Principles of high speed countercurrent chromatographs

1.Countercurrent chromatography was developed in the 50s of the 20th century from multipolar extraction techniques (discontinuity).

However, the multi-polar extraction equipment is large, complex, fragile, and the solvent system is easy to emulsify, with large solvent consumption and long separation time.

2.A two-dimensional force field generated by revolution and rotation (synchronous planetary motion) retains one of the two phases as the stationary phase.

Introduction: High-speed countercurrent chromatography is a liquid-liquid chromatography separation technology, its stationary phase and mobile phase are liquid, there is no irreversible adsorption, and it has the advantages of no sample loss, no pollution, high efficiency, rapid and large preparation separation. Due to the obvious advantages of HSCCC compared with traditional separation and purification methods, this technology has been widely used in the fields of traditional Chinese medicine component separation, health food, biochemistry, bioengineering, natural product chemistry, organic synthesis, environmental analysis and other fields.

China is the first country to carry out the application of countercurrent chromatography after the United States and Japan. Zhang Tianyou et al. were the first to develop analytical and preparative high-speed countercurrent chromatographs in China, and achieved remarkable results in the separation and preparation of functional components of traditional Chinese medicine in China. The high-speed countercurrent chromatograph produced by Shanghai Tongtian Biochemical Technology Co., Ltd. has a purity of 99% for separating traditional Chinese medicine components, which can be used for HPLC detection standards.

High-speed countercurrent chromatography is a continuous and efficient liquid-liquid partition chromatographic separation technology developed in the 80s of the 20th century, which does not use any solid supports or carriers. It uses a two-phase solvent system to establish a special unidirectional hydrodynamic equilibrium in a high-speed rotating spiral tube, where one phase acts as the stationary phase and the other phase acts as the mobile phase, retaining a large amount of the stationary phase during continuous elution.

Table of Common Basic Solvent Systems for High-speed Countercurrent Chromatography Types of Substances to be Separated Basic Two-Phase Solvent Systems Auxiliary Solvents Non-polar or Weakly Polar Substances N-Hexane-Methanol Chloroalkanes N-Hexane-Ethyl Chloroalkanes N-Hexane-Methanol (or Ethyl)-Water Chloralkanes Medium Polar Substances Chloroform-Water Methanol, N-Propanol, Isopropanol Ethyl Acetate-Water N-Hexane, Methanol, N-Butanol Polar Substances N-butanol-Water Methanol, Acetic Acid The above table lists some basic solvent systems that can be used for reference according to the polarity of the separated substances, including non-aqueous and aqueous systems.

The choice of solvent system is critical for HSCC separations. Unfortunately, so far there is no sufficient theoretical basis for the selection of solvent system, but it is based on the rich experience accumulated in practice. In general, solvent systems should meet the following requirements:

The solvent system will not cause the decomposition or denaturation of the sample, and each component in the sample has a suitable partition coefficient in the solvent system, and it is generally considered that the partition coefficient is more suitable within the range, and the partition coefficient value of each component should be sufficiently different, and the separation factor should be greater than or equal to; The solvent system does not interfere with the detection of the sample; In order to ensure that the retention rate of the stationary phase is not less than 50%, the stratification time of the solvent system is not more than 30 seconds; The volume ratio of the upper and lower phases is appropriate to avoid wasting solvents; Volatile solvents should be used as much as possible to facilitate subsequent processing and avoid the use of toxic solvents as much as possible. According to the polarity of the solvent system, it can be divided into three categories: weak polarity, medium polarity and strong polarity. Classic solvent systems include n-hexane-methanol-water, n-hexane-ethyl acetate-methanol-water, chloroform-methanol-water, and n-butanol-methanol-water.

In the experiment, according to the actual situation, we should summarize and analyze and refer to relevant monographs and literatures, start from the category of substances to be separated to find similar separation examples, select a solvent system with suitable polarity, adjust the relative proportion of various solvents, determine the partition coefficient of the target components, and finally select a suitable solvent system.

High-speed countercurrent chromatography (HSCCC) is a liquid chromatography technique that does not use any homomorphic carriers, and its principle is based on the relative movement of components in the rotating spiral tube and the different distribution between immiscible two-phase solvents to obtain separation, and its separation efficiency and speed can be comparable to HPLC. HSCCC has high separation efficiency and high product purity; There is no adsorption and contamination of the sample by the carrier; Large preparation volume and low solvent consumption; It is easy to operate and can separate specific components from very complex mixtures.

The application of HSCCC to the separation of natural products can achieve: (1) the preparation of high-purity reference substances for medicinal ingredients and impurities that must be controlled; (2) Cooperate with the activity tracking and the design of the drug site, and gradually separate and prepare the active part or active ingredient; (3) the establishment of fingerprints of Chinese herbal medicines and Chinese herbal formulas to provide richer information and data; (4) Carry out pilot mass production and industrial production. For example, the Institute of Engineering of the Chinese Academy of Sciences has explored the method of using HSCCC to develop the fingerprint of traditional Chinese medicine, and the HSCCC fingerprint of Salvia miltiorrhiza has been preliminarily established with the original medicinal material of Salvia miltiorrhiza as the model plant.

This technology is expected to become a new method for the research of quality standards of active ingredients in traditional Chinese medicine and a new separation technology for the production of traditional Chinese medicine.

There is a detailed systematic introduction to the knowledge of high-speed countercurrent chromatography in the learning and training column of "Chromatography World", you can take a look.

High-speed countercurrent chromatography (HSCCC) is a new, continuous and efficient liquid-liquid distribution chromatography technology developed by Dr. ITO of the National Institutes of Health in 1982.

Because it is different from the general chromatographic separation method, it can achieve gradient elution and reversed-phase elution, and can also perform repeated injections, making it especially suitable for preparative separation, with high product purity and large preparation volume.

(1) Separation and purification of known active ingredients of natural products.

2) Separation and purification of chemically synthesized substances.

3) The development of Class I and Class V new drugs of traditional Chinese medicine.

4) Research on fingerprinting and quality control of traditional Chinese medicine.

5) Separation and purification of antibiotics.

6) Separation and purification of unknown active ingredients of natural products (development of new compounds) and (7) Separation and purification of marine bioactive components.

8) Radioisotope separation.

9) Separation of biological macromolecules such as peptides and proteins, as well as chiral separation, etc.