the effect of temperature on short term...

THEEFFECTOFTEMPERATUREONSHORTTERMETHYLACETATEDEGRADATIONINBEER

MichellePather

ProjectSupervisor:AlisynNedoma

TheUniversityofAuckland,departmentofChemicalandMaterialsEngineering

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AbstractMacsHopRockerwassubjectedtoshorttermageingandthesubsequentchangesinflavourchemicalconcentrationswereinvestigated.Threedifferentageingconditions-cold,roomtemperature,andheatedwereused. Inparticular,ethylacetate,anester,was investigated.Ethylacetateprovidesapleasantfruityflavourinbeer,adecreaseinthisallowsstaleflavourstobemorepronounced.Gaschromatographycombinedwith flame ionisationdetection (GC-FID)wasused tomeasure thechanges in the level of ethyl acetate. The first step involved crafting a method for temperaturechanges in the GC-FID. Due to time constraints, once this method was developed, only threemeasurementsweremadeoveraoneweekperiod.Previousresearchshowedthatforotherbeertypes,ethylacetateconcentrationdecreasedovertime.Fromtheresultsobtainedinthisresearchnotrendwasfound.However,itissuggestedthatfurtherresearchisdone,involvingmoremeasurementsoveralongertimeperiod,beforeanyconclusionsaremade.

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TableofContentsAbstract.................................................................................................................................................III1.0Introduction.....................................................................................................................................1

1.1Projectobjectives.........................................................................................................................21.2Acknowledgements......................................................................................................................2

2.0LiteratureReview.............................................................................................................................42.1Thebeerbrewingprocess............................................................................................................42.2Flavourcomponentsofbeer........................................................................................................52.3Majorflavourcompoundsinbeer...............................................................................................62.4Unfavourableflavourcompoundsinbeer...................................................................................92.5Sensoryanalysis.........................................................................................................................102.6Methodsofidentifyingchemicalcompoundsinbeer...............................................................11

2.6.1Gaschromatography..........................................................................................................112.6.2MassSpectrometry.............................................................................................................122.6.3Flameionizationdetector...................................................................................................122.6.4Electroncapturedetector...................................................................................................12

2.7Problemstatement....................................................................................................................133.0Semesteroneprojectwork............................................................................................................143.1ExperimentalProcedure.................................................................................................................15

3.1.1MajorequipmentUsed...........................................................................................................153.1.2EarlierExperimentalwork.......................................................................................................153.1.3LaterExperimentalwork.........................................................................................................17

4.0ResultsandDiscussion...................................................................................................................205.0Futuredevelopments.....................................................................................................................246.0Conclusions....................................................................................................................................267.0References......................................................................................................................................288.0Appendices.....................................................................................................................................30

8.1AppendixA:Ethylacetateconcentrationcalibrationcurves.....................................................308.2AppendixB:GC-FIDspectrum....................................................................................................318.3AppendixC:Safety&LaboratoryUse........................................................................................32

TableofTablesTable1:Significantchemicalcompoundsinbeer..................................................................................6Table2:Ageingtemperatureeffectonethylacetatelevels..................................................................7Table3:Commonoffflavoursinbeer(Carr,2016;Meilgaard,1982)....................................................9Table4:Flavourthresholdsforbeertasting(Briggsetal.,2004)........................................................10Table5:WhiskeyGC-MSmethods.......................................................................................................15Table6:StoreboughtbeerGC-MSmethods.......................................................................................17Table7:Mac'sHopRockerbeerGC-FIDmethodsused.......................................................................18Table8:MeasuredethylacetateconcentrationsfromGC-FID............................................................21Table9:BeerdegradationHAZOPanalysis..........................................................................................32TableofFiguresFigure1:Beerflavourwheel(Hughes&Baxter,2001)..........................................................................5Figure2:Effectofethanolconcentrationonethylacetateformed.......................................................7Figure3:Effectoftemperatureonethylacetateformation..................................................................7Figure4:Reactionschematicforproductionofethylacetate...............................................................8Figure5:Tasteareasoftongue............................................................................................................10Figure6:Olfactorysystem...................................................................................................................10Figure7:Ethylacetatestandardsolutionsandpipette.......................................................................18

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Figure8:EthylacetateandbeersamplesinGC-FID............................................................................18Figure9:GC-FIDmachineusedformeasurements..............................................................................18Figure10:SpectrumfromGC-FIDfor200ppmethylacetatestandardsolution..................................20Figure11:Calibrationcurveproducedfromethylacetatestandardsolutions....................................20Figure12:RoomtemperaturebeersampleGC-FIDspectrum.............................................................21Figure13:ThirdtrialwatersamplespectrumfromGC-FID.................................................................22Figure14:Ethylacetatelevelsovertimewithdifferentstoragetemperatures..................................22Figure15:Firstethylacetatesamplecalibrationcurve.......................................................................30Figure16:Thirdtrialcalibrationcurve.................................................................................................30Figure17:FirstroomtemperatureGC-FIDspectrum..........................................................................31

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1.0IntroductionThebeerindustryThebeermanufacturingindustryiscontinuallygrowing,thereforeconsumershavealargevarietyofbeer to choose from. This leads to expectations of high quality, putting pressure on brewers toproducepremiumproduct.Productioncostsforbreweriesarehigh,hence,alotofvalueisputonthefinalproductproduced.BeerstabilityBeer ismadeupofhundredsofchemicalcompoundsthatofferdifferentflavourprofiles.Previousresearch has shown that the chemical compounds in beer will either increase or decrease inconcentration over time due to chemical reactions still occurring (Vanderhaegen et al., 2003).Different types and brands of beer age at varying rates and therefore undergo different flavourchanges.Ethylacetatewasanalysedinthisprojectasitprovidesacommonanddistinguishableflavourchangeinbeer.Thisdegradationhappensinashortertimeperiodcomparedtootherflavourcompounds,andsocouldbeobservedinthetimeperiodforthisproject.Beer flavour stability over time is important to maintain customer satisfaction. However, longtransportationtimesmeansthatbeercanundergovariablestorageconditionsbeforeitreachestheconsumer.Thevariationofstorageconditions,suchashumidity, light,andtemperature,havebeenshowntoaffectthechemicalreactions inbottledbeer.Temperaturewillbe investigated inthisreportas itsvariabilityismorerealisticforconsumers.Temperatureaffectsthereactionkineticsofchemicalsinbeer.Therefore,changesintemperaturewillaffectwhetherthesereactionsoccurornot,howfasttheyoccur,andhencehowfastabeerdegrades.1.1ProjectobjectivesPreviouspapershaveinvestigatedethylacetatedegradationinbeer.Overtimeitslevelshavebeenseentodecrease.However,mostofthesestudieshavebeendoneonAmericanandEuropeanbeer,andnoneonMac’sbrandbeer.Different typesofbeerareproducedbydifferenthops, strainsofyeast,andfermentationtemperatureswhichleadstodifferentchemicalcompoundsbeingproducedinvaryingamounts.Therefore,differentresultscouldbeobtained.Mac’sHopRockerbeerwillbeusedforanalysis inthisstudy. Itwillbeagedunderthreedifferenttemperatureconditions–cold,roomtemperature,andheated.Initiallyintheinvestigation,amethodofquantitativeanalysisofethylacetateinbeermustbedeveloped.Thechangeinconcentrationofethylacetatewillsubsequentlybeinvestigated.1.2AcknowledgementsThe author would like to recognise Alisyn Nedoma, Peter Martin, Matthew Sidford and MitchelWoodhouse for their knowledge and guidance in this project. The author would also like toacknowledge support from the University of Auckland Chemical and Materials Engineeringdepartment.

