安捷伦2007液相色谱高级研修班资料2.pdf

1Group/Presentation TitleAgilent RestrictedMonth#,200XPage 1A Guide To Speeding Up Your Separations加速分离指南加速分离指南Adam WoodhouseHPLC HPLC MasterClassMasterClass 20072007安捷伦安捷伦安捷伦安捷伦20072007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1200 Rapid Resolution System Agilent RestrictedPage 2Rule of Thumb经验规则经验规则Maybe not always 100%Technically Correct but a very good easy alternative可能不总是100%技术上正确，但是简单易行的好方法1200 Rapid Resolution System Agilent RestrictedPage 3Overview概要概要Choosing The Column 选择色谱柱选择色谱柱Reducing The Column Length降低色谱柱长Changing The Column Id改变色谱柱内径Adjusting The Injection Volume调整进样体积Transferring to 1.8um Columns转换成1.8um色谱柱Speeding It Up提速1200 Rapid Resolution System Agilent RestrictedPage 4RRHT 600 bar 色谱柱色谱柱DimensionsEclipse Plus C18Eclipse Plus C8Eclipse XDB-C18Eclipse XDB-C8Extend-C184.6 x 150959994-9024.6 x 100959964-902959964-906928975-902928975-906728975-9024.6 x 50959941-902959941-906927975-902927975-906727975-9024.6 x 30959931-902959931-906924975-902924975-906724975-9024.6 x 20926975-902926975-906726975-9023.0 x 150959994-3023.0 x 100959964-302959964-306928975-302928975-306728975-3023.0 x 50959941-302959941-306927975-302927975-306727975-3023.0 x 30924975-302924975-306724975-3023.0 x 20926975-302926975-306726975-3022.1 x 150959794-9022.1 x 100959764-902959764-906928700-902928700-906728700-9022.1 x 50959741-902959741-906927700-902927700-906727700-9022.1 x 30959731-902959731-906924700-902924700-906724700-9022.1 x 20926700-902926700-906726700-902DimensionsSB-C18SB-C8SB-PhenylSB-CNSB-AQRx-Sil4.6 x 150829975-902829975-906829975-912829975-905829975-9144.6 x 100828975-902828975-906828975-912828975-905828975-914 828975-9014.6 x 50827975-902827975-906827975-912827975-905827975-914 827975-9014.6 x 30824975-902824975-906824975-912824975-905824975-9144.6 x 20826975-902826975-9063.0 x 150829975-302829975-306829975-312829975-3053.0 x 100828975-302828975-306828975-312828975-305828975-314 828975-3013.0 x 50827975-302827975-306827975-312827975-305827975-314 827975-3013.0 x 30824975-302824975-306824975-3053.0 x 20826975-302826975-3062.1 x 150820700-902820700-906820700-912820700-9052.1 x 100828700-902828700-906828700-912828700-905828700-914 828700-9012.1 x 50827700-902827700-906827700-912827700-905827700-914 827700-9012.1 x 30824700-902824700-906824700-912824700-905824700-9142.1 x 20826700-902826700-906http:/ product page)http:/ List)推荐的推荐的2个起始选择个起始选择:959941-902 959741-902都是都是Eclipse Plus C18.1200 Rapid Resolution System Agilent RestrictedPage 5StableBond C18Extend C18Eclipse Plus/XDB C18SiOSiOC18C18Bonded Phase Selectivities键合相的选择性键合相的选择性StableBond CN1200 Rapid Resolution System Agilent RestrictedPage 6Choose Suitable Column Phase选择合适的色谱柱选择合适的色谱柱Mobile Phase流动相 pHStable Bond pH 1 4N.B.SB can use increased temperature up to 100CSB可以在升温至100C下使用Acidic酸性酸性Eclipse/Eclipse+pH 2 9N.B.Eclipse can use increased temperature up to 60CEclipse可以在升温到60C下使用Intermediate中等中等Extend pH 8 11.5N.B.Extend can use increased temperature up to 60CExtend可以在升温到60 C下使用Basic碱性碱性21200 Rapid Resolution System Agilent RestrictedPage 7液相柱液相柱-pHpH选择策略选择策略StableBond C18 pH 18Bonus-RP pH 29Extend C18 pH 211.5Eclipse PLUS/XDB C18 pH 29Agilent TC/HC pH 28/91200 Rapid Resolution System Agilent RestrictedPage 8Overview概要概要Choosing The Column 选择色谱柱Reducing