Thin-layer chromatography (TLC) of glycolipids

 Dissolve 200 mg of orcinol in 11.4 mL of H₂SO₄, and make up to 100 mL with DW.

 Store the reagent in a refrigerator.

 Dissolve 200 mg of resorcinol in 10 mL of DW, then add 80 mL of HCl and 0.25 mL of 0.1M CuSO₄.

 Make up to 100 mL with DW.

 Store the reagent in a refrigerator.

 Stock solution: Dissolve 100 mg of purimuline in 100 mL of DW.

 Dilute 1 mL of the stock solution with 100 mL of acetone-DW (4/1, v/v).

 Store the reagent in a refrigerator.

 Cut the TLC plate as necessary with a glass-cutting pen.

 Mark lines at the spot with a pencil.

 Dissolve the lipids in a small amount of CHCl₃/MeOH (1/2 or 2/1, v/v).

 Apply the samples to the TLC plate with a micro-syringe.

 Develop the TLC plate with an appropriate developing solvent as described below in a TLC chamber.

Solvent systems for analyzing neutral GSL and gangliosides

For neutral GSLs:

CHCl₃/MeOH/DW (65/35/8, v/v/v): for all neutral GSLs

CHCl₃/MeOH/DW (65/25/4, v/v/v): for non-polar neutral GSLs

CHCl₃/MeOH/DW (60/40/10, v/v/v): for polar neutral GSLs

For gangliosides:

CHCl₃/MeOH/0.02% CaCl₂ (5/4/1, v/v/v)

 Dry the plate and visualize the GSLs using appropriate staining reagents described below.

Visualization of GSLs with different staining reagents

Orcinol reagent:

Spray the reagent, and heat on a hot plate at 110°C until GSL bands are visible (~ 5 min).

Resolcinol reagent:

Place a clean glass plate on the hot plate at 95°C.

Spray the reagent on the TLC plate.

Put the TLC plate on a clean glass plate attaching the silica surface (sample-loading side) to the clean glass.

Heat at 95°C until GSL bands are visible (~ 10 min).

Purimuline reagent:

Spray the reagent until the TLC plate is wet.

Illuminate the plate with a UV transilluminator (~365 nm).