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

Area of Science:

• Biochemistry
• Analytical Chemistry
• Separation Science

Background:

• Sphingomyelin (SM) is valuable in food, cosmetic, and pharmaceutical industries.
• Traditional SM purification methods yield low purity and high solvent waste.
• Efficiently separating high-purity SM from sources like milk phospholipids (MPL) is challenging.

Purpose of the Study:

• To develop an efficient and reliable method for purifying sphingomyelin (SM) from milk phospholipids (MPL).
• To overcome the drawbacks of traditional purification techniques, such as low purity and high solvent consumption.
• To establish optimized conditions for silica gel column chromatography dynamically monitored by Thin-Layer Chromatography (TLC).

Main Methods:

• Optimized Thin-Layer Chromatography (TLC) conditions for SM separation using a specific solvent system (methyl acetate-isopropanol-chloroform-methanol-0.25% KCl).
• Utilized ImageJ software for relative quantification of SM, validating precision, accuracy, LOD, and LOQ.
• Employed gradient elution silica gel column chromatography with TLC dynamic monitoring and optimized eluent systems.

Main Results:

• Achieved SM recoveries exceeding 85% under optimized chromatographic conditions.
• The purity of the purified SM reached 90% (molar ratio) as confirmed by 31P NMR.
• Demonstrated a high percentage of esterified long-chain saturated fatty acids in the purified SM via UPLC-MS/MS.

Conclusions:

• The developed gradient elution silica gel column chromatography method with TLC dynamic monitoring is effective for high-purity SM purification from MPL.
• This method offers significant advantages over traditional techniques, providing high recovery and purity with reduced solvent usage.
• The purified SM, characterized by its high purity and fatty acid profile, is suitable for industrial applications in food, cosmetics, and pharmaceuticals.