Submitted to: High Performance Liquid Chromatography of Acyl Lipids
Publication Type: Book / Chapter
Publication Acceptance Date: 2/16/2004
Publication Date: 4/30/2005
Citation: Lin, J.T., Mckeon, T.A. 2005. Reversed-phase c18 hplc separations of free fatty acids and their methyl esters. High Performance Liquid Chromatography of Acyl Lipids.pp.1-16 (book chapter). Interpretive Summary: Ricinoleate (a hydroxy fatty acid) has many industrial uses such as the manufacture of aviation lubricant, plastics, paints, coatings and cosmetics. Its only commercial source is castor bean. Since castor bean contains the toxin ricin as well as potent allergens, it is hazardous to grow, harvest and process. It would be desirable to produce ricinoleate instead in a transgenic oilseed lacking these toxic components. We have recently reported the biosynthetic pathway of castor oil and identified the key enzymatic steps, which drive ricinoleate into castor oil in castor bean. In this report, we review the high-performance liquid chromatography of fatty acids and their methyl esters. The method has been used to establish the biosynthetic pathway of castor oil and can be used for the metabolic studies of fatty acids.
Technical Abstract: Reversed-phase C18 HPLC can be used to separate fatty acids (FAME and FFA) according to their structure differences. The factors decreasing retention times are: keto group, hydroxy group, epoxy group, triple bond, shortened chain length (C2H4 shorter), cyclopropenyl group, double bond and cyclopropanyl group, in approximate correspondence to the order of their decreasing polarity. Both double bond positional isomers and geometric isomers can be differentiated. The strength of eluent can be adjusted and the column can be shortened or lengthened to separate the FA of choice. For the separation of FA from naturally occurring sample, a gradient elution is recommended. C18 HPLC using methanol, isopropanol and/or water as eluent is particularly useful in metabolic studies using both the absorbance detector and flow scintillation analyzer.