現(xiàn)代儀器分析第十一章高效液相色譜備課講稿

現(xiàn)代儀器分析第十一章高效液相色譜injectorcolumnovendetectorpumppumpHIGH-PRESSUREGRADIENTSYSTEMpumpmixerdataprocessorShimadzuSolventDeliveryPumpPlungerReciprocatingPumpLC-10ADVP

:DualplungerwithparallelflowlineLC-10ATVP

:DualplungerwithtandemflowlineHigh/Lowpressure

gradientsystemmixerlowpressuregradientdevicehighpressuregradientsystemlowpressuregradientsystemDegasUnitExtremelysmall

fluctuationofpressurePressurefluctuationiscausedbyslowresponseofcheckvalve.Microcheckvaluedesigncanmakeresponsespeedhigh.FastResponseThisleakagewillcausepressuredrop.PARTSOFAHPLCSYSTEMrefillthesolventmotorSealsolventgearingThisvolumeisaround1L.A.SYRINGEPUMPmotorandcamplungerplungersealcheckvalvepumphead10-100uLPARTSOFAHPLCSYSTEMB.PLUNGERRECIPROCATINGPUMPPARTSOFAHPLCSYSTEMOUToilreserveoildiaphragmINpistonoilsealC.DIAPHRAGMPUMPPARTSOFAHPLCSYSTEMRheodyneManualInjectorINJECT/LOADPOSITION[INJECT]position[LOAD]position11.3固定相與流動相1液—液色譜2液—固色譜3離子交換色譜4凝膠色譜四類液相色譜1.反相色譜固定相為非極性物質疏水性流動相為極性溶液如甲醇或乙腈水溶液分離方式基于多次吸附-解吸的重復過程非極性化合物較極性化合物在柱中具有較強的保留2.正相色譜固定相是極性物質流動相是非極性或弱極性的溶液如正己烷或四氫呋喃如前所述分離過程也是基于被分離組分在流動相與固定相之間的分配平衡3.分子排阻色譜固定相由一些起分子篩作用的多孔材料組成較大的分子被較快地從色譜柱中洗脫較小分子則滯留在填充材料的細小孔徑中保留較長的時間。離子色譜法三種分離機理A.高效離子交換色譜法HPIC分離是基于發(fā)生在流動相和鍵合在基質上的離子交換基團之間的離子交換過程也包括部分非離子的相互作用這種分離方式可用于有機和無機陰離子和陽離子的分離B.高效離子排斥色譜HPICE分離是基于固定相和被分析物之間三種不同的作用-Donnan排斥空間排斥和吸附作用這種分離方式主要用于弱的有機和無機酸的分離C.流動相離子色譜法MPIC分離是基于被分析物在分析柱上的吸附作用分析柱的選擇性主要取決于可動相的組成和濃度流動相除了加入有機改進劑之外還需加入離子對試劑這種分離方式可用于表面活性陰離子和陽離子以及過渡金屬絡和物的分離。離子色譜的優(yōu)點1速度一般10min即可分別完成陰陽離子的剖面2靈敏度直接進樣可達ppb級用濃縮柱可達ppt級限制的因素是對普遍存在離子如Cl-和Na+的高靈敏度由此而存在的污染3選擇性Dionex已有多種成熟的固定相以及選擇性的檢測器4多組分同時測定但對樣品成分之間的濃度差太大的樣品如半導體級化學試劑中雜質的測定有一定的限制5運行費用非常低勿需特殊試劑6柱填料在廣的PH范圍內和對有機試劑的穩(wěn)定性廣的應用范圍及對復雜基體分析的可行性正相色譜NORMALPHASE硅膠Silicageltype :generaluse氰基Cyanotype :generaluse氨基Aminotype :forsugaranalysisSilicagelSiSi-Si-CH2CH2CH2CN-Si-CH2CH2CH2NH2-Si-CH2CH2CH2OCH(OH)-CH2(OH)ModifiedSiWHATISTHEINTERACTION?Silicagel(polar)HydrogenbondingNon-polarRETENTIONTIMEANDHYDROGENBONDINGOHHOSiOHSiOHStrongWeakWHATISTHEINTERACTION?HydrophobicinteractionNon-polarpolarsolventRETENTIONTIMEANDHYDROPHOBICITYOHOHC18(ODS)StrongWeak反相色譜REVERSEPHASEC18(ODS)typeC8(octyl)typeC4(butyl)typePhenyltypeTMStypeCyanotype-Si-C18H35SiNon-polarpropertyEFFECTOF

