Separation of Lipids: The Thin Layer Chromatography

SEPARATION OF LIPIDS: THIN LAYER CHROMATOGRAPHY
Abstract
A lipid is a general term that refers to all fatty acids as well as their derivatives. The term can also be used to refer to any other compound that is functionally or synthetically related to fatty acids. On the other hand, Thin Layer Chromatography is a separation technique that is widely utilized due to its relatively fast results and the fact that it is also quite inexpensive. Thin Layer Chromatography (TLC) is widely used in the separation of smaller, non-polar compounds. Indeed, the technique offers more advantages over the High Performance Liquid Chromatography (HPLC). As much as HPLC offers quantification ease and versatility, the Thin Layer Chromatography (TLC) technique has a very high rate of reproducibility. This main advantage, combined with the fact that the technique is inexpensive, makes researchers utilize it more often than not. This report discusses an experiment to separate different sets of lipids and determine unknown class of lipids from already known lipids. Based on migration coefficients (RF) values of known lipids on the TLC plates, it will be possible to identify the constituent lipids in the unknown lipid mixture.
Keywords: Lipids, Thin Layer Chromatography, TLC, High-Performance Liquid Chromatography, HPLC, TLC plates.
Introduction
Separation of lipids has gained interest from researchers worldwide owing to its importance in lipid analysis. In order for an unknown lipid mixture to be easily analysed, the lipid mixture must be separated into its separate fatty acid units. Analysis, then, can be carried out to determine individual components of the lipid mixture. In the history of lipid separation, chromatography has been at the heart of many laboratory procedures. Chromatography is the most preferred separation method because of its powerful and analytical resolution. Chromatographic separation utilizes the principle of selective adsorption of molecules (Henderson and Tocher 1992). A mixture of molecules to be separated is passed through a chromatographic column consisting of a matrix that selectively resists the flow of the molecules in the column. If a specific molecule has a greater affinity to the matrix, the movement of the molecule is more restricted. One major chromatographic technique that is widely utilized in lipid separation and characterization is Thin Layer Chromatography (TLC). This report will rely on Thin Layer Chromatography to separate an unknown lipid mixture based on four known lipids that include: oleic acid, triglycerides, phosphatidyl choline, and cholesterol (Christie and Han 2010). By knowing the migration distance (in mm) of the known lipids, the lipid mixture can be compared to them in order to determine its constituent parts and concentrations. The major hypothesis for this experiment is that the unknown lipid mixture has got one or more lipid constituents similar to the standards.
Materials and Methods
For this experiment, a Thin Layer Chromatography tank with solvent gel was used. Also, a TLC plate consisting of a glass plate coated in a white gel was utilized. The plate was meant to hold the standards (known lipids) and the unknown lipid mixture inside the Thin Layer Chromatography tank. Additional materi...