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©2007WatersCorporationWaters中国有限公司培训中心©2007WatersCorporation2内容提要ACQUITYUPLCTM色谱柱简介—BEH第二代杂化颗粒化学—Waters颗粒技术—色谱柱硬件—eCordACQUITYUPLCTM色谱柱使用指南©2007WatersCorporation3ACQUITYUPLC颗粒化学是什么在杂化的碳-硅基质内具有桥式乙烷的1.7µm颗粒—改善了高pH稳定性—改善了硅胶的柱效与反压三官能团键合C18配体—改善低pH稳定性专利端基封口技术—可能达到的昀佳峰形©2007WatersCorporation4创造全新的颗粒技术优势劣势无机(硅胶)•机械强度高•高柱效•保留时间可预测•有限的pH范围•碱性化合物拖尾•化学稳定性差聚合物(碳)•宽pH范围•没有离子相互作用•化学稳定性好•“软”,机械强度低•柱效低•保留时间不可预测杂化(硅-碳键合)颗粒技术©2007WatersCorporation5Tetraethoxysilane(TEOS)MethylPolyethoxysilane(MPEOS)Methyltriethoxysilane(MTEOS)第一代杂化技术(1999)(甲基-碳-硅)颗粒甲基在杂化颗粒表面(更好的峰形)并且在杂化颗粒内部(高pH条件下的长寿命)©2007WatersCorporation6第二代杂化技术简介桥式乙基硅氧烷/硅胶杂化颗粒Tetraethoxysilane(TEOS)Bis(triethoxysilyl)ethane(BTEE)Polyethoxysilane(BPEOS)+4SiEtOEtOOEtEtOSiEtOEtOCH2EtOCH2SiOEtOEtOEtSiEtOOCH2CH2SiOSiEtOOEtSiOOOEtOSiOSiOEtOOOEtEtEtn©2007WatersCorporation7SiSiSiSiSiOOOOOSiSiCCCCCC新的第二代桥式乙基硅氧烷/硅胶杂化颗粒©2007WatersCorporation8EthyleneBridgedHybrid(BEH)颗粒BEH颗粒的益处耐高压宽pH范围(1-12)高柱效(1.7µm)比硅胶低的硅醇基活性WallSurfaceOSiOSiOSiOSiOSiOSiOSiOSiOSiOOHOHOHOHOHH2CCH2OHOHOOOOOOOOSiOSiOSiOSiOSiOSiOSiOSiOSiOH2CCH2OOOOOOOSiSiOOOOHOCH2H2CH2CCH2硅胶基质中的乙基桥©2007WatersCorporation9优化的小颗粒解决方案1.7µm颗粒—新的专利桥式杂化颗粒o提供更高的机械强度同时改善色谱性能—适于保留和质量传递的多孔—创新的筛分技术色谱柱硬件—新的筛板技术以便保留小颗粒—新的端口接头技术以便高压下使用填充技术—色谱柱填充压力超过20,000psi—专利技术©2007WatersCorporation10ACQUITYUPLC色谱柱化学BEHC18三官能团键合C18专利的端基封口技术宽pH范围BEHC8三官能团键合C8专利的端基封口技术宽pH范围BEHShieldRP18单官能团键合内嵌极性基团BEHPhenyl三官能团键合C6Phenyl专利的端基封口技术BEHHILIC—未键合的BEH颗粒—HILIC用于极性更大的化合物HSST3—T3键合及封端—对极性化合物的优异保留两种UPLC®颗粒:•EthyleneBridgedHybrid(BEH)•HighStrengthSilica(HSS)不同基质的颗粒给出不同的选择性USPL1USPL7USPL1USPL11USPL1©2007WatersCorporation11UPLC®技术的HSS化学品HSST3—T3:极性组分保留—全水相兼容的C18化学品—为昀大化保留而设计HSSC18—高键合密度,三官能团键合C18化学品—通用的,高效C18化学品—为更好的峰形而应用的特定端基封口技术—硅胶颗粒性能HSSC18SB—SB:SelectivityforBases—无端基封口:昀佳的硅烷基选择性—为方法开发者而设计©2007WatersCorporation12UPLC®颗粒和化学品总结Sep2006Jun2007Jan2008Mar2004Mar2005Mar2005Mar2005Dec2005LaunchDate-方法开发常用©2007WatersCorporation13VanGuard™预柱特为UPLC®使用而设计第一个也是唯一能在高达15,000psi(1,000bar)下日常使用的保护柱专利的超低扩散设计有效地防护和保持UPLC®色谱柱的效能©2007WatersCorporation14UPLC™HILIC用于强极性碱性化合物的分离ConditionsColumn:ACQUITYUPLC™BEHHILIC2.1x50mm,1.7µmMobilePhaseA:ACN:10mMNH4CH2COOH,pH5.5(95:5)MobilePhaseB:ACN:10mMNH4CH2COOH,pH5.5(50:50)FlowRate:0.5mL/minGradient:TimeProfile(min)%A%B0.09912.01992.19912.5991InjectionVolume:2µLSampleDiluent:ACN:MeOH(75:25)SampleConcentration:25µg/mLTemperature:30oCDetection:UV@254nmInstrument:ACQUITYUPLC™SystemwithTUVCompounds1.Uracil2.5-Fluorocytosine3.CytosineAU0.000.100.200.30Minutes0.000.200.400.600.801.001.201.40Injection2,000