Revisiting the Development of the Bligh and Dyer Total Lipid Determination Method

The experiments leading to the development of the most well-known method for total lipid determination in marine biological tissues (Bligh, E. G. and Dyer, W. J. (1959) Can. J. Biochem. Physiol. 37, 911–917) were repeated in order to discover the secrets of its success. Along with measuring the phase volumes of the water/methanol/chloroform mixtures investigated by Bligh and Dyer, the phase compositions were determined by gas chromatograph (GC). An examination showed that, although Bligh and Dyer applied largely different solvent ratios, the composition of both phases varied only within a limited range resulting in an incomplete investigation of the effects of changing this factor. Using Bligh and Dyer’s solvent mixtures the recovered fraction of organic phase was found to be the key factor determining the extracted lipid yield. On its turn the recovered fraction was positively correlated to the size of the organic phase and its methanol content. Additional experiments applying new extraction points with higher methanol contents revealed an increase of extracted lipid. This increasing yield was mainly due to a better extraction of the phospholipids as could be deducted from lipid patterns recorded by normal phase high performance liquid chromatography (HPLC) using an evaporative mass detector.