless.Thissuggestsareactionoccurredbecauseiodineionsweredisplacedtothesulphuricacidandthedeepcoloursuggeststhestarchwaseffective.Afterthefirsttest,potentialproblemswereidentified:thestarch,asitdoesnotalwaysreactaspredicted,aswellasthemalonicacid,whichwasoldsomayhavedegraded,andthechosenvolumeofreactants.Subsequenttrialstestedanewstarchsolution,whichresultedinafaintchangefromambertocolourlessafteralongtime,suggestingtheiodineandiodideionswerepresent.Howeverwhentheblueberrysolutionwasadded,theoscillationappearedtostopsuggestingtheantioxidantsintheblueberrysolutionsloweddowntheoscillationtothepointwhereitwasunrealistictomeasureasthecolourchangewastoofainttodetect.FinallyItriedusinglargervolumesofsolutionsA,BandC(50ml)withasmallervolumeofblueberrysolution(1ml)andthereactionsolutionoscillatedfromambertocolourlesshowevernodeepbluecolourwasobserved.Duringtheactualexperiment,thedeepbluecolourwasnotobserved.


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DataCollection

Massofblueberryjuiceinblueberrysolution:

Whenpreparingtheblueberrysolutions,theoretically2gofblueberryjuicewasdilutedwith100mlofdistilledwater.Howevertheactualmassofblueberryjuiceinthesolutionvariedslightly.

WashingtonState(organic)

Canadian(wild) Spanish Moroccan

1.91g+/-0.01g 2.06g+/-0.01g 2.08g+/-0.01g 1.99g+/-0.01g

Figure6:Calculatinguncertaintyofblueberryjuiceinsolution

Figure5:Massofblueberryjuicein100mlofdistilledwaterinpreparationofblueberrysolution.Theuncertaintyof0.01gisduetothemeasuringuncertaintyoftheapparatus


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Testforanthocyaninlevelsindifferentblueberrysamples–Rawdata

Initialreactionused50mlofsolutionsA,BandCand1mlofblueberrysolutionina400mlbeaker.TheVerniermagneticstirrerwassettosettingnine.

TypeofBlueberry Timeforfouroscillations(s)

+/-0.5s

Observations

WashingtonState 183.0 Theblueberriesweresmallerwithdarkflesh.Theblueberrysolutionappearedaredtransparentcolourwithparticlessuspendedinthesolution.Aftertheblueberrysolutionwasadded,theinitialoscillationwassignificantlylongerthanthefollowingthree(almost150s).Thelastthreeoscillationsweresimilarinlength.

Canadian 240.5 Theblueberriesweresmallerwithdarkflesh.Theblueberrysolutionappearedadarkpinkish-purpletransparentcolourwithparticlessuspendedinthesolution.Aftertheblueberrysolutionwasadded,theinitialoscillationwassignificantlylongerthanthefollowingthree(almost210s).Theinitialoxidation/reductionwaslongest.

Spanish 35.8 Theblueberrieswerelargerwithpaleflesh.Theblueberrysolutionappearedapaleyellowwithsignificantamountofparticlesatbottomofsolution.Relativelyquickandsteadyoscillationsafterblueberrysolutionadded.

Moroccan 40.7 Theblueberrieswerelargerwithpaleflesh.Theblueberrysolutionappearedapaleyellowtransparentcolourwithparticlessuspendedinthesolution.Thiswasthefastestreactionoscillatingfromamber-colourlesstodeepblueaftersodiumthiosulfatewasadded.

Figure7:Theuncertaintyintimingisgreaterthantheuncertaintyoftheapparatus,+/-0.01s,becauseofhumanerrorwhichhasnotonlyadelayedreactiontimebutisbasedonobservationssoIjudgedtheuncertaintytobe+/-0.5s.

Generalobservations:

Throughoutthereaction,bubbleswereobservedandtherewerefizzingsounds.Thereactionsolutionoscillatedfromcolourlesstoamber.Thefirstoscillation,withoutblueberrysolution,wasverylong.


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Testforanthocyaninlevelsatdifferenttemperatures–RawData

Inthistest,theMoroccanblueberrysolutionwasusedbecausetheoscillationtimewasmostsuitable,shortenoughtoallowforarelativelyquickreactiontimebutlongenoughtoallowforaccuratetiming.50mlofsolutionsA,BandCwereusedwith1mlofblueberrysolutionwiththemagneticstirrersettosetting9.

Temperatureofblueberrysolution(°C)+/-2°C

Timeforfouroscillations(s)+/-0.5s

25 40.735 39.740 31.150 39.265 38.075 37.5Figure8:Theuncertaintyinthetemperaturewasincreasedfromtheapparatusuncertaintyof+/-0.1°Cto+/-2°Cduetohumanerror.Theuncertaintyintimingisgreaterthanthemeasuringuncertainty,+/-0.01s,becauseofhumanerror

(reactiontimeandobservations)

ProcessingData

Fouroscillationsweretimedtoreducetheuncertaintyinthetimeforoneoscillation.

Testforanthocyaninlevelsindifferentblueberries–ProcessedData

TypeofBlueberry Timeforoneoscillation(s)+/-0.1sWashingtonState 45.8Canadian 60.1Spanish 9.0Moroccan 10.2

Figure10:Theuncertaintyinthetimeforoneoscillationistakenfromthepropagatederrorinthedataprocessing

Figure9:Examplecalculationofprocessingoscillationtimeanduncertainty.Errormustbepropagated.


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Testforanthocyaninlevelsatdifferenttemperatures–ProcessedData

Temperatureofblueberrysolution(°C)+/-2°C

Timeforoneoscillation(s)+/-0.1s

25 10.235 9.940 7.850 9.865 9.575 9.4Figure11:Theuncertaintyinthetemperaturewasincreasedfromtheapparatusuncertaintyof+/-0.1°Cto+/-2°Cduetohumanerror.Theuncertaintyinthetimeforoneoscillationistakenfromthepropagatederrorinthedataprocessing

Graphs

45.8

60.1

910.2

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

1(WashingtonState- organic) 2(Canadian- wild) 3(Spanish) 4(Moroccan)

Timeforo

neoscillation(s)+

/-0.1s

TypeofBlueberry(bylocation)

Figure12:Howthetimeforoneoscillationchangeswiththetypeofblueberry


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7.0

7.5

8.0

8.5

9.0

9.5

10.0

10.5

20 30 40 50 60 70 80

Timeforo

neoscillation(s)+

/-0.1s

TemperatureofBlueberryJuice(°C)+/- 2°C

Figure13:Howthetemperatureoftheblueberrysolutionaffectsthetimeforoneoscillation


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Therearetwopossiblelinesofbestfitforthetimeforoneoscillationvs.temperaturegraph.Onepossibilityisthatthepointat40°Cisanoptimumtemperaturefortheantioxidantsinblueberries.