AU0.000.100.200.30Minutes0.000.200.400.600.801.001.201.40Injection10112233Nolossinefficiency,peakshapeorretentionoverthecourseof2,000injections.Injection2000Injection10©2007WatersCorporation15用户色谱柱寿命试验IsocraticAgingExperimentInitialinjectionInjection4,000AU0.000.200.40Minutes0.000.200.400.600.801.001.201.401.601.802.002.202.402.602.803.00AU0.000.200.40Minutes0.000.200.400.600.801.001.201.401.601.802.002.202.402.602.803.00加速色谱柱老化试验:1.Installandequilibratecolumn.2.Setflowrateto0.5mL/min(6,000psi),injectandrecorddata.3.Increaseflowrateto1.25mL/mintoachievebackpressureof13,000psi.4.Inject200to400times.5.Reduceflowrateto0.5mL/min(6,000psi),injectandrecorddata.6.Cycleandrepeatsteps2–5.Compounds1.Ketorolac(208μg/mL)inMeOH2.Naproxen(13μg/mL)inMeOH1221NOlossinefficiencyorpeakshapeafter4,000injectionsat13,000psiand65oCDatacourtesyofDr.KenWehmeyer,TheProctor&GambleCompanyACQUITYUPLC™BEHShieldRP182.1x50mm,1.7μm©2007WatersCorporation16UPLC™氨基酸分析应用方案©2007WatersCorporation17HPLC和UPLC氨基酸方法AMQAspSerGluGlyHisNH3ArgThrAlaProCysTyrValMetLysILeLeuPheMinutes8.0010.0012.0014.0016.0018.0020.0022.0024.0026.0028.0030.0032.0034.0036.0038.00HPLC50分钟周期时间UPLCTM10分钟周期时间AMQNH3HisSerArgGlyAspGluThrAlaProCysLysTyrMetValNVaILeLeuPheMinutes1.502.002.503.003.504.004.505.005.506.006.507.007.508.00©2007WatersCorporation18氨基酸分析的ESI-MS兼容性SerAlaMinutes1.502.002.503.003.504.004.505.005.506.006.507.007.508.003.416Peak1-ZQF1Scan100.00-600.00ES+,Con171.2276.1Intensity02x1074x1076x107m/z100.00200.00300.00400.00500.005.261Peak2-ZQF1Scan100.00-600.00ES+,C171.0260.1495.0Intensity0.02.0x1074.0x1076.0x1078.0x1071.0x1081.2x1081.4x108m/z100.00200.00300.00400.00500.00直接流速进入离子源700uL/min©2007WatersCorporation19用于肽谱图的ACQUITYUPLC柱肽分离技术—ACQUITYUPLC色谱柱经肽谱图QC检测oACQUITYUPLC™BEH130C181.7µm,2.1mmx50/100/150mmoACQUITYUPLC™BEH300C181.7µm,2.1mmx50/100/150mm用于ACQUITYUPLC肽谱图的高灵敏度UV检测器—仪器控制—混合器©2007WatersCorporation20固定相的选择性ConditionsColumns:ACQUITYUPLC®BEH2.1x50mm,1.7µmACQUITYUPLC®HSST32.1x50mm,1.8µmMobilePhaseA:0.1%HCOOHinH2OMobilePhaseB:0.1%HCOOHinACNFlowRate:1.0mL/minGradient:TimeProfile(min)%A%BCurveInitial982-1.50752551.519826InjectionVolume:1µLSampleConc:0.01-0.02mg/mLTemperature:35oCDetection:UV@240nmInstrument:ACQUITYUPLC®SystemwithACQUITYUPLC®PDACompounds1.Acetaminophen2.2-Acetamidophenol3.Caffeine4.Acetanilide5.AcetylsalicylicAcid6.SalicylicAcid7.PhenacetinHSST3©2007WatersCorporation21来自紫海胆的咖啡酸分离:不同UPLC色谱柱的选择性差异ConditionsColumns:ACQUITYUPLCTMBEHC182.1x50mm,1.7µmMobilePhaseA:0.1%CF3COOH
本文标题:UPLC-School-2-色谱柱
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