Improved column chromatography-thin layer chromatography densitometry method for purification of dairy sphingomyelin.

Sphingomyelin (SM) has the potential to be widely used in the food, cosmetics and pharmaceutical industries. However, SM is found in trace amounts in substances such as egg yolk and dairy products. Traditional purification methods, such as solvent extraction and column chromatography, have several drawbacks, including low purity, high loss and high solvent consumption, as well as complicated operation. These factors make efficiently separating high-purity SM difficult. In this work, a gradient elution silica gel column chromatography method dynamically monitored by TLC was developed for the purification of SM from milk phospholipid (MPL). First, the TLC elution conditions were optimized as follows: solvent methyl acetate-isopropanol-chloroform-methanol-0.25 % (w/v) potassium chloride (25:25:25:10:9, v/v/v/v/v/v), spot volume of 50-75 μg MPL or 5-7.5 μg SM sample, separation distance of 16 cm and 25 °C. Then, the relative quantification of SM was performed in combination with ImageJ software, and the method was confirmed to be accurate and reliable through a comprehensive evaluation of its precision, accuracy, LOD and LOQ. Finally, the optimized silica gel column chromatography method was used for the purification of SM by TLC dynamic monitoring, and the methyl acetate-isopropanol-dichloromethane-methanol-water system (25:25:25:10:11, v/v/v/v/v) was used as the first eluent, and the dichloromethane-methanol-water system (45:30:8.5, v/v/v) was used as the second eluent. SM recoveries exceeded 85 % under the optimal conditions of silica gel sample volumes of 3.125-3.75 mg/g and a diameter-to-height ratio of 1:5-1:6. The purity of SM prepared by the above method reached 90 % (molar ratio) as determined by 31P NMR, and SM had a higher percentage of esterified long chain saturated fatty acids (more than 20 carbon atoms) as determined by UPLC-MS/MS.