第十二章气相色谱法

Section1Overview第一节概述气相色谱法（gaschromatography,GC）是以气体为流动相的色谱法。当气化的被测物质随载气进入色谱柱时，基于被测物质在气相和固定相之间吸附-解吸附（气-固色谱）或分配（气-液色谱）差异进行分离。气液色谱：利用待测物在载气和固定在惰性固体表面的液体固定相之间的分配原理实现分离。Gaschromatography(GC)isagas-basedmobilephasechromatography.Whenthegasifiedtestsubstanceentersthecolumnwiththecarriergas,itisseparatedbasedonthedifferencebetweentheadsorptive-desorption(gas-solidchromatography)orthedistribution(gas-liquidchromatography)betweenthegasphaseandthestationaryphase.•Gas-solidchromatography:theuseofdifferentsubstancesinthesolidadsorbentonthephysicaladsorption-desorptioncapacityofdifferentmaterialseparation.气固色谱：利用不同物质在固体吸附剂上的物理吸附-解吸能力不同实现物质的分离。LiquidChromatography:Separationwasachievedusingthedistributionprincipleoftheanalyteinthecarriergasandtheliquidstationaryphaseimmobilizedontheinertsolidsurface.

AdvantagesofGaschromatographic

气相色谱优点

Goodseparationability:theoreticalnumberof103~106Highsensitivity:10-11~10-13gHighselectivity:separableenantiomersandotherdifficulttoseparatesubstancesFast:AnanalysiscanbedoneinsecondstotensofminutesCanbeusedfornon-chromophorecompositionanalysis分离能力强：理论板数103～106灵敏度高：10-11～10-13g选择性高：可分离对映体等难分离物质快速：一次分析可在几秒~几十分钟完成可用于无发色团组分分析DisadvantagesofGaschromatographic

气相色谱缺点

Onlyforheatstability,easytovaporizethematerialanalysisDerivativewhenanalyzingnonvolatilematter

Duetothesmallamountofinjection,quantitativeinjectionhassomedifficulties只适用于热稳定性好、易气化的物质分析分析非挥发性物质时需衍生化由于进样量少，定量进样有一定困难GasChromatograph气相色谱仪Schematicdiagramofagaschromatographic气相色谱流程示意图1.Gassupplysystem（载气系统）Includinggascylindersorgasgenerators,pressureregulators,purifiers,flowcontrollers,andsoon.包括气体钢瓶或气体发生器，压力调节器，净化管，流量控制器等。

Function:toprovideclean,constantflowofthecarriergas功能:提供清洁、流量恒定的载气2.Sampleinjectionsystem

(进样系统)Includingtheinjector(microinjectionorautosampler),septumandgasificationchamber.包括进样器(微量进样针或自动进样器)、隔垫和气化室。Function:IntroducethesampleintotheGCsystemandevaporateintothegas.功能:将试样引入GC系统并蒸发成气体。3.Columnsystem（色谱柱系统）

◆Column,columnoven(色谱柱，柱温箱)◆Columntype(色谱柱类型):Filledcolumn,columndiameter2~5mm(填充柱,柱内径2～5mm)Capillarycolumn,columndiameter0.1~0.53mm(毛细管柱,柱内径0.1～0.53mm)◆Columntemperaturecanbechangedaccordingtoprogram(programtemperature)柱温可按程序改变（程序升温）4.Detectionanddatasystem(检测和数据处理系统)◆Detector,dataacquisitionandprocessingdevice(检测器，数据获取和处理装置)5.Controlsystem（控制系统）Temperaturecontrol,carriergasflowcontrol,(automaticinjectioncontrol),signalcontrol温度控制，载气流量控制，（自动进样控制），信号控制

