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Chapter 4 High Performance Liquid Chromatography （ HPLC ）

Chapter 4 High Performance Liquid Chromatography （ HPLC ）. 高效液相色谱. 1952 ， A.J.Martin and R.L.M.Synge received the Nobel Prize for the discovery of partition chromatography. In 1941, they wrote:

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Chapter 4 High Performance Liquid Chromatography （ HPLC ）

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  1. Chapter 4High Performance Liquid Chromatography（HPLC） 高效液相色谱

  2. 1952，A.J.Martin and R.L.M.Synge received the Nobel Prize for the discovery of partition chromatography. • In 1941, they wrote: The mobile phase need not be a liquid but may be a vapour. We show below that the efficiency of contact between the phases is far greater in the chromatogram than in ordinary distillation or extraction columns. Very refined separation of volatile substances should therefore be possible in a column in which a permanent gas is made to flow over a gel impregnated with a nonvolatile solvent in which the substances to be separated approximately obey Raoults’ law.

  3. 4.1 Introduction of HPLC • Development of HPLCFrom LC to GC From LC to HPLCApplication of HPLC • Special characters of HPLCHigh pressure High FlowHigh performance Others • Types of HPLCChemical-bond (distribution), Adsorption, Ion exchange*, Exclusion

  4. 4.2 Discussion of LC from the Rate Theory • H=A+B/u+Cu • A=2λdp • B→0 • C=?

  5. Mass transfer resistance causing by the flowing mobile phase

  6. Mass transfer resistance causing by the stagnating（滞留）

  7. Equation of Rate-theory of HPLC 涡流扩散项 纵向扩散 流动相传质 滞留区传质 固定相内传质

  8. Conclusion • Fine stationary phase • big Dm • Low flow • Extra-volume of the column • Temperature

  9. Glossary • HPLC • Chemical-bond phase chromatography • Ion exchange chromatography • Exclusion chromatography • Stagnate • Isocratic and gradient elution

  10. 4.3 Instrument of HPLC • Typical HPLC chromatographs • Pumping and Solvent System • Sample Injection System • Columns • Detectors

  11. 4.3.1 Diagram of HPLC instrument

  12. Picture of HPLC instrument

  13. 4.3.2 Pumping and Solvent System Principle and Picture of reciprocating pump (往复泵）

  14. Gradient elusion （剃度洗脱） • Reason for gradient elusion • Compared with GC

  15. 溶剂A 溶剂B 溶剂A 溶剂B 电磁阀A 电磁阀B 高压泵A 高压泵B 混合室 混合室 泵 • Two types of gradient elusion Low-pressure mixing High-pressure mixing

  16. 4.3.3 Injection system Injection valve

  17. 4.3.4 Column • Size • Packing materials • Packing technology

  18. 4.3.5 Detectors • UV-detector (紫外检测器） • Differential refractive index detectorRI（示差折光检测器） • Fluorescence Detector (荧光检测器) • Electrical conductivity detector (电导检测器)

  19. UV detector Liquid flow cell for UV detection in HPLC

  20. Single wavelength detector （固定波长的紫外检测器） • Multiwavelength detector （可变波长的紫外检测器） • Photo-diode array detector （光电二极管阵列检测器）

  21. Single wavelength detector

  22. Multiwavelength detector

  23. DAD (二极管阵列) detector

  24. 3-D figure of photodiode-array detector (二极管阵列检测器之三维图)

  25. Example of Selecting different wavelength

  26. Characters of UV-detector • Sample should be UV-absorption • High sensitivity • Large linear range • Suitable for gradient elution • Most widely used

  27. Refractive index detector(折光检测器)

  28. Properties of RI detector • Non-specific • Low-sensitivity • Easily affected by temperature • Unsuitable for gradient elution

  29. Fluorescence detector

  30. Properties of Fluorescence detector • High selectivity ( for protein, drug) • High sensitivity

  31. Electrochemical detector (电化学检测器)

  32. Properties of Electrochemical detector • Selective to electro-active subject • Easily affected by some surface-active substances

