QUALITATIVE TEST OF LIPIDS AND PREPARATION OF CARBOXYLIC ACID

SALT FROM LIPIDS

INTRODUCTION
Lipids are a large organic molecules that contain carbons (C), hydrogens (H) and
oxygens (O). It is insoluble in water but soluble in organic solvent such as alcohol, ether and
chloroform. Lipids are significant as it acts as energy storage, a major components of cell
membrane, heat insulator in animal and human, transport fat soluble vitamins, as signaling
molecules and important for buoyancy. (Lim, n.d.)
There are six classes of lipids which includes fatty acids, triacylglycerols,
glycerophospholipids, sphingolipids, waxes and isoprene-based lipids including steroids
(UiTM, n.d.). Reaction between oxoacid and hydroxyl compounds such as an alcohol or phenol
in the presence of dehydrating agent produces an ester. Due to the presence of oxo (-COOH)
and hydroxyl (-OH) groups in molecular structure, lipids classified as an ester namely
triglycerides. (Hassan, Yahya, & Ariffin, n.d.)
In this experiment, three different assays which are ester assay, phosphate assay and
cholesterol assay were used to determine the presence of functional group or molecule in olive
oil, butter solution, methanol extract, acetone extract and unknown samples. The unknown was
one of the samples and it determined based on comparison of Table 1 result. Besides, glycerol
and carboxylic acid salt which commonly known as soap were produced from lipids via
saponification process. (Hassan, Yahya, & Ariffin, n.d.)

OBJECTIVES
i. To determine the presence of functional group or molecule in olive oil, butter solution,
methanol extract, acetone extract and unknown samples using ester assay, phosphate assay
and cholesterol assay.
ii. To prepare carboxylic acid salt from lipids
iii. To identify the unknown sample

MATERIALS
Extraction of egg yolk Chloroform
Chloroform solution of olive oil (1%) Methylene chloride
Chloroform solution of butter (0.5%) Acetic anhydride
Chloroform solution of X (unknown) Sulphuric acid
Ethanol
2M hydroxylamine hydrochloride
3.5M NaOH
HCL (5M and 1M)
Ferric chloride in 0.1M HCL (5%)
Ammonium Molybdate in 4M nitric acid
(6%)
PROCEDURES
1a. Sample preparation (extraction from egg yolk)
i. An egg was cracked open gently. Do Not Bust The Yolk
ii. The yolk was separated from the white using the shell. The white was discarded.
iii. A glass centrifuge tube was tared. The yolk was added and its mass and volume was
recorded.
iv. The yolk was shaken vigorously to homogenize it for one minute.
v. The yolk was split into 2 equal parts and the following steps were performed.

1b. Acetone Extraction

i. An equal volume of acetone was added for one half of the egg. This step was done in a
glass centrifuge tubes.
ii. The mixture was shaken vigorously for 2 minutes.
iii. The mixture was centrifuged.
iv. Acetone was decanted into the mixture.
v. Steps 6-9 were repeated 3 times.
vi. Acetone fractions were pooled and its was allowed to evaporate in the hood.
vii. The acetone extract was containing the cholesterol and yolk pigments. It was saved for the
cholesterol assay.

1c. Chlorofom:Methanol Extraction

i. An equal volume of chloroform/methanol (2:1) was added to the second fraction.
ii. The cap was closed and inverted and the tube was shaken vigorously for 3 minutes.
iii. The mixture was filtered using glass wool.
iv. The filtrate was pooled and the solvent was allowed to evaporate in the hood.
v. The chloroform/methanol extract was containing all the neutral and polar lipids in the egg.