(1)steampressure<10Paattheusetemperature(2)goodthermalstability,chemicalinertia(3)goodselectivity(4)themeasuredobjecthasacertaindegreeofsolubility第二节固定相stationaryphase一、Gas-liquidstationaryphase（气-液色谱固定相）气-液色谱固定相由固定液和载体组成.（一）stationaryliquid(固定液)Thestationaryliquidisgenerallyahighboilingsubstance.固定液一般为高沸点物质在使用温度下蒸汽压<10Pa热稳定性好，化学惰性选择性好对被测物有一定溶解度要求Itisconsistsofastationaryliquidandasupport.Stationaryliquidclassification(固定液分类)：●Classificationbychemicalstructure:alkanes,polysiloxanes,polydiols,estersandpolyesters

按化学结构分类:烷烃,

聚硅氧烷,聚二醇,酯和聚酯Alkanes:squalane,Apiezon,烷烃类:异三十烷,阿皮松Polysiloxanes(聚硅氧烷类):R=-CH3,methylsilicone,OV-1,OV-101,SE-30,non-polarR=94%methyl,5%phenyl,1%vinyl,SE-54,weaklypolarR=50%methyl,50%phenyl,OV-17,middlepolarR=50%methyl,25%cyanopropyl,25%phenyl,OV-225,polarPolyethyleneglycol(聚二醇类):polyethyleneglycol，PEG（聚乙二醇）PEG-6000PEG-20MEsters(酯类):R=octyl,DOP(dioctylphthalate)R=nonyl,DNP(dinonylphthalate)Polyester(聚酯类):Diethyleneglycolsuccinatepolyester，DEGS

(丁二酸二甘醇聚酯)Thepolarityofthefixativeisafunctionoftheinteractionbetweenthefixativeandtheanalyte,whichisoppositetothepolaritydefinedbythechemicalstructure.固定液的极性是指固定液与被测物之间相互作用力的函数，与纯粹的从化学结构定义的极性不同，它是相对的。●Sortbypolarity(按极性分类)Relativepolarity:相对极性:P0-10，+1；21-40,+2;……..;81-100,+5。P值越大，极性越强。Somecommonlyusedfixative一些常用的固定液Fixedliquidchoice(固定液的选择)Non-polarcomponents:Selectnon-polarfixative非极性组分：选择非极性固定液Dispersionmainlyinaccordancewiththeboilingpointfromlowtohighintheorderofthepeak

色散力为主，按照沸点从低到高的顺序出峰Polarcomponents:Selectpolarfixative

极性组分：选择极性固定液

Dipoleforcemainlyinaccordancewiththepolarityfromsmalltolargeintheorderofthepeak

偶极作用力为主，按照极性从小到大的顺序出峰无严格规则，一般可按“相似性原则”Nostrictrules,generallyaccordingtothe"similarityprinciple"Fixedliquidchoice(固定液的选择)Polarandnonpolarmixedcomponents:Selectpolarfixative

极性与非极性混合组分：选择极性固定液

Thenonpolarcomponentfirstpeaksandthepolarcomponentpeaksintheorderofpolarity

非极性组分先出峰，极性组分按极性顺序出峰Hydrogen-bondedcomponents:Selectpolyglycol-basedfixative氢键型组分：选择聚二醇类固定液

Accordingtotheformationofhydrogenbondcapacityintheorderofthepeak按照形成氢键能力大小顺序出峰

Whenselectingafixative,focusonwhatisthemajordifferencebetweentheseparatedcomponentsandchoosetheappropriatefixative.

选择固定液时，要重点关注被分离组分间的主要差异是什么，据此选择合适的固定液。Example:Theeffectofdifferentfixativeonseparation例：不同固定液对分离的影响(二)Carrier载体Largesurfacearea(比表面积大;)Chemicallyinert(化学惰性;)Goodthermalstability;(热稳定性好;)Haveacertainmechanicalstrength(有一定机械强度)basicrequirements基本要求Types类型diatomite(硅藻土)Non-diatomaceousearth(非硅藻土类)(Glassmicrospheres,Teflon(玻璃微球，特氟龙)ChromosorbW（白色载体，101等）ChromosorbP（红色载体，6201等）Passivationmethod钝化方法Pickling酸洗（AW）Alkaliwash碱洗(BW)Silylation硅烷化(DMCS,HMDS)Weakenthecarriersurfaceadsorptionactivity减弱载体表面吸附活性二、Gas-solidchromatographystationaryphase