Thesecondpossibilityisthatthepointat40°Cisanoutlier.Inwhichcasethelineofbestfitwouldappearsignificantlydifferent.


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ProcessingData

WashingtonState : Canadian : Spanish : Moroccan45.8s : 60.1s : 9.0s : 10.2s

45.8/9=5.088 : 60.1/9=6.677 : 9.0/9=1.000 : 10.2/9=1.1335.1 : 6.7 : 1.0 : 1.1

InterpretationandJustificationofData

Typeofblueberryaccordingtolocation

Thereisasizeabledifferenceinthetimeforoneoscillationdependingontheoriginoftheblueberries,fromtheCanadianwildblueberriesat60.1stotheSpanishblueberriesat10.2s.LookingattheratioofCanadian(6.7):WashingtonState(5.1):Moroccan(1.1):Spanish(1)clearlyshowsthattheCanadianblueberrieshadanoscillationtimenearly7timeslongerthantheSpanishones.Whentherearemoreanthocyaninsreactingwith,andstabilising(HusneyandRomito,2015),thefreeradicalspeciesofiodine(iodine,iodideandtri-iodide)createdduringtheBriggs-Rauscherreaction(Zaidan),ittakeslongertobuilduptohighenoughconcentrationstoeffectachangeinthesolution’scolour.Sothisrangeoftimeforoneoscillation,49.4s,suggeststhereisasignificantdifferenceinlevelsofanthocyaninsbetweensamples

TheWashingtonStateandCanadianblueberriesappeartohavesimilarlevelsofanthocyanins,withasignificantgapbetweenthesimilarlevelsoftheMoroccanandSpanishblueberries.

Thiscorrelateswiththegeographicaloriginoftheblueberries;NorthAmericaasopposedtoSouthernEuropeandNorthernAfrica(seeFigure17).Thisissignificantbecausegrowingconditions(climate,soilpHandfertiliser/soilnutrients)willbearedifferentsothesameblueberryspeciesareunlikelytogrownaturallyinthetwodifferentregions.

Figure16:Ratiotocomparetherelativetimeforoneoscillationinblueberriesfromdifferentlocations

Figure17:PoliticalMapofWorld(Geology.com,2007)showingtwodifferentregionsoforiginofblueberrysamples


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Bycomparingobservabledifferencesofcolourandsize(seeFigure7,pg12),itisevidentthatatleasttwoblueberryspeciesweretestedduringthisexperiment.AccordingtoRoutrayandOrsat(2011),‘Totalantioxidantcapacityandtotalphenolic…contenthavebeenfoundtobemoderatelyheritable.’andRiberaandothers(2010),‘Theevaluationofthetotalantioxidantactivityofhighbushblueberry…revealedthatfruittotalantioxidantactivityvariedamongdifferentblueberrycultivars.’Thissuggeststhatgeneticdifferencesbetweenvarietiesofblueberriesimpacttheanthocyanincontent,whichsupportsmyfindingssinceallthesampleshaddifferentoscillationtimes.

Cultivationpracticesalsoimpactthelevelsofantioxidants(RoutrayandOrsat,2011),ofwhichsoilpHisonefactor.Iattemptedtogetincontactwiththecompaniestodeterminethespeciesgrownandthesoilconditions.Disappointingly,noneofthecompaniesrepliedsoIcouldnotmakeadefinitelinktomyfindings.

Theuseoffertilisersalsoimpactlevelsofantioxidants.Organicfertilisersgenerallydonotprovidenitrogen,whichaffectslevelsofphenolicantioxidantsproducedbytheplant(MitchellandChassy,2004).However,studieshaveshownthatlevelsofphenolicantioxidantsdecreasewhenthereisanincreaseinnutrientavailability.Onetheorysuggeststhatthereisa,‘decreasedallocationofresourcestowardsproductionofexpendablemetabolitessuchasphenolicantioxidants,’whenplantgrowthisincreasedsothenaturalproductionofplant-defencemetabolitesisdisruptedbyexcessiveuseofpesticidesandinorganic,nitrogenrichfertilisers(MitchellandChassy,2004).Aseparatestudy(RoutrayandOrsat,2011),‘showedthetotalanthocyanincontentissignificantlyhigherinorganicallycultivatedblueberries.’Furthermore,accordingtoWebb(2014),antioxidantsareproducedasaresponsetoenvironmentalstressessuchasUVlight,coldtemperatureanddrought.Thiswouldsuggestthatwildblueberries,withfewernutrientsavailableandgreaterexposuretoenvironmentalstresses,havecomparablyhigherlevelsofphenolicantioxidantsthanorganicandinorganiccultivatedblueberries.Thissupportsmyfindingsasthewildblueberrieshavethehighestlevelsofantioxidantsfollowedbytheorganicblueberriesandthenthenitrogen-rich,stressfree,non-organic,cultivatedblueberries.

Whentheblueberrysampleswerepreparedtherewasasignificantdifferenceincolourbetweenthesolutions(seeFigure18).Thissupportstheideathatthesamplesaredifferentspeciesbutalsosupportsthedifferentlevelsofanthocyaninsfoundasdifferenttypesofanthocyaninsareresponsiblefordifferentcolours,mainlyred-orangeandred-blue(RoutrayandOrsat,2011).

Figure18:Differentcoloursoftheblueberrysolutions.

1isWashingtonState(organic),2isCanadian(wild),3isSpanishand4isMoroccan.


