This document describes experiments to extract and analyze lipids from serum. In the first part, lipids were extracted from serum using chloroform-methanol and separated via centrifugation. The organic phase containing lipids was evaporated, redissolved in chloroform, and observed as a yellow liquid, indicating the presence of lipids. In the second part, thin layer chromatography was used to separate lipids in the extract on silica plates developed in two solvent systems, revealing at least two lipid components with different polarities. The experiments demonstrated techniques for lipid extraction and separation.

1. KENYATTA UNIVERSITY
SCHOOL OF MEDICINE.
DEPARTMENT OF MEDICAL BIOCHEMESTRY
NAME: LANDO ELVIS OTIENO
REG NO: P29S/16344/2015.
COURSE: MBCHB
LECTURER: Dr OKUN/Prof NJAGI/Mr NJOGU
HANDING DATE: 14 /7/2016
SUBJECT: PRACTICAL REPPORT ON EXTRACTION
OF SERUM LIPIDS
AND ON TLC OF LIPID EXTRACT.
ELVIS L
©

2. Background:
Lipids are a heterogeneous group of compounds synthesized by organisms that are
present in all biological tissues. These compounds are characterized as being sparingly soluble in
water,but highly soluble in nonpolar solvents (e.g., chloroform, ether, hexane, etc.). We can use
this solubility difference to extract and separate lipid constituents from the many polar
compounds that are contained in biological tissues. Lipids can be classified by a number of
schemes,but looking at them according to their biological function is most useful. Triglycerides
(or triacylglycerols) are fats and oils that serve as storage and transport forms of metabolic
energy. These compounds contain fatty acids (generally 3 molecules of saturated and unsaturated
in various combinations) that are esterified to a 3-carbon glycerol molecule. Waxes (long chain
fatty acids esterified to a long chain alcohol), hydrocarbons (long chain alkanes) and fatty
alcohols form protective surfaces on many plant and animal tissues. Lipids that are membrane
components are usually amphipathic and complex (phospholipids, sphingolipids, glycolipids).
Finally, there are a large group of low molecular weight, non-polar compounds that are not
derived from fatty acids (e.g., steroids, vitamins, carotenoids, quinones, etc.) and function as
hormones, vitamins, photopigments and other specialized roles. In the first part of the
experiment, you will obtain a crude lipid fraction from either plant and/or animal tissues.
MATERIALS/REQUIREMENTS:
1.fresh serum
2.extraction solvenr; chloroform:methanol(2:4)
3.methanol
4.chloroform
5.extraction tube
6.centrifuge
PROCEDURE:
1.Place 1.0ml of serum in an extraction tube and add 7.0ml of chloroform;methanol(2:1) mixture.
2.shake vigorously for 3min( a white precipitate will form)
3.Transfer the mixture to a centrifuge tube and centrifuge for 10min ,the aqueous and organic phase will
separate into two layers with a dense white precipitate of protein at the interface.
4Take care not to mix the two phase,remove all the upper aqueous phase and the white precipitate.discard
small portion of the organic layer to ensure complete removal of all water and protein.
5.Transfer the organic phase to a clean,dry 50ml beaker and carefully evaporate to near dryness on a hot
plate
6.When nearly dry , add 1.0ml methanol to aid in moving traces of water.

3. 7.when dry , add 0.5ml of chloroform to beaker to dissolve lipids
OBSERVATIONAND RESULTS:
Yellow liquid is observed when chloroform is added to the dried organic phase solution. Indicating
presence of lipid(s).
Total cholesterol HDL-cholesterol LDL-cholesterol*
Desirable <5.2 mmol/l ³0.91 mmol/l <3.36 mmol/l
Borderline-High-Risk 5.2-6.2 mmol/l
<0.91 mmol/l
3.36-4.13 mmol/l
High-Risk 6.2 mmol/l 4.14 mmol/l
DISCUSION AND CONCLUSION:
Neutral lipids or generally storage lipids are extracted with relatively non-polar solvents such as
chloroform but membrane-associated lipids are more polar and require polar solvents such as ethanol or
methanol to disrupt hydrogen bondings or electrostatic forces.
Alcohols are good solvents for most lipids( methanol and ethanol )
Chloroform is a solvent, particularly for lipids of intermediate polarity and when mixed with methanol it
becomes a general extraction solvent.
In the presence of some water,small molecules are dissolved in organic solvents. Among these
contaminants there are pigments, lipophilic amino acids, polypeptides and some hydrophobic proteins.
The removal of contaminants can be done by various ways: one of the ways is to evaporate the lipid
extract under vacuum, add a small amount of methanol which is an excellent denaturation agent and
evaporate again. Redissolve the dried lipid extract with a small volume of chloroform/methanol (2/1) and
transfer into another clean tube. Also all operations should be carried out in glass equipment with, if
required, ground-glass joints. Therefore the high sensitivity of the analytical methods needed for low
amounts of extracted lipids requires the use of very pure solvents and clean glassware. The chemical
nature of the extracted lipids must also be taken into consideration.
Furthermore, all lipids must be protected against degradation through oxidation by solvent, oxygen,
enzymes in combination with temperature and light.
Lipid transport in the circulation occurs in the form of lipoproteins (protein shells surrounding a lipid
core). Lipoproteins are classified according to their source, composition and physiological action. The
two types considered to be most important for cardiovascular risk assessment are low density lipoproteins
(LDL) and high density lipoproteins (HDL). They are involved in the transport of cholesterol to and away
from the body tissues, which led to the suggestion that the ratio LDL/HDL is an important indicator for
cardiovascular risk. Besides cholesterol, triglycerides are considered to play an independent role in the
formation of atherosclerotic lesions. In a recent review of the epidemiological, clinical, cellular and