气-固色谱固定相Adsorbents:Graphitizedcarbonblack,silicagel,aluminafortheanalysisoflowmolecularweightalcohols,alkanesandaldehydes吸附剂:石墨化炭黑，硅胶，氧化铝，用于分析低分子量醇、烷烃和醛酮Molecularsieve:fortheanalysisofH2,O2,CO,N2,CO2,CH4andsoon分子筛:用于分析H2,O2,CO,N2,CO2,CH4等PolymerPorousMicrospheres(GDX):Analysisofcommonlyusedaminoacidsinethanolandorganiccompounds.高分子多孔微球（GDX）:药物分析中常用语分析乙醇、有机化合物中微量水分。例：废气成分检测固定相：5A分子筛;TCD检测器三、Carriergas（载气）N2:themostcommonlyusedgaschromatographiccarriergas;lowthermalconductivity,lowsensitivityforthermalconductivitytestingN2:最常用的气相色谱载气；热导率低，用于热导检测时的灵敏度

低H2:highthermalconductivity,theuseofthermalconductivitydetectorcommonlyusedinthecarriergas.Flammable,payattentiontosafetyH2:热导率高，使用热导检测器时常用该载气。

易燃，需注意安全He:highthermalconductivity,commonlyusedinGC-MSHe:热导率高，常用于GC-MSThethirdcolumntype

第三节柱类型◆Packedcolumn:填充柱:●Glassorstainlesssteel玻璃或不锈钢材质●Diameter2~4mm,length0.5~4m内径2～4mm,长0.5～4m●Fixedphaseparticles:60/80meshor80/100mesh

固定相颗粒：60/80目或80/100目●Commonlyusedcarriergasflowrate:20~60mL/min常用载气流速：20～60mL/min●Columnefficiency:800~1000/m柱效：800～1000/m●Conventionaldruganalysisiscommonlyused

常规药物分析时常用◆Capillarycolumn毛细管柱

●Quartzmaterial,coatedwithpolyimideprotectivelayer.

石英材质,外涂聚酰亚胺保护层.

●Diameter0.10~0.53mm,commonlyusedspecifications0.10,0.25,0.32,0.53mm.

内径0.10～0.53mm，常用规格0.10、0.25、0.32、0.53mm.

●Commontypes:常用类型：Coatedwithcapillary涂壁毛细管(WCOT)Carriercoatedcapillary载体涂层毛细管(SCOT)Cross-linkedcapillary交联毛细管（用于GC-MS）

●Carriergasflowrate:1~2mL/min

载气流速：1～2mL/min

●Applicationofsplit/splitlessinjection

应用分流/不分流进样Section4Detector

第四节检测器Concentrationtypedetector(浓度型检测器):TCD,ECDQualitydetector(质量型检测器):FID,FPD,NPDPerformance性能指标:Sensitivity(S):1mlofcarriergascarrying1mgofthemeasuredcomponentsintothedetectorwhenthevoltage(fortheconcentrationofthedetector),orcarry1mgpersecondofthemeasuredcomponentsintothedetectorwhenthevoltageValue(qualitytypedetector).2.Noise(N)anddrift

噪声(N)和漂移3.Detectionlimit(D):D=2N/S

检测限（D）:D=2N/S灵敏度(S):每1mL载气中携带1mg被测组分进入检测器时产生的电压值(对浓度型检测器),或者每秒钟携带1mg被测组分进入检测器时产生的电压值（质量型检测器）。一、Thermalconductivitydetector,TCD

