In this study, a novel technique for positional analysis of phosphatidylcholine (PC) was developed and investigated. This technique is based on a well-defined connection between two-dimensional chromatography and in situ enzymatic hydrolysis. Characterizations of PC composition mainly occurred on a thin layer chromatography (TLC) plate. This method could reveal the relative percentage of each fatty acyl residue on the sn-1 and sn-2 ester linkages of PC, respectively. PC was isolated from total lipids by TLC, and then its purity was confirmed by high performance liquid chromatography (HPLC). Without further extraction from the TLC plate, PC was directly hydrolyzed by phospholipases on the plate. A PLA1 enzyme loading of 0.12 mL appeared to be optimum for the in situ enzymatic hydrolysis of PC, with an 87.7% PC conversion; while for a PLA2 enzyme loading of 0.30 mL, the PC conversion was 84.8%. From HPLC, no acyl migration was observed above the HPLC detection limit in both in situ enzymatic hydrolysis reactions. Free fatty acids (FFAs) released from different positions of the PC were isolated from the product mixture by two-dimensional chromatography. The FFAs bands were chemically trans-esterified into fatty acids methyl ester (FAMEs) and analyzed by GC-MS to uncover the PC positional structure. Practical applications: This study developed a new method to directly perform enzymatic hydrolysis reaction on a TLC plate. With a connection between two-dimensional chromatography and in situ enzymatic hydrolysis, a TLC plate can play multiple roles, including a separation tool, an enzymatic reaction carrier, and an identification tool. Thus, TLC technique has high potential and this study helped extend its applications. Step 1: Total lipids were applied to a thin layer chromatography (TLC) plate, and then phosphatidylcholine (PC) is separated from other lipids. Step 2: Enzyme solution and wetting agent are applied to the PC band. Step 3: After in situ enzymatic reaction, PC is partly hydrolyzed into lysophosphatidylcholine (LPC) and free fatty acids (FFAs), and the hydrolysis rate are 87.7% (with 0.12 mL PLA1 enzyme) and 84.8% (with 0.30 mL PLA2 enzyme), respectively. Step 4: The TLC plate is rotated by 90°. Step 5: The FFAs released from PC are isolated from products mixtures by a second-dimensional chromatography.
Bibliographical noteFunding Information:
The authors are grateful for the financial support provided by Minnesota Legislative-Citizen Commission on Minnesota Resources (LCCMR), UMN MnDRIVE, and NIFA (2016-68003-24850), the Center for Biorefining. The authors have declared no conflict of interest.
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- In situ enzymatic hydrolysis
- Structural analysis
- Thin layer chromatography