In the Laboratory
Lipid Extraction and Cholesterol Quantification:
A Simple Protocol
M. Carmo Barreto
Departamento de Ciências Tecnológicas e Desenvolvimento, Universidade dos Açores, 9502 Ponta Delgada, Portugal;
barreto@notes.uac.pt
This experiment is of interest for introductory biochem-
istry classes, because it is simple and gives consistent, repro-
ducible results. The experiment involves disruption
techniques, extraction with solvents, separation with two-phase
systems, and a semi-quantitative analysis with a specific colo-
rimetric reagent. It was devised for a general biochemistry
course for first-year biology undergraduates. General biochem-
istry takes place in the second semester, after general chemis-
try (inorganic and organic). In the theoretical biochemistry
classes, these students are studying the classes of biological
molecules: carbohydrates, lipids, proteins, and nucleic acids.
Laboratory biochemistry classes, besides accompanying the
theory from the lectures, start with experiments that are more
qualitative and gradually advance to a more quantitative ap-
proach. This work is at an intermediate level, since it allows
for a semi-quantitative approach. It is designed for a two-hour
lab session, except for the evaporation of lipids to dryness,
which proceeds overnight. Similar articles reported in this Jour-
nal are designed for more advanced students and require sev-
eral three to four-hour sessions (e.g., 1–3).
As a result of an increasing concern about health issues,
comparison of cholesterol in different food sources has a spe-
cial attraction for most students. This practical experiment
provides an opportunity to discuss the influence of eating
habits on health. An example is the increased probability of
developing atherosclerosis in people with high cholesterol di-
ets, although other consequences of this type of diet include
cholesterol gallstones and liver dysfunction, among others (4).
The discussion on the advantages and disadvantages of ani-
mal versus vegetable sources of dietary fat also raises a lot of
interest, especially considering the fact that the number of
vegetarians and vegans has been steadily increasing in the stu-
dent population.
Basis
Isolation of lipids from natural sources is based on the
fact that these molecules are less polar than most cell com-
ponents and can therefore be selectively extracted with or-
ganic solvents. Egg yolk and walnut were chosen for their
high lipid content and also for their differences: (i) egg yolk
is very rich in cholesterol (5) while walnut is almost choles-
terol free (6) and (ii) egg yolk is fluid, which facilitates ex-
traction, while walnut has to be ground, to increase the
contact surface with the solvent mixture, and heated to help
release the lipids. The experiment can be enriched by includ-
ing other animal and vegetable sources.
www.JCE.DivCHED.org • Vol. 82 No. 1 January 2005
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Experiment
Lipid Extraction Method
The lipid extraction method was modified from Folch
et al. (7, 8). Before starting the work, the theory of lipid ex-
traction is discussed with the students, including safety pro-
cedures, stressing the fact that the experiment must be done
in a fume hood. Students work in pairs, so the extraction
step can be carried out at the same time for the egg yolk and
the walnut. An aliquot of the total lipid extract is collected
for cholesterol determination and the remaining “total lipid”
extracts are evaporated to dryness until the following day and
quantified by gravimetry.
Quantification of Cholesterol
Total lipids extracted as described above can be subse-
quently separated into lipid classes and quantified using chro-
matographic procedures. There are, however, specific tests
for some types of lipids that allow for a quantitative analysis
without a previous separation step. An example is the quan-
tification of “total cholesterol” (free cholesterol and choles-
terol esters) by the Liebermann–Buchard method. The basis
of this method is the reaction of cholesterol and cholesterol
esters with acetic anhydride and concentrated sulfuric acid,
resulting in the formation of a blue–green complex (9). This
reaction is used to determine cholesterol in the aliquots re-
moved at the end of the previous section, yielding results in
10–15 minutes. Students compare the colors resulting from
the Liebermann–Buchard reaction in egg yolk and walnut
lipid extracts with cholesterol standards and a chloroform
blank.
Hazards
Chloroform and methanol are flammable. Methanol is
toxic if inhaled, ingested, or absorbed by the skin. Chloro-
form is listed as irritant and possible carcinogenic. Inhala-
tion and ingestion are harmful and may be fatal.
Concentrated sulfuric acid is extremely corrosive. All work
must be done with acid-resistant gloves in a fume hood.
Discussion
After students have the results, including the mass of to-
tal lipid extracted, they write a brief report, including: (i) the
quantity of lipid per unit of mass of egg yolk or walnut; (ii)
the relative quantity of cholesterol in the samples assayed;
• Journal of Chemical Education 103
 In the Laboratory
and (iii) the advantages and disadvantages of vegetable and
animal food sources, considering cholesterol-related health
problems. This work can be expanded in further lab sessions:
(i) It can be done in a quantitative way, using a spectropho-
tometer; students can design a cholesterol standard curve and
measure the absorbance of the blue–green complex at 550
nm and (ii) lipids can be separated in lipid classes by TLC;
in that case, the lipid should be extract to dryness under ni-
trogen to avoid undesirable oxidations.
Conclusion
Although enzymatic methods are currently used to mea-
sure cholesterol levels, the method described here has the ad-
vantage of being simple and inexpensive, which is particularly
important when one has repeated lab sessions during the
week. Furthermore, it can be carried out on the organic phase
containing the lipids, without evaporating the solvent, yield-
ing almost immediate results. It is a experiment most stu-
dents find interesting and which leads to fruitful discussions
on scientific and health issues.
104 Journal of Chemical Education • Vol. 82 No. 1 January 2005
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Supplemental Material
Instructions for the students and notes for the instruc-
tor are available in this issue of JCE Online.
Literature Cited
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8. Cyberlipid Center–Resource Site for Fats and Oils. http://
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