The Column Length降低色谱柱长降低色谱柱长Changing The Column Id改变色谱柱内径Adjusting The Injection Volume调整进样体积Transferring to 1.8um Columns转换成1.8um色谱柱Speeding It Up提速1200 Rapid Resolution System Agilent RestrictedPage 9Reduce column length 缩短柱长缩短柱长Shorter Columns with Smaller Particles含有更小颗粒填料的更短的色谱柱含有更小颗粒填料的更短的色谱柱Rs=N4kk+1Plates塔板数塔板数Selectivity选择性选择性Retention保留保留LN dpTo Maintain Rs:e.g.:L/2 dp/2000990P2.PPTColumn Length =NParticle Size =NParticle Size =P-11200 Rapid Resolution System Agilent RestrictedPage 10N.A.N.A.4,20060008,50012,500ColumnEfficiency N(5 m)2,5002,100156,5004,2003012,0007,0005017,50010,5007523,25014,00010035,00021,000150ColumnEfficiency N(1.8 m)ColumnEfficiency N(3.5 m)ColumnLength(mm)Efficiency(N)PressureAnalysisTimePeakVolume-90%-80%-67%-50%-33%-Analysis Time*SolventUsageReduction in analysis time compared to 150 mm column;all columns 4.6-mm i.d.与150mm色谱柱相比，降低分析时间：所有色谱柱使用的是4.6mm内径Shorter columns with small particles provide the efficiency of longer columns with larger particles内装小颗粒的更短的色谱柱，可以获得与内装更大颗粒的长色谱柱的相同柱效内装小颗粒的更短的色谱柱，可以获得与内装更大颗粒的长色谱柱的相同柱效Reduce column length缩短柱长缩短柱长1200 Rapid Resolution System Agilent RestrictedPage 11N.A.=not available5um1.8umReduce column length by factor of 3N.B.Quite often original method will have more resolution than is actually needed and a reduction by 5 is possible原有方法常常分离度超出实际的需要，故可以柱长缩短5倍是可能的3.5 um1.8umReduce column length by factor of 2N.B.Quite often original method will have more resolution than is actually needed and a reduction by 3 is possible原有方法常常分离度超出实际的需要，故可以柱长缩短3倍是可能的Reduce column length缩短柱长缩短柱长1200 Rapid Resolution System Agilent RestrictedPage 12Overview概要概要Choosing The Column 选择色谱柱Reducing The Column Length降低色谱柱长Changing The Column Id改变色谱柱内径改变色谱柱内径Adjusting The Injection Volume调整进样体积Transferring to 1.8um Columns转换成1.8um色谱柱Speeding It Up提速31200 Rapid Resolution System Agilent RestrictedPage 13Change Column I.D.?改变色谱柱内径？改变色谱柱内径？Changing Column I.D.改变色谱柱内径 Reduce Mobile Phase Use降低流动相消耗 Can improve speed potential lower flow rate used higher overheads有改善速度的潜力使用更低的流速更长运转时间 Can improve sensitivity for same mass loading如果进样量相同，则可以改善灵敏度1200 Rapid Resolution System Agilent RestrictedPage 14How conversion works for flow流速如何转换流速如何转换Flow modification,for columns of different diameters流速变化，根据不同的色谱柱内径ml/minmmmmml/min42.04.63.01.0 i.e.2=2 col.2column1column21 col.Flow.DiamDiam.Flow=1200 Rapid Resolution System Agilent RestrictedPage 15Reduce column diameterand therefore flow rate降低色谱柱内径及流速降低色谱柱内径及流速N.A.=not available4.6 mm3 mmReduce flow rate by factor of 0.4N.B.reducing to 3mm i.d.column allows use of higher linear flow rates as the 1200 SL pump will pump at 5ml/min.1200 SL泵可以使用5ml/min流速，故内径降低到3mm的色谱柱可以使用更高的线速度2.1 mm4.6 mmReduce flow rate by factor of 0.21200 Rapid Resolution System Agilent RestrictedPage 16Overview概要概要Choosing The Column 选择色谱柱Reducing The Column Length降低色谱柱长Changing The Column Id改变色谱柱内径改变色谱柱内径Adjusting The Injection Volume调整进样体积调整进样体积Transferring to 1.8um Columns转换成1.8um色谱柱Speeding It Up提速1200 Rapid Resolution System Agilent RestrictedPage 17Conversion for injection volume进样体积转换进样体积转换Keep Injection volume proportional to column volume进样体积正比于柱体积2 col.column1column21 col.Inj.Vol.VolumeVolumeInj.Vol.