HPLC培训

1 Group/Presentation Title Agilent Restricted Month ##, 200XPage 1 Advantages of Using Sub-Two Micron Particles in HPLC 使用亚二微米颗粒在HPLC中的优势 Paul Barton HPLC HPLC MasterClassMasterClass 20072007 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 Masterclass 2007 Page 2 培训日程 T i me Ac t iv i t y C on t en t 9:00 Presentation讲座 Welcome 9:15 Presentation讲座 The Advantages Of Using Sub Two Micron Particle In HPLC 亚二微米颗粒在液相色谱分离中的优势 10:00 Lab Session实验一 Lab Practical One 实验一 11:00 Presentation讲座 A Guide To Speeding Up Your Separations 如何提高分离速度 Me t ho d tr a ns l at o r d e mo 11:45 Presentation讲座 How Can Temperature Help? 如何发挥温度的作用？ 12:15 Lunch Lunch 午餐 13:15 Lab Session实验二 Lab Practical Two 实验二 14:15 Break Break 茶歇 14:30 Presentation讲座 Increasing Efficiency, Increasing Resolution = Better Separations 提高柱效，改善分离度 14:50 Lab Session实验三 Lab Practical Three 实验三 15:30 Presentation/Q&A session讲座 Close小结，发结业证，结束 Masterclass 2007 Page 3 Outline概要 • Show how sub-2mm particle technology can be used to speed up LC analyses亚二微米颗粒技术如何能用于加速液相色谱分析 • Understand how silica manufacture has been adapted to lower backpressure whilst maintaining efficiency 理解硅胶制作技术如何能够降低反压但同时保持柱效 • Understand the impact of using short, highly efficient, columns packed with sub-2mm particles, on system dwell volume and extra-column effects 理解系统延迟体积和柱外效应对填装亚二微米颗粒的高效短柱的影 响 •Understand the impact of using short, highly efficient, columns packed with sub-2mm particles, on data acquisition rate 理解采样速率对填装亚二微米颗粒高效短柱分离的影响 Masterclass 2007 Page 4 Myths “神话” • Sub-2mm particle columns are New Technology亚二微米颗粒色谱柱是新技术 • The benefits of sub-2mm particle technology arise from the higher pressures they produce亚二微米颗粒技术的益处自高压而生 •Sub-2mm particle columns cannot be run on conventional HPLC systems and need special instrumentation亚二微米颗粒色谱柱不能用在常规HPLC系统上， 需要特殊的仪器配置 Ø Agilent introduced the first totally porous sub-2mm particle columns (RRHT columns) in early 2003 安捷伦早于2003年初就首推多孔亚二微米颗粒色谱柱 Ø High pressure is a result of the fundamental laws of physics. Efficiency is the result of improved mass transfer 高压是物理基本规律的自然结果。效率是改善传质的结果 Ø Our customers have been using sub-2mm particle columns since 2003 我们的客户自2003年就已经开始用亚二微米颗粒色谱柱 Masterclass 2007 Page 5 Resolution Equation分离度公式 a= Selectivity– influenced by mobile and stationary phase 选择性–受流动相和固定相的影响 N = Column Efficiency – influenced by length and particle size 柱效 –受柱长和颗粒度的影响 k = Capacity Factor (retention) – influenced by stationary and mobile phase, gradient slope and dwell volume (gradients) 保留因子 (保留) –受固定相和流动相，梯度斜率和延迟体积(梯度)的影响 Although we may be aiming for fast separations, we need to maintain or increase resolution 尽管我们的目标是快速分离，但需要保持或提高分离度 Masterclass 2007 Page 6 The Effect of Column Efficiency, Selectivity