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2.0LiteratureReview2.1ThebeerbrewingprocessMaltedbarleyisheatedbysoakinginhotwatertostartthegerminationprocess.Thismeansthatmaltsugarsarereleasedbythebarley.Thisprocessisstoppedpartwaythroughtoensurethebarleydoesnotfullyseed.Themaltsugarsolution,calledwort,isthenboiledwithhops,theflowerfromahopplant.Thehopsaddsbitterflavouringtobeerwhichbalancesoutthesweetnessofthemaltsugar(Coe,2003).Oncethissolutionhascooled,yeastisaddedwhichfermentsthemaltsugars,breakingthemdownintoalcoholandcarbondioxide.Thetopofthefermentationvesselisfittedwithanairlockthatishalffilledwithwater.Thisreleasescarbondioxidefromthevesselwhilesimultaneouslypreventingoxygenfromentering(Palmer,2006).The fermentation stage should theoretically be completed after about 10 days. To test this, ahydrometer reading is taken fromasampleofbeer.Thismeasures thespecificgravity (SG)of thesample. The sugars and the alcohol in the sample have a higher and lower density than waterrespectively.WhentheSGreadingisabout1.010itmeansthecorrectratioofsugarandalcoholhasbeenreachedandthebeerisreadytobebottled.Thebeeristhentransferredtoeitherkegsorbottles.Toprovidecarbonation,carbonationdropsorpriming sugarsareadded.These sugarsareagain fermentedby theyeast,however, this time thecarbondioxideistrappedinthevesselprovidingbeerit’sfizziness.

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2.2FlavourcomponentsofbeerTherearehundredsofdifferentfavourableandunfavourableflavourcombinationsthatcombinetocreate the different types and brands of beer, each with their own distinct taste (Meilgaard,1982).Theseareshownintheflavourwheelbelow.

Figure1:Beerflavourwheel(Hughes&Baxter,2001).

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2.3MajorflavourcompoundsinbeerTheyeastusedto fermentbeerproducesmanyotherchemicalcompoundsalongsideethanolandcarbondioxide.Theflavourcomponentsshownintheflavourwheelshowninfigure1areproducedby different combinations of chemical compounds A brief overview of two significant types ofcompoundsinbeer,estersandaldehydes,hasbeenprovidedintable1(Buiatti,2009;Hough,Briggs,Stevens,&Young,1982).Table1:Significantchemicalcompoundsinbeer

Type Name ChemicalStructure DescriptionEster Ethylacetate

The main ester present in beer is ethylacetate.Atlowlevelsethylacetateprovidesafruityflavour,however,athigherlevelsitproducesanunpleasantsolventlikeflavour.

Aldehyde Acetaldehyde

Thisisthemostcommonaldehydefoundinbeer–itisexcretedbyyeastintogreenbeerduringthefirstthreedaysoffermentation.It gives beer a ‘young’, ‘green’ flavour.Young beer usually contains about 20-40mg/L, whereas final beer productcontains about 5-15mg/L (Hough et al.,1982). Acetaldehyde can be reduced toethanol, but canalsobeoxidised to aceticacid.

Aldehyde Nonenal

Nonenal is regarded as one of the mainsourcesofflavourdegradationinbeer.Itisan alkyl aldehyde that gives beer apapery/wet cardboard taste.When a beerages,changesinthelevelofnonenalcausesbeer to have a stale taste.(Lentini et al.,2015).

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Theproductionanddegradationofethylacetatehasbeeninvestigatedfurtherasitisthechemicalcompoundsofinterestinthisresearch.ProductionofethylacetateEthyl acetate is produced during yeast fermentationthrough esterification of ethanol and an acyl CoAcompound (a long-chain fatty acid with a coenzymeattachedtotheend).Aschematicofthechemicalreactionpathwayshasbeenprovidedinfigure4(García,García,&Díaz, 1994). This also shows the complexity of chemicalreactionsundergoneduringbeerbrewing.An earlier paper shows that as ethanol concentrationincreasedinfermentation,sodidtheconcentrationofethylacetate(Nordstrom,1961).Thiscanbeseeninfigure2.Asethanol is readily available from the yeast fermentation,the concentration of esters is dependent on thefermentablesugarsavailable.ThesamepaperbyGarcia(1994)showedhowanincreaseintemperatureduringfermentationincreasedproductionofethylacetate,asindicatedinfigure3.(Garcíaetal.,1994)DegradationofethylacetatePrevious research has shown that as beer ages, theconcentration of esters, including ethyl acetate,decreases (Vanderhaegen et al., 2003). Extracellularesterasesfromtheyeastcanbreakdownesters,ortheycanbehydrolysed(brokendownwithwater).Thiscausesthefruityflavourofethylacetatetodecreaseandallowsunfavourable flavours to become more pronounced,thereforealteringthetasteofthebeer.The same report investigated whether storagetemperature and headspace gas affects the change inconcentration of ethyl acetate during ageing. Themeasurementsaftersixmonthsofstorageareshownintable2.Forallsamplestheconcentrationofethylacetatedropped.Thelargestdecreaseinconcentrationwasseeninthehighstoragetemperaturesamples.Table2:Ageingtemperatureeffectonethylacetatelevels

Freshbeer

0ºC 20ºC 40ºC

CO2 CO2 Air CO2 AirConcentration(µg/L)

28099 27578 27747 26711 22340 22650

Figure2:Effectofethanolconcentrationonethylacetateformed

Figure3:Effectoftemperatureonethylacetateformation

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Figure4:Reactionschematicforproductionofethylacetate

Vanderhaegen(2003)hasdoneextensiveresearchonhowchemicalcompoundsinbeerchangeovertime.Onepaperthatfocussedonanalemeasuredthelevelsofbothethylacetateandnonenaloversixmonths.Comparedtoothersimilarstudiesthistimeperiodissignificantenoughtoseeadifferenceincompoundlevels,butshortenoughtobearealistictimeframeforaconsumertohavestoredbeer.Thebeersstudiedinthispaperwereagedatvaryingtemperatureswhichallowsacomparisontobemadebetweenstoringconditions.Theethylacetatedecreasedinconcentrationinsamplesfromallthreeconditions,withthegreatestdecreaseintheelevatedtemperaturesample.Thenonenalconcentrationslightlydecreasedinthelowtemperaturesampleandslightlyincreasedinboththeothersamples.Thisveryslightdecreasedifferstotraditionalopinionthatnonenalprovidestheunpleasantcardboardflavourofstaledbeer.AlaterpaperalsoheadedbyVanderhaegeninvestigatedtheageingeffectsonflavourcompoundsineightdifferentbeersoveroneyear.Thismeantthatcomparisonscouldbemadebetweendifferentbeer types. Aswith his earlier paper, Vanderhaegen discusses how ester hydrolysis results in thedecreaseinconcentrationofmostestersinbeer.Forallbeertypes,theconcentrationofethylacetatewasshowntoremainconstantordecreaseovertheoneyearperiod(Vanderhaegen,Delvaux,Daenen,Verachtert,&Delvaux,2007).

