Separation principle of high performance liquid chromatograp

（1） Liquid liquid chromatography (or liquid-liquid partition chromatography)
Both the mobile phase and the stationary phase are liquid. After the sample is dissolved in the mobile phase, it enters the stationary phase (stationary phase) through the interface in the chromatographic column. Due to the difference in the relative solubility of the sample components between the stationary phase and the mobile phase, the solute is distributed between the two phases. Similar to gas-liquid partition chromatography, the separation sequence depends on the size of partition coefficient, and the components with high partition coefficient have higher retention value. The partition coefficient is the ratio of solute concentration in the stationary phase to the mobile phase. However, the property of mobile phase in gas chromatography has great influence on partition coefficient, while the type of mobile phase in liquid chromatography has a great influence on partition coefficient.
（2） Liquid solid chromatography (or adsorption chromatography)
The mobile phase was liquid and the stationary phase was adsorbent. It is separated according to the different adsorption of substances. The solute molecules adsorbed on the stationary phase will replace the solvent molecules on the surface of the stationary phase. If the solvent molecules have stronger adsorption, the adsorbed solute molecules will be reduced correspondingly. The solute with high adsorption will flow out at last.
Liquid solid chromatography is suitable for the separation of oil soluble samples with medium molecular weight, and has high selectivity for compounds and isomers with different functional groups. The compounds that can be separated successfully by TLC can also be separated by liquid-solid chromatography. The disadvantage is that the non-linear isothermal adsorption often leads to the tail phenomenon of the peak.
（3） Ion exchange chromatography
Ion exchange chromatography is based on the reversible exchange between the ionizable ions on the ion exchange resin and the solute ions with the same charge in the mobile phase. These ions are separated according to their different affinity to the exchanger. Ion exchange chromatography is mainly used to separate ions or dissociative compounds. It is not only used for the separation of inorganic ions, such as rare earth compounds and fission products, but also for the separation of organic substances. Around the 1960s, amino acids, nucleic acids and proteins have been successfully separated and widely used in the field of biochemistry.
(4) ion pair chromatography
Ion pair chromatography is to add one (or more) ions which are opposite to the molecular charge of solvent (called counter ion or counter ion) into the mobile phase or stationary phase to combine with solute ions to form ion pair compounds, so as to control the retention behavior of solute ions. Ion pair chromatography, especially reversed phase ion pair chromatography, has solved the separation problem of difficult mixtures in the past, such as acid, alkali and ionic and non-ionic mixtures, especially for the separation of some biochemical samples such as nucleic acid, nucleoside, catecholamine, alkaloids and drugs. In addition, UV absorption or fluorescence groups can be introduced into the sample by the formation of ion pairs to improve the detection sensitivity.
（5） Ion chromatography
Ion chromatography is the only method that can obtain fast, sensitive, accurate and multi-component analysis results, so it has been widely valued and developed rapidly. The detection method has been extended to other types of detectors, such as electrochemical detector, ultraviolet photometric detector, etc. The number of analyzable ions is increasing, from inorganic and organic anions to metal cations, from organic cations to sugars and amino acids, can be analyzed by ion chromatography.
（6） Steric exclusion chromatography
Space exclusion chromatography takes gel as stationary phase, and its separation mechanism is completely different from other chromatography. It is similar to the function of molecular sieve, but the pore size of gel is much larger than that of molecular sieve, usually from nanometer to hundreds of nanometer. Solutes are separated by molecular size rather than by their interaction forces. The separation is only related to the pore size distribution of the gel and the volume or molecular size of the solute.
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What is the standard curve of HPLC!
In chromatography, the peak area is proportional to the concentration. The standard curve is a curve with concentration on the horizontal axis and peak area on the vertical axis.
The conditions for making standard curve of high performance liquid chromatography are as follows
To make a standard curve, first you have a reference material, and then you have a concentration range of the curve.
For example, the reference material x, the concentration is 0.1mg/ml-2mg/ml.
When preparing the sample, take appropriate amount of X to prepare the mother liquor with the concentration of 10mg / ml, and then dilute it step by step to prepare the sample with the concentration of 0.1mg/ml, 0.2mg/ml, 0.5mg/ml, 1.0mg/ml and 2.0mg/ml, and then inject the samples from low to high for analysis. (from low to high is to avoid systematic error)
Note: here is a sample for preparation and dilution into several linear samples. Generally at least 5 concentrations.
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