and Retention upon Resolution 柱效，选择性和保留对分离度的影响 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Re so lu tio n Increase N Increase Alpha Increase k' Selectivity has the strongest influence on Resolution 选择性对分离度影响最大 • Stationary and Mobile Phase 固定相和流动相 • Temperature温度 Rs = N½/4 · (a-1)/a · k’/(k’+1) Plates: 5000 10000 15000 20000 25000 Alpha: 1.10 1.35 1.60 1.85 2.1 k’: 2.0 4.5 7.0 9.5 12.0 2 Masterclass 2007 Page 7 Identical Selectivity Independent of Particle Size 相同选择性：与颗粒度大小无关 5 µm 1.8 µm 3.5 µm α 2,3= 1.13 α 2,3= 1.13 α 2,3= 1.14 min1 2 3 4 5 min1 2 3 4 5 min1 2 3 4 5 min1 2 3 4 5 6 mAU 0 5 10 15 20 25 30 35 min1 2 3 4 5 6 mAU 0 5 10 15 20 25 30 35 min1 2 3 4 5 6 mAU 0 5 10 15 20 25 30 35 5 µm 1.8 µm 3.5 µm α 3,2=1.20 α 3,2=1.20 α 3,2=1.22 α 4,3=1.97 α 4,3=1.94 α 4,3=1.97 α 5,4=1.13 α 5,4=1.16 α 5,4=1.14 Eclipse XDB C18 – Barbitals巴比妥Eclipse XDB C18 - Xanthines黄嘌呤 Masterclass 2007 Normally measured in terms of :-通常以如下方式表达： Number of theoretical plates理论塔板数, N Theoretical plates per meter每米理论塔板, N/m Height equivalent to a theoretical plate理论等板高度: Hmm = Lmm/N NB: the smaller H, the more efficient the column H越小，色谱柱效越高 COLUMN EFFICIENCIES柱效 Masterclass 2007 Page 9 Van Deemter Curve : HETP vs. Volumetric Flow Rate 范德姆特曲线：板高与体积流速的关系 0.0000 0.0005 0.0010 0.0015 0.0020 0.0025 0.0030 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 Column: ZORBAX Eclipse XDB-C18 Dimensions: 4.6 x 50/30mm Eluent: 85:15 ACN:Water Flow Rates: 0.05 – 5.0 mL/min Temp: 20°C Sample: 1.0mL Octanophenone in Eluent Volumetric Flow Rate (mL/min) HE TP (c m ) 5.0mm 3.5mm 1.8mm Smaller particle sizes yield flatter curves, minima shift to higher flow rates 颗粒度越小，曲线越平，更高流速下曲线的上升最缓 H = A + B/u + Cu Masterclass 2007 Page 10 Sub-Two Micron Columns Can Be Used In Two Ways 亚二微米色谱柱可以两种方式使用 • Fast LC with short, highly efficient columns at conventional and high flow rates 在常规以及高流速下用高效短柱进行快速液相色谱分离 • High Resolution LC with longer (100mm-150mm) columns and conventional flow rates 在常规流速下使用较长色谱柱(100mm-150mm) 进行高分离度色谱分离 There is a trade-off between speed and resolution, but it is often possible to have a mixture of slightly faster analysis with more resolution在速度和分离度之间会有折衷，但常常可以实现更加快速 和分离度更好的结果 This comes at a price 上述结果的代价是:- Much higher system backpressure 更高系统压力 Masterclass 2007 Page 12 Pressure vs. Flow and Linear Velocity, <400 bar “ideal conditions”, 1.8um particle, visco.=0.7 cP 压力、流速和线速度的关系，< 400 bar 1.8um“理想条件” 1.734.92200.953.0x50 4.1 4.9220 2.25 4.6x50 0.854.92200.472.1x50 5 6123 2.25 4.6x30 2.85 8.11230.953.0x30 1.418.11230.472.1x30 Flow (ml/min) at max lin. vel. Max lin. vel. with 400 bar Pressure (bar) u=2.7mm/sec Flow (ml/min) u=2.7mm/sec Column Size 柱规格 Highlighted cells for required flow >2ml/min at 400 bar 突出的格中是在400bar下需要大于2ml/min流速的 Masterclass 2007 Page 13 Pressure vs. Flow and Linear Velocity, <1000 bar “ideal conditions”, 1.7um particle, visco.