=2 col.column1column21 col.42.04.020 i.e.lmlmll=Zorbax column volume=3.14 x r2x L x 0.6 (r and L in cm)1200 Rapid Resolution System Agilent RestrictedPage 18Reduce injection volume降低进样体积降低进样体积4.6 mm3 mmReduction to allow for diameter change内径变化导致的体积降低内径变化导致的体积降低2.1 mm4.6 mm=0.4 x Original=0.2 x Originalx150 mm50 mmReduction to allow for length change柱长度变化导致的体积降低柱长度变化导致的体积降低=0.33 x Originale.g.Original 4.6mm x 150mm transferred to 2.1mm x 50mm即：原有4.6mm x 150mm色谱柱 转换成2.1mm x 50mm时：=0.2 x 0.33=0.067x original injection volume（原有进样体积）100 mm50 mm=0.5 x Original41200 Rapid Resolution System Agilent RestrictedPage 19How conversion works for time如何转换时间？如何转换时间？Run Time or Gradient segment Time Adjustment运行时间或梯度时间调整.515050.15 i.e.minmmmmmin=*assumes flow is proportional for columns 1 and 22 col.column1column21 col.TimeLengthLengthTime=1200 Rapid Resolution System Agilent RestrictedPage 20Gradient slope vs.time and flow梯梯度度坡坡度与时间度与时间和和流速流速Gradient slope effects k*(“K star”)the gradient term for the isocratic capacity factor k(“K prime”)梯度坡度影响K*-与等度容量因子K相当Increasing k or k*generally increases resolution.This can be done by decreasing solvent strength or decreasing gradient slope,respectively 提高K或K*通常可提高分离度。可以分别通过降低溶剂强度或降低梯度坡度来实现=volumescol.1020%)-(100%8%i.e.#column volumes=(Flow x Gradient Time)/column volume柱体积个数=(流速x梯度洗脱时间)/柱体积Zorbax column volume=3.14 x r2x L x 0.6 (r and L in cm)=VolumesColumn#Start%)-(End%SlopeGradient%1200 Rapid Resolution System Agilent RestrictedPage 21Convert run time(and gradient)转换转换运行运行时间时间(梯梯度度)to shorter column至较至较短色谱柱短色谱柱with no flow increase流速流速不提不提高高150 mm50 mmReduction to allow for length change长度变化长度变化=1/3 x Reduction降低Flow Rate流速=As converted进行相应转换Injection Volume进样体积=As converted进行相应转换Run time 运行时间=Reduced by length change reduction factor根据柱长相应降低Gradient Times梯度时间=Reduced by length change reduction factor根据柱长相应降低e.g.15mins 5mins35-60%over 15mins 35-60%over 5mins1200 Rapid Resolution System Agilent RestrictedPage 22Overview概要概要Choosing The Column 选择色谱柱Reducing The Column Length降低色谱柱长Changing The Column Id改变色谱柱内径改变色谱柱内径Adjusting The Injection Volume调整进样体积Transferring to 1.8um Columns转换成1.8um色谱柱Speeding It Up提提速速1200 Rapid Resolution System Agilent RestrictedPage 23-0.00050.00000.00050.00100.00150.00200.00250.00300.00350.00400.00450.00.20.40.60.81.01.21.41.65.0 m 3.5 m 1.8 m 1mL/min2.0 mL/minHETP(cm/plate)Interstitial linear velocity(ue-cm/sec)Speeding It Up加速加速Increase Linear Velocity 提提高高线线速度速度(Flow Rate流速流速)Step 1(2x Original Flow)第一第一步步(将原将原始流速始流速提提高高一倍一倍)Increase the flow rate by 100%流速快一倍 Reduce run time by 50%运行时间缩短50%Reduce Gradient segments by 50%梯度时间缩短50%Example举例举例 1ml/min 2ml/min 30 minutes 15 minutes 35-65%over 30 mins over 15 mins1200 Rapid Resolution System Agilent RestrictedPage 24-0.00050.00000.00050.00100.00150.00200.00250.00300.00350.00400.00450.00.20.40.60.81.01.21.41.65.0 m 3.5 m 1.8 m 1mL/min3.0 mL/minHETP(cm/plate)Interstitial linear velocity(ue-cm/sec)Step 2(3 x original flow)第二步第二步(流速流速提提高高三倍三倍)Increase the flow rate by another 100%将流速再提高100%Reduce original run time to 1/3运行时间缩短至原来的三分之一.Reduce original Gradient time to 1/3梯度时间缩短到原来的三分之一Example举例举例 1ml/min 3ml/min 30 minutes 10 minutes 35-65%over 30 mins over 10 minsIs resolution OK？Is Pressure OK?Keep Going!分离度可以吗?压力可以吗？继续进行！