安捷伦2007液相色谱高级研修班资料4.pdf

1Masterclass2007Page 1Group/Presentation TitleAgilent RestrictedMonth#,200XPage 2Increasing Efficiency,Increasing Resolution-Better Separations改善分离效率与分离度，获得更好的分离结果改善分离效率与分离度，获得更好的分离结果Paul BartonHPLC 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Masterclass2007Page 3Outline概要概要Sub-2m particle columns can be used in two ways亚二微米颗粒色谱柱可以根据需要按两种方式选用：Fast LC with short,highly efficient columns at conventional and high flow rates用高效短柱在常规及高流速下进行快速液相色谱分析 High Resolution LC with longer(100mm-150mm)columns and conventional flow rates用较长(100mm-150mm)色谱柱在常规流速下进行高分离度液相色谱分析 Longer(100mm 150mm)sub-2m particle columns have much higher system backpressure,but this is less of an issue because we are looking to exploit the efficiency gains,not speed gains flow remains the same as for 5m or 3m column较长亚二微米颗粒色谱柱会产生比较高的反压，但此时我们关心的是分离效率而不是速度流速可以与5m or 3m 色谱柱保持相同Masterclass 2007Page 4Rapid Resolution HT Rapid Resolution HT RRHT Columns in 2007RRHT Columns in 2007RRHT ColumnsRRHT Columns600 bar Columns(no cartridges)600 bar Columns(no cartridges)400 Bar Columns/CartridgesLength:15,30,50 mmID:2.1,4.6mmBonded Phases:5Eclipse XDB-C18,C8SB-C18,C8Extend-C1820032007Length:20,30,50,100,150mmID:1.0,2.1,3.0,4.6mmBonded Phases:11Eclipse XDB-C18,C8SB-C18,C8,CN,PhenylExtend-C18,Rx-silEclipse Plus C18/C8Masterclass2007Page 5Effect of Column Length and Particle Size on Column Efficiency柱长和粒径对柱效的影响柱长和粒径对柱效的影响N.A.N.A.4,20060008,50012,500ColumnEfficiency N(5 m)2,5002,100156,5004,2003011,6507,0005017,50010,5007523,25014,00010035,00021,000150ColumnEfficiency N(1.8 m)ColumnEfficiency N(3.5 m)ColumnLength(mm)Compared with 5mm,1.8m columns have nearly 3x more efficiency与5um颗粒相比，1.8m色谱柱的柱效几乎是5m色谱柱的三倍Masterclass 2007Page 6Resolution Equation分离度公式分离度公式Although 1.8m columns have 3x more efficiency,we do not get 3x more resolution尽管柱效提高了三倍，但我们并尽管柱效提高了三倍，但我们并不能获得提高三倍的分离度，原因是：不能获得提高三倍的分离度，原因是：The expected increase in resolution is 分离度预期提高3 1.73 or 73%(actually 67%)2Masterclass2007Page 7Particle Size Comparison all 4.6mm i.d.columnsvan Deemter Curve for Methanol/Water粒径比较使用粒径比较使用4.6mm 内径色谱柱内径色谱柱在甲醇在甲醇/水体系中测得的范德姆特曲线水体系中测得的范德姆特曲线00.0050.010.0150.02Plate Height(mm)5.0 um SB-C183.5 um SB-C181.8 um SB-C18Lin.Vel.mm/sec123454.6 mmml/min0.71.42.12.83.53 mmml/min0.30.60.91.21.52.1 mmml/min0.140.290.440.580.731 mmml/min0.0330.0660.10.1330.166Masterclass 2007Page 8Ideal Flow Rates for 1.8m Columns 1.8m色谱柱的理想流速色谱柱的理想流速Because we are looking for maximum efficiency,we do not need high flow rates.We can use conventional flow rates因为我们希望获得最高效率，无需高流速。可以使因为我们希望获得最高效率，无需高流速。可以使用常规流速用常规流速 More viscous mobile phases will increase the pressures shown粘度大的流动相粘度大的流动相将将使使反压反压提高提高 Increases in pressure may be offset by increasing temperature压力压力的的升升高可用提高可用提高高温温度度来缓解来缓解4851.8150 x 4.63101.8100 x 4.63960.4100 x 2.15950.4150 x 2.14180.8100 x 3.06270.8150 x 3.0Pressure 0.7cP(bar)Ideal Flow(mL/min)Dimensions(mm)Masterclass2007Page 11min2468mAU020040060080010001200140016005um:Rs(4,5)=3.141.8um:Rs(4,5)=5.07+60%Resolution (Theory:67%)+160%Efficiency (Theory:177%)1200 RRLC System Low Delay Volume Configuration 120ul,no mixer,no damper 2.1 x 150mm SB-C18 ColumnImproved Resolution with 2.1mm i.d.Columns用用2.1mm内径色谱柱改善分离度内径色谱柱改善分离度Up to 60%higher Resolution分离度改善分离度改善达达60%Masterclass 2007Page 134.6 x 150,1.8um490 barN=28669 R_S=1.80(+57%)S/N=44 7 ImpuritiesAll 7 Baseline Separated!4.6 x 150,5um93 barN=7259R_S=1.15S/N=42 4 Impurities2 Not Baseline Separated!Customer Example Isocr.Impurity MethodZoom of critical time range 7min4.6 x 150,3.5um165 barN=14862R_S=1.37S/N=50 7 Impurities6 Not Baseline Separated!Improved Resolution Customer ExampleUp to 60%higher Resolution+Identical Selectivity for easy,fast,risk-free method transfer!