Separation of Phospholipids by HPTLC – An Investigation of Important Parameters

Numerous chromatographic systems have been reported in the literature for the separation of phospholipids by High Performance Thin-Layer Chromatography. We have noticed that the reproducibility of analytical results of most of these methods is not satisfactory over longer periods of time, even when modern instruments are employed. No theory explaining the reasons for this problem currently exists. We have investigated several parameters of the chromatographic process, including chamber saturation, derivatization, plate activity, and batch to batch consistency of the plates. This paper provides a summary of results obtained in our laboratory over more than six years. Separation of the phospholipids PA, PC, PE, PI, LPA, LPC, LPE, and LPI can be achieved on HPTLC silica gel 60 (Merck) with chloroform, methanol, water, ammonia 25% (60:34:4:2) as mobile phase. For reproducible results, the employed methodology must be strictly standardized. Most importantly, the developing chamber must be homogenously saturated for a specified time and the activity of the layer should be kept constant.