Abstract
Abstract The recommended method involves separation of the morphine from the mixed alkaloids in chloroform using 0·1n sodium hydroxide and separation of the codeine and thebaine by means of a buffer solution of pH 2·0. The two fractions are then chromatographed on paper and after suitable colour development the areas of the spots are measured planimetrically. Constant areas for the same quantities of alkaloid are obtained by ensuring that the initial spots of material applied to the starting line are of constant area and that the running conditions are rigidly controlled, especially the temperature. To overcome the variability due to differences between sheets of chromatographic paper the “4 point bioassay” technique is used (in which two dose levels of standard solution of alkaloids and two dose levels of the unknown fraction are applied for each assay). The coefficients of variation for individual assays are for morphine, 3 to 6 per cent; for thebaine 5 to 7 per cent. Replicate assays can readily be run on one sheet of paper resulting in increased accuracy. The method is particularly suitable for small quantities of raw material (100 to 200 mg.) and is two to three times quicker than other published methods.
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