High-Performance Thin Layer Chromatography: A Technique for Preliminary Separation and Quantification of Lipids From Neutrophils
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High-performance thin layer chromatography, HPTLC, is an advanced analytical technique for separating different analytes using fine-sized adsorbent particles as the stationary phase. This feature facilitates efficient packing, ensuring better separation.
To separate lipids using HPTLC, take a mixture of lipids with different polarities obtained from neutrophils. Now, pre-equilibrate the plate in an organic solvent for removing any traces of water vapors and dirt, then activate at a high temperature to prevent plate deterioration.
Apply the lipid mix and a standard as separate spots near the plate's base. Transfer the plate to a glass chamber saturated with a polar solvent – the mobile phase. Allow the solvent to move upward through capillary action.
While moving, more polar lipids migrate along the solvent front due to their affinity toward the polar solvent. Conversely, less polar lipids get adsorbed on the silica particles and lag. Run the lipids in an intermediate polarity solvent, followed by a completely non-polar solvent, resulting in further lipid separation.
As the solvent front reaches the desired distance, remove the plate and dry it. Expose the plate to a staining reagent, such as acidified copper sulfate, causing lipid charring. This helps visualize separated lipid spots, compared to the standard spots that help in preliminary lipid identification.
