Derivatization, Extraction and Purification of High Molecular Weight Mycolic Acid of Mycobacterium bovis BCG by HPLC

Abstract

Biochemical identification of Mycobacteria after primary culture is a laborious and timeconsuming procedure. One of the suitable ways for overwhelming meaning this problems is using high performance liquid chromatography (HPLC) of their Mycolic acids and one of the most important materials for this process is phenacyl derivatives of high molecular weight Mycolic acids which is used as standard in this method. This standard is synthesized by an American company and is not available every time. So, this study is done for extraction, derivatization and purification of high molecular weight Mycolic acids from Mycobacterium bovis BCG as standard in the identification of Mycobacteria by means of HPLC. Used bacterium in this study was M.bovis BCG and its high molecular weight Mycolic acids were derivatized by para bromophenacyl bromide and 18- Crown 6 and extracted by Lich. RP 18 column and methanol/chloroform gradient. Chromatographs of company synthesized standard and prepared one’s were compared and retention time of related peaks were recorded. Complete similarities between chromatographs of commercially produced standard and provided standard suggest their uniqueness. So, this prepared standard can be used in identification of Mycobacteria.