安捷伦高效液相色谱高级培训班.pdf

1Group/Presentation TitleAgilent RestrictedMonth#,200XPage 1How Can Temperature Help温度可怎样提供帮助温度可怎样提供帮助Adam Woodhouse,Agilent UK,Sept.2006edited by An Rong,Agilent China,Dec.2006HPLC HPLC MasterClassMasterClass 20072007安捷伦安捷伦安捷伦安捷伦20062006液相色谱技术高级研修班液相色谱技术高级研修班液相色谱技术高级研修班液相色谱技术高级研修班Page 2Overview概要概要Affect of Temperature on Pressure温度对压力的影响Speeding It Up 加速Thermal Stability 热稳定性Separation Robustness 分离稳定性/耐受性UV Sensitivity 紫外灵敏度LC/MS Sensitivity 液质联用灵敏度Page 3Pressure Equation压力公式压力公式P =FL/K0 r2dp2Viscosity粘度粘度Flow rate流速流速Column length柱长柱长Specific permeability比渗透性比渗透性Column radius柱半径柱半径Particle diameter粒径粒径Page 4Viscosity Plots粘度曲线粘度曲线ACN/water0.000.200.400.600.801.001.20050100150%ACNViscosity202530354045505560乙腈/水Page 5Pressure Equation压力公式压力公式P =Viscosity(cP)x 139Consider a 50 x 4.6mm Column with 1.8um particles at 1 ml/min考虑一根考虑一根内含内含1.8um颗粒的颗粒的50 x 4.6mm 色谱柱在色谱柱在1 ml/min流速下流速下P =FL/K0 r2dp2At 10%Acetonitrile 10%乙腈时乙腈时20oC P =1.12 x 139=156 Bar40oC P =0.76 x 139=105 Bar60oC P =0.43 x 139=72 Bar80oC P =0.33 x 139=46 Bar100oC P =0.24 x 139=33 BarPage 6Viscosity Plots粘度曲线粘度曲线Methanol/water0.000.501.001.502.00050100150%MethanolViscosity202530354045505560甲醇/水2Page 7Pressure Equation压力公式压力公式P =Viscosity(cP)x 139Consider a 50 x 4.6mm Column with 1.8um particles at 1 ml/min考虑一根内含考虑一根内含1.8um颗粒的颗粒的50 x 4.6mm 色谱柱在色谱柱在1 ml/min流速下流速下At 50%Methanol 50%甲醇时甲醇时20oC P =1.79 x 139=249 Bar40oC P =1.21 x 139=169 Bar60oC P =0.83 x 139=116 Bar80oC P =0.83 x 139=78 Bar100oC P =0.83 x 139=52 BarP =FL/K0 r2dp2If pressure is reduced then flow can be increased accordingly and analysis accelerated如果压力降低，则流速可以相应提高，加快分析速度Page 840C1.00ml/minmin0.20.40.60.810Speeding Up加速分析加速分析Column=2.1mm x 50mm 1.8mWater/Acetonitrile Gradient水水/乙腈梯度分离乙腈梯度分离0.200.4 min95C 2.40ml/min,2.5x faster快两倍快两倍Page 9High Temperature LC-MS/MS(HTLC-MS/MS)for High Throughput BioanalysisDaniel Tang,PhD,PDM,Michigan Laboratories,PGRD,Pfizer PharmaceuticalsT:+60C P:53%2x Speed!1570740Speeding Up加速分析加速分析Page 10Analyte Thermal Stability热稳定性热稳定性Zorbax RRHT 2.1 x 50 SB-C18如果在热的色谱柱中停留过久，样品会不稳定，Page 11Analyte Thermal Stability 热稳定性热稳定性No Compound Degradation at high flow rates/short run times!在较高流速下在较高流速下/短分析时间内无任何化合物降解！短分析时间内无任何化合物降解！Zorbax RRHT 2.1 x 50 SB-C18Page 12Long Lifetime of RRHT Column at 80C0200040006000800010000120001499718853988115313051506164117761911204621812319250826432778291330853220346836033738388740544189432445374756489250335201Injection NumberPlatesPlatesColumn Robustness 色谱柱稳定性色谱柱稳定性At elevated temp and flow在升高温度和流速下在升高温度和流速下Column:Zorbax RRHT StableBond C18,1.8 mPressure at 80C=355 barColumn:2.1 x 50mm,Flow Rate:1 mL/minMobile Phase:40%ACN:60%Water Initial Plates 10,010Final Plates 10,089Final Inj#54253Page 13Selectivity选择性选择性Temperature Effect on RT and Resolution温度对温度对RT和分离度的影响和分离度的影响Temperature=New degree of freedom during method development!温度温度=方法开发中的新的一方法开发中的新的一个自由个自由度！度！