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2.4UnfavourableflavourcompoundsinbeerA summary of some common off flavours in beer are presented in table 3. To include all flavourcompounds is beyond the scopeof this research.Mostof theseoff flavours arisedue tobrewingconditions,butdegradationofthesechemicalsafterbottlingwillalsoleadtoflavourchanges.Table3:Commonoffflavoursinbeer(Carr,2016;Meilgaard,1982)

Compound ChemicalStructure

Boilingpoint

Flavour Cause

Diacetyl(2,3-butanedione)

88ºC Buttery Producedduringfermentationbyyeast

3-methyl-2-butene-1-thiol

128-135ºC

Skunky Hop acids and riboflavin in thebeerreactwithsunlight

DimethylSulfide

37ºC Sweetcorn/Vegetables

Formed during malting fromgermination of grain where S-methyl methionine is brokendown

Aldehydes, mainlyisobutyraldehyde

63ºC Freshwheat/grains

Compoundreleasedbymaltwhencrushed,maltedorsoaked

IsoamylAcetate

142ºC Fruity/bananas

Yeast produces esters duringfermentation,anoverproductionof these will produce this offflavour

HydrogenSulfide

-60ºC Rotteneggs Again, yeast produces hydrogensulfide during fermentation, anover production of these willproducethisoffflavour

Isovaleric acid (3-Methylbutanoicacid)

175ºC Cheesy/dirtysocks

Whenhopalphaacidsareoxidisedisovalericacidisproduced.

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2.5SensoryanalysisInindustry,sensoryanalysisisusedbytastingpanelstoidentifyflavoursinbeer.Thisisdonetocheckthe quality of the beer, and to ensure that different batches are identical. Flavour sensation is acombinationofbothgustatory(taste)andolfactory(odour)perceptions(Briggs,Boulton,Brookes,&Stevens,2004).

Sensory tasting is a structured methodology for analysing food and beverage samples. A briefoverviewofitsguidelinesandoperationprocedurehasbeenincludedinthisresearchreportasthisishowflavourchangesarequantifiedinindustry.Therearedifferentthresholdsusedwhenmeasuringtheflavourcompositionsoffoodanddrink.Thefollowingdefinitionsareused,Table4:Flavourthresholdsforbeertasting(Briggsetal.,2004)

Detectionthreshold TheminimumvalueofasensorystimulusneededtogiveasensationDifferencethreshold ThesmallestperceptiblechangeinthephysicalintensityofastimulusRecognitionthreshold Theminimumvalueofasensorystimulusallowingidentificationofthe

sensationreceivedTerminalthreshold Themaximumvalueofasensorystimulusallowingidentificationofthe

sensationperceived.Differenttasterscanbepronetobias,andwillhavedifferentsensitivitiestoflavours.Tominimisethesefactorssensoryguidelinesshouldbefollowed;

1. Atastingpanelshouldcompriseof20-50subjects.2. Repeatedtestsshouldbedone.3. Individualtestboothsfortastersshouldbeused.4. Randomnumberlabelsshouldbeusedinsteadofbrandlabels.

(Lawless&Heymann,2010;Meilgaard,Carr,&Civille,1999)Newtechnologyhasarisentotryandminimisetasterbias.Thisincludesinstrumentssuchasaquartzmicrobalanceelectricnosethatmimicsthehumanolfactorysystemtoprovidedetectionofanarrayofvolatilecompoundspresentinthesamples(Bernaetal.,2005).Itisessentiallyheadspaceanalysiswithsensorsthatreactdifferentlydependingonthevolatilecomponentsitisexposedto.Thishastheadvantage of doing continuous routine assessments, and has decreased influence from theenvironmentandbias.

Gustatoryreceptorsarefoundinthemouthandare comprised of tastebuds and free nerveendings. The four main tastes are observed alloverthetonguebutmoststronglyinthespecificareasshowninfigure5(Briggsetal.,2004).

Odour is more complex than taste. There areolfactoryreceptors intheupperpassagesofthenostrilsthatpasssignalstotheolfactory lobe inthebrain.Thestrengthofanodourdependsonthevolatilityofthecompoundspresent(Dewey,2011).

Figure5:Tasteareasoftongue Figure6:Olfactorysystem

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2.6MethodsofidentifyingchemicalcompoundsinbeerThereareavarietyofmethodstoidentifydifferentchemicalcompoundsinsolutions.Eachtypehasspecialtyuses.- Gaschromatography,GC- Massspectrometry,MS- Flameionizationdetector,FID- Electroncapturedetector,ECD- Highperformanceliquidchromatography,HPLC- Sulfurchemiluminescencedetector,SCD- Ionchromatography,IC(Coe,2003)ForthescopeofthisreportHPLC,SCDandIChavenotbeendiscussedastheyaretheleastrelevantmethodsforsamplingbeerforflavourcompounds.2.6.1GaschromatographyGC uses a capillary column to separate a sample into its individual chemical components. Not allsamplesaresuitableforGCuse,theymustbesufficientlyvolatileandhavesomethermalstabilitytowithstandtheconditionsintheGC.GCanalysisisdonebyinjecting1-5µLofsampleintothesamplingportwhereitisinstantlyvaporisedbythehightemperature.Thevapour(mobilephase)thenmovesupthethincapillarycolumnthatisabout30minlength.Themobilephaseinteractswiththestationaryphasethatisinternallycoatedonthecolumn.Thedifferentcompoundsinthemobilephasestayonthestationaryphasefordifferentperiodsoftimeduetodifferencesintheirvolatility,massandlevelofinteractionwiththestationaryphase.Thismeanstheytakedifferentperiodsoftimetoreachtheendofthecolumnwheretheyarerecorded,thereforeseparatingthesampleintoitsdifferentcomponents.(Coe,2003;Jennings,1987)The‘method’foraGCisadescriptiononthespeedatwhichtheinstrumentstemperatureisrampedupandhowlongthesetemperaturesareheldfor.TrialanderrorisusedtodevelopaGCmethod.Itisrecommendedtostartwithatemperaturejustbelowtheboilingpointofthechemicalofinterestandrampupthetemperatureslowlytoabovetheboilingpointandthenholdthetemperature.ThismethodcanthenbetweakedfromtheresultingchromatogramproducedbytheGCsoftware.Achromatogramisanobservablespectrumthatdisplaysdifferentpeaksthatcorrespondtodifferentchemicalcompoundseluted.Anacceptablespectrumconsistsofnarrow,separated,clearpeakstopredictchemicalcompoundspresent.ChangestotheGCmethodwillresultindifferentspectraduetoalteredchemicalinteractionsandthereforeachangeinelutiontime.GCcolumnparametersListedbelowaresomefactorstoconsiderwhenchoosinganappropriateGCcolumnfordifferenttypesofsamples,- Thestationaryphaseshouldbechosen in relation to thesample.Thegeneral rule is that the

mobilephasewillreactwithastationaryphasethatissimilarinchemicalcharacter.- Thefilmthicknessofthestationaryphasealsoneedstobeconsidered.Athinfilmwillrelease

componentsquickerthanathickfilmwill.Highboilingpointcomponentswillonlyrequireathinfilmofabout0.2-0.5mm.Thickerfilmsofabout1-1.5mmwillberequiredforlowerboilingpointmaterials.Thickerfilmsarealsousedforgasesandsolventssothattheinteractionbetweenthemandthestationaryphaseisincreased.

