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1、PartAromaAnalysisTechniquesInjectorColumn OvenColumnDetectorGC ComponentsThe principle of gas chromatographylGas chromatograph is a tool for separation andanalysisofmulticomponentmixtures.Itusesthegasasthemobilephaseandisakindofelution.lWhenmulticomponentanalytesenterthecolumn,duetothedifferentparti
2、tioncoefficientsbetweenthegasphaseandthestationaryphase,therunningspeedofeach component in the column is different.Afterpassingthroughacertainlengthofcolumn,itleavesoutinsequenceandentersthedetector.lAfterdetection,chemicalisconvertedintoelectricalsignalandsenttodataprocessingworkstation.Thus,thequa
3、litativeandquantitativeanalysisofthetestedsubstanceiscompleted.The principle of gas chromatographylTheseparationofsamplesinchromatographiccolumnisbasedonthedifferenceofthermodynamicproperty.Thestationaryphasehasdifferentaffinitywitheachcomponentinthesample(forgas-liquiddistributionchromatography,the
4、solubilityisdifferent).lWhenthecarriergascarriesthesamplecontinuouslythroughthecolumn,thecomponentswithhighaffinitymoveslowly,whilethosewithlowaffinitymovefast.lThe detector gives a corresponding signal to eachincomingcomponent.The principle of gas chromatographylStartingfromthetimewhenthesampleisin
5、jectedwithcarriergas,thecomponentsareseparatedandcarriedintothedetectorsuccessively,tillthemomentthedetectorgivesthemaximumsignal corresponding to each component(often called peakvalue),thisperiodoftimeiscalledtheretentiontime(tr)ofcomponents.lThe practice has showed thatunder certain conditions(inc
6、luding carrier gas flow rate,materials and properties ofstationary phase),the retention time(tr)of differentcomponentsisalsocertain.Inturn,wecaninferwhatkindofsubstancethecomponentisfromtheretentiontime.lTherefore,theretentiontimecanbeusedasthebasisforthequalitativeanalysisofchromatography.Types:-Sp
7、lit/Splitless-On-column-Programmed temperature vaporizer(PTV)Ideal injection technique:-should result in no compound destruction-Cause no discrimination among compounds-Suitable for analysis of all sample types (either dirty or clean samples)GC InjectorsSplitlesslWhentheelectromagneticvalveisturnedo
8、ff,allsamplewillenterthecolumnofGC.lThesplitlessmodeisnotanabsolutelysplitless,itisacombinationofsplitandsplitless.Schematicdiagramofsplit/splitlessinletofHP4890(5890)1-totalflowcontrolvalve;2-inlet;3-regulatingvalveofdottlepinpurgegas;4-exportofdottlepinpurgegas;5-splitter;6-split/splilesssolenoidv
9、alve;7-controlvalveofpressureinfrontofcolumn;8dialofpressureinfrontofcolumn;9-splitvent;10-chromatographiccolumn;11-detectorinterfaceStructure of commonly used GC injector linerA-usedfortheinletofpackedcolumn;BG-usedforsplitinletofcapillarycolumn;GandH-usdforsplitlessinlet;G,HandI-usedfortheinletofp
10、rogrammedtemperaturevaporizerSteam expansion factorSolventDensityMolecular weight Estimated expansion factorisooctane0.89114138:1hexane0.6686174:1pentane0.6272198:1ethylacetate0.9088233:1chloroform1.48119284:1dichloromethane1.3385356:1methanol0.793253:1water1.000.18inlettemperature:250;pressureinfro
11、ntofcolumn:90kPa.Solvent effect of splitless injection(a)Completelysplitless;(b)InstantaneoussplitlessCool on columnlThesampleisinjecteddirectlyintoachromatographiccolumnatorbelowroomtemperature,andthenthetemperatureincreasesgraduallytomakethesamplecomponentsvaporizeinturnandseparatedbythecolumn.lRe
12、sultinnocompounddestruction,andcausenodiscrimination amongcompounds.Programmed temperature vaporizer(PTV)lAliquidorgassampleisinjectedintothelinerofinjectioninletatlowtemperature,thenthetemperatureoftheinjectioninletincreasesaccordingtotheprogrammingprocedure.lAcombinationofsplit/splitlessandcoolonc