=0.7 cP 压力、流速和线速度的关系，< 1000 bar 1.7um“理想条件” 1.9 10.92460.472.1x50 3.8 10.92460.953.0x50 1.9 5.54900.953.0x100 0.955.54900.472.1x100 6.4 18.41470.953.0x30 3.2 18.41470.472.1x30 Flow (ml/min) at max lin. vel. Max lin. vel. with 1000 bar Pressure (bar) u=2.7mm/sec Flow (ml/min) u=2.7mm/sec Column Size Highlighted cells for required flow >1ml/min at 1000 bar 突出的部分是在1000bar 条件下超过1ml/min流速的 3 Masterclass 2007 Page 14 Why Not Just Use 2.1mm i.d. Columns? 为什么不仅仅使用2.1mm内径色谱柱？ •Column void volumes become very small色谱柱空体积变得很小 • Extra-column effects become very significant (see next slide) 柱外效应 会相当显著 •Small column void volumes impose a significant gradient delay 小色谱 柱会有很显著的梯度延迟 • Injection volumes become a significant proportion of the column void volume, leading to overload 进 样体积会变得占柱体积很大的比例，导 致过载 7030 x 2.1 12050 x 2.1 240100 x 2.1 350150 x 2.1 14430 x 3.0 24050 x 3.0 480100 x 3.0 56050x 4.6 1130100 x 4.6 1700150 x 4.6 Void Volume (uL) Dimensions (mm) Masterclass 2007 Page 15 N Does Not Decline Linearly with Column Length - Dispersion Degrades N on Short, Low-volume Columns 柱效并非完全随色谱柱长线性降低－扩散使短柱、小体积色谱柱柱效降低 R as Affecte d by Efficie ncy 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 0 5000 10000 15000 20000 25000 30000 35000 Efficiency (N) R c on tr ib ut io n Sq R oo t( N )/4 R contrib. R = 2x N = 4x Typical data from 5um columns 2cm to 30cm in length, peaks with k’ >3 Masterclass 2007 Page 16 Outline概要 • Show how sub-2mm particle technology can be used to speed up LC analyses亚二微米颗粒技术如何能用于加速液相色谱分析 Ø Understand how silica manufacture has been adapted to lower backpressure whilst maintaining efficiency 理解硅胶制作技术如何能够降低反压但同时保持柱效 • Understand the impact of using short, highly efficient, columns packed with sub-2mm particles, on system dwell volume and extra-column effects 理解系统延迟体积和柱外效应对填装亚二微米颗粒的高效短柱的影 响 •Understand the impact of using short, highly efficient, columns packed with sub-2mm particles, on data acquisition rate 理解采样速率对填装亚二微米颗粒高效短柱分离的影响 Masterclass 2007 Page 17 What Can We Do To Make Columns Packed With Sub-Two Micron Particles More Usable? 我们用什么办法来使亚二微米颗粒填装的色谱柱更有用？ Masterclass 2007 Production of ZORBAX® Porous Silica Microspheres Zorbax多孔硅胶微球的生产 5 Particle Size ZORBAX Rx 1.8 µm 3.5 µm 5 µm 7 µm Silica Sol + Urea (pH-2) + CH2O D O2 Pore Agilent owns the patent for sol-gel HPLC packings Masterclass 2007 Page 19 Pressure Equation 压力公式 ΔP = ηFLK0πr2dp2 粘度 流速 长度 viscosity flow length specific Permeability column Radius 柱内径 particle Diameter 粒径 4 Masterclass 2007 Page 20 Pressure Equation 压力公式 ΔP = ηFLK0πr2dp2 粘度 流速 长度 viscosity flow length specific Permeability column Radius 柱内径 particle Diameter 粒径 Masterclass 2007 Page 21 Comparison of Typical Particle Size Distribution and Engineered PSD of sub-2mm particles 亚二微米颗粒的典型颗粒尺寸分布和 设计 领导形象设计圆作业设计ao工艺污水处理厂设计附属工程施工组织设计清扫机器人结构设计 分布的比较 0. 