Patent GB-730173-A (GB)
Title: Improved procedures for obtaining concentrates containing vitamin b
IPC: A61K31/00
Filed: 1950-09-21
Abstract: The invention comprises a process of eluting LLD and APF active substances from charcoal containing such substances adsorbed thereon with a two-phase system of water and an organic polar solvent incompletely miscible with water.  A combination of such an elution process with a treatment of the starting material with a substance furnishing cyanide ions as in the examples of Specification 681,503 is disclaimed.  From the eluate the product may be recovered by chromatrography on alumina, and the effluent may be treated with a substance yielding cyanide ions as in the process of Specification 681,503.  Suitable solvents yield a two-phase system on forming a 1 : 1 by volume mixture with water, e.g. n-, sec.- and iso-butyl alcohols, amyl, hexyl and octyl alcohols, ketones, e.g. methyl ethyl ketone and methyl isobutyl ketone, aralkyl alcohols, e.g. benzyl alcohol, esters, e.g. amyl acetate and phenols, e.g. cresol.  At least sufficient solvent to saturate the adsorptive capacity of the charcoal for the solvent should be used.  Preferably the charcoal with adsorbed active material is first agitated with the organic solvent using sufficient solvent to provide a slurry and then an equal volume of water is added.  After further agitation the liquid is separated and after separation of the phases, the active substances are recovered from the aqueous layer and the solvent is reused.  The active substances may be extracted from the separated aqueous solution by adding a salt, e.g. ammonium sulphate, aluminium sulphate, sodium sulphate, sodium chloride, calcium chloride or magnesium sulphate, and extracting with a solvent, e.g. benzyl alcohol.  This may be followed by extractions in turn with water in the presence of chloroform and from the water with further benzyl alcohol.  The benzyl alcohol solution of active substances may be chromatographed on alumina, preferably from benzyl alcohol, and the product is then either treated with cyanide ions to yield additional Vitamin B12, or is isolated as a complex of Vitamin B12 and Vitamin B12-like materials.  The complex is obtained by chromatographic adsorption from a solvent on to alumina and developed with methanol. Characteristic distribution coefficients for it are given.  In examples: (1) fermentation broth from the elaboration of a strain of S. griseus, after preliminary treatment with diatomaceous earth is slurried with active charcoal.  The charcoal, after drying, is divided into portions, each being then agitated with a solvent or solvent mixture, and then washed with water, the water layer being separated and assayed for LLD activity.  Results are given for mixtures of water with the following solvents (some included for comparison purposes): carbon tetrachloride, benzene, petroleum ether, methanol, ethanol, n-butanol, sec.-butanol, 2 - methyl - butanol - 4, benzyl alcohol, 2 - butanol, methyl ethyl ketone, methyl isobutyl ketone, cresol, amyl acetate and 1-hexanol, and also for methanol and ethanol used alone; (2) a 1.2 : 1 mixture of water with n-butanol, s-butanol or benzyl alcohol is used to elute LLD and APF active substances from charcoal on which they have been adsorbed, and the charcoal then washed with water.  The aqueous layer after saturation with ammonium sulphate is extracted with benzyl alcohol, the extract is chromatographed, eluted, precipitated with ether and then subjected to countercurrent distribution between water and benzyl alcohol.  The optical densities at 5500   before and after the distribution are mentioned; (3) activated charcoal is stirred with clarified fermentation broth from the elaboration of a strain of S. griseus. The separated charcoal is agitated with n-butanol alone, and then also with water and diatomaceous earth.  After filtration and washing the aqueous layer is separated, agitated with ammonium sulphate and benzyl alcohol and the solution chromatographed.  The column is developed with methanol, all the red-coloured effluent being collected.  After concentration, ether and acetone are added to give a reddish-brown precipitate.  The precipitate is extracted with methanol and the solution subjected to chromatographic purification. From the effluent a crystalline product (lambdamax. 3550  , E 1%/1 cm.=130, and 5350 A, E 1%/1cm.=48) is isolated, which is considered to be a complex of about one part of Vitamin B12 and two parts of Vitamin B12-like substances, as shown by the distribution constant of the product obtained after subjecting the complex to countercurrent distribution between water and benzyl alcohol.  The complex was also characterized by its conversion, on treatment with excess cyanide ion, to an equal amount of Vitamin B12.  Reference has been directed by the Comptroller to Specification 673,601.