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Temperature

Keepinginmindthatalongeroscillationtimecorrelateswithhigherlevelsofanthocyanins,thegeneraltrendintemperature(seeFigure15,pg16)suggeststhathighertemperaturesledtolowerlevelsofanthocyanins.At25°C,thetimeforoneoscillationis10.2sandat75°Cthetimeforoneoscillationis9.4s,arangeof0.8s.

Theresultssuggesttherecouldbeanoptimumtemperatureforanthocyaninsinblueberriesasthetimeforoneoscillationdecreasesrapidlyto7.8sat40°C(seeFigure14,pg16).However,thistemperaturehastheshortesttimeforoneoscillationbyasignificantamount(1.6s),suggestingthatthelevelofanthocyaninsdroppedsuddenly,thencamebackup.Althoughthisispossible,itseemshighlyunlikely.Toverifythisresult,Iwouldneedtorepeattheexperiment.Infact,Ibelievethisreadingat40°Cisanoutlier,andassuch,shouldbeignored(seeerrorindata,pg23).

Thechangeintimeforoneoscillationduetotemperatureiswithinonesecondsuggestingthattemperaturehasverylittleimpactonthelevelofanthocyaninswithinthistemperaturerange.Theslightchangeinoscillationtimecouldbecreditedtothehighertemperatureoftheblueberrysolution,increasingtherateofreactionandresultinginadecreasedtimeoverall,ratherthanrelatingtolevelsofanthocyaninswithinthereactionsolution(seesystematicerror,pg22).

Researchhasfoundanthocyaninstobethermo-sensitive,howeverdegradationoftheanthocyaninshasbeenobservedover70°Cwhileheatingbetween40°C-60°Cisreportedtonothaveasignificanteffect(RoutrayandOrsat,2011).Thissupportsmytrendoflowerlevelsofanthocyaninsathighertemperaturesandexplainswhyonlyaverysmalleffectwasnoticedwhenheatingtheblueberryjuicebetween25°Cand75°C.

Therearedifferingopinionsastowhytemperaturesabove70°Cdecreaselevelsofanthocyaninsinblueberries.Temperaturesover70°Ccanleadtohydrolysis(RoutrayandOrsat,2011),areactionwherebondsinamoleculearebrokenwithwater(KimandHehir).AccordingtoPatrasandothers(2010),‘Degradationismainlyduetooxidation,cleavageofcovalentbondsorenhancedoxidationreactionsduetothermalprocessing.’Thoughlittleisknownaboutthemechanismfordegradationofanthocyanins,onemechanismproposedisapossiblethermaldegradationmechanismfortwocommonanthocyanins.Withheating,anthocyaninswouldbreakdownintoachalconestructurethentransformtoacoumaninglucosidewiththelossoftheB-ring(Patrasandothers,2010).(SeeFigure19pg20)


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Conclusion

Inconclusion,myresultssuggestthattheoriginoftheblueberries,whichcorrespondstothevarietyofspeciesandthecultivationpractices,hasasignificantimpactonthelevelsofanthocyaninspresent.Wildblueberrieshavethehighestlevelsofantioxidantsfollowedbytheorganicblueberriesandthenthenon-organic,cultivatedblueberries.

Theresultsfortheimpactoftemperatureonthelevelsofantioxidantsinblueberriesareinconclusive.

Figure19:Thermaldegradationoftwocommonanthocyanins(PatrasandOthers,2010)


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UnansweredandAdditionalQuestions

Theexpecteddeepbluecolourneverappearedinthereactionoscillation.Iwasunabletodeterminewhysowouldliketoexplorethisfurther.

Toextendthefirstresearchquestion,Icouldinvestigatelevelsofanthocyaninsusingthesameblueberryvarietiesindifferentlocationsordifferentvarietiesinthesamelocation.

TheinvestigationonthecorrelationbetweentemperatureandlevelsofanthocyaninsinblueberrieswasinconclusiveandIwouldliketoextendtherangetohighertemperatures,from70°Cto200°C,tocorrelatewithresearchonthistopic,andalsotosatisfymyinterestinwhethercookingtheblueberriesinmuffinsdecreasesthehealthbenefitsoftheblueberries.Dealingwithhighertemperatureswouldhavesafetyimplicationsinvolvingmoresevereburns.Topreventthis,Icouldusesiliconglovesandtongstotransferhotmaterials.

FinallyIwouldliketotestthedifferenceinlevelsofanthocyaninsbetweencultivated,processedandwildblueberriesseparatelyasthiswasavariablethatIwasnotabletokeepconstantinthisexperiment.

Evaluation

Justifyingrange

Iusedfourtypesofblueberries,asthiswasthemaximumnumberofdifferentvarietiesIhadavailabletomeinlocalstores.

Thetemperaturerangewastestedfrom25°Cto75°Cwithincrementscloseto10°Callowingmetotesthowthelevelsofanthocyaninschangedoverrelativelysmalltemperatureincrements.

FouroscillationsoftheBriggs-Rauscherreactionweretimedratherthanone,reducingtheuncertaintyforthetimeofoneoscillationasthetimewasnowdividedbyfour.

Limitations

Unfortunatelytherewerelimitationstotheexperiment.Firstofall,therewaslimitedmaterialavailable,especiallythemalonicacid,whichrestrictedthenumberoftrialspossibleandlimitedtherangeoftemperaturesthatcouldbetested.Thisiseasilyimprovedhadtheschoolbeenabletoordermorechemicals.Also,therewerelimitedvarietiesofblueberriesavailable.Toacquiremoresamples,itmaybepossibletoorderfrozenvarietiesfromagreaterrangeoflocations.

Theblueberriestestedwereallpackagedandthereforeprocessed.AccordingtoRoutrayandOrsat(2011),thereisahighprobabilitythatprocessingresultsinlossofanthocyanins.TheCanadianwildblueberrieswereflashfrozen;theprocessofstoragethatcausestheleastdeteriorationofantioxidants(RoutrayandOrsat,2011).MyresultssupportthisstatementastheCanadianblueberriescontainedthemostanthocyanins.Sincetheblueberriestestedwerenotprocessedinthesamemanner,theaccuracyofthecomparisonisreduced.Toimprovethis,freshblueberries(perhapsfromu-picks)canbetestedsoprocessingwillnothavereducedtheirlevelsofantioxidants.Or,toallowforafaircomparison,alltheblueberriestestedcouldbeprocessedinthesamemanner,suchasflash-freezing.


