Biochem Lab 2

2.
EXTRACTION AND ESTIMATION OF TOTAL LIPID CONTENT
2.1.
OBJECTIVE
To extract and estimate total lipid content in a sample.
2.2.
INTRODUCTION
Lipids are relatively insoluble in water. They are soluble in non-polar solvents,
like ether, chloroform, methanol. Lipids have high energy content and are metabolized to
release calories. Lipids also act as electrical insulators, they insulate nerve axons. Fats
contain saturated fatty acids, they are solid at room temperatures. Example, animal fats.
Plant fats are unsaturated and are liquid at room temperatures. Pure fats are colorless,
they have extremely bland taste. The fats are sparingly soluble in water and hence are
described are hydrophobic substances. They are freely soluble in organic solvents like
ether, acetone and benzene. The melting point of fats depends on the length of the chain
of the constituent fatty acid and the degree of unsaturation. Geometric isomerism, the
presence of double bond in the unsaturated fatty acid of the lipid molecule produces
geometric or cis-trans isomerism. Fats have insulating capacity, they are bad conductors
of heat. Emulsification is the process by which a lipid mass is converted to a number of
small lipid droplets. The process of emulsification happens before the fats can be
absorbed by the intestinal walls. The fats are hydrolyzed by the enzyme lipases to yield
fatty acids and glycerol. The hydrolysis of fats by alkali is called saponification. This
reaction results in the formation of glycerol and salts of fatty acids called
soaps. Hydrolytic rancidity is caused by the growth of microorganisms which secrete
enzymes like lipases. These split fats into glycerol and free fatty acids.
Lipids are soluble in some organic solvents. This property of
specific solubility in non polar solvents is utilized for extracting lipid
from tissue. In biological materials, the lipids are generally bound to
proteins and they are, therefore, extracted either with mixture of
ethanol and diethyl ether or a mixture of chloroform and methanol.
Inclusion methanol or ethanol in the extraction medium helps in

breaking the bond between the lipids and protein.
2.3.
MATERIALS AND EQUIPMENT
Conical flasks
Separatory funnel
Seeds (mung bean)
Anhydrous sodium sulphate
Diethyl ether : ethanol mixture (2:1,v/v)
1% sodium chloride
Water bath
Rotavapor
Agitator
Pestle and mortar
2.4.
PROCEDURE
Take 2 g of the seeds and grind it in presence of 5 g anhydrous
sodium sulphate in a pestle and mortar.

Add 50 mL ethanol : diethyl ether mixture to it and transfer to an
air tight glass stoppered conical flask. Shake the content of the
flask on a mechanical shaker for 10 minute and then filter it.

Repeat the extraction of the residue and pool the filtrates.
Rotavap the filtrates to remove the solvent . Re-extract by
adding 9 mL of ethanol and 1 mL of diethyl ether containing 1%
sodium chloride.
Take the pooled fractions in a separatory funnel, shake it
thoroughly and allow it to stand for 5 minutes. The lipids will be
recovered in diethyl ether layer while soap, glycerol and other
water insoluble impurities move into another layer.

Collect the lipid containing fractions in a pre-weighed beaker.
Evaporate the solvent by keeping the beaker in oven-incubator
(65C) for 30 minutes.
2.5.
RESULTS
2.6.
2.7.
2.8.
Group
2.14. Weight of empty

beaker (g)
2.21. Weight of beaker

+ crude lipids (g)
Weight
2.28.
of
beaker
B 2.10. B 2.11. B 2.12. B 2.13. B

3
2.15. 6 2.16. 6 2.17. 6 2.18. 6 2.19. 6 2.20. 6

4.496
5.415
0.940
4.171
3.703
4.418
2.22. 6 2.23. 6 2.24. 6 2.25. 6 2.26. 6 2.27. 6

3.563
5.482
0.975
4.212
3.754
4.436
of
crude lipids (g) =

(weight
B 2.9.
crude lipid) (weight of
2.30. 0 2.31. 0 2.32. 0 2.33. 0 2.34. 0 2.35. 0