- Splitorsplitlessinjectorscanbeused.CapillarycolumnsforGCcaneasilybeoverloadedwithtoomuchsample.Onewayofavoidingthisistouseasplitinjector.Ifsplitinjectionisusedthena

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ratioissettostatetheamountofsampleenteringthecolumnversustheamountexitingthroughthevent.Splitinjectionshouldbeusedwhenthereisahighly-concentratedsolutionsothatthecolumndoesnotbecomeoverloaded.Ifsplitlessinjectionisused,thesplitlineventwillbeclosedandalltheinjectedsamplewillgointothecolumn.Splitlessinjectionshouldbeusedwhenthereis a low concentration of solute in the sample and so a larger volume is needed. (AgilentTechnolgy,n.d.;Chasteen,2000)

CompoundselutedfromaGCcanbefurtherprocessedbyanotherdevicesuchasMS,FIDorECD,asdiscussedfurtherbelow.2.6.2MassSpectrometryMS works by blasting the chemical components with electrons, breaking them up into chargedfragments.Theseionsthengothroughanelectromagneticfieldthatfiltersthembymeasuringtheirmasstochargeratios.Thenumberofeach ion is thencountedandamassspectrumcreated.Theindividual peaks can then be predicted from an inbuilt library on the GC-MS computer software(Vivian,Aoyagui,deOliveira,&Catharino,2016).2.6.3FlameionizationdetectorFIDworksbypassingthesamplethroughahydrogen/airflamewhichionisesthemolecules.Theyarethendetectedusingametalcollectorthathasahighvoltagedeliberatelyappliedacrossit.Thecurrentcanthereforebemeasured,whichisindicativeofthenumberofionsandthereforetheconcentrationof the chemical compound. FID is a very sensitive GC detector for organic compounds. For thisresearch,thisisnotalimitationasethylacetateisanorganiccompoundandispresentinverysmallamountsinbeer.2.6.4ElectroncapturedetectorECDbombardstheelutedstreamfromtheGCwithbetaparticles.Whenthesebetaparticlesimpactwithmolecules in themakeupgas,electronsare released.Theelectronsaredrawntoapositivelychargedanodeandaremeasuredasacurrent.Someorganicmoleculeswillcapturetheseelectrons.Thisresultsinareductioninthecurrentbeingmeasured.Theseabsorptionscanbetranslatedintopeaksthatcanbereadasthetypeofcompoundpresent.IncomparisontotheothermethodsECDisverysensitive,evenincomparisontoFID.Thismakesitidealformeasuringsamplesthatcontainverylowlevelsofcontaminants.QuantitativemeasurementswithGCAnexternalstandardneedstobeusedwhenmakingGCquantitativemeasurements.Atleastthreesolutionsofvaryingconcentrationofthepurechemicalsubstanceofinterestshouldbeusedtocreateacalibrationcurve.TheR2valuegeneratedbytheGCcomputersoftwaregivesanindicationofhowwell theplottedpoints fit the linear curve.AnR2 valueof>0.99 is generallyacceptable.Once thecalibration curve has been generated the samples of unknown concentration are run and arecompared to the calibration curve. From this a concentration valueof the chemical compoundofinterestcanbeevaluated.

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2.7ProblemstatementThisliteraturereviewhasshownthatthechemicalcompositionsinbeerchangeovertime,resultingin unfavourable flavour changes. These chemical compositionswill be different depending on thespecific type and brand of beer. In particular, ethyl acetate has been shown to decrease inconcentrationovertimeinarangeofdifferentbeers.ThisageingeffectwillbeinvestigatedinMac’sHop Rocker beer. The effects of different ageing temperatures will also be investigated. It ishypothesisedthattheconcentrationofethylacetatewilldecreaseovertime.Thisdecreasewillbemorepronouncedintheelevatedstoragetemperaturesample,and lesspronouncedinthecooledsampleduetothetemperatureeffectsondegradationreactionrate.

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3.0SemesteroneprojectworkLion brewery in East Tamaki agreed to offer support for this research project. A meeting withengineersthereresultedinamultitudeofresearchideasandindustryinsight.Thisvisitandatouroftheirtestinglaboratories,sensoryandtastinglab,pilotplant,andnewproductdevelopmentareasprovidedaninsightintotheissuesthatabreweryfaceseveryday,andthesolutionstheycameupwithtosolvethese.Italsoshowedthesignificanceofthisresearchprojectanditsreal-lifeapplications.Byunderstandingflavourdegradationinbeer,storageconditionscouldbetailoredtoeachbeertypetomaximisetheirqualityforalongerperiodoftime.MostofsemesteronewasspentdefiningaprojecttoundertakewiththehelpofLionbrewery.Thefollowinglistofprojectideaswereresearchedaspotentialprojects.- Theageingeffectofyeastinbottledbeer.- Homebrewingoffouridenticalales,anotherdifferentale,oneIPA,onestout,andonelager.The

effect of different storage conditions such as temperature, light and oxygen on beer flavourwouldthenbeinvestigated.

- Theeffectofdissolvedoxygenduringproductionofbeer,withacostbenefitanalysistoseeifchangingproductiontechniqueswouldbeeconomicallybeneficial.

- TheeffectofUVlightonskunkyflavourinbeer.- Theeffectofenzymesinthemashprofile,andhowtemperaturevariationwouldaffectthis.- Theeffectofyeastondifferentflavourcompoundsproduced.Thiswouldbeinvestigatedusing

differentyeaststrainsinfouridenticalbatchesofwort.- Measurement of hop content in beer to create a standard that future beer batches can be

comparedagainstsothataconsistentproductismade.

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3.1ExperimentalProcedureTheexperimentalprocedurehasbeensplit intotwosections.Theearlierprojectworkfocussedontrialsofwhiskey,storeboughtbeerandhomebrewbeerbatchesintheGC-MS,anddevelopingaGCmethodforbeeranalysis.Thesecondsectionhowever,usedtheGCmethoddevelopedinthefirstsectionand focussedon theMac’sHopRockermeasurements fromGC-FID.Thesecondsection ismostrelevanttothisresearchandisdiscussedfurtherintheresults.3.1.1MajorequipmentUsed- TheGC-MSusedforalltheexperimentsinthisreportwasaShimadzuGC-QP2010Ultra,withan

AgilentTechnologiescolumnHP-5MS,30mx0.320mm.- The GC-FID used for the Mac’s Hop Rocker beer was a Shimadzu GC-2010, with an Agilent

Technologiescolumn,HP-INNOWAX,30mx0.320mm.3.1.2EarlierExperimentalworkGC-MSWhiskeyrunInordertopracticeusingtheGC-MSanditssoftware,storeboughtwhiskeywasrunwiththetwomethodsoutlinedintable5.Table5:WhiskeyGC-MSmethods

MethodNumber Method1 50ºC,holdfor10min

50-180ºCat1ºC/min,holdfor3min2 Asplitratioof25:1

35ºC,holdfor5min35-100ºCat10ºC/min,holdfor1.5min100-220ºCat15ºC/min,holdfor3min220-250ºCat25ºC/min,holdfor2.8min

Themethodsintable5and6wereusedforthefollowingreasons.- Theboilingpointofethylacetateis77ºC.TheGCtemperaturestartsbelowthisboilingpointso

thatthetemperaturecanrampupthroughit.- Theinitialholdingtemperatureandtimewasreducedfrommethodonetomethodtwointable

5toimprovetheresolutionoftheearlierelutingpeaksasthelessvolatilecompoundscaninteractwiththestationaryphaselonger.