热导检测器R1/R2=R3/R4,i=0R1/R2≠R3/R4,i≠0被测组分通过热导池时，由于其热导率与载气不同，带走的热量不同，导致电桥不平衡，检流计中有电流通过。将微电流通过电阻转换成电压，即为色谱信号。Wheatstone电桥Whenthemeasuredcomponentpassesthroughthethermalconductivitycell,becauseofitsthermalconductivityandthecarriergasisdifferent,takeawaytheheatisdifferent,resultinginbridgeimbalance,galvanometerflowthrough.Themicrocurrentthroughtheresistanceintovoltage,thatis,thechromatographicsignal.Thermalconductivitydetectorcharacteristics热导检测器的特点：Belongtotheuniversaldetector,aslongasthethermalconductivityandcarriergascanbedetected.属于通用性检测器，只要热导率与载气不同即可检测.Comparedwithotherdetectors,thesensitivityislow.与其他检测器相比，灵敏度较低.ResponseWidedynamicrange(105)响应动态范围宽(105)Nodamagetothecomponentundertest对被测组分无破坏性Factorsthataffectthesensitivityofthermalconductivitydetectors影响热导检测器灵敏度的因素1.Bridgecurrent(电桥电流)Theresponsevalueisproportionaltothecubicpowerofthebridgecurrent,sotheincreaseofthebridgecurrentcansignificantlyincreasetheresponsesignal,butthebridgecurrentcannotbearbitrarilyincreased,thecarriergasN2:100-150mA,H2docarriergas:150-200mA.响应值与桥电流的三次方成正比，故增加桥电流可显著增加响应信号，但桥电流不可任意增大，

载气N2时:100-150mA,H2做载气时:150-200mA。Note:addthebridgebeforethefirstneedtopassthegas,soastoavoidhotwireblown加桥电流前需先通载气，以免热丝烧断2.Detectortemperature

(检测器温度)Notlowerthanthecolumntemperature,oftenhigherthanthecolumntemperature20~50℃.不得低于柱温，常比柱温高20~50℃.3.Carriergas

(载气)Sensitivityisproportionaltothethermalconductivityofthecarriergasandthecomponentundertest,H2>He>N2灵敏度正比于载气与被测组分的热导率差，H2>He>N24.Hotwireresistancevalue(热丝电阻值)载气热导率

载气热导率

H2224.3CH445.8He175.6propane26.4N231.5ethanol22.3Air31.5acetone17.6

二、Flameionizationdetector，FID(氢焰离子化检测器)Structure(结构)Carbon-containingorganicmatterisburnedintheH2-Airflametoproducedebrisions,andanioncurrentisformedundertheactionofanelectricfield.Theseparatedcomponentsofthecolumnaredetectedbasedontheintensityoftheelectricalsignalgeneratedbytheioncurrent含碳有机物在H2-Air火焰中燃烧产生碎片离子，在电场作用下形成离子流，根据离子流产生的电信号强度，检测被色谱柱分离的组分。Ionizationmechanism(离子化机理)

Themechanismisnotveryclearthatchemicalionizationisapossibleionizationmechanism.机理不十分清楚，化学离子化是可能的离子化机理.Takebenzeneasanexample:以苯为例:FactorsthataffectFIDsensitivity:影响FID灵敏度的因素：1.Gasflowrateratio(气体流速比例):

N2:H2=1:1~1:1.5；H2:Air=1:102.Detectortemperature(检测器温度):>100℃（常用200-250℃）FIDFeatures(FID特点)•Qualitydetector质量型检测器•Simplestructure,durable结构简单、耐用•Highsensitivity(10-13g/s)灵敏度高(10-13g/s)•ResponseWidedynamicrange(107)响应动态范围宽(107)•Theresponsevalueisrelatedtothenumberofcarbonatomsintheorganicmatter响应值与有机物中含碳原子的数目有关•Noresponsetobustiblecompounds(H2O,CO2,SO2,CS2,NOx)对不可燃化合物（H2O,CO2,SO2,CS2,NOx）无响应三、Electroncapturedetector，ECD（电子捕获检测器）63Nior3HStructure:结构:Detectionprinciple(检测原理:)•Theβ-electronsemittedbytheradioactivesourceionizethecarriergasmoleculesandformastablebasestreamundertheactionofanelectricfield.放射源发出的β-电子使载气分子离子化，在电场作用下形成稳定的基流；•Organicmoleculeswithstrongelectronegativeelementscaptureβ-electrons,reducingthebaseflowandformingapeak.具有强电负性元素的有机分子捕获β-电子，使基流降低，形成倒峰。Characteristicsofelectroniccapturedetectors

