Improved thin-layer chromatographic method for the separation of cholesterol, egg phosphatidylcholine, and their degradation products.
Degradation products of egg phosphatidylcholine (EPC) and cholesterol were analyzed with different normal- and reversed-phase thin-layer chromatography (TLC) systems. The best separation, in terms of the highest number of degradation products from both analytes, was obtained with a reversed-phase system, using butanol-methanol-water-96-98% (v/v) acetic acid (40 + 40 + 20 + 4, v/v/v/v) as the mobile phase after overnight saturation at 25 degrees C. A special development technique was used. After a first development, the plate was dried and a second development was performed in the same direction. This method enabled us to separate lysophosphatidylcholine, several free fatty acids and hydroperoxides, and several undefined degradation products of EPC and cholesterol. All products were visualized after the plate was dipped in a 1% (v/v) solution of 4-methoxybenzaldehyde in 98% sulfuric acid-96-98% (v/v) acetic acid-ethanol-water (2 + 10 + 60 + 30), presenting a blue color or a white spot against a colored background. After activation at 110 degrees C, a stable color for both analytes was reached after 12 min. Precision of <5% was obtained at 2 levels of analysis. Good linearity was obtained in the range of 5-30 microg for EPC (r = 0.991) and 5-40 microg for cholesterol (r = 0.991). These results show that TLC can be an inexpensive and easy alternative for the analysis of EPC and cholesterol.
Gabriëls
Margriet
M
VUB Pharmaceutical Technology and Physical Pharmacy, Brussels, Belgium. apogama@yahoo.com
Camu
Frederik
F
Plaizie-Vercammen
Jacqueline
J
D002138:Calibration; D002784:Cholesterol / Q000302:isolation & purification*; D002855:Chromatography, Thin Layer; D019327:Copper Sulfate; D004531:Eggs / Q000032:analysis*; D006851:Hydrochloric Acid; D006861:Hydrogen Peroxide / Q000737:chemistry; D007202:Indicators and Reagents; D019787:Linoleic Acid / Q000737:chemistry; D008081:Liposomes; D010713:Phosphatidylcholines / Q000302:isolation & purification*; D011786:Quality Control; D012015:Reference Standards; D012996:Solutions; D013056:Spectrophotometry, Ultraviolet; D013535:Suspensions
D007202:Indicators and Reagents; D008081:Liposomes; D010713:Phosphatidylcholines; D012996:Solutions; D013535:Suspensions; D002784:Cholesterol; D019787:Linoleic Acid; D006861:Hydrogen Peroxide; D019327:Copper Sulfate; D006851:Hydrochloric Acid