- Thetemperatureramprateaffectstheresolutionofthepeaks.Forthewhiskeyrunsintable5,theresolutionwassufficient,sotherampratewasincreasedsothatanalysistimedecreased.Ifthe resolutionwaspoor, the ramprateshouldbedecreased.Thiswouldallowmore time forinteractionsbetweenthemobilephaseandthestationaryphase,increasingresolution.However,abalanceisrequired,asincreasingtherampratealsomeansthatmorevolatilecompoundseluteveryquickly,sometimesresultinginpeaksjoiningtogether(AgilentTechnologies,2007).

- Amidramphold,asshownformethodtwointable5,isusedtotryandimprovetheresolutionofthepeaksinthemiddleofthisramp

- Theholdingtimeattheendoftheruntriesremovetheremainingcompoundsonthecolumn.Thisholdingtimecanbereducedifallsolutescomeoffthecolumninashortamountoftime.

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GC-MSStoreboughtbeerrunTofurtherpracticeusingtheGC-MSandtoadaptaGCmethodforbeeranalysis,storeboughtbeerwastrialled.Beck’s(aGermanpilsner),Mac’s3Wolves(apaleale),Mac’sSpringtide(alowercarblager),andMac’sSassyRed(anamberale)wererunthroughtheGC-MS.Themethodusedforthesebeerswas;65ºC,holdfor5min65-150ºCat4ºC/min,holdfor5min.The initial spectra produced from thewhiskey and beer runswere unclear and unusable as theyshowedmanywidepeakslayeredovereachother.ChangestotheGC-MSmethodwererequired,asdiscussedlaterinthissection.GC-MSHomebrewrunInitially,homebrewedbeerwasgoingtobeusedforanalysisinthisprojectinsteadofstoreboughtbeer.Therefore, twobatchesofMangrove Jack’spalealewerebrewed in23Lbatches frommaltextracts,andstoredinamberbottlesinlightproofboxes.Unfortunately,variationintheconcentrationofyeastby-products,suchasestersandaldehydes,canariseinhomebrew.Therefore,commerciallyproducedbeerwasusedforthisresearch.TwobottlesfromthehomebrewbatchwereselectedatrandomtobetrialledwiththeGC-MS.Threesamplesof2mLweredrawnfromonebottleandfilteredthrough0.45µmfiltersintocleanvials.Twosamplesweretakenfromthesecondbottleviathesameprocess.Repeatedsamplesofthesamebeerwereusedsothatthespectraresultscouldbecompared.Themethodusedforthesebeerswas;50ºC,holdfor5min50-250ºCat3ºC/min,holdfor5min.Thismethodwasusedbecause itwasbelievedthata long,slowrampingthrougha largerangeoftemperaturescouldshowmorechemicalcompounds.However,asshownpreviously,theresultsfromtheGC-MSsoftwareindicatedunclearandindistinguishablepeaks.ThisindicatedthatmoreworkwasrequiredtorefinetheGC-MSmethod.PreparationofstandardsolutionsToperformquantitativeanalysis,standardsolutionswererequired.Multiplevolumetricflaskswerepreparedbythoroughlyrinsingwithethanol,togetridoforganicmolecules.Theywerethenrinsedwithmilli-Q(ultrapure,Type1water)waterfollowedbyacetone.Theglasswarewasthendriedwithnitrogengastoensureallresiduewasremoved.Theaccuracyofthepipetteusedinsolutionpreparationwastestedbyweighingthewaterdrawnupthe pipette with an electronic scale to see if they were the same value. Only pipettes that gaveaccuratereadingswereusedforsolutionpreparation.Thesolubilityofethylacetateinwateris8.3g/100mL.Therefore,astandardsolutionof83,000ppm(partspermillion)wasproducedina100mLvolumetricflask.Thissolutionwasthendilutedtentimestogetan830ppmsolution,andsoon,downto8.3ppm.This8.3ppmsolutionwasusedsoasnottooverload the GC-MS. The same pure ethyl acetate solution was used to make all the standardsolutions. This reduced variation due to preparation as therewas no difference in initial solutionconcentrationorpurity.

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GC-MSmethodswithMac’sHopRockerThe8.3ppmsolutionofethylacetateandaroomtemperaturesampleofMac’sHopRockerwererunthroughtheGC-MSusingthemethodsintable6.Table6:StoreboughtbeerGC-MSmethods

MethodNumber Method1 60ºCfor4min

60-200ºCat6ºC/min200ºCfor2min

2 35ºC,holdfor5min35-100ºCat10ºC/min,holdfor1.3min100-200ºCat15ºC/min,holdfor3min220-250ºCat25ºC/min,holdfor3min

3 60ºC,holdfor5min60-200ºCat6ºC/min,holdfor2min



Asexplained for table5, themethods in table6wereadaptedafterviewing thespectra fromthepreviousrun.Theinitialholdtimewasreducedfrommethodonethroughfiveintable6.Thiswastotryto increasetheresolutionoftheinitialpeaks,asethylacetate isoneofthefirstcompoundstoelute.Thetemperaturerampratewasalsoreducedtotryandimprovethepeakresolution.The first method was suggested by Agilent Technologies (Technologies, n.d.). This method wassuggestedforalcoholbeveragestandards,includingdetectionofethylacetate.However,thecolumnusedforthisanalysiswasHP-FFAPwhichdifferedfromthecolumninthe laboratories.Thismeantthat different chemical compounds in the samplewould interact differentlywith the column andthereforewillnotproducethesamespectraofresults.Aspreviously,thesemethodsresultedinspectrathathadwideandindistinguishablepeaks,meaningchemicalcompoundscouldnotbepredicted.Therefore,GC-FIDwasusedfortherestofthisresearchprojecttotryanddetectethylacetateandobtainreadableresults.ThecolumnontheGC-MSdiffersfromthatontheGC-FID.ThismeansethylacetatewillhaveadifferentaffinitywiththechemicalsoftheGC-FID’sstationaryphaseresultinginadifferentelutiontime.3.1.3LaterExperimentalworkStorageconditionsofstoreboughtbeerAftertheinitialunsuccessfulGC-MStestingwasdone,theMac’sHopRockerwasplacedindifferentstorageconditionsandlefttoageforoneweekbeforethefirstGC-FIDmeasurement.Twobottlesofthebeerwereplacedinthelaboratoryfridgethatwassetat4ºC.Twomorebottleswereplacedinthechemicalanalysisroomthatwaskeptatroomtemperatureof19ºC.Thelasttwobottleswereplacedinanelevatedtemperatureenvironmentontopofanelectronicwaterboiler.Theelevatedtemperatureofthiscouldnotbemeasured,butheatedthebeerover30ºC.Thisvariableelevatedtemperaturecouldhavebeenasourceoferrorinmeasurementsasthebeerwaskeptatanunknownelevatedtemperature.Iffutureexperimentsweretobedone,thewaterincubatorinthelaboratoryshouldbeusedasthiswouldgiveamorereliableelevatedstoragecondition.

