This article discusses the use of high-performance thin layer chromatography (HPTLC) for the separation of lipids from neutrophils. The technique involves the application of lipid mixtures on a pre-treated silica gel plate and utilizes capillary action to achieve separation based on lipid polarity.
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.
To begin, fill up to 5 milliliters of each solution in the corresponding glass chamber. Add any kind of filter paper to increase the running speed in each chamber. Pre-incubate a 20 x 10-centimeter HPTLC silica gel 60 glass plate in the first running solution. Once the running solution reaches the top of the plate, dry it for 10 minutes at 110 degrees Celsius.
Dissolve the liquid pellet previously obtained after drying with a vacuum concentrator, in the desired volume of 1:1 chloroform/methanol solution, and incubate for 15 minutes at 37 degrees Celsius to dissolve. Next, use a ruler and a soft pencil to mark the loading spots for the desired number of samples, plus at least one standard. Mark the running distance at approximately 4 centimeters for the first running solution, and at approximately 6 centimeters for the second running solution.
To load the samples, wash the 10-microliter syringe three times, in 1:1 chloroform/methanol, prior to loading each new sample. Load 10 microliters of each sample dropwise, trying to concentrate the sample on as small an area as possible. Place the plate vertically into the first chamber with running solution #1. Ensure that the plate is parallel to the wall of the glass chamber, to achieve a uniform migration speed.
Together the plates are placed parallel to the back wall of the glass chambers, and the solvent fronts do not run too far to ensure that the lipids are efficiently separated.
Once the solvent line has reached the first mark, remove the plate, dry it, and place it in the second solution. Repeat similar steps for the second, and for the third running solutions. Leave the plate in the solution until the solvent front reaches the top of the plate. Then, remove the plate, and dry it at room temperature for one minute.
Place the plate in the copper sulfate solution for seven seconds. After drying the plate thoroughly, bake it in an oven for seven minutes at 170 degrees Celsius. Remove the plate from the oven and allow it to cool.
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