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RandomError

Randomerrorispresent,ascanbeseenbytheerrorbarsonthegraphshowinghowtemperatureaffectsthetimeforoneoscillation(seeFigures13,pg15).Theseareduetouncertaintiesintheapparatusandhumanerror.Thevolumesofreactantsareapossiblesourceofrandomerrorasthe50mlmeasuringcylindershaveanuncertaintyof+/-2mlwhiletheblueberryjuicewasmeasuredusinga5mlmeasuringcylinderwithanuncertaintyof+/-0.5ml.Humanerrorthroughinaccuratepouring,measuringandtransferringofsolutionscanalsoincreasetherandomuncertainty.Thiscanbereducedbymeasuringcarefullyandslowlyforagreaterdegreeofaccuracywhennearingthedesiredvolume.Forsmallervolumes(lessthan25ml)glasspipettescouldbeused,whicharemoreprecise,furtherreducinghumanerror.Althoughthereisopportunityforrandomuncertaintywhenpreparingthereactants,thisdoesnothaveaneffectonthecomparisonsmadeasthesamesolutionswereusedthroughouttheexperiment.

Significantrandomerror,causedbyhumanerror,isresponsibleforthevariationinmassofblueberryjuiceintheblueberrysolution(2.00g+/-4.5%.)Itwasdifficulttomeasureaccurately2gofblueberryjuicebecause,whenstrained,theblueberryjuicecamethroughinspurts.Toimprovethis,alargervolumeofblueberryjuicecouldbeblended,usingablender,makingitmoreviscousandallowingittopourmoresmoothlysothemasscanbemeasuredmoreaccurately.

Timingtheoscillationswasafurthersourceofrandomerror.Firstlythereisrandomerrorduetohumanreactiontimes.Thenthereishumanerrorassociatedwithstarting(andstopping)thestopwatchsinceitwasbasedonobservationswhenthesolutionchangedcolour(difficulttojudgeasthecolourchangewasnotdistinct)increasinguncertainty.Finally,thereisarangeoftimeratherthanapointintimewhentheblueberrysolutionwaspouredintothereaction,whichisasystematicerrorleadingtorandomerrorasitrequireshumanjudgementtodecidewhentostartthestopwatchsotherecordedtimecouldbeaboveorbelowthe‘true’time.Toreducetheseinaccuracies,theblueberrysolutioncouldbepouredquickly(toreducetheperiod)andthestopwatchstartedhalfwaythroughpouring.Inaddition,Icouldtime10oscillationstoreduceerror.Afinalimprovementwouldbetodeterminetheproportionsandvolumesofreactantsthatleadtoadistinctcolourchangetherebyimprovingtheaccuracyintimingoneoscillationbymakingthecolour-changemorerecognizable.

Duetothelimitationinmaterial,itwasnotpossibletodomultipletrialsforeachexperiment,leadingtoanincreasedrandomerrorasnoaveragecouldbecalculated.Thiscanbeimprovedbyhavingaccesstomorechemicals,asmoretrialswouldthenbepossible.

SystematicError

Systematicerrorwaspresentintheheatingmethod.Itwasdifficulttomaintainaconsistenttemperaturewhenusingthehotplate,whichmeanstheactualtemperaturesoftheblueberrysolutionswereconsistentlyabovetherecordedtemperatures,resultinginthetimeforoneoscillationtobefasterthanthe‘true’value.Onepossiblesolutionistoheattheblueberrysolutionsusingahotplate,onalowheat,sothetemperatureincreaseisgradual,makingiteasiertotakeareadingatthecorrecttemperature.

Solutiontemperatureisalsoasourceofsystematicerror.Astheheatedblueberrysolutionwasaddedtothecolderreactionsolution,thermalenergywouldbetransferredtothereactionsolution,sotheactualtemperatureislessthantherecordedtemperature.Inaddition,theslightincreaseintemperatureofthereactionsolutioncouldincreasetherateoftheBriggs-Rauscherreaction,thereforethetimeforoneoscillationwouldbefasterthanexpected.Howeveronlysmallvolumesof


23

blueberrysolutionwereused,andallsamplesweretreatedthesameway,thereforethetemperaturechangeisnotgreatlysignificanceinthisexperiment.Athighertemperatures,moreenergywouldbetransferredtothereactionsolutionandthereforethetemperaturewouldincreasemoresignificantlytothepointwhereitmaybecomeaconcern.

Themajorityoftheblueberryskindidnotgrindintoajuicesoanyantioxidantpresentintheskinwillnotbepresentintheblueberrysolution.AccordingtoRibera(2010),‘Skinextractshavesignificantlyhighertotalantioxidantactivitythanthosedetectedforwholefruitandpulp.’Thisisasignificanterrorinthemethodbecauseitmeanstheresultsarenotanaccuraterepresentationofthelevelofantioxidantsintheentireblueberrybutonlyofthepulp.Thiscouldbeimprovedbyblendingtheentireblueberryinablendersotheantioxidantsintheskincouldalsobetested.However,asIammakingacomparison,andallsamplesweretreatedthesameway,myconclusionsarestillaccurate.

Anotherproblemencounteredwasthedifficultyinsynchronisingtheoscillationswithheatingtheblueberryjuice.Whenheatingwiththehotplate,thetemperaturewasalwaysincreasingsoIhadtojudgewhentostartthereaction(bycombiningsolutionsA,BandC)sothatthesecondoscillationcoincidedwiththecorrecttemperatureoftheblueberrysolution.Thissystematicerrorleadstorandomerrorastheactualtemperaturecouldbeaboveorbelowtherecordedtemperature.Asmentionedpreviously,animprovementwouldbetoheattheblueberryjuiceslowlysothatthetemperatureoftheblueberrysolutioncanbemaintainedwhenthereactionisstarted.

Errorindata

Thetemperaturerangewasnotbigenoughtoestablishastrongcorrelationwithexperimentaldata,asresearchsuggestsmostdegradationofanthocyaninsoccursattemperatureshigherthan70°C,whichwasanerrorintheplanning.

Thetrialat40°Cismostlikelyanoutlier,becauseIbelieveIonlytimedthreeoscillationsratherthanfour.HowevertherewasnotenoughmaterialtoretestwhenIsuspectedmyerror.

