Application of the hottest high speed countercurre

Application of high-speed countercurrent chromatography

China is the country that has carried out research on Countercurrent Chromatography Technology earlier in the world. Its application range is very wide, such as bioengineering, medicine, natural product chemistry, organic synthesis, environmental analysis, food, geology, biochemistry, medicine, agriculture, environment, materials, chemical industry, marine biology, inorganic ions, health care raw materials, food additives, cosmetics and many other fields

1. Natural products

hsccc can adopt solvent systems with different physicochemical characteristics and diverse operating conditions, which has strong adaptability, and provides favorable conditions for extracting effective components with different characteristics (such as different polarity) from complex crude natural products. Therefore, in the late 1980s, under the sweeping tide of returning to nature around the world, HSCCC was widely used for the analysis, preparation and separation of chemical components of natural products, and it is also the most reported at present

for example, use n-hexane/ethyl acetate/methanol/water (3:7:5:5) to separate the crude extract of dry roots of tetrandria tetrandra; The crude extracts of Taxus chinensis were separated by n-hexane/ethyl acetate/ethanol/water (6:3:2:5) or n-hexane/ethyl acetate/methanol/water (1:1:i:i); The system of petroleum ether (℃)/ethyl acetate/methanol/water (50:70:80:65) is suitable for separating paclitaxel mixture; Hexane/ethyl acetate/methanol/water (3:7:5:5) was used to effectively separate the mixture of cinnamic acid, ferulic acid and caffeic acid; Taxol and caphalornannine were separated by hexane/ethyl acetate/methanol/ethanol/water (5:7:5:1:1.5); Brucine and strychnine were separated and prepared by chloroform/0.07 mol/l sodium phosphate 0.04 mol/L citric acid buffer system (ph:

5.08, 1:1); The crude extracts of Norway spruce needles were separated by chloroform/methanol/acetone/water (5:6:1:4); Using n-Heptane/ethyl acetate/methanol/water (3:l0:io:7) to separate the crude extract of Hybrid Tomato branch seeds, etc., the lower phase is generally used as the mobile phase

others, there are also two-step separation of paclitaxel homologues (eaphalonmaine, 7-epi-10-deaeetyltaxo1) in n-hexane/ethyl acetate/methanol/water, etc., the first step is (1:1:1), the second step is (3:3:2:3 or 4:4:3:4) and realizes the preparation separation, and it is found that the methanol content has a great influence on the separation effect; In addition, isorhamnetin and 4,5,7-trihydroxyflavonol quercetin were continuously separated and prepared by multidimensional high-speed countercurrent chromatography (multidimensional HSCCC), using chloroform, methanol/water (4:3:2) system J. In a word, HSCCC separation and preparation of natural products is very suitable, which is not only suitable for polar compounds, but also suitable for non-polar compounds; It can be used to remove impurities from crude extracts of natural products, refine the final products, and even get pure products in one step. When the rotating speed of analytical HSCCC instrument reaches more than 2000r/rain, and the solvent system is appropriate, the separation speed can be comparable to that of HPLC

traditional Chinese medicine comes from natural plants or animals. HSCCC can also be applied to the separation of the effective components of single herbs or prescriptions of traditional Chinese medicine. For example, the chloroform extract of Polygonum multiflorum Thunb was separated by n-hexane/ethyl acetate/methanol/water (1:1:1) and chloroform/methanol/water (4:3:2) systems to obtain emodin and physcioue. The alcohol extract was separated to obtain 1,2-stilbene glycosides, etc. The methanol extract of Radix Polygoni Multiflori was separated with ethyl acetate/methanol/water (50:1:50), and the lower phase was used as the mobile phase; The alkaloids of Coptis chinensis were separated and prepared with chloroform/methanol/0.1 mol/L hydrochloric acid (4:1.5:2), and the lower phase was used as the mobile phase; Angelica sinensis extract was separated with r134a/methanol/water system, rlim-a as mobile phase and methanol/water (65:30 or 70:30) as stationary phase; The extract of Ginkgo biloba leaves was separated with chloroform/methanol/water (4:3:2) to obtain 4,5,7-trihydroxyflavonol, isorhamnetin and quercetin. For this, we have developed new materials that benefit the public as much as possible. In conclusion, HSCCC is still blank in the separation and preparation of traditional Chinese medicine prescriptions, so it is of great significance to carry out HSCCC in this area