电子捕获检测器的特点•(10-15g/s)withelectronegativegroups(halogen,nitro,peroxide)

对含电负性基团（卤素、硝基、过氧化物）具有选择性响应(10-15g/s)•Alkali,alcohol,hydrocarbonsarebasicallyinsensitive

对胺、醇、碳氢化合物基本不敏感•Responsetonarrowdynamicrange(102)

响应动态范围较窄(102)•Non-linearresponse:非线性响应:•RequireshighpurityN2(>99.999%)ascarriergas

需用高纯N2(>99.999%)作为载气•Non-destructivetesting非破坏性检测Otherimportantdetectors

其他重要检测器•Flamephotometricdetector，FPD(火焰光度检测器)

HasaselectiveresponsetothecompoundscontainingSandP对含S和P化合物具有选择性响应•thermionicdetector，TID;orNitrogenphosphorusdetector,NPD

热离子检测器;也称氮磷检测器HasaselectiveresponsetoNandPcontainingcompounds

对含N和P化合物具有选择性响应•Massspectrometerdetector,MSD(质谱检测器)•Fouriertransforminfrareddetector

傅里叶变换红外检测器（FTIR）Section5Selectionofchromatographicconditions

第五节色谱条件选择一、thebasisforselectionofchromatographicconditions色谱条件选择的依据Whereαisseparationfactor,k2isretentionfactor(capacityfactor)ofcompound2whichisretainedstrongerthancompound1.1.Fixedphaseselection(固定相选择)

SeesectionII.(见第二节).2.Columntemperature(柱温)Aftercolumnselection,columntemperatureisthemostimportantfactoraffectingseparation.Columntemperaturedecreased,theseparationincreased,butthepeakwidthincreased,theanalysistimeislonger.色谱柱选定后，柱温是影响分离的最重要条件。柱温降低，分离度提高，但峰宽增加，分析时间加长。Selectionprinciple:inthedifficulttoseparatesubstancestogetagoodseparation,analysisofappropriatetime,peakshapesymmetryunderthepremise,asfaraspossiblewithlowcolumntemperature.选择原则：在使难分离物质对得到良好分离，分析时间适宜，峰形对称的前提下，尽量用低柱温。

•Isothermal:thecolumntemperatureremainsconstantduringtheanalysis.等温:在分析过程中柱温保持不变.

•temperatureprogramming:Duringaperiodofanalysis,thecolumntemperaturechangesaccordingtoacertainproceduretofacilitateproperseparationofcomponentswithlargedifferencesinproperties.Themethodisapplicabletotheanalysisofcomplexcomponents(suchaswideboilingrangesampleswithlargepolarities)程序升温(temperatureprogramming):在一个分析周期中，柱温按照一定程序改变，以利于性质差异大的组分达到合适的分离度。该法适用于复杂组分（如宽沸程样品、极性差异大的样品）分析。二、Selectionofexperimentalconditions(实验条件的选择)例：等温和程序升温对分离效果的影响Example:Theeffectofwarmerprogramtemperatureontheseparationeffect3.

Carriergas(载气)

Lowwirespeed,withN2(低线速时，用N2)Highspeed,withH2orHe(高线速时，用H2或He)WithTCD,useH2orHetoincreasesensitivity(用TCD时，使用H2或He可提高灵敏度)4.