Reliabilityofdata

Thereissignificanterror,bothrandomandsystematic,inthisexperiment,decreasingdatareliability.However,theseuncertaintieshadlessofanimpactontheresultsthantheycouldhavehadbecausedatacomparisonsweremadewithnoabsolutevaluescalculated.Thismeansmyconclusionscanbeconsideredreliable.Noconclusioncouldbemadeontheeffectoftemperatureonlevelsofantioxidants,butthiswasduetoinconclusivedatanotunreliabledata.

Whenevaluatingtheimpactoftemperatureonlevelsofantioxidants,theoutlier(at40°Cwithatimeof31.1s)reducesthereliabilityofthedatabecauseitindicatesthepresenceofeitherhumanerrororanerrorinthemethod.Whenprocessingthedataanddrawingmyconclusions,Ichosetoignoretheoutlier.


24

Bibliography

Journals

Nijveldt,Randothers(May14,2001)‘Flavonoids:areviewofprobablemechanismsofactionandpotentialapplications’,TheAmericanJournalofClinicalNutrition,volume74(4),pp.418-425

Ribera,A.Eandothers(2010)‘Antioxidantcompoundsinskinandpulpoffruitschangeamonggenotypesandmaturitystagesinhighbushblueberry(VacciniumcorymbosumL.)growninsouthernChile’,Journalofsoilscienceandplantnutritionvolume10(4),pp.509-536

Routray,W.andOrsat,V.(2011)‘BlueberriesandTheirAnthocyanins:FactorsAffectingBiosynthesisandProperties’,ComprehensiveReviewsinFoodScienceandFoodSafety,volume10,pp.303-320

Thalheimer,J(July2015)‘CelebrateSummerFruit’,TodaysDietitian,volume17(7),pp.20

Webb,D.(2014)‘Anthocyanins’,TodaysDietitian,volume16(3),pp.20

Websites

Andrew,J.AreAntioxidantsDestroyedbyHeat?Availableat:www.healthyeating.sfgate.com/antioxidants-destroyed-heat9263.html[Accessed:30July2015)

Bickelhaupt,D.SoilpH:WhatitMeans.Availableat:http://www.esf.edu/PUBPROG/brochure/soilph/soilph.htm[Accessed:13August2015]

Farusi,G.(09/12/2009)LookingforAntioxidantFood.Availableat:http://www.scienceinschool.org/2009/issue13/antioxidants[Accessed:01/06/2015]Foss,K.(April1,2014)CanblueberryextractpreventParkinson’s.Availableat:http://www.mun.ca/science/news.php?id=3305[Accessed:18August,2015]

Husney,A.andRomito,C.(11June2013)FreeRadicals.Availableat:www.webmd.com/hw-popup/free-radicals[Accessed:22July2015]

Kim,P.andHehir,G.Hydrolysis.Availableat:http://chemwiki.ucdavis.edu/Physical_Chemistry/Equilibria/Solubilty/Hydrolysis[Accessed:13August,2015]

MITopencourse.TheSplendorofOneChemicalReaction.Availableat:http://ocw.mit.edu/high-school/chemistry/demonstrations/videos/briggs-rauscher-reaction/briggs_rauscher.pdf[Accessed18August,2015]

Mitchell,A.E.andChassy,A.W.(2004)OrganicAgriculture:Doesitaffectantioxidantsandnutritionalquality.Availableat:www.soyconnection.com/newsletters/soy-connection/health-nutrition/articles/Organic-Agriculture-Does-It-Affect-Antioxidants-and-Nutritional-Quality[Accessed:1August2015]

Patras,A.andothers(2010)Effectofthermalprocessingonanthocyaninstabilityinfoods;mechanismandkineticsofdegradation.Availableat:www.acedemia.edu/1179480/Effect_of_thermal_processing_on_anthocyanin_stability_in_foods_mechanisms_and_kenetics_of_degredation[Accessed3August,2015]

Robertson,S.(December2,2014)WhatareFlavonoids.Availableat:www.news-medical.net/health/What-are-Flavonoids.aspx[AccessedAugust3,2015]


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UCSB.Briggs-RauscherOscillatingReaction.Availableat:www.people.chem.ucsb.edu/feldwinn/darby/DemoLibrary/DemoPDFs/Demo005.pdf[Accessed8September,2015]

WebMD.(July142014)Antioxidant–TopicOverviewAvailableat:http://www.webmd.com/food-recipes/antioxidants-topic-overview[AccessedJuly17,2015]

Zaidan,G.Briggs-RauscherReaction.Availableat:http://ocw.mit.edu/high-school/chemistry/demonstrations/videos/briggs-rauscher-reaction/[Accessed:13August,2015]

Books

Prasad,K.N.(2014)FightHeartDiseasewithvitaminsandantioxidants.Rochester,Vermont:HealingArtsPress

Smythies,J.(1998)EveryPerson’sGuidetoAntioxidants.NewJersey:RutgersUniversityPress

Snyder,M.andClum,L.(2011)Theantioxidantcounter:ApocketguidetotherevolutionaryORACscaleforchoosinghealthyfoods.UlyssesPress.

Illustrations

Figure1.Routray,WandOrsat,V(2011)SkeletalStructureofAnthocyaninsIn:BlueberriesandTheirAnthocyanins:FactorsAffectingBiosynthesisandProperties.[Diagram](Accessed2August,2015)

Figure2.Routray,WandOrsat,V(2011)Anthocyaninscommonlypresentinblueberries,theirstructures,sugarmoieties,andcolour.In:BlueberriesandTheirAnthocyanins:FactorsAffectingBiosynthesisandProperties.[Diagram](Accessed2August,2015)

Figure3.UCSB(Unknowndate)SummaryoftheBriggsRauscherreaction[Diagram]At:http://people.chem.ucsb.edu/feldwinn/darby/DemoLibrary/DemoPDFs/Demo005.pdf(Accessed9September,2015)

Figure17.Geoglogy.com(2007)PoliticalMapofWorld[Diagram]At:http://geology.com/world/world-map.shtml(Accessedon11.08.2015)

