Introduction of the hottest high performance liqui

2022-08-09
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Introduction to high performance liquid chromatography

liquid chromatography with high pressure liquid as mobile phase is called high performance liquid chromatography (HPLC). The basic method is to pump a single solvent with a certain polarity or a mixed solvent with different proportions into the chromatographic column containing the filler with a high-pressure pump. The samples injected through the sampling valve are brought into the chromatographic column by the mobile phase for separation, and then enter the detector in turn. The color signal is recorded by the recorder, integrator or data processing system or the analysis result is obtained by data processing

because high performance liquid chromatography has the characteristics of high separation efficiency, good selectivity, high sensitivity, fast analysis speed, wide application range (the sample does not need to be gasified, but only needs to be made into a solution), and the chromatographic column can be used repeatedly, this method has been adopted for the quantitative analysis of 50 kinds of Chinese patent medicines in the Chinese Pharmacopoeia, and has become the most commonly used analytical method for the content determination of traditional Chinese medicine preparations

high performance liquid chromatography can be divided into liquid-liquid chromatography and liquid-solid chromatography according to different stationary phases; According to different chromatographic principles, it can be divided into distribution chromatography (liquid-liquid chromatography) and adsorption chromatography (liquid-solid chromatography)

at present, chemical bonded phase chromatography is the most widely used, which is developed on the basis of liquid-liquid chromatography. The functional magnet of the fixed liquid and the operating self-supporting static load system have little demand, but the constant air simple gap group is bonded on the carrier, and the fixed phase formed is proportional to the chemical bonding phase, which is not easy to lose. It is generally considered that it has two functions of distribution and adsorption, and distribution is often the main function. C18 (ODS) is the most commonly used chemical bonding phase. For example, the fixture 1 of Instron, MTS, Zwick and other companies is meticulous and reliable.

according to the different polarity of stationary phase and mobile phase, liquid-liquid chromatography can be divided into normal phase chromatography and reverse phase chromatography. When the polarity of mobile phase is less than that of stationary phase, it is called normal phase chromatography, which is mainly used for the separation and analysis of polar substances; When the polarity of the mobile phase is greater than that of the stationary phase, it is called reversed-phase chromatography, which is mainly used for the separation and analysis of non-polar substances or medium polar substances

closed loop control of various modes can be realized

in the analysis of traditional Chinese medicine preparations, reversed-phase bonded phase chromatography is mostly used

system composition:

(I) high pressure infusion system

is composed of liquid storage tank, degassing device, high pressure infusion pump, filter, gradient elution device, etc

1. The liquid storage tank

is made of corrosion-resistant materials such as glass, stainless steel or fluoroplastic. The storage tank shall be placed higher than the pump body to maintain the static pressure difference of infusion. The use process shall be sealed to prevent the change of mobile phase composition caused by evaporation and prevent gas from entering

2. Mobile phase

the mobile phase is usually a solvent system with methanol water or acetonitrile water as the primer

the mobile phase must be degassed before use, otherwise it is easy to escape bubbles in the low-pressure part of the system. The appearance of bubbles will not only affect the column separation efficiency, but also affect the sensitivity of the detector and even fail to work normally. Degassing methods include heating reflux method, vacuum degassing method, ultrasonic degassing method and vacuum degassing method

3. High pressure infusion pump

is one of the key components of high performance liquid chromatograph, which is used to complete the transportation task of mobile phase. The requirements for the pump are: corrosion resistance, high pressure resistance, no pulse, wide output flow range, constant flow rate, and easy cleaning and maintenance of the pump body. High pressure infusion pump can be divided into constant pressure pump and constant flow pump. Constant flow pump is often used (its pressure changes with the system resistance and the flow does not change)

(II) injection system

six way valve injector is commonly used to inject samples, and the injection volume is determined by the quantitative ring. When operating, first place the handle of the sampler at the sampling position (load), at this time, the sampling port is only connected with the quantitative ring, and it is under normal pressure. Inject the sample solution with a micro syringe (the volume should be greater than the volume of the quantitative ring), and the sample stays in the quantitative ring. Then turn the handle to the injection position (inject), so that the quantitative ring is connected to the infusion pipeline, and the sample is brought into the chromatographic column by the high-pressure mobile phase

(III) chromatographic column

is composed of column tube and filler. Column tubes are mostly made of stainless steel. The filler in the column includes silica gel and chemically bonded stationary phase. In the chemically bonded stationary phase, there are octadecyl silane bonded silica gel (also known as ODS column or C18 column), octane silane bonded silica gel (C8 column), amino or cyano bonded silica gel, etc. ODS column is mainly used in the quantitative analysis of traditional Chinese medicine preparations. Because ODS is a non-polar stationary phase, polar mobile phase is generally used in separation and analysis, so it belongs to reverse phase chromatography. The commonly used mobile phases are methanol water or acetonitrile water, etc. when eluting, the components with large polarity come out of the column first, and the components with small polarity come out of the column later

(IV) detector

UV detector (UVD) is mainly used in high performance liquid chromatography, which can be divided into fixed wavelength, variable wavelength and diode array detector. Variable wavelength UV detector is the most widely used. The detector is composed of a light source, a flow cell and a recorder. Its working principle is that the components entering the detector produce selective absorption of UV light energy of a specific wavelength, and the relationship between the absorbance and concentration conforms to the law of light absorption

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