Speeding It Up加速加速Increase Linear Velocity 提提高高线线速度速度(Flow Rate流速流速)51200 Rapid Resolution System Agilent RestrictedPage 25-0.00050.00000.00050.00100.00150.00200.00250.00300.00350.00400.00450.00.20.40.60.81.01.21.41.65.0 m 3.5 m 1.8 m 1mL/min4.0 mL/minHETP(cm/plate)Interstitial linear velocity(ue-cm/sec)Step 3(4 x original flow)第三步第三步(流速是流速是原原流速的流速的4倍倍)Increase the flow rate by another 100%流速再提高100%Reduce original run time to 1/4.运行时间降低到原来的四分之一 Reduce original Gradient time to 梯度时间降低到原来的四分之一Example举例举例 1ml/min 4ml/min 30 minutes 7.5 minutes 35-65%over 30 mins over 7.5 minsReducing Column Length by x3柱长降低3倍Increasing Velocity by x4 线速度提高4倍=x12Increase in method speed方法速度提高12倍Speeding It Up加速加速Increase Linear Velocity 提提高高线线速度速度(Flow Rate流速流速)Is resolution OK？Is Pressure OK?Keep Going!分离度可以吗?压力可以吗？继续进行！1200 Rapid Resolution System Agilent RestrictedPage 26 When flow limit of pump is reached当达到泵的流速极限时Consider using a smaller i.d.column 考虑使用小内径色谱柱 When pressure is approaching maximum(e.g.550 bar)当压力达到最大值时Remember to allow for increase in pressure at non-ideal mix记住压力在溶剂混合时会升高 When resolution is no longer satisfactory当分离度不再满意时When to stop何时何时停止停止Methanol/water 40C0.000.200.400.600.801.001.201.40020406080100120%MethanolViscosityACN/water 40C0.000.200.400.600.80020406080100120%ACNViscosity1200 Rapid Resolution System Agilent RestrictedPage 27Rapid Resolution Compendium CD-ROM光盘(Please Ask For A Copy Pub No 5989-5130EN)Tools工具工具 Application Examples应用实例 Technology Overview技术概述 Rapid Resolution in The Scientific and Trade Press and Conference Posters高分离度快速液相色谱在科学展会和会议上的展报 System Configurator(guide to reduce dead volume and extra column effects)系统配置转换工具 Method Translator Program方法转换工具1200 Rapid Resolution System Agilent RestrictedPage 28Mini-Demo Method Translator方法转换工具小型演示Tools工具工具1200 Rapid Resolution System Agilent RestrictedPage 29System constraints:系统限制系统限制Peak dispersion,Flow and pressure limitations色谱色谱峰扩散峰扩散，流速，流速和压力限制和压力限制 Increased flow,along with decreased particle size,will significantly increase operating pressure-check with supplier for specifications,precautions提高流速，加之降低粒径，将会显著提升操作压力注意供应商的指标和注意事项 Reducing the column volume increases the negative effect of system bandbroadening on the separation降低柱体积会使系统谱带展宽对分离的负面影响更显著 Ensure all sources of extra column broadening are minimised(e.g.tubing,flow cell volume)确保所有柱外展宽最小化(如，管线，检测池等)Reduced peak durations will need faster data acquisition to maintain at least 20 points across the peak 峰宽降低需要较高的采样速率以保持每个色谱峰至少20个采样点 Possibly at cost of extra noise on older systems旧系统上会有噪音加大的可能性 Increased flow rate may affect degasser performance,pressure stability check with supplier for specifications,precautions提高流速会影响脱气机性能，压力稳定性注意供应商的指标和注意事项1200 Rapid Resolution System Agilent RestrictedPage 30Summary小小结结 Shorter 1.8um columns allow faster analysis with same resolving power as longer 5um columns1.8um的较短色谱柱可以与较长5um色谱柱保持相同的分离能力，但分析时间更短 Change column diameter if necessary to allow for lower solvent consumption and higher accessible velocity如需更低的溶剂消耗和更高的速度，可以改变色谱柱内径 Adjust the injection volume to maintain the same mass loading on the smaller column调整进样体积以保持小色谱柱上的相同载样量 Adjust run time and gradient to allow for the shorter column调整运行时间和梯度以适合小色谱柱分离 Speed up the flow rate until max flow or max pressure or loss of resolution 提高流速直至流速或压力达到最大值或分离度降低61200 Rapid Resolution System Agilent RestrictedPage 31Questions?问题问题？