Chromatography of Phospholipides and Related Compounds on Glass Paper Impregnated with Silicic Acid MONA BROWN, D. A. YEADON, L. A. GOLDBLATT, and J. W. DIECKERT Southern Regional Research laboratory, U. S. Department o f Agriculfure, New Orleans, l a .
,A rapid and convenient chromatographic procedure for the separation of phospholipides and some of their hydrolytic cleavage products is described. Glass fiber paper impregnated with silicic acid is used as the chromatographic medium, and a solvent system composed of phenol, ethyl ether, acetone, and water is the developing solution. Choline-containing compounds are detected on the chromatogram with a phosphomolybdic acidstannous chloride reagent or with a modified Dragendorff reagent. Compounds containing primary amino groups are located with a ninhydrin reagent. Substances that char are detected also by spraying the chromatogram with concentrated sulfuric acid and heating over a hot plate.
A
micro procedure is needed for the examination of miutures of phospholipides and their hydrolytic cleavage products in connection with studies in this laboratory on fat emulsions for intravenous alimentation. Several paper chromatographic procedures for resolving certain phospholipide mixtures have been described (6-8, IO). Separation of phospholipides has also been achieved on glass fiber paper impregnated with silicic acid (s),using a methanol-ethyl ether solvent system as the developing solution. This method is relatively rapid, and less than 2 hours is required for the development of a 10- to 12-inch chromatogram. The use of the glass paper offers the additional advantage of permitting the visualization, by charring with hot sulfuric acid, of compounds for rvhich no simple spot test spray reagents are available. The sulfuric acid reagent is more sensitive for the phospholipides tested than are the usual spot test reagents. I n the present investigation a phenol-ethyl ether-acetone-water solvent system, devised originally for the separation of sugars (b), was found to have certain advantages over the methanol-ether solvent for separating phospholipides and related hydrolytic cleavage products. SENSITIVE
APPARATUS A N D REAGENTS
Chromatographic chamber, similar to those described by Dieckert and Reiser ( 3 ) as modified by Dieckert and Morris (3). Reagents and solvents, commercial
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ANALYTICAL CHEMISTRY
products of reagent grade unless otherwise specified. Developing solution, 200 grams of phenol dissolved in 400 ml. of ethyl ether followed by addition of 250 ml. of acetone and 50 ml. of distilled water. Ninhydrin, 0.270(w./v.) ninhydrin in acetone (11). Phosphomolybdate reagent, 2% (w./v.) phosphomolybdic acid in distilled water; 0.4% stannous chloride in 3-47 hydrochloric acid (9). Dragendorff reagent, as described by Bregoff and coworkers (1). Glass fiber paper, S o . X-934-.4HJ 12 X 15 inches, H. Reeve Angel and Co., Inc., 52 Duane St., New York, N. Y. I t was impregnated with silicic acid essentially as described by Dieckert and Reiser (S), except that the papers were washed with water six times.
Sphingomyelin, courtesy H. E. Carter. Dimyristoyllecithin, courtesy Ericb Baer. Stearic acid, Eastman grade. Linoleic acid, Hormel Foundation. PROCEDURE
The chromatographic tank containing the developing solvent was allowed to equilibrate for 2 or 3 hours before the paper strips were introduced. The solutions of the test materials were spotted in 1- to 10-pl. quantities on the dry silicic acid-impregnated paper about an inch from the bottom of the strip and about 0.75 inch apart. Typically four spots were applied to a strip 4 inches wide. The solvent mas allowed to evaporate. The strip was then attached to the rod in the tank by means of a binder clip clamped t o the upper edge of the paper and was developed by the ascending technique at room temperature. It was not found necessary to equilibrate the paper in the tank prior t o development. Approximately 25 minutes were required for the development of chromatograms 6 inches long and less than 90 minutes for IO-inch chromatograms. K h e n the solvent front had advanced to Tvithin about an inch of the top, the strip was removed and hung in a fume hood to allow the ether and acetone to evaporate. The phenol was then removed by heating the strip in the hood over a n open coil hot plate heated to red heat. Care must be taken to ensure complete removal of the phenol lest it interfere with subsequent tests. After the developing solvents were removed the compounds were located on the chromatogram by applying the following spot test reagents. Sulfuric Acid. The chromatogram was sprayed evenly with concentrated sulfuric acid using a glass nebulizer and then was heated over a n open coil hot plate until sulfuric acid fumes were no longer evolved. Most of the compounds tested produced charred spots on a white background which could easily be observed by transmitted
TEST SUBSTANCES
Egg lecithin, prepared by fractionation of egg phospholipides on an alumina column ( 4 ) . Lysolecithin, prepared by the action of cobra (Nuiu naiu) venom on purified egg lecithin (6). Phosphatidylethanolamine, prepared by fractionation of egg phospholipides on a silica column (8). Ethanolamine, Eastman grade. Choline chloride, Eastman grade. Sodium glycerophosphate, Eastman grade. Glycerol, Baker’s reagent grade.
Table I. Typical R j Values of Some Phospholipides and Hydrolytic Cleava g e Products
Compound Sodium glyceiophosphate Ethanolamine Choline chloride Lysolecithin Sphingomye!in Dimyristoyllecithin Egg lecithin Glvcerol Phosphatidylethanolamine Linoleic acid Stearic acid
I? f Value 0 0 0 0 0 0 0
05
11
20 31 50
65 66 0 T6 0 85 0 95 0 95
Table II. Average R, Values and Standard Deviation Using Different Preparations of Silicic Acid-Impregnated Glass Paper Batch 2 Several Batches Batch 1 (5 Trials) (17 Trials) (7Trials)
Rf
Phospholipide .~ Lysolecithin Sphingomyelin Lecithin Phosphatidylethanolamine
R/
value, av.
Std. dev.
value, av.
Std. dev.
0.33 0.51