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GC-FIDstandardsolutionpreparationVolumetricflasks(three5mL,two25mL,one50mL,andone100mL)werecleanedas described above. 0.111 mL of ethyl acetate was pipetted into the 100 mLvolumetric flask and toppedwithmilli-Qwater tomake a 1000ppm solution. This1000ppm solution was then used to produce 500, 200, 100, 40, 20 and 10ppmsolutionsintheothervolumetricflaskswhichcanbeseeninfigure71mLofeachofthestandardsolutionswassyringedthrougha0.45µmPTFEfilterintosampling bottles. A 1 mL sample of cold, room temperature and elevatedtemperatureMac’sHopRockerbeerwerealso filtered through the0.45µmPTFEfilter intoseparatesamplingbottles.Thesewereplaced intheGC-FIDasshowninfigure8.Only5mLofbeerfromthebottleswasusedforthissampling,therestwas poured into plastic vessels and kept under the same storageconditions until the next sample was taken. This could have affectedresultsasafterabeerbottleisopenedthebeerisexposedtooxygenintheairwhichcanbegintobreakdownsomechemicalcompounds.Inreallife applications, in contrast to this experiment, beer would be agedwhileinanunopenedbottle.The samples were run through the GC-FID in the following order; allstandardsolutions,watersample,beersamples.Thisorderistotryandreduceanyeffectofbuild-upofchemicals inthecolumn,called ‘carryover’. These could be eluted in the beer sample and therefore giveinaccuratemeasurements. To further try and reduce carry over, eachbeersamplewasrunthroughtwice.Thefirstsampleshouldclearoutanyresidualchemicalsleftonthecolumnsothatthesedidnotcontaminatethenextrun.Anaverageofthesetworunswasusedintheresults.Thefollowingmethods,shownintable7,weretheninputintotheGC-FID.Table7:Mac'sHopRockerbeerGC-FIDmethodsused

MethodNumber Method1 25ºC,holdfor20min

25-100ºCat2ºC/min,holdfor5min2 27ºC,holdfor20min

27-100ºCat2ºC/min,holdfor5minAninjectiontemperatureof250ºC,andinjectionvolumeof3µLwasusedforbothmethods.Themethodwaschangedto27ºCfrom25ºCbecausetheGC-FIDstruggledtoreach25ºC.Thishadnoeffectontheresultsobtained.Thestandardsolutions,waterandbeersampleswererunthroughtheGC-MSthreetimeswithinoneweek.Thesamestandardsolutionswereusedforthefirstandsecondrun,withfreshonesproducedforthethirdrun.

Figure7:Ethylacetatestandardsolutionsandpipette

Figure8:EthylacetateandbeersamplesinGC-FID

Figure9:GC-FIDmachineusedformeasurements

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AnalysisofGC-FIDresultsAfterthesampleswererunthroughGC-FID,spectrawereproducedanddisplayedonthepostrunanalysisscreen.Manualintegrationwasusedtoanalysethepeaksbyselectingthebaseeachsideofthepeak.ThefunctionontheGC-FIDsoftwarethencalculatedtheareaoftheethylacetatepeakineachofthestandardsolutions.Theseareaswerethenplottedagainsttheirconcentrationstoformacalibrationcurve.ThetimeatwhichtheethylacetateinthesolutionselutesisalsonotedbytheGC-FID.Whenthebeersamplesarerun,apeakatthesametimeintervalisexpectedtobeobserved,thisislikelytobeethylacetate.Theareaofthispeak iscalculatedandcomparedagainstthecalibrationcurvetogivetheconcentrationofethylacetateinthesample.Theconcentrationinthesampleisfoundinppm.Toconvertthistoapercentageofthebottleofbeer10,000ppm=1%.Therefore,25ppm=0.0025%.

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4.0ResultsandDiscussionThepostrunanalysissoftwarefortheGC-FIDproducesaspectrumforeachsamplerun.Thespectruminfigure10 is fromthe200ppmstandardsolution.Anarrowethylacetatepeakcanbeseenatanelutiontimeof6.2minutes.Thenoisefrom35-40minutescanbeignored.

Figure10:SpectrumfromGC-FIDfor200ppmethylacetatestandardsolution

These standard solution areas are plotted against the appropriate concentrations to produce acalibrationcurve.Thecalibrationcurveusedforthefirstandsecondtrialisshowninfigure11.TheR2valueforthiscurveis0.99998,meaningthattheconcentrationsoftheethylacetatesolutionsweresufficiently accurate, and can be used for quantitative analysis. A second calibration curve wasproducedforthethirdtrial,ithadanR2valueof0.99992.ThiscanbefoundinappendixA.

Figure11:Calibrationcurveproducedfromethylacetatestandardsolutions

For the first successful use of the GC-FID only one room temperature beer sample was tested.25.4ppmofethylacetatewaspredictedtobepresent in thissample fromcomparisonagainst thecalibrationcurve.Thisisabout0.00254%.ThislikenstoVanderhaegen’spaper(2003)where28ppmofethylacetatewasmeasuredinfreshbeer.Thefirstrunwithacoldsample,aroomtemperaturesampleandheatedsamplewasdoneonthe20thSeptember. The same standardswere run through theGC-FID anda similar calibration curvewasproduced.Theremayhavebeensomedegradationoftheethylacetatewhenitwasstoredforthetwo days. The cold, room temperature, and heated beer all had slightly different spectra. Thespectrumforthefirstsampleoftheroomtemperaturebeerfromthefirsttrialisshowninfigure12.TheethylacetatepeakcanbeidentifiedasitelutesfromtheGCatthesametimeastheethylacetateinthestandardsolutions.

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Figure12:RoomtemperaturebeersampleGC-FIDspectrum

Forthesecondandthirdtrialonthe25thand26thofSeptemberduplicatesamplesofthebeerateachstorage condition were put through the GC-FID. The GC ran samples from the same vial twice.However,thefirstandduplicatesamplesdidnothaveidenticalresults,soanaverageofthetwowastaken.Thesecanallbeseenintable8.Table8:MeasuredethylacetateconcentrationsfromGC-FID

Thedifferenceinethylacetateconcentrationbetweenthefirstsampleandtheduplicatesamplecouldbeduetoresidualchemicals leftontheGCcolumn.Thiscouldbefromtheethylacetatestandardsolutions,orotherchemicalsleftbyresearchersusingthesameGC-FID.Someofthesecompoundscouldhavestayedonthecolumnwallandelutedwithoneofthesamples.Itwasoriginallythoughtthatthesechemicalswouldcomeoffinthefirstsample,andnotaffecttheduplicatesample.However,forsixoutoftheninerunstheduplicatesamplehadahigherlevelofethylacetatecomparedtothefirstsample.Furthermore,thewatersamplespectrumfromthethirdtrialisshowninfigure13.Thewatersamplewasrunaftertheallstandardsolutionsbutbeforeanybeersamples.Itcanbeseenthatthereisstillanethylacetatepeakandpeaks fromothercompoundspresent.Thesechemicalswouldnothave

Firstsample(ppm)(mg/L)

Duplicatesample(ppm)(mg/L)

Average(ppm)(mg/L)

1sttrial20thSeptember

Cold 14.4 14.7 14.5Roomtemperature 14.4 14.5 14.5Heated 12.0 13.5 12.7

2ndtrial25thSeptember


3rdtrial26thSeptember


22

beenpresentintheMilli-Qwaterused.ThisindicatesthattherewasethylacetateandotherchemicalcompoundsleftontheGCcolumnfrompreviousruns. Ifthiswaterrundidnotflushoutalloftheresidualchemicalsthentheycouldhaveelutedinthesubsequentbeersampleruns.

