LOCALIZATION AND IDENTIFICATION

OF PHOSPHOLIPIDS ON TLC PLATES

Phospholipids can be located on TLC plates first by non-specific techniques such as primuline spray but other reagents are needed to identify accurately individual components.
While primuline detection is non-destructive, the others are destructive and no fatty acid composition can be determined. They can be used after a primuline spray.

Detection of phosphorus

Many methods are available but we have selected two recipes which are simple and efficient to assert that one TLC spot contains a phosphorylated molecule. The second one is more sensitive than the first sine 0.01 mg of phosphorus can be detected in a 5 mm diameter spot.

1. Dittmer reagent:

solution A: dissolve by heating 4 g MoO₃ in 100 ml conc. H₂SO₄
solution B: dissolve by heating 0.18 g Mo (metallic molybdene) in 50 ml of solution A
Stock solution: after cooling, mix 50 ml solution B with 50 ml solution A
This mixture is stable at least one year in a closed vial.

Procedure:

When necessary, mix 2.5 ml of the stock solution with 5 ml water and 7.5 ml ethanol
Spray lightly the TLC plates in an efficient fume hood, phospholipids appear within 5-10 min at room temperature as blue spots on a white background. Less than 1 µg P can be detected with this reagent.

It must be noticed that a simple modification of the previous method allows it to be used also as a specific detection reagent for phosphonolipids (Stillway LW et al., J Lipid Res 1980, 21, 1141). Briefly, after viewing the phospholipid spots, the plates are heated at 100°C for 15 min. The background of the plate became a dark blue color, and the lipid spots became a dark brown color. Upon cooling for 15-30 min the blue background became colorless. The phosphonolipid spots appeared a brilliant blue color, but all the others remained dark brown. The blue color remained stable for several days.

2. Malachite green reagent:

Add a solution of Na₂MoO₄ (5g) in 2 M HCl (150 ml) to a solution of malachite green (1 g) in 2M HCl (100 ml); mix and dilute to 500 ml with water. After 1-2 h, filter the mixture and keep the reagent in the dark, it remains stable for several months. Filter the solution before use.

Procedure:

Dry completely the TLC plates
Spray the plates with 60-70 % HClO₄, place the plates on a hot plates and warm until a surface temperature of 250°C. 
After the disappearance of dark spots and evaporation of the acid, remove the plates and, after cooling, spray them thoroughly with the malachite reagent so as to obtain a uniform orange background. After a few minutes, bright green spots of phosphorus compounds will appear.

Detection of choline

Phosphatidylcholine, its lyso derivative and sphingomyelin give a positive orange reaction with the Dragendorff reagent.

Dragendorff reagent:

Solution A: Dissolve 0.5 g bismuth nitrate (Bi(NO₃)₃ 5H₂O) in 20 ml of 20% acetic acid
Solution B: 5 ml of a 40% KI solution in water

Procedure:

Before use, mix 20 ml solution A, 5 ml solution B and 70 ml water
Spray the dry TLC plates with the working solution in an efficient fume hood. Choline-containing lipids appear as orange spots within some minutes.

Detection of amino groups

Phosphatidylethanolamine, phosphatidylserine and their related lyso compounds can be detected with the help of a ninhydrin reagent since they contain a free amino group.

Ninhydrin reagent:

Dissolve 0.2 g of ninhydrin in 100 ml of butanol saturated with water (agitate butanol with water and separate the lower phase).

Procedure:

Spray the TLC plates with the ninhydrin reagent in an efficient fume hood, cover with a glass plate and heat in an oven at 100°C. Lipids with amino groups appear as red-violet spots. As little as 1 µg of lipid can be detected.
Use gloves to prepare the reagent, spray and manipulate the plates.

Detection of plasmalogens

Plasmalogen phospholipids are not separated from the diacyl or alkyl forms by TLC but their presence can be detected with a simple reagent.

Reagent:

Dissolve 0.4 g of 2,4-dinitrophenylhydrazine in 100 ml of 3 M HCl.

Procedure:

Spray the TLC plates with the reagent in an efficient fume hood and warm in an oven at 100°C. Spots containing plasmalogens appear yellow-orange on a white background.