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Abstract:
CPC of an extract of Streptomyces tsukubaensis fermentation broth was subjected to two consecutive CPC runs. In the Fig. 1 there is shown a TLC plate with analysis of the eluate. In an absence of a FK-506 standard at the beginning of experiment, the CPC solvent system was selected with a goal of obtaining even distribution of the mass of the mixture components between two phases. The components represented a wide range of polarities and thus, the use of an isocratic solvent system led to the heaviest elution of components at both extremes, front end of the mobile phase as well as the end of the stationary phase. Such a situation is sub-optimal if a component of interest is found there. In an absence of standards, however, such results are expected to occur. A gradient solvent system would not be necessarily a more advantageous at this stage.
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The plate shows that four components gave the same color reaction as the spot of FK-506 (marked with a star), indicating potential similarity of the components. The fraction # 8, containing FK-506, was further purified using another, specifically composed solvent system.
Second CPC purification step, illustrated with Fig. 2, involved only the components from the fraction # 8. FK-506 eluted in the fraction # 2. All components shared similar polarity, and as a result were not as widely spread, however, many contaminants with the same Rf values on silica gel were finely separated. The new solvent system, being more focused, more fully exploited subtle polarity differences between the remaining components. For example, in the fractions 3 and 4 a structurally related to FK-506 compound was separated. It was not seen in the first CPC run because it co-eluted with FK-506. The third and final purification step (not shown) was achieved on a silica gel column. The silica gel column easily produced high purity FK-506 because the two CPC runs removed many closely eluting contaminants.
In retrospect, the analysis of both CPC purification runs reveals that the CPC purification of FK-506 can be improved by modifying the CPC solvent systems. A single step CPC purification appears to be plausible.
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