NINHYDRIN TEST

~Dissolve residue B in 2.0 ml of water. Add 1 ml of 0.1%ninhydrin reagent. Mix thoroughly and heat in
boiling water bath for two minutes.

~Note the color of the resulting solution.

a. Pupose: test for amines including alpha amino group.

b. Principle involved in the test: oxidative decarboxilation, deamination and condensation.
c. Observations: blue-violet solution.
d. Compound responsible for the visible result: blue-violet complex.
e. Sample equation using a lipid sample that gave a positive result.

f. Structural formulas of other lipids that will be tested positive in test.

glycine

Phosphatidyl-Serine
alanine

Leucine
SODA LIME TEST

~Grind a pinch amount of soda lime and residue B. Transfer the mixture in a dry test tube and heat
gently over a small flame. Test vapor by placing moistened litmus paper at the tip of the stirring rod.

~note the change in color of the stirring rods.

a. Pupose: test for amino and imino group.

b. Principle involved in the test: deamination.
c. Reagent: NaOH and CaO
d. Observations: R to B (lithmus paper)
e. Compound responsible for the visible result: hydroxyl group (OH)- basic
f. Sample equation using a lipid sample that gave a positive result:

Soda lime removes the amino group from an amino acid. Ammonia, the gas evolved during heating
reacts with water in the moistened lithmus paper to form ammonium hydroxide (responsible for R to B
change)

NH3 + H2O NH4 + + OH-

Possible identity of residuge B

From the above observations, we can say that the following lipids may be in the residue:

Sphingolipids, phosphatydylserine or any lipid with amino group.

g. Structural formulas of other lipids that will be tested positive in test.

sulfatides
LEIBERMAN BURCHARD TEST

~Prepare two clean and dry test tubes. In first tube, dissolve residue C in 1 ml methylene chloride. In
second test tube, place 1ml vegetable oil. Add 5 drops of acetic anhydride followed by three drops of
conc. sulfuric acid into both tubes. Mix thoroughly.

~Compare resulting intensity and color of solution.

a. Pupose: Unsaturated steroids (cholesterol)

b. Principle involved in the test: Oxidation
c. Reagents: Acetic anhydride and sulphuric acid
d. Observations:
Residue C: emerald green
Vegetable oil: blue green
+ VR: emerald green
e. Compound responsible for the visible result: steroid nucleus
f. Sample equation using a lipid sample that gave a positive result.

Possible identity of residue C: unsaturated steroid cholesterol

steroid cholesterol

g. Structural formulas of other lipids that will be tested positive in test.