Chapter 3 High Performance Liquid Chromatography (HPLC)
Chapter 3 High Performance Liquid Chromatography (HPLC)
Outline of chapter 3 • Characteristics of HPLC(特点) • Structure of high performance liquid chromatographer (仪器构造） • Types and separation principles of HPLC methods(方法的类型及其 分离原理） • Factors influencing broadening of chromatographic peaks and separation(影响色谱峰扩展及色谱分离的因素) • Stationary phase of HPLC(液相色谱固定相) • Mobile phase of HPLC(液相色谱流动相) • Selection of chromatographic methods(色谱方法的选择) • Requirements:要求掌握HPLC法的基本原理 • Emphases: 重点学会色谱分析方法的选择
Outline of chapter 3 • Characteristics of HPLC(特点) • Structure of high performance liquid chromatographer (仪器构造） • Types and separation principles of HPLC methods(方法的类型及其 分离原理） • Factors influencing broadening of chromatographic peaks and separation(影响色谱峰扩展及色谱分离的因素) • Stationary phase of HPLC(液相色谱固定相) • Mobile phase of HPLC(液相色谱流动相) • Selection of chromatographic methods(色谱方法的选择) • Requirements:要求掌握HPLC法的基本原理 • Emphases: 重点学会色谱分析方法的选择
§3-1 Characteristics of HPLC ●High pressure Because carrier liquid moving through the column endures high resistance, high pressure must be provided Usually The pressures for supplying carrier liquid(供液压力）and for injection sample(进样压力）reach 150 ~ 350×105 Pa ●High velocity Flow rate of carrier liquid: 1 ~ 10mL.min-1 , much faster than classical liquid chromatography. Analytical velocity is much faster than classical LC. It usually takes less than 1 hour
§3-1 Characteristics of HPLC ●High pressure Because carrier liquid moving through the column endures high resistance, high pressure must be provided Usually The pressures for supplying carrier liquid(供液压力）and for injection sample(进样压力）reach 150 ~ 350×105 Pa ●High velocity Flow rate of carrier liquid: 1 ~ 10mL.min-1 , much faster than classical liquid chromatography. Analytical velocity is much faster than classical LC. It usually takes less than 1 hour
●High efficient Due to use of new-type of stationary phases, column efficiency of HPLC improved further (number of theoretical plates per meter is more than 3×104 /m ●High sensitivity Highly sensitive detectors’ application improves the analytical sensitivity. Minimum detectable quantity UV-detector: down to 10-9g Fluorescence detector: 10-11g Sample amount needed: Very little, micro-liter of test sample is enough for full analysis ●Wide application fields Available for organic compounds with high boiling point, poor thermal stability and high molecular weight(>400) and some of inorganic compounds
●High efficient Due to use of new-type of stationary phases, column efficiency of HPLC improved further (number of theoretical plates per meter is more than 3×104 /m ●High sensitivity Highly sensitive detectors’ application improves the analytical sensitivity. Minimum detectable quantity UV-detector: down to 10-9g Fluorescence detector: 10-11g Sample amount needed: Very little, micro-liter of test sample is enough for full analysis ●Wide application fields Available for organic compounds with high boiling point, poor thermal stability and high molecular weight(>400) and some of inorganic compounds
§3-2 Factors influencing broadening of chromatographic peaks and separation Diffusion coefficient Dm/cm2 .s-1 : 10-1 (gas)/10-5 (liquid) Density /g.cm-3 : 10-3 (gas)/1(liquid) Viscosity /gcm-1 .s-1 : 10-4 (gas)/10-2 (liquid) = 2l dp + Cd Dm u + Cm dP 2 CSmdP 2 Cs df 2 Dm Dm Ds + + u H = A + + Cu B u ● 由于柱内色谱峰扩展所引起的塔板高度的变化为：
§3-2 Factors influencing broadening of chromatographic peaks and separation Diffusion coefficient Dm/cm2 .s-1 : 10-1 (gas)/10-5 (liquid) Density /g.cm-3 : 10-3 (gas)/1(liquid) Viscosity /gcm-1 .s-1 : 10-4 (gas)/10-2 (liquid) = 2l dp + Cd Dm u + Cm dP 2 CSmdP 2 Cs df 2 Dm Dm Ds + + u H = A + + Cu B u ● 由于柱内色谱峰扩展所引起的塔板高度的变化为：