2. Antibiotics

because crude products can be directly introduced into HSCCC, HSCCC is also used for the analysis, preparation and separation of antibiotics. The injection volume is 1 mg-5 G

generally, hydrophobic systems can be used to separate the physical units of antibiotics (2 and 3) and password protection. For unknown samples, the strong polar components use the system containing n-butanol, the medium hydrophobic system uses the system containing chloroform, and the hydrophobic system uses the system containing n-hexane. For example: use chloroform/ethylene chloride/n-hexane/methanol/water (1:1:1:3 5:1) system to separate daunorubicin derivatives. The upper phase is used as the mobile phase; Use benzene/chloroform, methanol/water (15:15:23:7) to separate the mixture of ilomycin (Nain), the injection volume is 100mg, and the upper phase is used as the mobile phase; Hexane was identified with ether, and actin compounds were separated with methanol/water (5:1:4:5). The injection amount was 83 mg, and the upper phase was used as the mobile phase; The derivatives of candicidin were separated by chloroform/methanol/water (4:4:3) system. The injection amount was 100 mg, and the upper phase was used as the mobile phase; The mixture of niddamycins was separated with carbon tetrachloride/methanol/0.01 mol/L potassium phosphate buffer (ph=7) (2:3:2). The injection volume was 100mg; Ivermectin was separated with n-hexane/L ethyl acetate, methanol/water (19:1:lo:lo), the injection amount was 25 mg, and the lower phase was used as the mobile phase; Using chloroform/ethyl acetate/methanol/water (3:1:3:2) to separate pristinamycins and so on

3. protein and peptide

the recent progress in the separation and preparation of protein or peptide mixtures by HSCCC mainly depends on the following three factors: the solvent system with low viscosity is adopted, and most of the solvent system contains n-butanol; The emergence and development of new pH zone extraction countercurrent chromatography; Comprehensive application of HSCCC and ion pair countercurrent chromatography. The latter two countercurrent chromatographic techniques will be described in detail below. For example, chloroform/benzene/methanol/water (15:15:23:7) is used to separate Brevibacterium peptide, the injection amount can reach 100 mg, and the upper phase is used as the mobile phase; Chloroform/ethanol/water (5:4:3) or chloroform/ethyl alcohol/methanol/water (5:3:3:4), which is large but not strong in China's new material industry, is used to isolate baeitracin, and the injection amount can reach 50mg; Colistin was separated with n-butanol/o.03 mol/L trifluoroacetic acid (1:1); Dipeptide mixtures can be separated by n-butanol/dichloroacetic acid/0.1 mol/L ammonium formate (1:o.o1:0.01), tert butyl methyl ether/acetonitrile/trifluoroacetic acid (O.5% - 5%) (2:2:3), tert butyl methyl ether/acetonitrile, trifluoroacetic acid (1%) (2:2:3) or tert butyl methyl ether/n-butanol/acetonitrile/Z FLUOROACETIC acid (1%) (2:2:1:5). The injection amount can reach 3 mg, and the lower phase is used as the mobile phase; Cholecystokinin was separated by n-butanol/0.2 mol/L ammonium formate (ph:8.5) (1:1, 5O ℃) or n-butanol/0.2mol/L ammonium formate (ph=9) (1:1)

4. Food

hsccc has the greatest advantage in the application of food separation, which is that crude or complex samples can be injected directly, which is generally used for food detoxification and impurity removal or the preparation of bioactive substances. For example, use HSCCC to detect the toxin content in food and separate sea (a common toxin that causes food pollution) [28]; Falcarind and falcarindeil were separated from parsley with n-hexane/acetonitrile/tert butyl methyl ether (10 ∶ 10 ∶ 1) [29]; Sugar and PNP derivatives were separated by HSCCC orthogonal spiral tube planetary centrifugation. The solvent system was n-butanol/acetic acid/water (4 ∶ 1 ∶ 5) or n-butanol/methanol/water (4 ∶ 1 ∶ 4) The separation system can increase the hydrophobicity of molecules through derivatization, so as to realize the separation with HSCCC

5. Inorganic matter

the application of HSCCC in the separation of inorganic matter mainly focuses on rare earth elements or heavy metal elements. For example, use miscible 0.5mol/ldepha (diethylhexyl phosphate) and dodecane as stationary phase and hydrochloric acid as mobile phase to enrich rare elements; The lanthanide elements SM, Gd, TB, Dy, ER and Yb were separated by hydrochloric acid and chloroform (with 0.15mol/l depha dissolved) (1:1) solvent system, and the effect was good; Using the toluene solution system of ehpa (ethyl hexyl phosphate) and mono-2-ethyl hexyl ether as the stationary phase, the solution containing lanthanide elements such as holmium and erbium (equivalent to the mobile phase) is eluted from the top, and the metal elements are enriched in the stationary phase. It is found that the retention value of the complex of rare elements is greatly related to the pH of the mobile phase. The pH increases and the retention volume increases, but the number of theoretical trays decreases. In addition, Adding ammonium salt to the mobile phase does not affect the retention value of the stationary phase. According to the above methods, HSCCC can be used for environmental analysis and detection or pollution control. Although the sensitivity is low, the stationary phase can enrich interfering metal ions

6. Other

with the development of technology, the application scope of HSCCC is gradually expanded. Some people have tried to use HSCCC to separate racemic compounds. For example, n-dodecyl-l-proline-3,5-xylidine has been successfully used in the separation of amino acid derivatives. Some people use HSCCC to separate lac dyes. The solvent system is tert butyl methyl ether/n-butanol/ethanol/water (2 ∶ 2 ∶ 1 ∶ 5). The purity of the obtained material is about 95%. Ma and ITO found that increasing the content of chiral selective reagents in the organic stationary phase and increasing the hydrophobicity of the solvent system can improve the resolution of chromatographic peaks