0 0. 2 0. 4 0. 6 0. 8 1. 0 1. 2 1. 4 1. 6 1. 8 2. 0 2. 2 2. 4 2. 6 2. 8 3. 0 3. 2 3. 4 3. 6 3. 8 4. 0 Particle Size (um) Engineered PSD Narrower PSD Lower level of fines 较少细微颗粒 Small amount of slightly larger particles 少量较大颗粒 Goal: Achieve lower backpressure vs. a typical PSD Engineered Bimodal Distribution: Only 3% compromise in efficiency 25% gain in back pressure reduction 特别设计的双分布 : 分离度仅仅降低3% 反压降低25% Masterclass 2007 Page 23 Back Pressure for Narrow vs. Production Particle Size Distribution 1.8-mm SB-C18 不同粒径分布的1.8-mm SB-C18颗粒反压比较 0 50 100 150 200 250 300 0 1 2 3 4 5 Columns: ZORBAX SB-C18 Dimensions: 4.6 x 30mm, 1.8 mm Eluent: 85:15 ACN:Water Flow Rates: 0.05 – 5.0 mL/min Temp: 20°C Sample: 1.0mL Octanophenone in Eluent Detection: UV 245nm Production 1.8mm – Wider PSD Narrow 1.8mm – Narrow PSD System Pressure (No Column) Flow Rate (mL/min) Pr es su re (B ar ) 25% HIGHER Pressure at Optimum Flow (2.0 mL/min) Masterclass 2007 Page 24 Van Deemter Curves for Narrow vs. Production Particle Size Distribution 1.8mm SB-C18 不同粒径分布的1.8-mm SB-C18颗粒的曲线比较 0 1 2 3 4 5 6 7 8 9 10 0 2 4 6 8 10 12 14 16 18 20 Reduced Linear Velocity (n) Re du ce d Pl at e H eig ht (h ) Production 1.8mm – Wider PSD Narrow 1.8mm – Narrow PSD 7793 Plates @ 1.6 mL/min (-3.4%) 8065 Plates @ 2.0 mL/min Columns: ZORBAX SB-C18 Dimensions: 4.6 x 30mm, 1.8 mm Eluent: 85:15 ACN:Water Flow Rates: 0.05 – 5.0 mL/min Temp: 20°C Sample: 1.0mL Octanophenone in Eluent Detection: UV 245nm h vs. u Fit Coefficients Production PSD Narrow PSD VD A: 0.795 0.760 B: 3.97 4.26 C: 0.085 0.077 r2: 0.9994 0.9998 较低压力下，实现小颗粒填料的 高性能 Masterclass 2007 Page 25 Rapid Resolution HT Rapid Resolution HT –– RRHT Columns in 2007RRHT Columns in 2007 快速高分离度高通量色谱柱－快速高分离度高通量色谱柱－RRHTRRHT种类种类 RRHT ColumnsRRHT Columns 600 bar Columns (no cartridges) 600 bar Columns (no cartridges) 400 Bar Columns/Cartridges Length: 15, 30, 50 mm ID: 2.1, 4.6mm Bonded Phases: 5 Eclipse XDB-C18, C8 SB-C18, C8 Extend-C18 2003 2007 Length: 20, 30, 50, 100, 150mm ID: 1.0, 2.1, 3.0, 4.6mm Bonded Phases: 10 Eclipse XDB-C18, C8 SB-C18, C8, CN,Phenyl, Aq Extend-C18 Eclipse Plus C18/C8, Rx-sil Masterclass 2007 Page 26 Lifetime with Clean Samples – Thousands of Injections干净样品的色谱柱寿命－上千次进样 Resolution of Benzophenone and Butyrophenone 0 1 2 0 1000 2000 3000 4000 5000 Injection Number Re so lu tio n Rs Plates for Benzophenone 0 5000 10000 0 2000 4000 Injection Number Pl at es Plates (Benzo) Column Pressure 0 200 400 600 0 2000 4000 Injection Number