On silica-based solid phase extraction techniques for isolating microbial membrane phospholipids: Ensuring quantitative recovery of phosphatidylcholine-derived fatty acids

A silica-based solid phase extraction (SPE) protocol commonly used to isolate phospholipids from total lipid extracts failed to quantitatively recover phosphatidylcholines (PC) from three commercial SPE columns because a polar eluent volume of 5 mL methanol per 0.5 g silica was shown to be insufficient. Phosphatidylcholines, which are an important component of some fungal and bacterial cell membranes, were completely recovered when a larger ratio of 20:1 v/w methanol (mL) to silica (g) was used. Separation of phospholipids from a soil sample showed that a methanol:silica ratio of 20:1 recovered substantially greater amounts of phospholipids and resulted in a different phospholipid fatty acid (PLFA) structural profile than a 10:1 ratio. This study also confirmed that methanol preconditioning of the manufactured SPE columns studied is necessary for quantitative recovery of phospholipids.