1d. Preparation of Carboxylic Acid Salt from lipid

i. 170g NaOH was added to 590ml of distilled water. This was the lye.
ii. 12ml lye was added slowly with 27g of olive oil and mixed.
iii. Fragrance and food colouring was added.
iv. The product was weighted and was keeps for 2 weeks.
v. After 2 weeks, slice of the soda ash was ready to use.
2a. Ester Assay
i. 2 drops of sample were put into a test tube.
ii. The solvent was dry off with a gentle stream of air.
iii. The sample was dissolved with 0.5 mL of ethanol.
iv. 2 drops each of 2M hydroxylamine hydrochloride and 3.5M of NaOH were added.
v. The solvent was mixed well and the test tube was heated in a boiling water bath for 30
seconds.
vi. 2 drops of 5M HCl and 1 drop of 5% ferric chloride were added in 0.1M HCl.
vii. The solvent was mixed well and a magenta-colored complex that indicates a positive result
was observed.
2b. Phosphate Assay
i. 2 drops of sample were put into a test tube.
ii. The solvent was dry off with a gentle stream of air.
iii. 2 drops of 10% magnesium nitrate were added in 95% ethanol.
iv. The test tube was heated over a flame in the fume hood until the solvent evaporates.
v. The test tube was allowed to cool when brown gas stops evolving.
vi. 6 drops of 1M HCl were added and the test tube was heated in a boiling water bath for 15
minutes.
vii. 6 drops of 6% ammonium molybdate were added in 4M nitric acid.
viii. The solvent was mixed well and a fine yellow precipitate that indicates a positive result
was observed within 10 minutes.
2c. Cholesterol Assay
i. 2 drops of sample were put into a test tube.
ii. The solvent was dry off with a gentle stream of air.
iii. The sample was dissolved with 10 drops of methylene chloride.
iv. 6 drops of acetic anhydride and 2 drops of concentrated sulphuric acid were added.
v. The solvent was mixed well and a greenish color that indicate a positive result was
observed after a few minutes.
RESULTS
Table 1
Results ( positive (+) or negative (-) )
Sample Ester Assay Phosphate Assay Cholesterol Assay
Olive oil - - +
: The colorless : The colorless : The colorless
solution unchanged solution unchanged solution turned into
greenish
Butter + - -
: The colorless : The colorless : The colorless
solution turned into solution unchanged solution unchanged
magenta-colored
complex
Chloroform - - -
(Methanol extract) : The colorless : The colorless : The colorless
solution unchanged solution unchanged solution unchanged
Unknown + -
: The colorless : The colorless
solution turned into solution unchanged
magenta-colored
complex
Acetone extract +
: The colorless
solution turned into
greenish

i. Why soap is referred as a ‘salt’?

Because sodium hydroxide or potassium hydroxide is used to neutralize the fatty acids and
convert it into salts. Thus, soap is a sodium or potassium salts of long chain fatty acids that
produced via saponification of fat.

ii. What kind of molecular properties enable substances to dissolve in water?

The existence of attractive force known as dipole dipole forces between water and solutes
molecules allowing the substances to mix properly.

iii. Show the structures of reaction for saponification of ethyl acetate?

DISCUSSION
In this experiment, three qualitative tests of lipids which are ester assay, phosphate assay
and cholesterol assay were performed on different samples which are olive oil, butter solution,
methanol extract, acetone extract and unknown samples. The purpose is to determine the presence
of functional group or molecule in olive oil, butter solution, methanol extract, acetone extract and
unknown samples. Besides, the saponification process of ester produces glycerol.and carboxylic
acid salt which commonly known as soap.
In practical 2, the ester assay is used because it able to determine the presence of ester
bonds in different samples. It is because the reaction between the hydroxyl group of glycerol in
the sample with the carboxyl group formed ester bonds. Then, the ester reacted with 2M
hydroxylamine hydrocloride and converted to hydroxamic acids. The reaction between
hydroxamic acids and 5% of ferric chloride in 0.1M HCl produced magenta-coloured complex
which indicates positive result. The colour of sample changed from colorless into magenta. Based
on Table 1, only butter and unknown show positive results. Thus, the functional group or molecule
that present in butter and unknown samples is ester.
In practical 3, the phosphate assay used to determine the presence of phospholipids in
different samples. Each sample is acidified with 4M nitric acid and 6% ammonium molybdate.
The formation of a bright yellow precipitate layer of ammonium phosphomolybdate indicates the
presence of phosphate ions. A fine yellow precipitate which indicating the presence of
Phospholipid biosurfactant may formed slowly afterward. Based on the Table 1, there is no sample
reacted with the test hence the color of sample remain colorless. It is expected for the phospholipid
sample to test positive, but they did not. This is maybe because of several error that occur during
conducting this experiment which is the phospholipid sample are not homogenize properly thus
resulting in suspension at the bottom of the bottle. This because the concentration of sample is low
therefore difficult to identify the colour change.
In practical 4, the cholesterol assay used to determine the presence of cholesterol in
different samples. When a mixture of concentrated sulphuric acid and acetic anhydride is added to
a cholesterol sample in the methylene chloride, steroid that containing either two double bond in
the same ring or a double bond adjacent to the ring containing a double bond or hydroxyl group
changed the colorless solution to greenish after a few minutes. This indicates that the result is
positive. Based on Table 1, only olive oil and acetone extract show positive results. Thus, , the
functional group or molecule that presents in olive oil and acetone extract is cholesterol.
Based on Table 1, the unknown sample was identified as butter because it has same results
as butter.
CONCLUSION
At the end of experiment, the functional group or molecule presents in olive oil, butter
solution, methanol extract, acetone extract and unknown samples were able to determine using
three different assays which are ester assay, phosphate assay and cholesterol assay, carboxylic acid
salt also known as soap were able to prepare from lipids and the unknown sample was identified
as butter.

REFERENCES

Hassan, W. M., Yahya, M. R., & Ariffin, Z. Z. (n.d.). Biochemistry laboratory manual. Universiti
Teknologi MARA.
Lim, C.Y. (n.d.). Biology Unit Labuan Matriculation College. Labuan Matriculation College.
UiTM. (n.d.). Biochemistry notes.