4. molecular evidence of the role of triglycerides in athlerogenicity, conclusion is that high levels of
triglycerides are an important risk factor,especially for mild cases of atherosclerotic lesions. Fasting
triglycerides or the postprandial rise of triglycerides in response to a test meal have been found to be risk
factors.
2.PRACTICAL REPPORT ON TLC OF LIPID EXTRACT.
BACKGROUND:
TLC is somewhat similar in principle to the paper chromatography technique.the main difference is that
in place of the hydrated cellulose fribres of ,the absorbent or stationary phase in TLC is in the form of a
very thin film of powder material fixed on an inert rigid support such as glass plate.Silica and alumina are
relatively polar stationary phases. Both have OH groups on their surfaces that interact strongly with polar
compounds. Such compounds are adsorbed strongly and therefore move along the plate slowly, while
non-polar compounds are absorbed only weakly and are therefore carried along the plate more quickly. Of
course, solvent polarity also affects how fast compounds travel. Polar compounds are carried along
quickly by polar solvents, but move slowly or not at all with non-polar solvents. Because non-polar
compounds don't adhere strongly to the silica, they tend to move more quickly in most solvents.
Principles ofTLC.
Solvent front
Neutral lipids Polar lipids
Free fatty acids phosphatidylethanolamine
Cholesterol ester Phosphatidyl srine
triglycerides Phosphatidyl choline
cholesterol sphingomyelin
(non polar solvent) origin (polar solvent)
TLC is normally done on a small glass or plastic plate coated with a
thin layer of a solid — the most common are silica (SiO2) or alumina (Al2O3). This is the
stationary phase. The mobile phase is an organic solvent or solvent mixture. The sample
mixture is applied near the bottom of the plate as a small spot, then placed in a jar containing a

5. few ml of solvent. The solvent climbs up the plate by capillary action, carrying the sample
mixture along with it. Each compound in the mixture moves at a different rate,depending on its
solubility in the mobile phase and the strength of its absorption to the stationary phase. When
the solvent gets near the top of the plate, it is allowed to evaporate,leaving behind the
components of the mixture at various distances from the point of origin. The ratio of the distance
a compound moves to the distance the solvent moves is the Rf value (retention factor). This
value is characteristic of the compound, the solvent, and the stationary phase.
MATERIALS:
.chromatography solvent.
.chloroform: acetic acid(99.5 : 0.5)
.chloroform: methanol:acetic acid: water(25:14:4:2)
.TLC(2): silica gel on glass, 5 x 20cm
.capillary pipette
.chromatography jars(2)
.iodine vapour chamber.
PROCEDURE
1.Place two TLC plates on a clean dry work surface with coated side up.Handle plates only by the edges
and take care not to disturb the coating.
2.On each plate,make a single faint pencil mark approximately 2cm from one end and 3cm from either
side. Where sample spot will be made.
3. Carrefully apply the lipid extract to each plate at this spot, by dipping a capillary pipette into the extract
until the tip fills to a depth of about 1cm.this volume is about 10µl. Let the spot dry between each
application and take care to keep the spot small, repeat until the volume of about 20µl has been applied to
each plate.
4.Place one plate in the chromatography jar containing chloroform and acetic acid and the other in
chloroform,methanol,acetic acid and water mixture.Ascend about two thirds of the length of the
plate.(about 45minutes)
RESULTS:
Rf = Distance traveled by solute
Distance traveled by solvent band
-Rf of spots formed by the extract on plate paced in chloroform: acetic acid(99.5 : 0.5) containing jar were
0.12 and 0.97 respectively.

6. -Rf of spots formed by the extract on plate paced in chloroform: methanol:acetic acid: water(25:14:4:2)
were 0.24 and 0.94 respectively.
-Therefore the least polar phospholipid(lipid) in the extract was 0.12 in non polar solvent chloroform:
acetic acid(99.5 : 0.5) and the least polar being 0.97. While in polar solvent were 0.24(least polar) and
0.94( polar), i.e least polar as it was on the bottom of the chromatograph plate ,and the most polar
molecule was furthest up on the chromatograph silicon plate.
Discussion:
In this practical a technique called thin layer chromatography (TLC) was used. TLC is a form of
adsorption chromatography where the substances that are being separated are absorbed on to the matrix
and allow a solvent to pass through the thin layer matrix.
Thin layer chromatography can be performed on a sheet of glass, plastic or alluminium foil, which is then
coated with a thin layer of adsorbent material, usually silica gel, alluminium oxide or cellulose. The layer
of adsorbent is known as the stationary phase. After the sample has been applied on to the plate the
mobile phase (solvent mixture) is drawn up the plate via cappilary action.
As substances will interact with the matrix and the solvents in a specific manner, they are able to be
separated. In the case of lipids, they move at a rate which depends on their relative absorption to the
hydroxyl groups attached to the silica atoms and their solubility in the organic solvents.
Conclusion
Thin-layer chromatography (TLC) is an extremely valuable analytical technique in the analyzing polarity
absorbance of phospholipids. It provides a rapid separation of compounds, and thereby gives an indication
of the number and nature of the components of a mixture. TLC can also be used to identify compounds by
comparison with known samples, to check the purity of a compound, or to monitor the progress of a
reaction, an extraction, or a purification procedure.

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