State of problem
The principle of HPLC is to make use of the difference in the polarity between the stationary phase and the flow phase, and to achieve the purpose of dispersing the material.
(1) the elucidation process of high performance liquid chromatography
The solvent in the liquid storage bottle is inhaled by the pump, then the output, after the measurement of flow and pressure, is introduced into the injector. The analyte is injected by the injector, and with the mobile phase through the column, were dispersed in the column after entering the detector, the detection signal from the data processing equipment acquisition and processing, and record the chromatogram. Waste liquid flow into the waste liquid bottle. A gradient controller can be used for gradient elution when the complex mixture is dispersed (with a wide range of polarity). This is similar to the gas chromatograph, which is different from the gas chromatograph transition temperature.
The change of HPLC is the polarity of the mobile phase, which causes the samples to be dispersed under the best conditions.
High performance liquid chromatography can be divided into liquid solid adsorption chromatography, liquid-liquid partition chromatography (positive and reverse), ion exchange chromatography, ion pair chromatography and exclusion chromatography.
1. liquid solid chromatography with solid adsorbents, the dispersion principle of the dispersed components on the chromatographic column is based on the dispersion of the adsorption capacity of the fixed relative components. The dispersing process is a equilibrium process of adsorption desorption. The commonly used adsorbents are silica gel or alumina, the size of 5~10μ m. It is suitable for the components of the dispersive mass of 200~1000, most of which are used for nonionic compounds, and the ionic compounds are easy to produce trailing. It is often used to disperse isomers.
2., liquid-liquid chromatography uses stationary phase that is coated on the surface of a carrier or chemically bonded to the surface of a carrier. The dispersion principle is based on the dispersion of dispersed components in the mobile phase and the stationary phase. The dispersal process is a distribution balance process.
Coating type fixed response has outstanding inertia; mobile phase must be used to reduce the fixed phase saturation, stationary phase from the carrier surface erosion; difference between temperature change and different batches of the mobile phase is often caused by changes in another column; the mobile phase stationary phase and the dispersed collection of complex samples. It is very difficult to avoid the loss of fixed liquid because of the coated stationary phase, which is now rarely accepted. It is now widely accepted as chemically bonded stationary phases, such as C18, C8, amino column, cyanic column and phenyl column.
Liquid liquid chromatography can be divided into positive and reverse phase chromatography (NPC) and reverse phase chromatography (RPC), according to the different polarity of the stationary phase and the mobile phase.
Normal phase chromatography accepted polar stationary phase (such as polyethylene glycol, amino and cyano bonded phase); the mobile phase is relatively non hydrophobic solvent polarity (alkanes such as hexane, cyclohexane), often adding ethanol, isopropanol, tetrahydrofuran, chloroform and other components to adjust the retention time. Compounds commonly used in dispersed and highly polar compounds (such as phenols, amines, carbonyl groups and amino acids).
Reversed-phase chromatography usually uses nonpolar stationary phases (such as C18 and C8). The mobile phase is water or buffer solution, often adding methanol, acetonitrile, isopropanol, acetone, tetrahydrofuran and other organic solvents to adjust the retention time. It is suitable for dispersing compounds with weak and weak polarity. RPC in modern liquid chromatography is the most widely used, according to statistics, it accounts for about the application of HPLC 80%.
With the rapid growth of column packing, the application of reversed phase chromatography has been gradually expanded, and has been applied to the elucidation of some inorganic samples or easily dissociated samples. In order to control the dissociation of the sample during the elucidation process, the pH value of the liquid phase is controlled by the commonly used buffer solution. However, it is important to note that the pH value used by C18 and C8 is usually 2.5~7.5 (2~8), and the high pH value will dissolve the silica gel, and the low pH value will cause the bonding of the alkyl to fall off. It is reported that the new commodity column can be operated in the pH 1.5~10 range.
Comparison between positive phase chromatography and reversed phase chromatography
Positive phase chromatography
Reversed-phase chromatography
Fixed phase polarity
High to middle
Middle to low
Mobile phase polarity
Low middle
Middle to high
Component elution order
Very small first wash out
Wash out the polarity first
It can be seen from the above table that there is no boundary between the positive phase chromatography and the reverse phase chromatography when the polarity is medium (such as the amino bond stationary phase).
The 3. ion exchange chromatography stationary phase is ion exchange resin, commonly used styrene and two ethylene cross-linked polymer backbone, and on the surface of the aromatic ring is connected with carboxyl group, sulfonic group (called cation exchange resin) or quaternary ammonium (anion exchange resin). The dispersed component is dispersed on the chromatographic column. The principle is that the ionizable ions on the resin can be reversibly exchanged with the ions with the same charge in the mobile phase and the ions of the tested components, and disperse according to the different charge magic of each ion and the ion exchange group.
The buffer solution is often used as the mobile phase of the ion exchange chromatography. The retention time of the dispersed components in the ion exchange column is not only related to the strength of the ion exchange group of the dispersant and the resin, but also by the pH value and ionic strength of the mobile phase. The pH value can change the degree of dissociation of the compound and continue to affect its effect on the stationary phase. The salt concentration in the mobile phase is large and the ion intensity is high, which is not conducive to the dissociation of the sample, which leads to the rapid flow out of the sample. Ion exchange chromatography is mainly used to elucidate organic acids, amino acids, peptides and nucleic acids.
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