Figure13:ThirdtrialwatersamplespectrumfromGC-FID

ThisthereforeindicatesthattherecouldbeerrorsintheGC-FIDreadingsasotherchemicalsthatarenotpresentinthesamplesweredetectedandrecorded.Toreduceresidualchemicalsonthecolumn,multiplewaterrunscouldbedoneuntilthechemicalresponsesrecordedreducestozero.Anotherpossiblecleaningmethodistoholdthecolumnatahightemperaturesothatthechemicalsgetburntoff.Thelevelofethylacetateatthethreedifferentstoragetemperaturesforeachtrialhasbeenplottedonthegraphshowninfigure14.

Figure14:Ethylacetatelevelsovertimewithdifferentstoragetemperatures

0

5

10

15

20

25

30

35

1sttrial 2ndtrial 3rdtrial

Ethylacetatelevel(pp

m)

Trialnumber

Ethylacetatelevelsovertime

Cold Roomtemperature Heated

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Figure14showsnotrendforchangeinethylacetatelevelsinMac’sHopRockerwithrespecttotime.However,thisresearchwasconductedoveraveryshorttimeperiodof lessthanaweek,andonlythreedatapointsweremeasured.Therefore,thisdataisnotstatisticallysignificantasthesamplesizeisfartoosmall.Toimprovethisexperimentalmethodandgetstatisticallysignificantresultswhereatrendcouldpotentiallybeseen,itisrecommendedtomeasureethylacetatelevelsoverasixmonthtimeperiod.Thisisarealistictimeperiodforafourthyearprojectandalsoarealistictimeframeforageingbeer.Thebeerusedforthissamplingwasboughtfourmonthsbeforethefirstethylacetatemeasurementwasmade.Itwasthoughtthatethylacetatecouldhavedegradedexponentiallyovertime.Thereforethemeasurementsmadeshouldnotshowmuchvariationastheyarelateinthedegradationstages.However,Vanderhaegen(2003)measuredethylacetateconcentrationovertwohundreddaysandfoundalineardecreaseinethylacetate.Forallthreetrials,thecoldbeersampleshowedhigherlevelsofethylacetatethanboththeroomtemperatureandheatedsamples.Thisispossiblyduetothelowertemperaturereducingtherateatwhichtheesterasesbreakdowntheethylacetate,andreducingtherateofthehydrolysisreactionofethylacetate.However,duetothesmallnumberofmeasurementsthisconclusioncannotbemadeconfidently.Figure14showslargevariationbetweentrials,butlittlevariationwithintrials.Itisunlikelythattheconcentration of ethyl acetate increased significantly (12.7ppm to 31.6 ppm) in 5 days and thendecreasedsignificantlyagainonedaylater(13.6ppmto18.2ppm).ThiscouldindicateerrorduetotheGC-FIDequipmentused.Eachtrialwasrunoverafullday.Therefore,itispossiblethatondifferentdaystheGCgaveinaccurateresultsforallthreesamplesofasingletrial.Thefirsttwotrialsusedtheexactsamestandardsolutions,whereasthethirdtrialusedfreshstandardsolutions.Thiscouldhaveaffectedtheresultsasadifferentcalibrationcurvewasproducedforthethirdtrial.However,usingthesamestandardsforthesecondtrialfivedaysaftertheywereproducedcouldalsohaveaffectedtheresults.Thisisbecausethestandardscouldhavedegradedandchangedincompositionovertime,thereforeresultinginaninaccuratecalibrationcurve.

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5.0FuturedevelopmentsIfanyfutureresearchisdoneinrelationtothisproject,thefollowingisrecommended,- Therearemanyflavourchemicalcompoundsinbeerthatchangeovertime.Anyfurtherresearch

onMac’sHopRockerbeercouldalsoincludeanalysisofothercompounds,suchasnonenal.- Noconclusionscanbemadefromtheresultsofthisresearchastoofewmeasurementsweremade

overtooshortatimeperiod.Anyfutureresearchonethylacetatedegradationshouldbedoneoveralongertimeperiod,forexample,sixmonths.

- ThestationarysolutionontheGCcolumnisimportantincolumnselectionasitdependsontheapplicationtheGCcolumnisusedfor.Stationaryphaseshavevaryinginteractionswithdifferenttypesof chemicals.Adifferent stationaryphasemay separate twocompounds that co-elute inanother column. TheGC-FID column available in the labs, HP-INNOWAX,was used during thisprojectbutwasnotideallysuitedforanalysisofflavoursandfragrancesinthefoodandalcoholicbeverage industry. For future researchers, it is recommended to use an SPB or CP-WAX-52-CBcolumn(SigmaAldrich,2010).

- The elevated temperature beer samples were not kept in a constant heated environment. Toimprovetheaccuracyoftheresults,thebeersamplesshouldbeplacedinthewaterincubatorinthelaboratorieswheretheywillbekeptataconstanttemperature.

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6.0ConclusionsTheconcentrationofethylacetateinMac’sHopRockerbeerwasnotconstantwithtime.However,duetothelimitednumberofmeasurementsmade,notrendcouldbefoundwiththeresults.Furtherresearchisrequiredtofindtheeffectofageingtemperatureonethylacetateconcentration.Themainconclusionsthatweremadefromthisresearchprojectaresummarisedbelow,- Theresultsobtainedinthisresearcharenotstatisticallysignificantasthesamplesizewastoo

small.Notrendcouldbeseenfromtheresultsasthetimeperiodthisresearchwasconductedinwastooshort.

- Theoretically,comparedtosimilarresults fromdifferentbeertypes, the levelofethylacetateshoulddecreaseinconcentrationasbeeragesasitisbrokendown.

- At elevated temperatures, this decrease in concentration should be accelerated due to theincreasedrateofbreakdownreaction.Atlowertemperatures,theoppositeistrue.

- Futuretestingshouldbedoneoveralongertimeperiodofaboutsixmonthssothatanageingtrendcanbeseen.

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7.0ReferencesAgilentTechnolgy.(n.d.).Choosingtherightcolumn.Retrievedfrom

http://www.chem.agilent.com/cag/cabu/gccolchoose.htmAgilentTechnologies.(2007).GCMethodDevelopment.GCMethodDevelopment.Retrievedfrom

www.chem.agilent.comBerna,A.Z.,Buysens,S.,DiNatale,C.,Grün,I.U.,Lammertyn,J.,&Nicolaï,B.M.(2005).Relatingsensory

analysiswithelectronicnoseandheadspacefingerprintMSfortomatoaromaprofiling.PostharvestBiologyandTechnology,36(2),143–155.https://doi.org/10.1016/j.postharvbio.2004.12.006

Briggs,D.E.,Boulton,C.A.,Brookes,P.A.,&Stevens,R.(2004).Beerflavourandsensoryassessment.InBrewingScienceandPractice(pp.716–758).