Otherconditions(其他条件)(1)Inlettemperature:shouldensurethatthesampleinstantaneousgasification进样口温度：应保证样品瞬间气化(2)Detectortemperature:higherthancolumntemperature20-50°C检测器温度:高于柱温20-50℃1.positionmethod(分解法)•Pyrolysis:Suitableforpolymerandothermacromoleculeanalysis热裂解法:适用于高聚物等大分子分析•Hydrolysis:themacromoleculeshydrolyzeintosmallmolecules,theproteinhydrolyzedintoaminoacids水解法:将大分子水解成小分子,蛋白质水解成氨基酸2.Derivatizationmethod衍生化法•esterification(酯化):-COOH-----COOR•silanization(硅烷化):-OH------O-Si(CH3)3-COOH------COOSi(CH3)3-NHR------NRSi(CH3)3•acylation(酰化):-OH------OCOR-NN2-------NHCOR三、

Samplepretreatment

(样品预处理)Section6QualitativeAnalysisandQuantitativeAnalysis

第六节定性分析和定量分析

一、Systemsuitabilitytest(系统适用性试验)

Testwithaprescribedreferencesolutionorsystemsuitabilitytestsolutiontodetermineifthechromatographicsystemusedisspecified.

用规定的对照品溶液或系统适用性试验溶液进行试验，以判定所用色谱系统是否规定的要求。1．Numberoftheoreticalplatesofthecolumn(n)Thisparameterisusedtoevaluatetheseparationefficiencyofthecolumn.

色谱柱的理论板数（n）此参数用于评价色谱柱的分离效能。2．Separation(R):Requires>1.5(分离度（R）:要求>1.5)3．Repeatability:Usedtoevaluatetherepeatabilityofthechromatographicsystemresponsevaluesincontinuousinjections.

重复性:用于评价连续进样中色谱系统响应值的重复性。4．TheThetailingfactor(T)isusedtoevaluatethesymmetryofthechromatographicpeaks.

拖尾因子（T）用于评价色谱峰的对称性。

Unlessotherwisespecified,Tshouldbebetween0.95and1.05.

除另有规定外，T应在0.95~1.05之间。

Section6QualitativeandQuantitativeAnalysis

第六节定性分析和定量分析

二、Qualitativeanalysis(定性分析)

Knownreferencesubstancecontrolmethod(已知对照品对照法)Usingtherelativeretentionvalue(采用相对保留值定性)UseKovatstoretainexponentialqualitative(用Kovats保留指数定性)GC-MS,GC-FTIRcombinedwithqualitative

以GC-MS、GC-FTIR联用法定性三、Quantitativeanalysis(定量分析)(一)Correctionfactor(校正因子)(二)Quantitativemethod(定量方法)1.Normalizationmethod(归一化法)

•Allcomponentsshouldbeseparatedanddetected

所有组分均应被分离并被检测到•Theamountofinjectionisnotcritical对进样量要求不严格2.Externalstandardization（外标法）（2）Onepointexternalstandardmethod（外标一点法）Suchasthecalibrationcurvethroughtheorigin,availableexternalstandardmethod如校正曲线通过原点，可用外标一点法（1）Standardcurvemethod,orCalibrationcurvemethod

（标准曲线法或校正曲线法）Note:Theinjectionvolumemustbestrictlyaccurate进样量必须严格准确3.Internalstandardmethod

(内标法)Requirementsforinternalstandards:对内标物的要求：Asubstancethatdoesnotexistinthesample

样品中不存在的物质Andthemeasuredcomponentsarecompletelyseparated,buttheretentiontimeisclose;whenthevariouscomponentsarequantitativelyquantified,theinternalstandardpeakispreferablybetweenseveralmeasuredcomponentpeaks.与被测组分完全分离，但保留时间接近；多种组分同时定量时，内标峰最高Highpurity纯度高3.Internalstandardmethod

(内标法)（1）Internalstandardcorrectionfactormethod

内标校正因子法（2）Internalstandardcal