High-performance liquid chromatography analysis principle and process

High performance liquid chromatography is based on classical liquid chromatography and is a chromatographic process using a liquid under high pressure as the mobile phase. Commonly referred to as column chromatography, thin layer chromatography or paper chromatography is a classic liquid chromatography. The stationary phase used is an adsorbent (silica gel, alumina, etc.) greater than 100 um. This traditional liquid chromatography uses a large particle size of the stationary phase, slow mass transfer diffusion, low column efficiency, poor separation capability, and separation of simple mixtures only. The stationary phase used in the high-performance liquid phase has a small particle size (5um-10um), fast mass transfer, and high column efficiency. High-performance liquid chromatography (HPLC) was an analytical method developed in the late 1960s. In recent years, it has been widely used in the separation and determination of functional ingredients, nutritional enhancers, vitamins, and proteins in health foods. About 80% of the world's organic compounds can be analyzed by HPLC.

Principles of High Performance Liquid Chromatography (I) The process of high-performance liquid chromatography is performed by pumping the solvent in the reservoir into the chromatographic system and outputting it. After measuring the flow rate and pressure, it is introduced into the sample injector. The analyte is injected by the sampler and passes through the column with the mobile phase. After separation on the column, it enters the detector. The detection signal is collected and processed by the data processing equipment, and the chromatogram is recorded. Waste liquid flows into the waste bottle. The complex mixture separation (wider polarity range) can also be achieved using gradient controller gradient elution. This is similar to the gas chromatographic program, except that gas chromatography changes the temperature, while HPLC changes the polarity of the mobile phase, allowing the components of the sample to separate under optimal conditions.

(B) The separation process of high-performance liquid chromatography Like other chromatographic processes, HPLC is also a continuous multiple exchange process between a stationary phase and a mobile phase. It separates the different solutes by the difference in the exclusion effect caused by the difference in partition coefficient, affinity, adsorption force or molecular size of the solute. The initial sample is added to the stigma. Assume that the sample contains three components, A, B, and C, which enter the column along with the mobile phase and begin to distribute between the stationary phase and the mobile phase. The component A, which has a small distribution coefficient, is not easily retained by the stationary phase and flows out of the column earlier. The component C with a large partition coefficient has a longer residence time on the stationary phase and elutes later than the column. The partition coefficient of component B is between A and C, and the second is out of the column. If a mixture containing multiple components enters the system, the components in the mixture flow out of the column one after the other according to the difference in partition coefficient between the two phases for separation purposes.

The separation of different components in the chromatographic process depends first of all on the differences in the partition coefficients, adsorption capacities, and affinities between the two components. This is a problem of thermodynamic equilibrium and is also the primary condition for separation. Second, when the different components move in the column, the band broadens with the length of the column. The separation is related to the diffusion coefficient between the two phases, the size of the stationary phase, the filling of the column, and the flow rate of the mobile phase. Therefore, the ultimate effect of separation is the combined benefits of thermodynamics and kinetics.

Types of High-Performance Liquid Chromatography (I) Adsorption Chromatography In adsorption chromatography, the polar functional groups of the sample are firmly retained in the active adsorption center of the filler, and non-polar hydrocarbon groups are hardly retained. Therefore, clearly identify the type, number and location of polar functional groups. In general, samples that can be separated by adsorption chromatography should be soluble in organic solvents and non-ionic, and strong ion samples are not suitable.

The mobile phase used for the adsorption chromatography is based on n-hexane, chloroform, and dichloromethane, and the polarity of the sample is added to ethanol. However, it is preferable that the concentration of the added alcohol is 10% or less. If possible, the percentage can be further reduced. Because the high concentration of alcohol will reduce the adsorption activity of the filler, weaken the adsorption capacity, and make it difficult to reproduce.

(B) Partition Chromatography 1. Normal Phase Partition Chromatography Normal Phase Partition Chromatography is suitable for samples that are insoluble in water but soluble in organic solvents and have polar groups, but normal phase partition chromatography is not suitable for ionic substances.

2. Reversed-phase partition chromatography This method is currently used in a very wide range of applications, and in the reversed-phase distribution chromatography, the non-polar part of the sample retains its effect.

By using a mobile phase of water-methanol and water-acetonitrile, the separation is adjusted by the amount of methanol or acetonitrile added, but if the sample has an ionic group, it is necessary to add salt to the mobile phase or to adjust the pH of the mobile phase. Values, for example, if the sample has a -COOH group, make the pH of the mobile phase acidic, and enhance retention by suppressing the ionization of -COOH groups. This method is called the ion suppression method. If the sample has a strong ionic group, an ion pair method in which an appropriate counter ion is added to the mobile phase to form an ion pair is sometimes used.

In the adjustment of PH value, keeping the PH value in the range specified in the filler specification manual, most of the chemical bonding silicas are used at pH=2-9, however, when adding salt, it is best to make it PH=7.5 For -8 or smaller, porous polymer fillers can be used for a very wide range of pH values.

(III) Ion Exchange Chromatography In this method, ion exchange between the ion exchange group of the stationary phase of the filler and the ionic group of the sample is used to separate the sample components, which are divided into cation exchange and anion exchange according to the exchanged ions.

Ion exchange chromatography is used for ionic substances that are soluble in water. In ion exchange chromatography, the concentration of salt in the mobile phase, the pH, and the type of salt have a great influence on the retention value. The salts used in the ion exchange of high performance liquid chromatography include phosphates, acetates and borates. Because chlorides can attack stainless steel instruments, NaCI or other chloride salts cannot be used in HPLC. Some salts cannot be used depending on the measurement wavelength. For example, the absorption of acetic acid is about 210 nm. When the detection is at the short-wave end, it is not suitable to use acetic acid as the mobile phase.

(D) Gel chromatography Gel chromatography is different from the above three separation methods. Gel chromatography separates sample components by molecular sieve effect according to the size of the molecule. This method is also called size exclusion chromatography or size exclusion chromatography. Porous synthetic polymers having a certain pore size are often used as fillers.

Because in the sample, small-sized molecules penetrate deeply into the micropores, they flow out late, and large-sized molecules do not penetrate into the micropores and quickly flow out. Usually the separation of synthetic resins uses an organic solvent as the mobile phase, which is called gel permeation chromatography.

Gel chromatography can be divided into gel permeation and gel filtration depending on the nature of the sample.

1. Gel Permeation Chromatography (Gel Permeation Chromatography), abbreviated as GPC. This type of chromatography is used in samples of organic solvents such as PVC, PS, ABS, etc. The eluents used are THF, Chloroform, and others.

2. Gel Filtration Chromatography (Gel Filtrrion Chromatography), referred to as GFC. This type of chromatography is used in aqueous media such as proteins, starch, and aqueous synthetic polymers, and the solutions used are water, buffers, and the like.

There are many types of gels, which can be divided into organic and inorganic glues according to their source of raw materials. According to its preparation method can be divided into uniform, semi-uniform and non-uniform three kinds of gel. According to the strength of the gel can be divided into three categories: soft, semi-rigid and hard plastic. According to the scope of its application to the solvent can be divided into hydrophilic, lipophilic and amphoteric gels and so on.