• Small quantities of lipid material extracted from plant tissues may be tested by simple tests such as checking the greasy spot left on paper when stained with it, or by checking its insoluble nature in water.
• When obtained in quantity specific gravity of lipids is usually in the range 0.87–0.97.
• Fixed oils, fats and waxes in general are defined by their solubility, consistency, hardness, colour, specific gravity, viscosity, melting point, solidifying point, optical rotation, and refractive index.
• Some well-known chemical tests may be used to identify fixed oils like Halphen test for cottonseed oil, Baudouin test for sesame oil, and Fitelson test for tea seed oil.
• On account of the complexity of mixture of fatty acids normally present in fixed oils, no single method is available for their separation or analysis. Chemical parameters like acid value, iodine value, saponification value, acetyl value, Reichert-meissl value, and peroxide value are used as indicators for the quality of fixed oils.

Plant lipids may be conveniently identified by chromatographic techniques. For a preliminary analysis and to identify lipid fractions or products of saponification, TLC is best suited. Isolated lipid components or products of hydrolysis can be derivatized into methyl esters and analyzed directly by GLC.

1. Preliminary identification of the total lipids of plant tissue may be undertaken by TLC using appropriate marker lipids. Neutral lipids such as oils and fats may be analyzed by development on silica gel using isopropyl ether-acetic acid (24:1) and petroleum ether-ethyl ether-acetic acid (90:10:1) as successive solvents systems on the same TLC. For detection the plate is sprayed with 25% sulphuric acid, followed by heating the plate to 230°C. Glycerides and hydrocarbons are seen as pale brown-coloured spots. Hydrocarbons are seen near the solvent front and phospholipids remain near the origin. Triglycerides move ahead of diglycerides, which are ahead of monoglycerides.
2. Phospholipids and glycolipids may be identified on TLC in silica gel plates developed in chloroform-methanol-acetic acid-water (170:30:20:7). The plate is sprayed with a solution of 0.2% ethanolic 2′,7′-dichlorofluorescein. Presence of fluorescent green spots in UV light, against a purplish violet background indicates the presence of phospholipids and glycolipids.
3. To identify the lipid composition, the total lipids isolated-neutral or polar lipids are subjected to alkaline or acid saponification. Acid hydrolysis is carried out with 2M sulphuric acid under nitrogen at 100°C for 6 hours. Upon dilution with water, the fatty components are extracted into chloroform.
   • The aqueous portion is neutralized and further analyzed by TLC for the presence of glycerol, galactose, phosphates, etc. Paper chromatography in n-butanol-pyridine-water (7:3:1) for about 40 hours along with the authentic sample of glycerol or sugars will reveal their presence when activated with alkaline silver nitrate reagent. For phosphate detection, TLC of the neutralized aqueous portion on silica gel in methanol-1M ammonia-10% trichloroacetic acid-water (10:3:1:6) is done and activated by spraying with 1% aqueous ammonium molybdate followed by 1% stannous chloride in 10% hydrochloric acid. Presence of blue spots indicates the presence of phosphate and hence phospholipids.
   • The chloroform portion is analyzed for the presence of amines and fatty acids. Alternatively, the lipid may be saponified by heating under reflux with 5% ethanolic potassium hydroxide on a water bath for 1 hour. It is then filtered and acidified. This or the chloroform layer obtained in the acid hydrolysis may be analyzed by GLC for the determination of its fatty acid composition.
4. Fatty acids may also be isolated from the saponification mixture by fractional crystallization, fractional distillation, preparation and debromination of polybromides, column chromatography, or by the formation of urea-fatty acid complexes.
5. Plant waxes on the other hand may be directly extracted by quickly dipping the unbroken leaves or stems in hexane or ether. In so doing, the surface waxes are extracted into the solvent without disturbing the cellular contents.
   • On concentration the extracted wax may be further fractionated by passage through an alumina column to separate into alkanes or related hydrocarbons.
   • Direct TLC of the wax dissolved in chloroform on silica gel in chloroform-benzene (1:1) and development by spraying with 0.5% ethanolic Rhodamine B fluorescein will indicate the presence of hydrocarbons and other fractions as yellowish spots against a pink background.
   • Constitution of waxes is deduced by studying the products of hydrolysis. Saponification of waxes is generally more difficult than that of saponification of fats.