Buiatti,S.(2009).BeerComposition:AnOverview.InBeerinHealthandDiseasePrevention(pp.213–225).ElsevierInc.https://doi.org/10.1016/B978-0-12-373891-2.00020-1

Carr,N.(2016).18Common“Off”FlavorsInBeer.Retrievedfromhttps://learn.kegerator.com/off-flavors-in-beer/

Chasteen,T.G.(2000).Split/SplitlessGasChromatographyInjection.DepartmentofChemistry,SamHoustonStateUniversity.

Coe,M.(2003).IGBExam.Dewey,R.(2011).Olfaction.Retrievedfromhttp://www.intropsych.com/ch04_senses/04nasal.jpgGarcía,A.I.,García,L.A.,&Díaz,M.(1994).Predictionofesterproductioninindustrialbeer

fermentation.EnzymeandMicrobialTechnology,16(1),66–71.https://doi.org/10.1016/0141-0229(94)90111-2

Hough,J.S.,Briggs,D.E.,Stevens,R.,&Young,T.W.(1982).ChemicalandPhysicalPropertiesofBeer.InMaltingandBrewingScience(pp.776–838).https://doi.org/10.1007/978-1-4615-1799-3_11

Hughes,P.S.,&Baxter,E.D.(2001).Beer :Quality,SafetyandNutritionalAspects.TheRoyalSocietyofChemistry.https://doi.org/10.1039/9781847550224

Jennings,W.(1987).AnalyticalGasChromatography.AcademicPressInc.Lawless,H.T.,&Heymann,H.(2010).Introduction.InSensoryevaluationoffoodprinciplesandpractices.

https://doi.org/10.1007/978-1-4419-6488-5Lentini,A.,Nischwitz,R.,Rigoni,P.,Goldsmith,M.,Duan,D.,Fogarty,L.,&Rogers,P.(2015).Further

StudiesontheImpactofCommercialMaltingandBrewingPracticesonBeerFlavourStability,(November),1–16.

Meilgaard,M.C.(1982).Predictionofflavordifferencesbetweenbeersfromtheirchemicalcomposition.JournalofAgriculturalandFoodChemistry,30,1009–1017.https://doi.org/10.1021/jf00114a002

Meilgaard,M.C.,Carr,B.T.,&Civille,G.V.(1999).IntroductiontoSensoryTechniques.InSensoryEvaluationTechniques(pp.1–6).https://doi.org/doi:10.1201/9781439832271.ch1

Nordstrom,K.(1961).FormationofEthylAcetateinFermentationKineticsofFormationFromEthanoland,188–196.

Palmer,J.J.(2006).HowtoBrew:EverythingYouNeedtoKnowtoBrewBeerRighttheFirstTime.Retrievedfromhttp://howtobrew.com/book/introduction

SigmaAldrich.(2010).GCColumnSelectionGuide,1–24.Technologies,A.(n.d.).GCandGC/MS.Vanderhaegen,B.,Delvaux,F.,Daenen,L.,Verachtert,H.,&Delvaux,F.R.(2007).Agingcharacteristicsof

differentbeertypes.FoodChemistry,103(2),404–412.https://doi.org/10.1016/j.foodchem.2006.07.062

Vanderhaegen,B.,Neven,H.,Coghe,S.,Verstrepen,K.J.,Verachtert,H.,&Derdelinckx,G.(2003).EvolutionofChemicalandSensoryPropertiesduringAgingofTop-FermentedBeer.JournalofAgriculturalandFoodChemistry,51(23),6782–6790.https://doi.org/10.1021/jf034631z

Vivian,A.F.,Aoyagui,C.T.,deOliveira,D.N.,&Catharino,R.R.(2016).Massspectrometryforthecharacterizationofbrewingprocess.FoodResearchInternational,89,281–288.https://doi.org/10.1016/j.foodres.2016.08.008

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8.0Appendices8.1AppendixA:EthylacetateconcentrationcalibrationcurvesFirstsample(onlyroomtemperaturebeer)calibrationcurve.R2=0.99997

Figure15:Firstethylacetatesamplecalibrationcurve

Thirdtrialcalibrationcurve.R2=0.99992

Figure16:Thirdtrialcalibrationcurve

31

8.2AppendixB:GC-FIDspectrumGC-FIDspectrumoffirstsuccessfulmeasurementwithonlyroomtemperaturebeermeasurement.Thisshowsanethylacetateconcentrationof25.4ppm.

Figure17:FirstroomtemperatureGC-FIDspectrum

32

8.3AppendixC:Safety&LaboratoryUseHAZOPProject:ShorttermethylacetatedegradationinbeerandeffectoftemperatureProjectObjectives:ToinvestigatethechangeinconcentrationofethylacetateinMac’sHopRockerbeerovertimeafterithasbeenagedinthreedifferenttemperatureconditions–cold,roomtemperature,andheated.Equipmentused:1. ShimadzuGC-QP2010UltraGC-MS2. ShimadzuGC-2010GC-FID3. Glassvolumetricflasks,pipette4. 5mLplasticsyringes5. 0.45µLfilters6. Smallglassvials

Chemicalsused:1. Ethylacetate2. Milli-Qwater3. Acetone4. Ethanol5. Beer

Experimentalprocedure:1. Produceethylacetatestandardsolutionsbyfirstrinsingvolumetricflaskswithethanol,milli-Q

water,andacetone.2. Pipettecorrectproportionsofethylacetateandmilli-Qwaterintovolumetricflasks.3. Syringestandardsolutionsandbeersamplesthroughfilterandintosmallglassvials,tothenbe

putintheGC-MSorGC-FID.4. InputmethodandrunGC-MSorGC-FIDthroughoutday,orovernight.Personalprotectiveequipment(PPE)required:1. Labcoat2. Safetyglasses3. CoveredshoesEmergencyshutdownprocedure:Turnoffmachinewithpowerbuttononbottomright.Switchpoweroffatwall.HAZOPanalysisTable9:BeerdegradationHAZOPanalysis

PotentialHazard Consequence SuggestionsElectrocutionfrommixtureofwaterandelectricalequipment

Electrocution KeepwaterawayfromGCequipment

Prolongedexposuretochemicals(Beersamplesdonotcarrythishazard,onlyethylacetate)

Fatigue,headaches,ornausea Chemicalsamplingtobeperformedinfumehood

Sprayofchemicalsfromsyringeontoface/skin/eyesduetopressureappliedtopushsamplethroughfilter(Beersamplesdonotcarrythishazard,onlyethylacetate)

Irritationandpossibledamagetoskinandeyes

Wearsafetyglassesandlabcoatatalltimes

Brokenglassware Minorcutstoskin Usefirstaidkitanddisposeofbrokenglasscarefully

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ProjectCompletionSignoffsheet

the effect of temperature on short term...

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