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General and Specific Tests

General Tests
- Tests for Lipids

A. Solubility in Polar and Nonpolar Solvents

- Lipids are insoluble in polar solvents and soluble in nonpolar solvents. For this
test, the polar solvent is water; the nonpolar solvent is olive oil.

Distilled water
Olive oil

1. Add 1mL (20 drops) of the sample in each pair of liquids indicated.
2. Mix the contents of each tube by agitating.
3. Wait for 2 minutes.
4. Examine the tube carefully. Has the sample dissolved in the solvent or do you
see two separate layers in the tube?
5. Record your observations.
6. Save the tube for the Sudan Red test described below.

Positive Result:
Two separate layers in water and dissolved in olive oil

Solubility, Polarity

B. “Grease Spot” Test

- Lipids make unglazed paper (brown paper, writing paper) translucent.

1. Put a drop of the sample on a piece of unglazed paper.
2. Draw a circle around the spot with a soft pencil.
3. Allow the spot to dry thoroughly.
4. Hold the paper in front of a light source and observe the spot.

Positive Result:
C. Physical Properties
Color= palest yellow to greenish yellow
Appearance= mobile liquid
Odor= typical grapefruit

D. Refractive index at 20°C= 1.472-1.479

- As this test requires very little of the liquid, and is quickly performed, it is one of
the first test carried out with volatile oils and many other substances. Deviation from the
official range is prima facie evidence of adulteration, and where it is merely a question
of acceptance or rejection of the oil, there is no call for further detailed examination.

Positive Result:
Within the range of 1.472 – 1.479

Determination of concentration of volatile oils. Determination of oil whether it falls
at the expected range.

E. Differentiation test of Volatile oils from Fixed oils (Spot Test)

- Place a drop of the sample on a filter paper and let it dry.

Positive Result:
No stain in volatile oil

Principle: Volatile oils do not produce permanent spot unlike fixed oils.

Specific tests
-Test for Aldehyde

A. Baeyer’s Test
-Test for unsaturation
-Test for Multiple Bonds

Standards: Cyclohexane, Cyclohexene and Bromobenzene.

potassium permanganate
unknown in 0.5 mL reagent grade acetone

1. Dissolve 1 drop or 0.02 grams of the unknown in 0.5 mL reagent grade
2. Add a 1% aqueous solution of potassium permanganate dropwise with
3. If more than one drop of reagent is required to give a purple color to the
solution, unsaturation or an easily oxidized functional group is present.

Positive Result:
The disappearance of the KMnO4's purple color and the appearance of a brown
suspension of MnO2 is a positive test.

Principle: Oxidation

B. 2, 4- Dinitrophenylhydrazone Test
Test for aldehydes & sterically unhinedered ketone

Regent/s :
2,4-DNP reagent
95% ethanol

Procedure in making the reagent:

Add a solution of 3 grams of 2,4-DNP in 15 ml conc. H2SO4 slowly and with
stirring to a mixture of 20 ml of water and 70 ml of 95% ethanol.

1. Dissolve 2-3 drops of your test compound in 2 ml 95% ethanol in a small test
2. Mix this solution with 2 ml of the 2,4-DNP reagent.
3. Look for the formation of an orange-yellow precipitate to indicate the presence of
an aldehyde or ketone.

Positive Result:
Orange hydrazone ppt. indicates the presence of a ketone (conjugated) , yellow
hydrazone ppt. indicates an aldehyde (non-conjugated)


C. Fehling’s Test

Fehling’s A- 7 g copper II sulfate dissolved in 100 ml water
Fehling’s B- 35 g potassium sodium tartrate and 10 g NaOH in 100 ml water


1. With equal amounts of Fehling’s A and Fehling’s B to make 1 ml, add 3 drops of
2. Water bath for 10-15 minutes.

Positive Result:
Brick red precipitate

Oxidation of strong alkaline medium

D. Benedict’s Test

Benedict’s solution

Procedure in making the reagent:

1. Dissolve copper sulfate, sodium citrate and sodium carbonate in water. The
Cu2+ cation is solubilized by citrate. When the copper is reduced to form Cu +, it
precipitates as brick-red cuprous oxide

1. Add 2 ml Benedict’s solution to the test tubes
2. Add 2-3 drops of test compound to each test tube.
3. Heat the test tubes in boiling water for 10-15 mins.
4. Look for the formation first of a pale green color followed by the formation of
the reddish precipitate of cuprous oxide.

Positive Result:
Pale green color followed by the formation of the reddish precipitate

Oxidation in less basic medium

E. Bromine Test
Test for unsaturation

0.5% Br2 in CCl4

1. Place 5 drops of the sample in a dry test tube. Add 10 drops 0.5% Br2 in CCl4
reagent. Shake the test tube vigorously and observe the rate and extent
(colour change) by which the reagent is decolorized; the bromine color is
discharged. OPTIONAL: Place moistened blue litmus paper on a glass rod
across the mouth of the test tube and note any color change.

2. Compare with water as a negative control. If the reagent failed to decolorize
within 1 minute, expose the reaction mixture to sunlight. Note down the

Positive Result:
Colorless - Discharging of the bromine color without the evolution of hydrogen
bromide gas

Radical substitution reaction

F. Nitration Test
Test for aromaticity
Nitrating Mixture

1. Place 2 mL of conc. HNO3 in an Erlenmeyer flask. Immerse the flask in a water
bath and gradually add 2 mL conc. H2SO4. Cool the resulting mixture to room
temperature. This will serve as the nitrating mixture.

2. Place 5 drops of the sample in a dry test tube. Add 8 drops of the nitrating
mixture and shake the test tube to ensure complete mixing. Note the formation of
a yellow oily layer or droplet. Dilute with 20 drops of water.

3. If there is no apparent reaction observed within a minute, place the test tube in a
water bath (~50°C) for 10 minutes. Dilute with 20 drops water. Note down the

Positive Result:
Yellow oily layer


Experimental Result
General Tests

Experimental Result
Specific Test
Name of the Test (+) Result Experimental Actual Figure
1. Solubility in Two separate Two separate
Polar And Non layers in layers in
Polar Solvents water and water and
dissolved in dissolved in
olive oil olive oil

2. Grease Spot Translucent Translucent


3. Physical Color= palest Color= palest

Properties yellow – yellow –
greenish greenish
yellow yellow
Appearance= Appearance=
mobile liquid mobile liquid
Odour= Odour=
lemon-like lemon-like
4. Refractive Index Within the 1.472
range 1.472-

5. Differentiation No stain in No stain in

Test of volatile oil volatile oil
Volatile Oil from
Fixed Oil
(Spot Test)

Name of the (+) Result Experimental Actual Figure
Test Result
1. Bayer’s Test Disappearance of Disappearance of
purple color and purple color and
appearance of appearance of
brown suspension brown suspension
of MnO2 of MnO2

2. 2,4 DNP Yellow precipitate Yellow precipitate


3. Fehling’s Brick Red Brick Red

Test Precipitate Precipitate

4. Benedict’s Greenish to red Greenish to red


5. Bromine Colorless Colorless


6. Nitration Yellow oily layer Yellow oily layer