13、olumn.Injector and Injection technologyCharacteristicPackedcolumninjectorThisisthesimplestinjector.Allvaporizedsamplesenteredthechromatographiccolumn.Theinjectorcanbeconnectedwiththeglassandstainlesssteelpackedcolumn.ItalsocanbeconnectedwithlargecalibercapillarycolumnfordirectinjectionSplit/splitles
14、sinjectorThisisthemostcommonlyusedcapillarycolumninjector.Splitinjectionisthemostcommon,anditsoperationissimple,buttherearesplitdiscriminationandsamplesmightbedecomposed.Theoperationofsplitlessinjectioncomplicated,buttheanalysissensitivityishigh,anditisoftenusedfortraceanalysis.CooloncolumninjectorT
15、hesampleentersthechromatographiccolumndirectlyinliquidform.Thereisnodiversiondiscrimination.Thisinjectorhashighanalyticalaccuracy,goodreproducibility,andespeciallyissuitableforsampleswithwideboilingpointrangeorthermalinstability,andoftenusedfortraceanalysis(columnconcentration)Programmedtemperaturev
16、aporizer(PTV)injectorThisinjectorcombinessplit/splitlessandcooloncolumn.Ithasmanyfunctionsandawiderangeofapplications,makingitanidealGCinjector.BulkinjectionPTVorcooloncolumninjectorisusedwithsolventemptyingfunction.Theinjectionvolumecanreachseveralhundredmicrolitersorevenhigher,whichcangreatlyimpro
17、vethesensitivityofanalysis.Itiswidelyusedinenvironmentalanalysis,buttheoperationiscomplex.ValveinjectionSix-wayvalveiscommonlyusedtoquantitativelyintroducethegasorliquidsamples.Itsreproducibilityisgoodanditiseasytoautomate.However,thisinjectionhasgreatinfluenceonpeakbroadening.Itisoftenusedfortheana
18、lysisofpermanentgasandthemonitoringofmaterialflowinchemicalprocessHeadspaceinjectionOnlythegasportionabovethecomplexsampleisanalyzed.Therearestaticheadspaceanddynamicheadspace(Purgeandtrap).Itissuitableforenvironmentalanalysis,foodanalysisandvolatileanalysisofsolidmaterialsCrackinginjectionThistechn
19、iquesplitsnon-vaporizableorpartiallynonvaporizablesamplesintovaporablesmallmoleculecompoundsathighlycontrolledtemperaturesforGCanalysis.ItissuitablefortheanalysisofpolymerormineralsamplesCommon used GC injector and injection technologyJ&W Scientific Columns-Recommendations Column dimensions depend o
20、n application Polar-DB-WAX(Carbowax)use long,narrow bore,thin film columns with GC-MS(30 m x 0.25 mm i.d.x 0.25 m film)-best efficiency under reduced flow rate Nonpolar-DB-5 Two column*phase types are all you need*beware of limitations(see reference 7)use medium length,medium bore,medium film column
21、s in routine GC applications(30 m x 0.32 mm i.d.x 0.5 m film).Retention characteristics of 0.25 mm i.d.x 0.25 m film column are equivalent those of a 0.32 mm i.d.x 0.5 m film column.respond only to a particular class of compoundsGC DetectorsUNIVERSAL respond to all compounds including mobile phase S
22、ELECTIVE or SPECIFICGENERAL TYPES Thermal Conductivity Detector(TCD)Flame Ionization Detector(FID)Photoionization Detector (PID)Mass Selective Detector(MSD)Fourier Transform Infrared Detector(FTIF)Olfactometer (O)Flame Photometric Detector(FPD)Nitrogen-phosphorus(NPD)Atomic emission detector(AED)-su
23、lfur and phosphorus-nitrogen and phosphorus-element specific:N,C,O,S-aroma-active compounds-structural information-structural informationPhoto Ionization DetectorslPhotoIonizationDetectors(PID)isusedtodetectverylowconcentrations(0-1000ppm)ofVOCs(volatileorganiccompounds)andothertoxicgases.lManyhazar
24、doussubstancesinaccidentsareVOC,therefore,PIDwithhighsensitivitytoVOCdetectionhasanirreplaceableuseinemergencydetection.Nitrogen Phosphorus Detector(NPD)lNPDisalsocalledThermionicDetector(TID).AglassbeadcontainingrubidiumsilicateisplacedbetweenthenozzleandthecollectorofFID.Undertheactionofrubidiumbe
25、ads,manyelectronsaregeneratedfromthermaldegradationofthenitrogen-andphosphorus-containingcompounds,thesignalintensityismuchhigherthanthatwithoutrubidiumbeads,therefore,thesensitivityofthedetectorisimproved.lNPDismostlyusedinanalysisoftracenitrogen-andphosphorus-containingcompounds.Flame photometric
