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Preparative-scale separation of colistin sulphate bulk sample was carried out on a preparative poly(styrene-divinylbenzene) stationary phase. Isocratic elution with acetonitrilesodium sulphate solution (0.7% m/v; pH adjusted to 2.5 with TFA) -water (16 : 50 : 34, % v/v/v) was carried out at a flow rate of 4.0ml min"1. Six colistin componentswere isolated and characterized using *H and 13C NMR.The molecular weights were confirmed by mass spectrometry. The structures of 2 components were determined for the first time. Polymyxin E7 was identified as having the same composition as polymyxin E1? except that the fatty acid moiety was 7-methyloctanoic acid. Isoleucine polymyxin E8 was characterized as having the same composition as isoleucine polymyxin Ex with 7-methylnonanoic acid as the fatty acid moiety.Colistin, also known as polymyxin E, is a complex mixture of closely related polypeptides isolated from Bacillus polymyxa var. colistinus.Colistin