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Protocol for High-Performance Chromatographic Separation of Cerebrosides

1 HPTLC Development Tank Setup

a) Add freshly prepared Developing Solvent to the bottom of the tank to a depth of about 1 cm (usually 30–50 mL).

b) Close the tank using the fitted glass lid and allow the solvent system to equilibrate for at least 1 h.

2 HPTLC Setup

a) Always handle HPTLC plates with gloves to avoid any contamination that could interfere with TLC staining.

b) HPTLC plates should be kept in a dry environment, if it is not the case the plates can be placed in an oven for about 10 min at 60–70 °C.

c) Use a soft pencil to indicate the sample position on the HPTLC. Samples and standards should be located at 1.5–2 cm above the bottom of the plate and at least 2 cm from each side to avoid any edge effects. Samples and standards (0.5–1 cm) should also be separated 1 cm from each other.

d) To improve repeatability between separate plates, a line is drawn at 1–1.5 cm from the top of the HPTLC plates and will be used to remove the plate when the solvent front reaches this line.

3 Sample Loading

a) If using dried samples, resuspend them in chloroform:methanol solution in 2:1 volume ratio.

b) Load your standards and samples using microcapillaries or fine gel-loading tips. Between each application evaporate the solvent using hair-dryer. Load samples evenly along the marked line drawn to improve reproducibility. Depending on the concentration of the sample a volume between 10 and 50 μL can be deposited.

4 Chromatogram Development

a) After removing the lid, carefully place the plate into the developing chamber. The solvent (or mobile phase) is drawn through the plate by capillary action and hence should start uniformly to avoid differences in running behavior between samples.

b) Remove the plate once the solvent front reaches the upper line of the HPTLC.

c) Dry the HPTLC plate using the hair dryer using cold air.

5 Visualization and Quantitative Determination

a) Place the HPTLC plate upright in a TLC spray box under a chemical hood.

b) Apply Orcinol Reagent using the fine mist sprayer in a zigzag pattern from one edge to another until the whole surface is covered.

c) Carefully remove the HPTLC from the spray box and place it on the heat plate or the oven at 150 °C for about 10 min.

d) Scan the HPTLC plate for further quantitation and analysis.


  1. Bhattacharya, S. K. (2017). Lipidomics. Methods in Molecular Biology, 1609.
* For Research Use Only. Not for use in diagnostic procedures.
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