  33. RI UV Flu. EC selectivity General Special High Special Special Gradient elution no yes yes no Linear range 104 105 103 104 Detection limit μg ng pg ng Sensitive to temperature high low low middle Property of mobile phase Unlimited limited limited limited Comparison of the detectors

  34. Glossary • Pump • Valve • Loop • Gradient elution • Refractive index • Fluorescence • Cell • DAD (photodiode array detector)

  35. 4.4 Types of HPLC (HPLC modules) • Chemically bonded phase HPLC • Adsorption HPLC • Ion pair chromatography • Ion exchange and ion chromatography • Size exclusion chromatography

  36. 4.4.1 Chemically bonded phase chromatography (化学键合相色谱法) • Liquid-liquid Partition chromatography (液-液分配色谱） • Normal phase liquid chromatography：polar stationary phase and less polar or nonpolar mobile phase • Reverse phase liquid chromatography:Polar mobile phase and less polar or nonpolar stationary phase • Disadvantage of the partition chromatography

  37. Solid phase of chemical-bonded phase Chro.(1)Reversed phases: silica gel surface is modified with alkyl group(non-polar, such as n-octadecyl，C18), becoming hydrophobic.

  38. (2) More about the solid phase of RP-HPLC • Principle of the retaining of the analyte • Alkyl group and other groups • Stability of the solid phase (3) Normal phase: polar functional groups chemically modified on the surface of silica gelThe group could be diol, cyano, amino, etc.

  39. Mobile phase of HPLC • General ruleMobile phase play an important role on kSuitable k is between 2 and 5 • Eluotropic seriesPolarity and polarity index of solventsPolarity of solvent mixtures • Elution strengthRelationship between polarity of solvent used as mobile phase and solid phaseElution behavior of reversed phase chro. and normal phase chro.

  40. Other properties of solvent used as mobile phase:chemical stablecompatible to detectorlow viscosity (粘度)others (boiling point, toxicity and expense) • Strategy of choosing mobile phaseOptimizing k and α through experience or systematic procedure according to some rules (typical solvents trial).

  41. In reversed-phase Chro.: mixture of the solvents methanol, acetonitrile and tetrahydrofuran with water.In normal-phase chro.：mixture of diethyl ether, methylene chloride and chloroform with n-hexane • Isocratic elution and Gradient elutionTroubles of gradient elusion (column, detector and pump)

  42. 4.4.2 Adsorption chromatography of HPLC (liquid-solid chromatography) Mechanism of the separation By using solid absorbent as stationary phase and solvent as mobile phase, the solute of being analyzed could be absorbed by the active centers on the surface of the stationary phase and then desorbed by the solvent. Difference of this procession could cause the separation of different analyte.

  43. Solid phase Polar phase：silica gel，alumina etc. non-polar phase：active carbon, polymer micro-bead

  44. Mobile phase • ε° of the solvent • Mixture of the n-alkyl(正构烷烃）and methylene chloride • Effect of water and other solvent

  45. Effecting of water to the adsorption HPLC

  46. （4）Application of adsorption HPLC • Suitable to nonpolar substances which are difficult to dissolve in water, such as fattiness and oil • Positional isomers or stereoisomers

  47. Glossary • Chemically bonded phase HPLC • Reverse Phase HPLC (RP-HPLC) • Normal phase HPLC • Liquid-liquid Partition HPLC • C18,C8 ,-NH2, -CN • Alkyl group • Elution strength • Methanol, acetonitrile • absorbent

  48. 4.3.3 Ion pair chromatography(离子对色谱法) （1）Mechanism • If the analyte is ion（A+）, which can not be retained by the solid phase, an counter ion (B-) is added into the mobile phase to combine with the ion（A+）,forming an neutral molecule （ion-pair complex) and then retained by the solid phase.

  49. （2）Solid phase, moble phase and ion-pair reagent • Solid phase：C18,C8 • Mobile phase：water solution • Ion-pair reagent：(C4H9)4N+(四丁基铵正离子)、十六烷基三甲基铵正离子，ClO4-，十二烷基磺酸根等

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