Optimization of the fractionation and recovery of polyketide antibiotics by countercurrent chromatography

The polyketide antibiotics are a large and diverse range of natural product s exhibiting a wide range of antimicrobial activities. The biosynthesis of these compounds results in the formation of analogues of the main antibioti c that are structurally and chemically very similar. These pose a significa nt separation problem, particularly on a large scale. In this work, we have investigated the fractionation and recovery of erythromycin A (EA) from it s analogues by countercurrent chromatography (CCC). In particular, we studi ed the effect of increasing mobile phase flow rate (2-10 mL.min(-1)) and so lute loading (0.1-1.0 g) on various chromatographic parameters, such as sta tionary phase retention, solute partition coefficients, and column efficien cy, together with the estimated throughput of the process. Experiments were performed on a Quattro J-type coil planet centrifuge (94.3 or 101.1 mL PTFE coil, 800 rpm) using a quaternary phase system, comprisin g hexane/ethyl acetate/methanol/water (1.2/2.0/2.0/1.0 v/v). Under optimal conditions, at a mobile phase flow rate of 8 mL.min(-1) and an injected sol ute mass of 0.6 g, EA could be obtained with a purity and yield of 97% w/w and 100% w/w, respectively. The maximum solute throughput in this case was estimated to be 0.96 Kg.(L-coil.day)(-1), which represented a 17 fold incre ase over the starting conditions identified during earlier method developme nt studies. The results provide an encouraging basis for the subsequent app lication of CCC technology to the separation of novel recombinant polyketid es, currently being developed by combinatorial biosynthesis techniques.