改善分离度改善分离度 用用户样品户样品分分析实例析实例分离度改善分离度改善达达60%Masterclass2007Page 14Maximizing Peak Capacity最大的最大的峰容量峰容量 Peak capacity is the number of peaks which can be separated with defined resolution(example:R=1)in a certain period of time for a given system(column length and particle size).峰容量是对于指定系统(柱长和颗粒度固定)在特定时间内以一定分离度(如，R=1)能够分离色谱峰的个数。Peak capacity is another measure of separation efficiency and is especially useful for complex chromatograms.峰容量是分离效率的另一种尺度，尤其对于复杂色谱图而言Rs=+50%5m1.8m2 Peaks 3 Peaks 50%more!Masterclass 2007Page 15Calculating Peak Capacity计算峰容量计算峰容量klast:Retention factor for last peakN :EfficiencyRs:Minimum Required Resolution(Baseline:Rs=1.5)Pc:Peak CapacityEquation for Gradient Elution 梯梯度度洗脱洗脱公式公式Pc=ln1+klastN4RS1+w is influenced by N 峰宽峰宽w受受柱效柱效N的影响的影响N depends on Column length and Particle size柱效柱效N取决于取决于柱长和粒径柱长和粒径Equation for Isocratic Elution 等等度度洗脱洗脱公式公式Pc:Peak CapacitytG:Gradient Timew:Peak WidthPc=1+tG1nwn1=1+tGwav3Masterclass2007Page 16Peptide Map of BSA on Three SB-C18 Columns with Different Particle Sizes不不同同粒径色谱柱分离粒径色谱柱分离BSA的的肽肽谱谱Gradient Time=30 minTemp.=80C238Peak Capacity391540StartingPressure51103340min468101214161820mAU051015202530min*468101214161820mAU01020304050min*468101214161820mAU0102030405060SB-C18,2.1x150mm,5SB-C18,2.1x150mm,3.5SB-C18,2.1x150mm,1.8Masterclass 2007Page 17Improved Resolution Complex Samples改善分离度改善分离度复杂样品复杂样品”LC with GC Resolution”Pat Sandra&Frank David,RICColumn:150mm RRHT SB-C18Efficiency 30,000Masterclass2007Page 18HPLC分分析析：欧亚甘草根欧亚甘草根提提取物取物m in2 5303 54 04 55 0m A U010203040506070 M W D 1 B,S ig=2 54,1 6 R e f=4 5 0,1 0 0 (B U D L IC 00 0 0 28.D)2 2.01 6min01020304050mAU050100150200250300350400450 MWD1 B,Sig=254,16 Ref=450,100(BUDLIC000028.D)Analysis of licorice root extract B using a Zorbax SB-C18 4.6 x 250 mm column,5-m.Peak capacity(Rs 1.0)=306GA 41.40 minG 22.8 minGA 306 Peaks in 60 minsMasterclass 2007Page 19RRLC分分析析：欧亚甘草根欧亚甘草根提提取物取物改善速度改善速度、分离度与分离度与灵敏灵敏度度min051015202530mAU050100150200250300350400450 MWD1 B,Sig=254,16 Ref=450,100(BUDLIC000025.D)Peak capacity(Rs=1.0)=426 G 14.6 minm in161 82 02 22 42 62 83 03 23 4m A U2 03 04 05 06 07 08 09 0 M W D 1 B,S i g=2 5 4,1 6 R e f=4 5 0,1 0 0 (B U D L IC 00 00 2 5.D)GA 25.5 minGAAnalysis of licorice root extract B,using a Zorbax SB-C18 Rapid-Resolution HT,4.6 x 150 mm column.426 Peaks in 30 minsMasterclass2007Page 20Conclusions结结论论Longer(100mm and 150mm),sub-2m particle columns can be used to significantly increase resolution较长色谱柱的亚二微米色谱柱可以显著改善分离度 Zorbax silica is manufactured the same way,regardless of particle size,ensuring no change in selectivity无论粒径大小如何，Zorbax硅胶以相同方法制作，确保选择性无变化 Using sub-2m particle columns for complex samples can yield very high(GC-like)peak capacities使用亚二微米色谱柱分离复杂样品可以获得很高的色谱峰容量(类似GC)Peak Capacity calculations may be used to estimate resolution gains for complex chromatograms峰容量计算可用于估计复杂色谱图的分离度改善 It is sometimes possible to realise BOTH speed and resolution gains using sub-2m particle columns 使用亚二微米颗粒的色谱柱可能实现速度和分离度的同时改善Masterclass 2007Page 21Questions?问题？问题？4Masterclass2007Page 22RRLC workshop-what we learned与含与含5um颗粒的色谱柱相比，在保持流速不变颗粒的色谱柱相比，在保持流速不变的情况下，使用小颗粒色谱短柱会改善色谱性能的情况下，使用小颗粒色谱短柱会改善色谱性能 L/dp保持相同时，小颗粒短柱可以在保持分离效率的同时，加快分离速度 L/dp提高时(降低粒径)，含小颗粒的色谱柱能够在保持或提高分离速度的同时，改善分离度。超快液相色谱分析可以通过以下途径实现超快液相色谱分析可以通过以下途径实现 大幅度提高流速 升高柱温，降低黏度，以达到升高流速的目的