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Browsing by Subject "Phospholipases"

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    Development of a thin-layer chromatography based method for structural analysis of phosphatidylcholine (PC)
    (2015-09) Tan, Guiwei
    Structural analysis of phosphatidylcholine (PC) plays an important role in omega-3 fatty acids enriched products assessment, developing structured omega-3 fatty acids– containing PC, metabolic processes studies and diseases diagnosis. In this thesis, a new positional analysis of PC is described. Its methodology is based on a smart connection between two-dimensional chromatography and in-situ enzymatic hydrolysis. Most characterizations of PC composition happened on a thin layer chromatography (TLC) plate. Firstly, PC was isolated from total lipids by TLC and its purity was confirmed by high performance liquid chromatography (HPLC). Without further extraction from the TLC plate, PC was hydrolyzed by phospholipases on the plate directly. The key step of the in-situ enzymatic reaction was to promote interactions between PC and phospholipases, because this in-situ enzymatic hydrolysis occurred on a silica gel matrix. These increases can be accomplished via adding a wetting agent consisting of chloroform/ methanol/ water (65:24:4, v/v/v) onto the reaction area. Free fatty acids (FFAs) released from different positions of PC were then isolated from products mixture by second-dimensional chromatography and were chemically transesterified into fatty acids methyl ethers (FAME). With help of gas chromatography- flame ionization detector (GC-FID), the presented method could reveal relative percentage of each fatty acid on sn-1 and sn-2 positions of PC with 91.59% and 84.80% accuracies, respectively. Therefore, separation of PC from total lipids, enzymatic conversion of PC to FFAs and lysophosphatidylcholine (LPC), and separation of FFAs from products mixture can be performed on one TLC plate. This will remove the need to extract the separated PC from the TLC plate for the enzymatic reaction, avoid the risk of losing materials during the extraction processes; saving time, labor and cost.