26、detector(FPD)lDetectionofphosphorus:phosphorusoxidesaregeneratedduringthecombustionoforganicphosphides,itisthenreducedtoHPObyhydrogeninthehydrogenrichflame.Whenitreturnstogroundstate,theexcitedstatephosphorusfragment(HPO*)excitedbythehightemperatureoftheflame,emitsaseriesofwavelengthsoflightof480-58
27、0nm(maximum526nm).A526nmfiltercanbeusedtomakethemaximumemissionlightofphosphoruspassthrough,filteringoutotherwavelengthsofemittedlight,themthepurposeofselectingdetectionisachieved.lDetectionofsulfur:Sulfurcompoundsentertheflametoformexciteds*molecules.Whenitreturnstothegroundstate,emitslightwithawav
28、elengthof320-480nm(maximum394nm).Using394nmfilter,Itcanonlybetransmittedbylightnear394nm。Mass Selective DetectorGas Chromatography-Mass Spectrometry(GC-MS)Molecules travel from end of GC column into ion source Ion Source,molecules are broken down(fragmented)when bombarded by high-energy electrons(ty
29、pically70 eV for EI-MS)or gas molecules(CI-MS)Electrically charged fragments areseparated according to mass/charge(m/z)ratio and detected by electron multiplier A plot of relative intensity versus m/z yields a mass spectrum for the moleculeStructural InformationMolecular WeightFingerprint Informatio
30、n(Searchable)Reference 8Quantitative analysis method of GC-MSlThecommonlyusedquantitativemethodsareareanormalizationmethod,externalstandardmethodandinternalstandardmethod.lTheadvantageofareanormalizationmethodis:Theresultsaresimple,accurateandThequantitativeresultsareindependentoftherepeatabilityoft
31、heinjectionvolume(withintherangeofnooverloadofchromatographiccolumn);Whentheoperatingconditionsareslightlychanged,ithaslittleinfluenceontheresults.lItsdisadvantageis:Allcomponentsmustflowoutofthecolumninoneanalysiscycle,andthedetectorgeneratessignalsforallofthem.Itisnotsuitableforthedeterminationoft
32、raceimpurities.Area normalization methodlThetotalcontentofallcomponents(havingchromatographicpeaks)iscalculatedas100%.Whenallcomponentsinthesamplecanflowoutofthecolumnandgeneratesignalsonthedetector,thenareanormalizationmethodcanbeused.Themassfractionofcomponenticanbecalculatedasfollows:Where:Ai-the
33、peakareaofcomponenti;fi-masscalibrationfactorWhenfiisthemolarorvolumecalibrationfactor,theresultismolarorvolumepercentagerespectivelyFigure1ThechromatogramofShedianlaojiuliquorExternal standard method lTheexternalstandardmethodissimpleinquantificationandcalibrationfactorisnotneeded.Whetherothercompo
34、nentsinthesamplehavepeakornot,itcanbeusedforquantitativeanalysisoftestedcomponents.However,theaccuracyofthismethodisaffectedbytherepeatabilityofinjectionandthestabilityofexperimentalcondition.Internal standard methodlThepuresubstancenotcontainedinthesampleisselectedasthereferencesubstanceandisaddedi
35、ntothesamplesolutiontobetested.Comparedwiththeresponsesignalsofthecomponentstobetestedandthereferencesubstance,thecontentofcomponentstobetestedisdetermined.lWhenselectingtheinternalstandard,itshouldbenotedthat(1)theinternalstandardisacomponentnotcontainedintheoriginalsample;(2)theretentiontimeofthei
36、nternalstandardissimilartothatofthecomponenttobetested,buttheycanbecompletelyseparatedfromeachother;(3)thepurityoftheinternalstandardshouldmeettherequirements.Thismethodislessaffectedbytheoperatingconditions.Internal standard methodTheconcentrationofanodorant(Cx)=theconcentrationofinternalstandard(C
37、IS)thepeakareaofanodorant(Ax)thepeakareaofinternalstandard(AIS)Stable isotope dilution array(SIDA)lTheproportionofvolatilesubstancesinfoodisverysmall,therefore,itisverydifficulttoquantifyitaccurately.Theexternalstandardmethodiscomplicated,whilethemethodofaddingoneorseveralinternalstandardshasalarged
