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High pressure liquid chromatography


Martin and Synge proposed the idea of ​​high performance liquid chromatography in 1941, but it was not until the late 1960s that high performance liquid chromatography was realized due to the development of various technologies. This chromatographic technique was once called High Speed ​​Liquid Chromatography, High Parss-ure Lipuid Chromatography, and the most widely used name is High Pe-rformance Liauid Chromatography (HPLC). ). High performance liquid chromatography has been widely used as an indispensable technology. Its main advantages are (1) higher resolution than other chromatography; (2) fast, ten minutes to tens of minutes can be completed; (3) high repeatability; (4) high-efficiency phase column can be used repeatedly; (5) automated operation, high analytical accuracy . According to the difference between the interaction of solute molecules and stationary phase in the separation process, high performance liquid chromatography can be divided into four basic types, namely liquid-solid chromatography, liquid-liquid chromatography, ion exchange chromatography and size exclusion chromatography. High performance liquid chromatography is widely used in the biological field for the isolation and identification of the following products: (1) amino acids and their derivatives; (2) organic acids; (3) steroids; (4) biopterin; (5) antibiotics; (6) saccharides; (7) porphyrins; Nucleic acids and degradation products thereof; (9) proteins, enzymes and polypeptides; (10) lipids and the like.
I. Classification
High performance liquid chromatography can be divided into four basic types: liquid-solid chromatography, bonded phase chromatography, ion exchange chromatography and size exclusion chromatography.
(1) Liquid-solid chromatography
Liquid-solid chromatography is generally called adsorption chromatography. The adsorbent has activated carbon, alumina and silica gel. The carrier used in liquid-solid chromatography is silica gel. The adsorption capacity of silica gel on solute and molecules is not evenly distributed over the entire surface of silicon. Some areas on the surface of silica gel interact strongly with solute molecules. These regions are active sites. The main interaction between silica gel and solute molecules is dipole moment hydrogen. Bond and electrostatic interaction. The stronger the polarity, the longer the residence time of the compound on the silica gel column. In liquid-solid chromatography, the mobile phase solvent molecules compete with the solute molecules to fix the mutual active sites, thereby allowing the solute to elute from the column. A solvent that binds strongly to the surface active site of silica has a strong ability to elute solute molecules, and thus is called a strong solvent, and vice versa. Liquid-solid chromatography is suitable for separating chromatographic geometric isomers and can be used for fat-soluble compounds such as phospholipids, steroids, fat-soluble vitamins, prostaglandins and the like.
(2) Bonded phase chromatography
Bonded phase chromatography was developed by liquid-liquid chromatography, ie, partition chromatography. Bonded phase chromatography covalently binds the stationary phase to the carrier particles, overcoming the slight loss of the stationary phase in the flow due to the stationary phase, and the mechanical impact of the mobile phase as it passes through the column. The stationary phase is continuously lost. Shortcomings such as gradually changing nature. Bonded phase chromatography can be divided into normal phase chromatography and reverse phase chromatography.
Normal phase chromatography
The groups covalently bonded to the support in normal phase chromatography are all polar groups such as primary amino, cyano, diol, dimethylamino and diamino. The mobile phase solvent is a non-polar solvent similar to the mobile phase in adsorption chromatography, such as heptane, hexane, and isooctane. Since the stationary phase is polar, the more polar the flowing solvent is, the stronger the elution ability is, that is, the solvent having a large polarity is a strong solvent. This relationship between the stationary phase and the mobile phase is exactly the same as that of liquid-solid chromatography, which is called normal phase chromatography. Nevertheless, the separation principle of normal phase chromatography is mainly based on the partition coefficient of the compound in the stationary phase and the mobile phase, and it is not suitable for separating geometric isomers.


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