Figure19.Patras,Aandothers(2010)Thermo-degradationoftwocommonanthocyanins.In:Effectofthermalprocessingonanthocyaninstabilityinfoods.[Diagram]At:http://www.acedemia.edu/1179480/Effect_of_thermal_processing_on_anthocyanin_stability_in_foods_mechansim_and_kenetics_of_degredation(Accessedon2August2015)


26

AppendixI

Method:

Apparatus

- 4differentvarietiesofblueberrieso FromSpaino FromMoroccoo FromWashingtonState(organic)o FromCanada(wild)

- Distilledwater(3litr)- Potassiumiodate(43g)- Malonicacid(16g)- Hydrogenperoxide(410mlof30%)- Sulfuricacid(4.3mlof18M)- Manganese(II)sulphatemonohydrate(3.4g)- Sodiumthiosulfate(20g)- Solublestarch(0.3g)- Volumetricflasks

o 31Lglassvolumetricflasko 4-640mlglassvolumetricflask

- Glassbeakerso 10mlbeakero 2100mlbeakero 7200mlbeakero 10400mlbeaker

- GlassMeasuringCylindero 1Lo 3500mlo 650mlo 10mlo 45ml

- MetalSpatula- 2-decimalbalance- Hotplate- 3Glassstirringrods- Magneticstirrerandstirrod(2”)- Smallbowl- Finestrainer- Pestleandmortar- DigitalThermometer- Boardmarker- Stopwatch- Fumehood- Largebucketandlid- Safetygogglesandgloves- SodiumBicarbonate


27

JustificationofApparatus

- Distilledwater–ratherthantapwatertoreducechemicalsthatarepresentintapwater(suchaschlorine)thatcouldpotentiallyaffectthereaction.

- Volumetricflask–ratherthanabeakerbecausethevolumetricflaskonlyhasonelineindicatingthevolume,solesslikelytomeasuretothewrongline,andvolumetricflasksalsoallowformoreaccuratemeasurementsofvolume.

o Thevolumesofthevolumetricflaskswerechosentocorrespondtotheonemarkedline,sinceonlyonevolumecanbeaccuratelymeasuredusingaspecificvolumetricflask.

- Glassbeakers–usedinsteadofplasticbeakersbecausethegraduatedscaleontheglassissharpersoitiseasiertogetamoreprecisemeasurement.

o Volumeswerechosensothatthesolutionsmeasureddidnotfillthebeaker,toreduceriskofspilling,andsmallenoughtohaveappropriateincrementsinthescale.

- Glassmeasuringcylinder–usedinsteadofaplasticmeasuringcylinderbecausethegraduatedscaleontheglassissharpertomakethemeasurementsmoreprecise.IusedaglassmeasuringcylinderratherthanpipettesbecauseIcouldthenleavethepreparedsolutioninthemeasuringcylinderuntilIneededit

o Thesizewaschosentothesmallestpossiblevolumeforthemeasurementsothattheincrementsweremostappropriate,andcouldbeaspreciseaspossible.

- Metalspatula–usedtotransfersolidstoweighingboatsratherthanrisktouchingthechemicalswithmyhands.

- Two-decimalplacebalance–usedinsteadofaoneorzerodecimalplacebalancebecauseitismoreaccurate.Iusedthisinsteadofamoreaccuratebalancebecausetherandomuncertaintywaslargerthanthemeasuringuncertaintyoftheapparatus.

- Hotplate–usedinsteadofaBunsenburnerbecauseitiseasiertocontrolthetemperature.- Glassstirringrods–ratherthanmetalsoitdoesn’treactwiththereactionsolution- Magneticstirrer–ratherthanstirringrodbecauseitstirsmoreconsistentlyandallowsmeto

performothertasksatthesametime- Finestrainer–ratherthanfilterpaperbecausetheblueberrypulpwasnotliquidenoughto

strainthroughfilterpaper.- Pestleandmortar–ratherthanblenderbecausetheamountofblueberriesbeingcrushed

wastoosmallforablendertobeaffective.- Digitalthermometer–ratherthanaglassthermometerbecauseitismoreaccurate.- Stopwatch–ratherthanaclockasitishandheldsoeasiertomanoeuvreanditismore

accurate.- Sodiumbicarbonate–toneutralisesulphuricacidspills


28

Procedure

Note:mustwearsafetygoggles/glassesandgloves,havebucketandlidready

1) Label450mlbeakerswiththevarietyofblueberrytobetested(onevarietyforeachbeaker)usingaboardmarker.

2) Mashonesampleusingapestleandmortar.Placea200mlglassbeaker,labelledwiththecorrespondingtypeofblueberry,onabalanceandzerothebalance.Straintheblueberryjuicefromthemortarintothebeakerusingafinestrainuntilthebalancereads2.00g.Setthesampleaside.Rinseanddrythepestleandmortarthenrepeatfortheothersamplesofblueberries.

3) Usinga100mlglassmeasuringcylinder,measure100mlofdistilledwaterandaddtothe200mlglassbeakerlabelledwiththefirstsampleandstirwithaglassrod.Rinseanddrytheglassrod.Repeatfortheremainingsamples.Thesearethefourblueberrysolutionstobetested.

4) PrepareSolutionAinaventedfumecupboard.- Label1Lvolumetricflask‘SolutionA–hydrogenperoxide’withaboardmarker.Label

one500mlglassmeasuringcylinder‘distilledwater’andanother‘30%hydrogenperoxide’.

- Measure400mlofdistilledwaterintothe500mlglassmeasuringcylinderlabelled‘distilledwater’andaddtothe1Lvolumetricflasklabelled,‘SolutionA–hydrogenperoxide.’

- Measure410mlof30%H2O2intothe500mlglassmeasuringcylinderlabelled‘30%hydrogenperoxide’andaddtothe1Lvolumetricflasklabelled,‘SolutionA–hydrogenperoxide.’

- Adddistilledwatertothe1Lvolumetricflaskuptothe1litremarkthengentlyswirlflask.

- Keepthesolutioninthefumecupboarduntilrequired.5) PrepareSolutionBinaventedfumecupboard.

- Labeladifferent1Lvolumetricflask‘SolutionB–Potassiumiodateandsulfuricacid’withaboardmaker.

- Labelthe1000mlglassmeasuringcylinder‘distilledwater’anduseittomeasure800ml,thentransferintothevolumetricflasklabelled‘SolutionB.’

