The principle of high performance liquid chromatography and how to use it

High performance liquid chromatography is an important branch of chromatography, using liquid as the mobile phase, using a high-pressure infusion system, a single solvent with different polarity or different proportions of mixed solvents, buffers and other mobile phases are pumped into the chromatographic column equipped with stationary phase, and after the components in the column are separated, they enter the detector for detection, so as to realize the analysis of the sample.

Usage: The packing material of the column and the composition of the mobile phase should be specified under each variety. Commonly used column chemistries are silica gel and chemically bonded silica.

The latter is octadecylsilane bonded silica gel, followed by octyl bonded silica gel, and cyano or amino-bonded silica gel is also used; Ion exchange resins are used in ion exchange chromatography; gels or glass microspheres, etc., for molecular exclusion chromatography, etc. The sample size is usually a few microliters. Unless otherwise specified, the column temperature is room temperature and the detector is an ultraviolet absorption detector.

When using UV absorption detectors, the flow used correspondingly meets the solvent requirements for UV spectrophotometry.

In addition to the type of stationary phase, mobile phase composition, and detector type, the rest of the conditions specified under the items in the text include the inner diameter of the chromatographic column, the length, the grade of the stationary phase, the particle size of the carrier, the flow rate of the mobile phase, the proportion of each component of the mixed mobile phase, the column temperature, and the chemical bonding of the stationary phase reaction.

The sample size, the sensitivity of the detector, etc., can be changed appropriately.

In order to adapt to specific varieties and meet the requirements of system suitability test. Typical chromatograms are recorded in about 20 minutes. 2.System suitability testing.

The applicability test of the instrument shall be carried out according to the requirements of each variety, that is, the instrument shall be tested and adjusted with the specified reference substance, and the specified requirements shall be met; or specify the minimum theoretical plate count, resolution, and tailing factor of the column in the analytical state.

How to use high-performance liquid chromatography (HPLC).

Chromatography is a separation technique in which the mixture of a sample is separated by a continuous distribution of the components of the sample between two phases in a column called a chromatographic separation column.

One of the phases is fixed and is called the stationary phase;

The other phase is a fluid (gas or liquid) that carries a specimen mixture through this stationary phase, called the mobile phase.

As the mixed stream carried in the mobile phase passes through the stationary phase, it interacts with the stationary phase. Due to the differences in the properties and structure of each component in the mixture, and the difference in the magnitude and strength of the force generated between the stationary phases, with the movement of the mobile phase, the mixture is repeatedly distributed and balanced between the two phases, so that the components are retained by the stationary phase for different times, so that they flow out of the stationary phase in a certain order. Combined with appropriate post-column detection methods, the separation and detection of components in the mixture can be achieved.

Classified by two-phase state.

Chromatography in which the gas is a mobile phase is called gas chromatography (GC) g-s & g-l