Pr es su re (b ar ) Actual Pressure Peak Width of Benzophenone 0 0.002 0.004 0.006 0 2000 4000 Injection Number Pe ak W id th (1 0% ) PW (benz) Note: pressure is 585 bar 5 Masterclass 2007 Page 27 Stability with Protein Precipitated Samples “Dirty Sample”– Resist Column Plugging蛋白沉淀”脏”样品－色谱柱耐阻塞 Pressure vs. Injection # 90 100 110 120 130 140 150 0 100 200 300 400 500 600 700 Inj # ba r End Press Column: ZORBAX RRHT SB-C18 2.1 x 30mm, 1.8 mm Pressure Increase of only 8 bar over 600+ injections!! 600余次进样，压力仅仅提高8 bar!! Masterclass 2007 Page 28 Column Length and Particle Size on Column Efficiency 柱长和粒径对柱效的影响 N.A. N.A. 4,200 6000 8,500 12,500 Column Efficiency N(5 µm) 2,5002,10015 6,5004,20030 12,0007,00050 17,50010,50075 23,25014,000100 35,00021,000150 Column Efficiency N(1.8 µm) Column Efficiency N(3.5 µm) Column Length (mm) Efficiency (N) Pressure Analysis Time Peak Volume -90% -80% -67% -50% -33% - Analysis Time* Solvent Usage •Reduction 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 • 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 Masterclass 2007 Page 29 Reduce Particle Size to Improve Efficiency and Resolution 降低粒径改善柱效与分离度 High Speed Separation of Analgesics on Columns with Different Particle Sizes 不同粒径色谱柱高速分离镇痛药结果比较 min0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 mAU 0 25 50 75 100 125 150 175 mAU min0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 0 25 50 75 100 125 150 175 min0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 mAU 0 25 50 75 100 125 150 175 1.8 um 3.5 um 5 um 1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 4-Acetamidophenol对乙酰氨基酚 2 Caffeine 咖啡因 3 2-Acetamidophenol 2-乙酰氨基酚 4 Acetanilide 乙酰苯胺 5 Acetylsalicylic Acid 乙酰水杨酸 6 Phenacetin非那西汀 7 Salicylic Acid 水扬酸 LC Conditions Column: SB-C18, 4.6 x 30 mm Detector: 254 nm Injector: 1 ul, Mobile Phase: 1% Formic Acid Acetonitrile (82:18) Flow: 2.0 ml/min Masterclass 2007 Page 30 Chromatographic Comparisons on Columns with Different Particle Sizes 不同粒径色谱柱的色谱结果比较 Separation of Analgesics镇痛药分离结果 6474 6049 3170 3430 1980 1917 Efficiency (plates)– Acetylsalicylic acid Efficiency (plates)– Salicylic acid 483527Peak Capacity (kmax=10, Rs =1, n=1+(ÖN/4)ln(1+kmax) Particle Size 粒径 8.5 2.25 6.0 2.07 4.7 1.61 Resolution – Acetylsalicylic acid/ Phenacetin Resolution – Phenacetin/Salicylic acid 0.028 0.049 0.043 0.059 0.056 0.077 Peak width, mL – Acetylsalicylic acid Peak width, mL – Salicylic acid 1.8 um3.5 um5 umChromatographic Parameter色谱参数 • The 1.8 um particles provided the best results, measured by any parameter. •所有参数衡量的结果是：1.8 um颗粒填料可以获得最好的结果 Masterclass 2007 Page 31 Update Validated Isocratic USP Methods with Rapid Resolution HT Columns – 10X Faster 用RRHT色谱柱更新经验证的等度USP方法－快10倍 min0 2 4 