- Placea50mlbeakeronathreedecimalbalanceandcalibrate(zero)thebalance.Then,usingametalspatula,weigh43gofpotassiumiodate(KIO3)intothe50mlbeakerandthentransferintothe1Lvolumetricflasklabelled‘SolutionB.’

- Addamagnetstirbartothe1Lvolumetricflasklabelled‘SolutionB’andplaceonmagneticstirapparatus(settomediumspeedsoitisnothittingside).

- Labela5mlglassmeasuringcylinder‘sulfuricacid,’thenuseittomeasure4.3mlof18Msulfuricacid.

- Heatthesolutioninthe1Lvolumetricflasklabelled‘SolutionB’onlowheatusingahotplate.Whilestirring(withthemagneticstirbar),addthe4.3mLofconcentratedsulfuricacid.

- Adddistilledwaterupto1L(todilutethesolution)andstir,usingaglassrod,untilalltheKIO3isdissolved.

- Removetheflask(labelled‘SolutionB’)fromtheheatandleavetocoolinfumehooduntilrequired.

6) PreparesolutionCintheventilatedfumecupboard- Labelathird1Lvolumetricflask,‘SolutionC–malonicacid,manganese(II)sulphate

monohydrateandstarch’usingaboardmaker.- Usingthe500mlglassmeasuringcylinderlabelled‘distilledwater’measure500mlof

distilledwaterandtransferintothe1Lvolumetricflasklabelled‘SolutionC’.


29

- Placea50mlbeakeronathreedecimalbalanceandcalibrate(zero)thebalance.Then,usingametalspatula,weigh16gofmalonicacidintothebeakerandtransferintothe1Lvolumetricflasklabelled‘SolutionC’.

- Placeaplasticweighingboatonthebalanceandcalibratethebalance.Weigh3.4gofmanganese(II)sulphatemonohydrateintotheweighingboatandtransfertothe1Lvolumetricflasklabelled‘SolutionC.’

- Placethe1Lvolumetricflaskonamagneticstirrerplate(withamagneticstirbaraddedtothebeaker)setatmediumspeedsoitisnothittingthesides.

- Usingthe50mlglassmeasuringcylinderlabelled‘distilledwater’,measure50mlofdistilledwaterintoanew100mlglassbeaker(labelled‘starchsolution’)andbringtoaboilusingahotplate.

- Placeasecondplasticweighingboatonthebalanceandzeroit,thenweigh0.3gofstarchandtransferintoanother50mlbeaker(labelled‘starch’).Usingthesecond5mlglassmeasuringcylinder(labelled‘distilledwater’)measure5mlofdistilledwaterandtransferintothe50mlbeakerlabelled‘starch’.Usingaglassrod,stirthewaterandstarchintoapaste(slurry)

- Pourthestarchslurry(fromthebeakerlabelled‘starch’)intothe100mlglassbeaker(labelled‘starchsolution’)ofboilingwaterandheatfor2-3minutesuntilthestarchisdissolved.

- Pourthesolutioninthebeakerlabelled‘starchsolution’intothe1Lvolumetricflasklabelled‘SolutionC’

- Adddistilledwatertothe1Lvolumetricflasklabelled‘SolutionC’untilthe1Lmarkandleavetocoolinthefumehooduntilrequired.

7) Placea400mlbeaker(labelled‘reactionbeakertemperatureofblueberriestrial1’)onmagneticstirplateandputamagneticstirbarinthebeaker.

8) Label350mlglassmeasuringcylinders,‘SolutionA’,‘SolutionB’or‘SolutionC’withaboardmarker

9) Usinga50mlglassmeasuringcylinderlabelled‘SolutionA’,measure50mlofsolutionAandsetaside.RepeatforsolutionBandC(usingtheirrespective50mLmeasuringcylinders).

10) Usinga5mlglassmeasuringcylindermeasure1mloftheblueberrysampletobetested(andkeepinmeasuringcylinder.)

11) Transferthe1mlofblueberrysampleintoa10mlbeaker.Usingahotplate,heatuptheblueberrysampleto25°C,trackingthetemperatureasitincreaseswiththedigitalthermometer.

12) Whenthetemperatureoftheblueberryjuicenearsthetargettemperature,combinethepremeasuredsolutionsA,BandCintothebeakerlabelled‘reactionbeaker’andstartthemagneticstirrertosetting9.Letthereactiongothroughtwooscillations,thenaddtheheatedblueberrymixture(whenthesolutionturnsfromambertocolourlessthesecondtime)tothebeakerlabelled‘reactionbeaker’andstartthestopwatch.Lettheoscillationgothroughfouroscillationsaftertheblueberrysampleisadded,thenstopthestopwatchandrecordthetimeinatablewiththecorrelatingvarietyofblueberry/temperaturedependingonthetest.Thenstopthemagneticstirrer.

13) Putthereactionbeakerintothebucketandputonthelidtomoveintothefumecupboard,thensprinklesodiumthiosulfateintoreactionbeakertoreduceiodinetoiodideionsfordisposal.

14) Rinsethe5mlglassmeasuringcylinderandthe10mlglassbeaker(thatcontainedtheblueberrysample.)

15) Repeatsteps7to14forthesameblueberrysampleatdifferenttemperatures(35°C,40°C,50°C,65°Cand75°C).

16) Repeatsteps7to14fortheremainingthreeblueberrysamples,omittingstep10(donotheattheblueberryjuice)astrialsaretobeperformedatroomtemperature.

17) Cleanupexperimentareaandwipedowndesks.


30

Disposal(MIT,2012)

- Reactionproducesalargeamountofelementaliodinethatmustbereducedtoiodideionsfordisposal.

o SprinklesodiumthiosulfateintothebeakercontainingtheBriggssolution(thereactionbeaker).Thiswillcausethesolutiontofoamup.Thereaction(betweentheiodineandthiosulfate)isveryexothermicsothebeakerwillbecomehot.Continuetosprinklethesodiumthiosulfateuntilthefoamingstops,indicatingthereactionhasstopped,andthesolutionwillbecomecolourless

o Waitfortheneutralisedsolutiontocoolthenthesolutioncanbedisposedofinaccordancetodisposallawsofyourspecificcountry.

