Bligh and Dyer Method

Bligh and Dyer (Can J Biochem Physiol 1959, 37, 911) introduced a method where extraction and partitioning are simultaneous, the precipitated proteins are isolated between the two liquid phases. This method is particularly suitable for lipid extraction of incubation medium, tissue homogenates or cell suspensions. The extraction can be carried out in a single tube where previous studies took place.

It must be pointed out that this extraction method was shown to give significantly lower estimates of lipid content in samples containing more than 2% lipid (mainly triacylglycerols) and this underestimation increased with increasing lipid content of the sample (Iverson SJ et al. Lipids 2001, 36, 1283). Thus, the total lipid content of fatty samples are accurately determined using the Folch extraction method.

Procedure: To a sample containing 1 ml water (1 ml cell suspension, homogenized tissue, plasma…), add 3.75 ml of a mixture chloroform/methanol (1/2) and vortex during 10-15 min, then add 1.25 ml chloroform with mixing 1 min and 1.25 ml water with mixing another minute before centrifugation. Discard the upper phase and collect the lower phase through the protein disk with a Pasteur pipette. For large volumes of liquid, it is advisable to filter the mixture to remove the insoluble parts of the sample and to centrifuge the liquid phase to allow the formation of the two liquid phases. After evaporation, the lipid extract (lower phase) will be redissolved in a small volume of chloroform/methanol (2/1).

The basic procedure was improved to increase the yield of lipids. One of the most common modifications is to replace water by 1M NaCl. This addition blocked the binding of some acidic lipids to denatured lipids. If necessary, the addition of 0.2 M phosphoric acid to the salt solution is possible (Hajra, lipids, 1974, 9, 502) to improve their recovery. In this case, plasmalogens are converted to lyso lipids.
If an exhaustive extraction is necessary, an extraction with two steps can be used.
Similarly, it was described that the addition of acetic acid (0.5% v/v) in the water phase significantly increased the recovery of acidic phospholipids (Weerheim AM et al., Anal Biochem 2002, 302, 191). Another modification has been proposed, in comparison with the traditional extraction method (Jensen SK, Lipid Technol 2008, 20, 280). Acidification with HCl improved the extraction of lipids in a shorter time.