Page 14Selectivity选择性选择性Temperature Effect on RT and Resolution温度对温度对RT和分离度的影响和分离度的影响20C60C40C30C90CSalicylic acid水扬酸min0120120120120123 4 534Salicylic acidSalicylic acidSalicylic acidSalicylic acidColumn:RRHT SB-C184.6 x 50mm,1.8umConditions:A:0.1%formic acid B:ACN w/0.1%formic acid(85:15)Detection:UV 254 nmPhenacetinPage 15不同不同温度比较温度比较:Rg1与与Remin5.866.26.46.66.877.2mAU01234567*DAD1 B,Sig=203,16 Ref=off(XIYANGSHENGSB-10-18-000007.D)*DAD1 B,Sig=203,16 Ref=off(XIYANGSHENGSB-10-18-000008.D)*DAD1 B,Sig=203,16 Ref=off(XIYANGSHENGSB-10-18-000010.D)下下，两两个个色谱色谱峰峰分离分离最好最好40 30 25 ReRg1Page 17min9.259.59.751010.2510.510.751111.25mAU246810121416*DAD1 B,Sig=203,16 Ref=off(XIYANGSHENGSB-10-18-000007.D)*DAD1 B,Sig=203,16 Ref=off(XIYANGSHENGSB-10-18-000008.D)*DAD1 B,Sig=203,16 Ref=off(XIYANGSHENGSB-10-18-000010.D)不同不同温度比较温度比较:Rb140下下，Rb1色谱色谱峰与峰与两两侧峰侧峰分离分离最好最好40 C30 C25 CRb1Page 18Performance Robustness性性能耐受能耐受性性Maintaining Retention Time Precision at High Analysis Speed在高分析速度下在高分析速度下保持保留保持保留时间时间精密精密度度Low flow F=0.35ml/minLow Temp.T=40Cmin0.511.522.53.0mAU050100150200250300350400High FlowF=2.00ml/minHigh Temp.T=80Cmin0.10.20.30.40.5mAU050100150200250300350RT Precision0.067%RSDRT Precision0.134%RSDRT Precision(n=10)Page 19Area Precision(n=10)Inj.Vol.:3.0l 0.307%RSDInj.Vol.:1.0l 0.569%RSDInj.Vol.:0.5l0.672%RSDmin1.271.281.291.31.31mAU020406080100120140ButyrophenoneBenzophenoneValerophenoneHexanophenoneHeptanophenoneOctanophenoneAcetanilideAcetophenonePropiophenonemin0.20.40.60.811.21.4Performance Robustness性性能耐受能耐受性性Maintaining Area Precision at High Analysis Speed在高分析速度下在高分析速度下保持峰面积精密保持峰面积精密度度4Page 20Noise of 1200 RRLC up to more than 10 x lower compared to 1100 at high temp and flow.与与1100相比相比，1200 RRLC在高温和高流速下的在高温和高流速下的噪音低噪音低10倍倍Npe ak-to-pe ak ACN 0.1%TFA 230 nm mAU for the 1100F mL/min12345T oC203040506070805.60.08Npea k-to-pea k ACN 0.1%TFA 230 nm mAU for the 1200F mL/min12345T oC203040506070800100.03Customer Evaluation用户评估结果用户评估结果1100 Binary Systemwith DAD“B”配有配有DAD”B”的的1100二元泵系统二元泵系统1200 Rapid Resolution Systemwith DAD SL带有带有DAD SL的的1200高分离度快速液相色谱高分离度快速液相色谱系统系统Affect of Temperature on UV温度对温度对紫外紫外的影响的影响Maintaining Sensitivity at High Analysis Speed Post Column Cooling(PCC)在高分析速度下在高分析速度下保持灵敏保持灵敏度度柱柱后冷却功能后冷却功能Page 211200 RRLC 柱温柱温箱箱柱柱前前加热加热及及柱柱后冷却模块后冷却模块precolumn Heater and postcolumn CoolerL-shaped pre-columnheater(1.6 uL)L形的柱前加热模块(1.6 uL)U-shaped Post-column cooler(1.5 uL)U形柱后冷却模块(1.5 uL)Page 22801000.41.01.82.40100200300400500600Temperature CFlow ml/minNoise P-to-PAU4040Hz80Hz20Hz40Hz80Hz20Hz40Hz80Hzpccpccpcc20Hz40Hz80Hzpccpccpcc80Hz40Hz20Hz20Hz40Hz80HzPost Column Cooling PCC柱柱后冷却功能后冷却功能Noise Reduction under extreme UFLC conditions 在极端超快LC条件下降低噪音 2.1x50 SB-C18 RRHT column:Noise 10 for 0.05%-level Impurities(1mAU)under anycondition!在任何条件下，可以大于10倍信噪比检测出0.05%水平的杂质！mi n02.557.51012.51517.5mAU-0.400.4mi n02.557.51012.51517.5mAU-0.400.4mi n02.557.51012.51517.5mAU-0.400.4Affect of Temperature on UV 温度对温度对紫外紫外的影响的影响Maintaining Sensitivity at High Analysis Speed Post Column Cooling(PCC)在高分析速度下在高分析速度下保持灵敏保持灵敏度度柱柱后冷却功能后冷却功能Page 24MS Sensitivity 质质谱谱灵敏灵敏度度在电喷雾质谱中，超热溶剂有助于离子形成过程中的雾化和脱溶剂效率，因而改善灵敏度。Page 25Summary小结小结 Use of elevated temperatures reduces viscosity of mobile phase and therefore backpressure提高温度可以降低流动相粘度及其反压 Higher linear velocities are achievable with reduced backpressure and therefore faster analysis times随着反压的降低，可以实现更高的线速度，因而分析时间可以更短 Selectivity can vary with Temperature allowing temperature to bemanipulated to achieve better separations选择性随温度变化，使得温度可以成为实现更好分离的选择性调节手段 Using post column cooling drastically reduces the UV detector noise seen when a hot mobile phase cools in the flow cell使用柱后冷却功能可以使热流动相在检测池中冷却，显著降低紫外检测器的噪音 LC/MS sensitivity can be increased by using elevated temperatures into the MSLC/MS灵敏度可以因液相色谱使用较高的温度而提高Page 26Questions?问题？问题？