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Abstract
Highly polar organic‐aqueous two‐phase and three‐phase solvent systems were applied to the countercurrent chromatographic (CCC) separation of biotic compounds. The ethanol/2 M ammonium sulfate (3∶5) system was used for protein separation between human serum albumin and lysozyme, and also for separations of sugars including D‐(+)‐glucose and L‐(−)‐fucose, D‐(+)‐xylose and α‐L‐rhamnose, and α‐D‐galacturonic acid, mannuronic acid lactone and D‐(+)‐glucoronolactone. When using the acetonitrile/1 M sodium chloride (5∶4) system for applying water‐soluble carboxylic acids, 2‐naphthoic acid, p‐methyl hippuric acid and p‐amino hippuric acid were well separated with upper phase mobile, and maleic acid and fumaric acid were resolved with lower phase mobile.
An organic‐aqueous three‐phase solvent system composed of n‐hexane/methyl t‐butyl ether/acetonitrile/water (5∶5∶7.5∶5) was also applied to the simultaneous CCC separation of fat‐soluble vitamins and water‐soluble vitamins. Using the middle phase as the stationary phase, the separation of thiamine hydrochloride and nicotinamide was achieved first with the lower mobile phase and then vitamin K1 and K3 were eluted after the mobile phase was switched to the upper phase reversing the direction of elution.
The overall results demonstrated that both extremely hydrophilic organic‐aqueous two‐phase solvent systems and organic‐aqueous three‐phase solvent systems are useful for the CCC separations of polar compounds.
Acknowledgments
The authors would like to thank Misses Yukiko Ishiguro, Miho Sugisaki, Sakura Kaieda and Sayaka Kato for their technical assistance. This work was supported in part by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and from College of Pharmacy, Nihon University, Chiba, Japan.