Chromatography for the extraction of antibiotics

With many novel antibiotics being discovered in the marine system, new methods are needed to isolate these specific compounds for further use. Using high-speed counter-current chromatography (HSCCC) He et al (2013) developed a new method for isolation of Macrolactin antibiotics from marine bacteria. As I understand it HSCCC is a form of liquid chromatography utilising a helical coil being spun on a planetary axis while, at the same time, being spun on a solar axis. The stationary and mobile phase are both liquids, with the mobile phase being lighter than the stationary phase. Using gravitational forces the less dense or lighter mobile phase will move through the stationary phase and cause separation of the sample.

He et al (2013) grew their sample of Bacillus amyloliquefaciens on a rather large scale, using a 30L broth culture which was known to contain macrolactin A and macrolactin B. Using centrifugation bacterial cell mass was removed and treated, resulting in 3.2g of crude sample. 300mg of crude sample was prepared and injected into the HSCCC machine. Two separate mobile phases we used: n-hexane-ethyl acetate-methanol-water at a 1:4:1:4 ratio for the first phase, and at a ratio of 3:4:3:4 for the second phase. After detection of the first peak corresponding to macrolactin B the mobile phase was switched from the 1:4:1:4 solution to the 3:4:3:4 solution for elution of the second peak which corresponded to macrolactin A. The effluent was monitored by a UV detector set to 280nm for the detection of the peaks. Effluent was collected and purity of the isolated compounds was determined.

After running the chromatograph He et al (2013) found that analysis of 300mg of crude sample resulted in the isolation of 40.4mg of macrolactin A and 22.7mg of macrolactin B, both with purities around 95%.  While this is mostly a technical paper outlining the chromatography method and optimization, I think it may still be relevant. As I discussed in my previous post, new methods for the detection of antibiotics produced by marine bacteria are continuously developed. This of course may require new and efficient methods to extract these compounds. Especially since relatively large amounts were extracted at a relatively high degree of purity. Although adjustments would need to be made to the extraction procedure, this paper may be able to combine quite nicely with the paper discussed in my previous post, combining new methods for the detection of antibiotics with efficient methods for consequently extracting them. As He et al (2013) suggest these successful methods to extract compounds may help alleviate problems with sample availability for further development with newly discovered compounds.  

He, S., Wang, H., Yan, X., Zhu, P., Chen, J., Yang, R.. (2013). Preparative isolation and purification of macrolactin antibiotics from marine bacterium Bacillus amyloliquefaciens using high-speed counter-current chromatography in stepwise elution mode. Journal of Chromatography A. 1272  15-19.