Separation of aflatoxins by column chromatography.

Several column chromatographic methods are available for the separation of aflatoxins (l-6). Most of these procedures are elaborate and involve several steps of purification. A rapid and simple method of obtaining pure aflatoxins was reported by Steyn (5); however, with this method, we were not able to obtain pure crystalline aflatoxins B, and G1 for our experimental studies on animals. When purified chloroform was used in the eluting solvent system, good resolution of the toxins could not be achieved. Moreover B, always remained as a contaminant with B1. Similarly B2 and G, were present in G1. These contaminants were not removed from B, or G, by crystallisation from benzene as was described in the procedure. Hence a number of solvent systems were tried for the preparation of pure aflatoxins B, and G,; and this communication describes the best solvent system that consistently gave pure crystalline B, and Gr. Aspergillus parasiticus (ATCC no. 155 17) was grown as surface culture at 27°C for 5 days. The chloroform extract of the media and myCelia was concentrated in vucuo and dissolved in a small volume of chloroform. The aflatoxin content of the solution was estimated spectrophotometrically by the method of Nabney and Nesbitt (7). Basic alumina (100 g of aluminium oxide active, basic) for chromatographic analysis, standardized according to Brockman (BDH) containing 6% oxalic acid was prepared and packed in a column of 50 x 2 cm as described by Steyn (5). The chloroform extract containing 200 mg of aflatoxins was applied to the column. After complete absorption of the toxins, the column was washed with 100 ml of benzene at a flow rate of 50 ml/hr. The aflatoxins were then eluted using 300 ml of benzene : acetone : ethanol (97 : 2 : 1, v/v/v) at the same flow rate. Fractions (4 ml) were collected and monitored by thin-layer chromatography on silica gel plates using toluene : isoamyl alcohol : methanol (90 : 32 : 3, v/v/v) (8). Fractions containing only B, and G, were pooled separately and concentrated to a small volume at 35-40°C. Colourless, shining, needleshaped crystals were formed when the solutions were allowed to stand for a few hours at room temperature. Purity of these samples was assessed by thin-layer chromatography and molar absorptivity. B, and G1 gave single spots on plates which resolved a mixture of standard B1, BZ, G1, and Gz (obtained through the kind courtesy of Dr. L. A. Goldblatt, New Orleans, Louisiana) into four distinct spots. The molar ab-