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APPLICATION OF CPC AND RELATED METHODS FOR THE ISOLATION OF NATURAL SUBSTANCES

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APPLICATION OF CPC AND RELATED METHODS FOR THE ISOLATION OF NATURAL SUBSTANCES ( application-cpc-and-related-methods-forisolation-natural-sub )

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Table 1. Parameters of alkaloids isolation. Application of CPC and related methods for the isolation... 225 Type of alkaloids Used phases stationary / mobile Parameters: Speed of rotation / flow rate Ergoline alkaloids lysergol, chanoclawine (10) methyl tert-butyl ether, acetonitrile, water (4 : 1 : 5 v/v/v) 1250 rpm 3 mL/min Indole alkaloids vindoline, vindolinine, catharanthine and vincaleukoblastine (11) methyl tert-butyl ether, acetonitrile, water (4 : 1 : 5 v/v/v) 800 rpm 3 mL/min Isoquinoline alkaloids palmatine, jatrorrhizine, columbamine, pseudocolumbamine (12) dichloromethane, methanol, water (12 : 4 : 9 v/v/v) 700 rpm 9 mL/min (first stage) 3 mL/min (second stage) Isoquinoline alkaloids palmatine, berberine, worenine, epiberbine, coptisine, jatrorrhizine (13) n-hexane, ethyl acetate, methanol, water (2 : 5 : 2 : 5 v/v/v/v) 850 rpm 2 mL / min and then 650 rpm 5 mL / min protopine, tetrahydropalmitine, bicuculline (14) methyl tert-butyl ether, acetonitrile, water (2 : 2 : 3 v/v/v) + triethylamine and hydrochloric acid 800 rpm 2 mL / min huperzine A , huperzine B (15) N-heptane, ethyl acetate, n-propanol, water (5 : 15 : 35 : 45 v/v/v/v) 1400 rpm 6 mL / min michellemine B (16) chloroform, methanol, 0.5% hydrobromic acid 300 rpm 15 mL / min Tropane alkaloids darlirgine (17) (5 : 5 : 3 v/v/v) chloroform, methanol, water (13 : 7 : 8 v/v/v) No data such as high performance liquid chromatography (HPLC). In contrast to the HPLC, CPC gives com- plete recovery of the sample introduced. There is no denaturation of the molecules, no irreversible absorption on the solid absorbent and the solvent consumption is very low (7). The main advantage of this technique is due to significant reduction of analysis costs, allowing the use of this method on a larger scale. CPC has a high capacity. Relatively large volume of stationary phase makes the separa- tion of substances effective (4). Survey on using isolated alkaloids CPC CPC is an analytical method used increasingly for the separation and purification of individual sub- stances from raw plant material. Below an overview of research aimed at different groups of alkaloids separated using centrifugal partition chromatogra- phy is presented. Due to the similarity of CCC to the CPC method, several examples of the isolated alka- loids will be also presented by this method. Summary of methods used for the isolation of alka- loids is presented in Table 1. The first example of the preparation of the alka- loid purification with CPC is a separation of clavine alkaloids from methanolic extract of Ipomoea muri- cata L. seeds. The separation of these substances was not easy because of their sensitivity to temperature and light, however, Mauri and Srivastava coped with this problem by using a modified CPC method, which separated the substances with an electric charge depending on the pH value. They managed to separate the two alkaloids: lysergol and chanoclavine from each other. CPC apparatus used for the separa- tion contained 1320 cells (total column capacity of 200 mL) with a modification of rotation speed from 200 to 2000 rpm. As the stationary and the mobile phases used methyl tert-butyl ether, acetonitrile and water in a ratio (4 : 1 : 5 v/v/v) were used. Separation process was performed in the descending mode under the rotor speed of 1250 rpm and at the flow rate of 3 mL/min. From 4 grams of dry extract, 210 mg lysergol (97% purity) and 182 mg chanoclavine (79% purity) were obtained (10). Renault and co-workers (11) implemented pH- zone refining mode in their separation for the

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