.0673
.0670
.0359
.0406
.0516
.0180
empty beaker)
2.29.
2.36.
2.37.
Table 1 : The weight of beaker and amount of crude lipids.
2.38.
2.39.
Grou
p
2.46.
Perc
2.40.
2.41.
2.42.
2.43.
2.44.
2.45.
B1
B2
B3
B4
B5
B6
2.47.
2.48.
2.49.
2.50.
2.51.
2.52.
3.36
3.35
1.79
2.03
2.58
0.90
entage of
crude
lipid
5
weight of crude lipid
100
2 g of seed
2.53.
2.54.
Table 2 : Percentage of crude lipid in 2 g mung bean
2.55. DISCUSSION
2.56. From the experiment, lipid can be extracted efficiently
using rotavapor which than the fat content can be determined by
filtrates to remove the solvent. Our group had chosen mung bean as
the sample for the experiment. The sample is prepared by mixing
with anhydrous sodium sulfate using a ratio 4 to 1. Sodium sulfate is
important to absorb any water from the sample. This mixture is
inserted into cellulose made thimble and placed inside the
rotavapor. The solvent used in this experiment is hexane which has
boiling point at 70 C. The rotavapor glassware with a roundbottomed flask is filled with boiling chips so that no splashing during
the evaporation. The evaporation and extraction took about an hour
and then the round-bottomed flask is removed and placed in the
heated water bath of the concentrator to remove all the solvent. The
fat content is calculated by this formula;
2.57.
Percentage of crude lipid =
weight of crude lipid

100
2 g of seed
2.58. The fat content of mung bean is 2.580 % of the sample.

2.59. Here are some important concept and principle in the
vacuum evaporators as a class function because lowering the
pressure above a bulk liquid lowers the boiling points of the
component liquids in it. Generally, the component liquids of interest
in applications of rotary evaporation are research solvents that one
desires to remove from a sample after an extraction, such as
following a natural product isolation or a step in an organic
synthesis. Liquid solvents can be removed without excessive
heating of what are often complex and sensitive solvent-solute
combinations.
2.60. Rotary evaporation is most often and conveniently
applied to separate "low boiling" solvents such a n-hexane or ethyl
acetate from compounds which are solid at room temperature and
pressure. However, careful application also allows removal of a

solvent from a sample containing a liquid compound if there is
minimal co-evaporation, and a sufficient difference in boiling points
at the chosen temperature and reduced pressure.
2.61. Evaporation under vacuum can also, in principle, be
performed using standard organic distillation glassware i.e.,
without rotation of the sample. The key advantages in use of a
rotary evaporator are that the centrifugal force and the frictional
force between the wall of the rotating flask and the liquid sample
result in the formation of a thin film of warm solvent being spread
over a large surface and the forces created by the rotation suppress
bumping.
The
combination
of
these
characteristics
and
the
conveniences built into modern rotary evaporators allow for quick,

gentle evaporation of solvents from most samples, even in the
hands of relatively in experienced users. Solvent remaining after
rotary evaporation can be removed by exposing the sample to even
deeper vacuum, on a more tightly sealed vacuum system, at
ambient or higher temperature.
2.62.
CONCLUSION
2.63.
2.64.
Our objectives are fulfilled because we managed to
extract the total lipid content from the sample of mung bean.
Furthermore we are also succeeded to determine the fat content which
is 2.580 % of that sample. From the experiment we know how to use
the rotavapor, its function and principle so that we can determine fat
content from any sample by ourselves.
2.65.
REFERENCES
2.66.
2.67.
McKee, T and Mckee, J.M. (2003). Carbohydrates in Biochemistry The
molecular basis of life, Third Edition. McGraw-Hill. New York. pp. 201-231.
2.68.
2.69.
Sawhney, S.K. and Randhir, S. (2005). Introdcutory practical Biochemistry.
Alpha Science International Ltd.,Harrow. U.K. pp.15-32.
2.70.
Harwood,
Laurence
M.;
Moody,
Christopher
J.
(1989).
Experimental organic chemistry: Principles and Practice (Illustrated

ed.). pp. 4751.

Biochem Lab 2

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2.

EXTRACTION AND ESTIMATION OF TOTAL LIPID CONTENT

To extract and estimate total lipid content in a sample.

Inclusion methanol or ethanol in the extraction medium helps in

MATERIALS AND EQUIPMENT

Take 2 g of the seeds and grind it in presence of 5 g anhydrous

sodium sulphate in a pestle and mortar.

flask on a mechanical shaker for 10 minute and then filter it.

water insoluble impurities move into another layer.

2.14. Weight of empty

2.21. Weight of beaker

B 2.10. B 2.11. B 2.12. B 2.13. B

2.15. 6 2.16. 6 2.17. 6 2.18. 6 2.19. 6 2.20. 6

2.22. 6 2.23. 6 2.24. 6 2.25. 6 2.26. 6 2.27. 6

crude lipids (g) =

crude lipid) (weight of

2.30. 0 2.31. 0 2.32. 0 2.33. 0 2.34. 0 2.35. 0

Table 1 : The weight of beaker and amount of crude lipids.

Table 2 : Percentage of crude lipid in 2 g mung bean

Percentage of crude lipid =

weight of crude lipid

2.58. The fat content of mung bean is 2.580 % of the sample.

pressure. However, careful application also allows removal of a

conveniences built into modern rotary evaporators allow for quick,

Our objectives are fulfilled because we managed to

Experimental organic chemistry: Principles and Practice (Illustrated

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