Colistin (CL) is a peptide antibiotic produced by Bacillus polimyxa var. Colistinus that inhibits the growth of Gram-negative organisms. CL is a mixture of many components (Figure 10) where two main components are colistins A (CL-A) and B (CL-B). As shown in Figure 11, CLs-A and -B are linear-ring peptides that differ only in their N-terminal fatty acid. CL is used as a feed additive for domestic animals such as calf and pigs for preventing bacterial infection and/or improving feed conversion efficiency. CL is soluble in water, slightly soluble in alcohols, but insoluble in nonpolar solvents such as hexane and chloroform. From this property, we selected n-bu-

Figure 9 HSCCC separation of ivermectin components. Apparatus, HSCCC-1A; revolution, 800 rpm; solvent system, n-hexane/ethyl acetate/methanol/water (19:1:10:10) mobile phase, lower aqueous phase; flow rate, 2mLmin~1; detection, 245 nm. (Reproduced with permission from Oka H et al. (1996, 1998).)

Figure 10 HPLC separation of commercial CL. Column, Chromatorex Ph (5 ^m, 4.6x250 mm); mobile phase, acetonit-rile/0.01 mol L~1 TFA aqueous solution (24:76); flow rate, 1.0mLmin"1; detection, 210nm. (Reproduced with permission from Ikai Y etal. (1998) and Oka H etal. (1998).)

tanol and water as a basic solvent system. However, this combination was not suitable by itself, because the CL components were entirely partitioned into the aqueous phase. In order to partition the CL components partly into the n-butanol phase, various salts (NaCl and Na2SO4) or acids (HCl, H2SO4 and CF3COOH or TFA) were added as a modifier. The desired effect was produced from TFA where the partition coefficients of CL components rose as the concentration of TFA in the solvent system was increased. This effect may be explained as follows: as shown in Figure 11, CLs-A and -B have five free amino groups in l-diamino-butyric acid (l-Dab), and these amino groups dissociate in the aqueous phase under neutral to acidic conditions. Since TFA forms an ion pair with these amino groups, the hydropho-bicity of the CL components increases with the concentration of TFA resulting in their partition toward the organic phase. In order to determine the optimal concentration of TFA in the solvent system, K values of five components were measured at various TFA concentrations. As shown in Figure 12, the K value of each component increases with the TFA concentration, and at 40mmolL~1 TFA concentration, K values of CL-A and CL-B reached 1.5 and 0.6, respectively. At this TFA concentration, the a values between the adjacent peaks are all greater than 1.5, promising a good separation for all components. The settling time of the solvent system was 28 s, which is within the acceptable range. Therefore, we selected a solvent system of n-butanol/40 mmol L_1 TFA aqueous solution (1:1) for the HSCCC separation.

Using the above solvent system, a 20 mg quantity of commercial CL was separated by HSCCC. The retention of the stationary phase was 45%. The

Figure 11 Structures of colistin components. (Reproduced with permission from Ikai Y et al. (1998) and Oka H et al. (1998).)

elution curve monitored at 220 nm is shown in Figure 13. According to the results of HPLC analysis and the elution curve, all collected fractions were combined into five large fractions as shown in Figure 13. The yields of CL-A and CL-B were 9 mg each and those of other minor components were 0.5-1.0 mg. HPLC analysis was performed for each fraction; as shown in Figure 14, the fractions of CLs-A and -B each produced a peak with a purity of over 90%.

Solar Panel Basics

Solar Panel Basics

Global warming is a huge problem which will significantly affect every country in the world. Many people all over the world are trying to do whatever they can to help combat the effects of global warming. One of the ways that people can fight global warming is to reduce their dependence on non-renewable energy sources like oil and petroleum based products.

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