Patent GB-625820-A (GB)
Title: Purification of antibiatic substances
Filed: 1946-06-12
Abstract: Water-soluble basic antibiotic substances elaborated by micro-organisms of the genus Actinomyces are purified by dissolving an acid salt thereof (e.g. a hydrohalide, picrate or lower aliphatic carboxylic acid salt of streptothricin or streptomycin) in a solvent for such salt or a solvent mixture in which it is at least moderately soluble, adding the solution to a column of adsorbent material, washing the column with a solvent or solvent mixture for the salt to develop zones of adsorbent material containing the antibiotic substance in differing amounts and degrees of purity, and selectively recovering the antibiotic substance from these zones.  Suitable adsorbents are charcoal, alumina, diatomaceous silica and especially acid washed charcoal and acid washed alumina, e.g. the product obtained by treating aluminium oxide with sulphuric acid, washing with water until the pH of the washings is about 4.5 and drying at elevated temperature.  The column, which may contain 10-30 grams of adsorbent for each gram of antibiotic salt to be treated, is preferably of the chromatographic type and preferably wet packed, and if desired the rate of flow of eluting solvent can be modified by adding a material such as filter paper pulp to the adsorbent. Suitable solvents for the preparation of the original solution, and for the development and elution (through the two need not be identical), are water, lower aliphatic alcohols and mixtures thereof, or, for somewhat known solubility (which is sometimes advantageous in the adsorption step), mixtures of these solvents with acetone or chloroform, and the concentration of the initial solution is advantageously about 10-50 per cent.  When the adsorption step is completed, fresh solvent is added to the top of the column and the eluate from the bottom is collected and tested until solids are found to be present, after which the eluate is collected in fractions which may be arbitrarily chosen or empirically predetermined or, in the case of coloured salts such as picrates, may be determined by observing the movement of coloured bands down the column. Alternatively, after development, the column is freed from solvent and divided into separate sections known or predetermined to contain optimum or predominant amounts of antibiotic salt, which is extracted therefrom with a solvent.  The distribution of antibiotic salt in the column differs with different compounds, streptothricin salts being mainly found in later fractions of eluate or higher zones of the column (with impurities in the earlier fractions or lower zones), whereas with streptomycin salts the purer salt is generally found in the early fractions or lower zones.  The recovery of the antibiotic salt from the various eluate fractions or extracts may be effected by partially concentrating them, either separately or combined, and mixing them with a miscible solvent in which the antibiotic is insoluble, e.g. acetone, ether or mixtures thereof, causing precipitation of the antibiotic salt. Alternatively the concentrates may be subjected to a further purification by adsorption and elution. Examples describe the purification of streptothricin hydrochloride and picrate and streptomycin hydrochloride.  Specification 625,821 is referred to.