38、eviation,especiallywhenthetargetcontentisveryloworunstable.lIsotopesarethenucleiofchemicalelementsthatcontainthesameprotonsbutdifferentnumbersofneutrons,sothemassnumberisdifferent,suchas1H/2Hand12C/13C.Becausetheisotopesofchemicalelementshavethesameprotonandelectron,thereforetheyhavethesamechemicalp
39、roperties.Comparedstableisotopelabeledaromacompoundswiththoseoneswithoutstableisotopelabeling,inadditiontoslightlydifferentmassnumbers,theirphysicochemicalpropertiessuchasvolatility,reactivity,partitioncoefficient,etc.arethesame.lTheprincipleofaccuratequantificationinstableisotopedilutionanalysisis,
40、comparedstableisotopelabelledcompoundwiththesamecompoundwithoutisotopiclabeling,duetothedifferenceofmassnumber,theycanbepartiallyseparatedonthecapillarycolumn.Whenaknownamountofisotopelabeledcompoundsareadded,underthesameGC-MSconditions,byanalyzingtheSIMmodeofanalyte,theycanbeaccuratelyquantified.lI
41、sotopicdilutionanalysisinvolvesthesynthesisofquasiquantitativearomacompoundslabeledwithstableisotopes,spikedtosampletobeanalyzed,aromaextraction/isolation,thenquantitativelyanalyzedbyGC-MS.lItshouldbenotedthattheprotonsatthecarbonylpositioncanbeenolizedinquantitativeanalysis,whichleadstotheexchangeo
42、fprotonsinthesample,thuserrorsgenerates.Therefore,itisnecessarytousestandardcompoundstochecktheexchangeofD/Hinstableisotopelabelledcompound,toensuretheaccuracyofquantification.Thetechniqueofodorantquantification-SIDALeft:TheMS-EIdiagramsof2H3-acetylpyrazine(a)andacetylpyrazine(b)Right:SIMchromatogra
43、msof1:1mixed2H3-acetylpyrazine(m/z125)andacetylpyrazine(m/z122)uBystableisotopedilutionanalysis,theaccuratelyquantificationofthetargetodorsubstancescanbemadeviathedifferentTICdiagramsofsubstances.New tools for flavor chemistry studyGCO and AEDAlAccording to literature,till now more than 8,000(or10,0
44、00)volatile compounds have been identified fromvariousfoods.Nowitisgenerallyrecognizedthatonlyfewofthemareodor-active,havingcontributiontothefoodaroma,notallthevolatiles.Moreover,theconcentrationsofsomecompoundswithstrongodorareverylow,soasnottobeidentifiedbyGC-MS.Sothesearoma-impactkeycompoundscann
45、otbeidentifiedonlybyGC-MS.lGC-O(gas chromatography-olfactometry)and AEDA(aromaextractdilutionanalysis)techniquescreateaneweraofmodernflavorchemistry,andaremoreandmorewidelyusedinthearomaanalysisoffood.(RIvalue,se-54column)(a)Gaschromatogram(RIvalue,se-54column)(b)FDofvolatilesinfreshwhitebreadcrumbO
46、dor detection threshold of some common soybean odor compoundsCompounds(odor)Odor threshold(ppb,ng/L)Hexanal(fresh,grassy)0.41-Hexanol(fresh)500(E)-2-Nonenal(hay,fatty)0.5-11-Octen-3-ol(mushroom)1(E,E)-2,4-Decadienal(fatty,fried)0.07Dimethyl trisulfide(garlic,cooked cabbage)0.01GCO(Gas Chromatography
47、-Olfactometry)lGC-Otechniqueismoreandmoreused,itisveryusefulandeffectiveforthearomaanalysisoffood.TherearesomekindsofGC-OtechniquessuchasCHARManalysis,AEDA(aromaextractdilutionanalysis),OSMEandDF(detectionfrequency),theyareallemployedfortheanalysisofaroma-activeorodor-activecompoundsandtherankingoft
48、heircontributiontothearomaprofileoffood.lGC-Otechniqueisagoodcombinationofstrongseparationcapacityofgaschromatographyandsensitiveolfactorysensationofhumannose.Gas chromatographolfactometertriple quadrupole mass spectrometerAroma extract dilution analysis(AEDA)lThemethodofaromaextractdilutionanalysis
49、(AEDA)wasfirstlyreportedbyProfessorW.Groschandco-workersfromGermanyin1987.Theoriginalaromaextractisanalyzedontwocolumnsofdifferentpolarities(e.g.DB-WaxforpolarandDB-5fornon-polar)byGC-O.lUsuallysniffing(GC-O)ofaserialofdilutionoforiginalaromaextractaredoneonthepolarcolumnsuchasDB-WaxorDB-FFAP,forthe
50、investigationofitscontributionofidentifiedodoranttothearomaprofileoffood,thisiscalledAEDA.lThenGC-Ooforiginalaromaextractisdoneonnon-polarcolumnsuchasDB-5.TheRIs(bothpolarDB-Waxandnon-polarDB-5columns)arecalculatedaccordingtotheformula,andtheunknowncompoundsareidentifiedtentativelyaccordingtotheinfo