STATIONARYPHASEC18(ODS)StrongC8samplesamplesampleC4MediumWeakREVERSEPHASE

ION-PAIRCHROMATOGRAPHYIon-PairReagent離子交換色譜IONEXCHANGEMODEN+RRRSampleSO3-Sample+++++++++++++IonAttractionForceRELATIONSHIPBETWEEN

MWANDRTMolecularWeight(LogMW)GPCColumnExclusionlimitPermeationlimitTimeUltraviolet/Visibledetector(UV/VIS)PhotodiodeArraydetector (PDA)Fluorescencedetector(RF)Conductivitydetector(CDD)RefractiveIndexdetector(RID)Electrochemicaldetector(ECD)MassspectrometerdetectorEvaporativeLightScatteringdetector液相色譜檢測器的類型TYPESOFHPLCDETECTORSPMTPhotodiodeGratingXeLampHighSensitivityandExcellentRepeatability

MirrorFlowCellq紫外--可見檢測器EinEoutA=eCl

=-log(Eout/Ein)lC:concentrationLambert-Beer'slaw(A:absorbance)CellUVDETECTOR8~12μlUV--VISDetectorSampleCellReferenceCellPhotodiodePhotodiodeEinEinEinGratingD2/WlamplEoutPARTSOFAHPLCSYSTEM275nm208nm275nm208nmSelectionofwavelength:quantitative/preparative?

PhotodiodeArraydetectorSampleCell512ElementsPhotodiodeArrayGratingD2/WlampOneelementcandetectoneabsorbanceatonewavelength.二極管陣列WavelengthScanModePARTSOFAHPLCSYSTEMPhotodiodeArraydetector:3DChromatogramTimeWavelengthAbsorbanceChromatogramSpectrumPARTSOFAHPLCSYSTEMCelldesignofFluorescencedetector熒光檢測器Fluorescencedetector+hn1

hn2+*AA*Ahn1

hn2ExcitationWavelengthEmissionWavelengthFluorescence*PARTSOFAHPLCSYSTEMRefractiveIndexdetectorOpticalSystem折射示差檢測器Conductivitydetector

IV

LAAK(conductivity)=I[A]/E[V]=A[cm2]/L[cm]*k

(k:specificconductivity)

k=(I/E)*(L/A)electrode電導檢測器

ChromatogramofAnioninthebrineAnalyticalConditionsColumn:Shim-packIC-A3Mobilephase:8.0mMp-hydroxybenzoicacid3.2mMBis-Tris*Flowrate:1.5mL/minTemperature:40CInjectionVolume:100uLPeaks1.F (1.4ppm)2.Cl (10200ppm)3.NO2 (10ppm)4.Br (43ppm)5.NO3 (44ppm)6.SO4 (431ppm)Bis-Tris:bis(2-hydroxyethyl)iminotris(hydroxymethyl)methanePARTSOFAHPLCSYSTEM海水中陰離子分析ElectrochemicaldetectorWorkingelectrodeAUXelectrodeReferenceelectrode電化學檢測器ChromatogramofCatecolaminesAnalyticalConditionsColumn:Shim-packCLC-ODSMobilephase:80mMphosphatebuffer(pH=2.7)100mMNaNO3,200mg/lSOS5mg/lEDTA,4%acetonitrileFlowrate:1.0mL/minAppliedPotential:+0.8VTemperature:40CInjectionvolume:10uLPeaks1.Noradrenalin(5ppb)2.Adrenalin(5ppb)3.Dopamine(5ppb)PARTSOFAHPLCSYSTEM兒茶酚胺1去腎上腺素2腎上腺素3多巴胺SECONDARYRETENTIONEFFECTSSilanolGroupEvenmodifiedsilicagel(e.g.ODS,C8),residualsilanolgrouparestillremainedonthesurfacearea.Silanolgroupwillstronglyabsorbaminecompounds,thereforetailingwillbehappened.C18C18C18C18silicacoreOHO-SilanolgroupnegativechargeHPLC方法的