Separation of polar lipids by two‐dimensional thin layer chromatography providing resolution of all the lipid classes commonly encountered in animal cells and a sensitive, rapid, reproducible procedure for determination of phospholipids by phosphorus analysis of spots are described. Values obtained for brain and mitochondrial inner membrane phospholipids are presented.

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Two new schemes for fractionation of complex lipid mixtures are presented. Their use for the study of lipids of beef brain, beef liver, and the sea anemone are described. Apparatus and techniques for working in an inert atmosphere, evaporation of solutions in the cold under nitrogen, use of infrared spectroscopy for examination of lipids and their hydrolysis products, preparation and clution of diethylaminoethyl (DEAE) cellulose and silicic acid‐silicate columns and general column combinations that can be used to fractionate complex lipid mixtures are considered in detail. The first scheme, employing DEAE cellulose columns followed by thin layer and paper chromatographic examination of the fractions, was applied to liver lipids. The many components, some of them new lipids not previously detected, are clearly seen with this technique but are not seen when paper or thin layer chromatography alone or silicic acid chromatography are used. The second scheme employing DEAE for initial fractionation, followed by complete separation on silicic acid and silicic acid‐silicate columns, was applied to lipids of the sea anemone and beef brain. Typical lecithin and phosphatidyl ethanolamine were isolated, but sphingomyelin was not found. A new sphingolipid, ceramide aminoethylphosphonate, with a free amino group and a direct carbon to phosphorus bond was isolated and characterized. The methods used for quantitative isolation, the infrared spectra, and the amounts of cholesterol, ceramide, cerebroside, galactosylglyceride, sulfatide, sphingomyelin, lecithin, phosphatidyl ethanolamine, phosphatidyl serine, phosphatidyl inositol, triphosphoinositide, phosphatidic acid, cardiolipin, and ganglioside of beef brain are presented. Finally, the types of lipid‐nonlipid interactions disclosed by column chromatography and their potential application to biological problems are discussed.

A column chromatographic procedure is reported utilizing a dextran gel (Sephadex) for the complete separation of the major lipid classes from water‐soluble nonlipids. Lipids other than gangliosides are eluted first with chloroform/methanol 19/1 saturated with water, gangliosides with chloroform/methanol/water containing acetic acid, and water‐soluble nonlipids with methanol/water 1/1. Results for adult human whole brain, grey and white matter, and normal infant whole brain lipids are presented. With beef brain lipid as sample the ganglioside fraction is essentially pure, but with human brain lipid samples only about 70% of the second fraction is ganglioside. All ganglioside and water soluble nonlipid of a human spleen chloroform/methanol extract was separated from lipids with the procedure. Control studies with P32O4蠁 and C14 labelled glucose showed that all counts were present in fraction 3. Similar studies with C14 labelled amino acids (glycine, serine, alanine, phenylalanine) showed that only phenylalanine counts were eluted in fraction 2 along with the gangliosides. The procedure was applied for removal of large amounts of ammonium acetate from DEAE cellulose column fractions and for complete removal of adsorbent and salts from lipids eluted from thin‐layer chromatograms. After passage through the dextran gel columns, lipids eluted from thin‐layer chromatograms were found to give infrared spectra identical to those of pure samples obtained by other procedures.

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