6 8 10 12 USP L1 Column: ZORBAX Eclipse XDB-C18 4.6x250mm, 5mm Eluent: 40% Methanol:60% Water:1.5% Glacial Acetic Acid Flow: 1.0 mL/min, Injection: 8mL, Temp: 25 °C Peak TR N Rs 1 6.63 12,737 0 2 11.19 18,552 15.8 1 2 min0 2 31 1 2 min0 21 1 2 Rapid Resolution HT Column (L1) ZORBAX Eclipse XDB-C18, 4.6x50mm, 1.8mm Eluent: 40% Methanol:60% Water:1.5% Glacial Acetic Acid Flow: 1.0 mL/min, Injection: 2mL, Temp: 25 °C Rapid Resolution HT Column (L1) ZORBAX Eclipse XDB-C18, 4.6x30mm, 1.8mm Eluent: 40% Methanol:60% Water:1.5% Glacial Acetic Acid Flow: 1.0 mL/min, Injection: 2mL, Temp: 25 °C Peak TR N Rs 1 1.40 11,421 0 2 2.33 12,909 12.3 Peak TR N Rs 1 0.85 5,855 0 2 1.43 7,300 8.6 USP Analysis of Guaifenesin愈创甘油醚的美国药典分析方法 Sample: 1. Guaifenesin – 0.04 mg/mL, 2. Benzoic Acid – 0.10 mg/mL ØKey Parameters-Changed Column Length柱长 Particle Size颗粒度 ØKey Parameters-Same Flow Rate流速 Eluent洗脱液 Injection Size相对进样体积 Temperature温度 Masterclass 2007 Page 32 Outline概要 • Show how sub-2mm particle technology can be used to speed up LC analyses亚二微米颗粒技术如何能用于加速液相色谱分析 • Understand how silica manufacture has been adapted to lower backpressure whilst maintaining efficiency 理解硅胶制作技术如何能够降低反压但同时保持柱效 Ø Understand the impact of using short, highly efficient, columns packed with sub-2mm particles, on system dwell volume and extra-column effects 理解系统延迟体积和柱外效应对填装亚二微米颗粒的高效短柱的影 响 •Understand the impact of using short, highly efficient, columns packed with sub-2mm particles, on data acquisition rate 理解采样速率对填装亚二微米颗粒高效短柱分离的影响 6 Masterclass 2007 Page 33 Gradient Elution – What Went Wrong? 梯度洗脱－那儿有问题了？ Mobile Phase: 80% Water (0.1% TFA) : 20% ACN Gradient: (20 - 60% B), 30 min. Column: ZORBAX SB-C18, length – see fig. Flow: 0.2 mL/min. Temperature: 35°C Detection: UV 254 nm 2.1mm x 150 mm Part No. 883700-922 Injection volume: 5 mL 2.1mm x 50 mm Part No. 860975-902 Injection volume: 2 mL 1. Tebuthiuron特丁噻草隆 2. Prometryn扑草净 3. Simazin西玛津 4. Atrazin 阿特拉津 5. Bentazon苯达松 6. Diuron敌草隆 7. Propazin扑灭津 8. Propanil敌稗 1 2 3 4 5 6 7 8 1 2,3 4 5 6 7 8 Time, (min) Time, (min) Coelution 共流出 梯度坡度: 2.1%/column volume; 0.7%/column volume Masterclass 2007 Page 34 Gradient Elution梯度洗脱 This relationship says上述关系表明: If “b” is kept constant from run-to-run, peaks will elute in the same relative pattern.如果b值保持恒定，色谱峰以相同的分离模式洗脱。 b =Gradient Steepness 梯度坡度 S · DF · Vm tG · F S = Constant常数 DF = Change in % organic有机相变化% (i.e. 10-90%, DF = 0.8) Vm = Volume of THE Column 色谱柱体积 tG = Gradient time 梯度洗脱时间 F = Flow 流速 Masterclass 2007 Page 35 Time (min) 0 5 10 15 20 25 1 2 4 5 3 Time (min) 0 5 10 15 Column: StableBond SB-C8 4.6 x 150 mm, 5 mm Gradient Time: 30 min. Flow Rate: 1.0 mL/min Analysis Time: 24 min 分析时间：24分钟 8 6 7 1 2 4 5 3 8 6 7 Column: StableBond SB-C8 4.6 x 75mm, 3.5 mm Gradient Time: 15 min. Flow Rate: 1.0 mL/min Analysis Time: 12 min 分析时间：12分钟 Sample: 1. Tebuthiuron 2. Prometon 3. Prometryne 4. Atrazine 5. Bentazon 6. Propazine 7. Propanil 8. Metolachlor 样品: 1.