Safety(MIT,2012)

- Iodineisproducedduringthereactionandvapourwillbegintoescapewhenreactionstopsoscillating.

o Vapourcanbeextremelyirritatingtoeyes,skin,mucousmembranesandlungso Asasafetyprecaution,havealargebucketwithlidhandy,andsetthereaction

beakerinthebucketwiththelidandplaceinthefumecupboardassoonasithasfinishedoscillating.

§ Quenchthereaction(instructionsin‘disposal’)- 30%hydrogenperoxideisastrongoxidizingagentthatcancauseseriousburnsifitcomesin

contactwithskin.o Rinseaffectedareaswithlotsofwaterfor15-20minutesthengetemergency

attentiono HydrogenPeroxideincontactwithotherchemicalscanresultinveryexothermic

(explosive)reactions.Keepaway(andstore)awayfromotherchemicals.- Sulfuricacidisastrongacidandcancauseburns.Ifthevapourisinhaled,itcouldcause

seriouslungdamage.o Neutralisespillswithaweakbase(ie.Sodiumbicarbonate)

- Malonicacidisastrongeye,skinandrespiratoryirritanto Wearsafetygogglesandworkinawell-ventilatedroom,keepingsolutionsinafume

cupboardwhennotinuse- Asasafetyprecaution,preparethesolutionsinaventedfumecupboardwearingsafety

gogglesandgloves.- DuringtheexperimentIamworkingwithboilingsubstances

o Becautiouswithequipmentandkeepawayfromtheedgeofdesks.o Ifyouspillinonyourskinrinsewithcoldwaterimmediatelyandseekmedical

attention.- Hotplateishotsomaycauseburns

o Asaprecaution,keepawayfromtheedgesoftablesandturnoffwhennotinuse.o Treatforburn(putaffectedareaundercold,runningwaterimmediately)andseek

medicalattentionifsevere.- Workingwithglasswarewhichmayshatterintosharpshards

o Asaprecaution,keepawayfromedgeso Sweepuppiecesaftermakingsurethechemicalsaresafe(neutralisethechemicals

first)o Ifinjuredseekmedicalattention.


31

Preliminary

IusedtheMoroccanblueberrysolution(2.00g+/-4.5%in100mlofdistilledwater)forallpreliminarytests.10mlofsolutionA,BandCwereusedwith3mlofblueberrysolution,unlessotherwisementioned.

First,Icrushedtheblueberries–usingthepestleandmortar,andthenstrainedthemwithastrainerratherthantheblenderbecauseonlysmallmassesofblueberrieswereavailable.

Thefirsttrialwasunsuccessfulastherewasnocolourchange,sonovisibleoscillations.Whensodiumthiosulfatewasadded(toquenchthereaction)attheendofthefirsttrial,thereactionsolutionturnedreddish-brownwithsomepurple-lilacareasthateventuallyturnedcolourless.Thecolourchangesuggeststheiodineionsweredisplacedtothesulphuricacid,suggestingareactiondidoccur,anditturnedadeepbluecolourwhenthesodiumthiosulfatewasaddedsuggestingthestarchwasalsoeffective.Thismeanttherecouldbeaproblemwiththestarch,withthemalonicacid,orwiththevolumeofreactants,whichcouldbetoosmalltoallowoscillationstobeseen.

Thereforethesecondtrialusedanewstarchsolutionandeverythingelsewaskeptconstant.Initially,theresultswerethesameasthefirsttrial,withnocolouredoscillations.However,afterasignificantamountoftime,thereactionsolutionchangedfromfaintambertocolourlesssuggestingthatiodineandiodideionsarepresent.Whensodiumthiosulfatewasaddedtothereactionsolution,itturnedbrown-redwithapurpletinge,asbefore.

Howeverwhentheblueberrysolutionwasadded,thereactionappearedtostoposcillating.Toconfirmthereactionwasoscillating,Iwaiteduntilthereactionsolutionwasclearlyoscillating(movingbetweenpaleambertocolourless)beforeIaddedtheblueberrysolutionasecondtime.Whentheblueberrysolutionwasadded,acolourchangewasnotobserved.Eithertheantioxidantintheblueberrysloweddowntheoscillationtothepointwhereitwasunrealistictomeasureorthecolourchangewastoofainttodetect.

AtthispointIknewthatthereactionsolutionwasoscillating,withoutgoingthroughthedarkbluestage,buttherewasaproblemwhenIaddedtheblueberrysolution.

PossiblechangesthatIcouldmakeincludedusinglargervolumesofreactants(solutionsA,BandC),smallervolumesofblueberrysolutions,addingsodiumthiosulfatetothereaction(asacolourchangewasobservedwhenitwasadded)orchangingthemalonicacidtoacetone(becausethemalonicacidwasoldandmaybeoxidisedorgoneoff).

Whenonesmallpieceofsodiumthiosulfatewasaddedtothereactionsolution,beforetheblueberrysolutionwasadded,thesolutionchangedfromcolourlesstodarkpurplebecauseofthereactionbetweentheiodineandthiosulfate.Therewasafaintoscillationbetweendarkandslightlypalerpurplebeforebecominglighterandfinallyturningcolourless,suggestingtheoscillationreactionwasoccurring.Ibelievethisisbecausethesodiumthiosulfatehadcompletelyreactedatthatpoint.

FinallyItriedusinglargervolumesofthesolutionsA,BandC(100ml),withoutaddingthesodiumthiosulfate.Thereactionsolutionoscillatedfromcolourlesstodeeperamber.When3mlofblueberrysolutionwasaddedtothereactionsolution,thereweresmallbubblesandafaintyellowtingeafterasignificantamountoftime.Thenthereactionsolutionturneddarkblue.Therewasnooscillation.Thismaybebecausethelevelofantioxidantswastoohighsotheionswereallscavengedbeforethesolutioncouldoscillate.ThereforeIdecidedtousesmallervolumesofblueberrysolution(1ml)fortheactualexperiment.Duringtheactualexperiment,thedeepbluecolourwasnotobserved.

How do levels of anthocyanins in blueberries change due … · How do levels of anthocyanins in blueberries ... How do levels of anthocyanins in blueberries change due to temperature

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