特丁噻草隆 2.扑灭通 3.扑草净 4. 阿特拉津 5.苯达松 6.扑灭津7.敌稗 8.异丙甲草胺 Two Chromatograms Both Having the Same Gradient Steepness拥有相同梯度坡度的两张色谱图比较 b = S · DF · Vm tG · F Masterclass 2007 Page 36 Dwell Volume 延迟体积 Gradient Separations – Dwell Volume Effects 梯度分离－延迟体积效应 { • Dwell volume = volume from formation of gradient to the column 延迟体积＝梯度形成点到达柱头的体积 • Behaves as isocratic hold at the beginning of gradient. 表现为梯度起点的等度保持 Masterclass 2007 Page 37 High-Throughput Gradient HPLC 高通量梯度HPLC Key Issue = Instrument Dwell Volume 关键因素－仪器延迟体积 VD If the Dwell Volume of an instrument is 1.0 ml 如果仪器的延迟体积 是1.0mL Then this volume is equivalent to则该体积等于 3.3 column volumes (Vms) of a 4.6 x 30 mm column 4.6 x 30 mm 色谱柱体积的3.3倍 14.3 column volumes (Vms) of a 2.1 x 30 mm column 2.1 x 30 mm色谱柱体积的14.3倍 Therefore, if any compound in your mixture elutes with a k of 13.3 or less under the initial gradient conditions, the peak(s) will elute isocratically on the 2.1 x 30 mm columns, as broad bands.因此，在起始梯度条件下，如 果混合物中任意个化合物K值小于等于13.3，则该化合物色谱峰在 2.1x30mm色谱柱上以宽峰被洗脱下来。 Masterclass 2007 Page 38 Effect of Dwell Volume 延迟体积的影响 0 10 20 30 40 0 10 20 30 40 VD = 0.43 mL VD = 2.0 mL Column: ZORBAX Rapid Resolution Eclipse XDB-C8 4.6 x 75 mm, 3.5 µm Mobile Phase: Gradient, 0 - 100 %B in 52.5 min. A: 5/95 methanol/ 25 mM phosphate pH 2.50 B: 80/20 methanol/25 mM phosphate pH 2.50 Flow Rate: 0.5 mL/min Temperature: 25°C Injection: 5 mL Detection: 250 nm Sample: Mixture of antibiotics and antidepressants Upper trace simulates actual run data entered into DryLab® 3.0 software Lower trace is simulated chromatogram for larger VD 7 Masterclass 2007 Page 40 min0.1 0.2 0.3 0.4 0.50 80Hz PW=0.30sec 40Hz PW = 0.33 sec 20 Hz PW=0.42sec 10Hz PW=0.67sec 5HzPW=1.24sec • Sample: Phenone Test Mix • Column: Zorbax SB-C18, 4.6x30, 1.8um • Gradient: 50 -100% ACN in 0.3min • Temperature: 50°, Flow Rate: 5ml • Flow cell: 5ul • Slit: 8 nm • Signal: 245nm, Bandwith: 10nm • Reference: 360nm, Bandwidth: 80nm 80Hz versus 20Hz – 30% Peak Width + 30% Resolution + 40% Peak Capacity + 70% Apparent Column Efficiency Effect of Data Acquisition Rate (time constant) Peak Width, Resolution and Peak Capacity in Ultra-Fast LC 采集速率的影响：超快LC分析中的峰宽，分离度和峰容量 80Hz versus 10Hz – 55% Peak Width + 90% Resolution + 120% Peak Capacity + 260% Apparent Column Efficiency Masterclass 2007 Page 41 Conclusions 结论 • Short, efficient, sub-2mm particle columns can be used to dramatically speed up separations without sacrificing resolution 内装亚二微米颗粒的高效短柱可以显著加速分离，而不会牺牲分离度 • Innovative particle ma