AC T IVIT Y 12: L IPIDS

LIEBERMANN-
BURCHARD TEST
FOR CHOLESTEROL
BY SHERWIN CARL R. DIMACUTAC

G R OU P 3
 PROCEDURE:

01 The Test tubes must be dry.

02 Prepare cholesterol solution

0.5 mL Pork Lard 5 mL Chloroform Cholesterol solution

ACTIVITY 12: LIPIDS | GROUP 3
 PROCEDURE:

03 Measure 2 mL of cholesterol solution and transfer to a different test tube.

04 Add 12 drops of acetic anhydride, mix

carefully.

ACTIVITY 12: LIPIDS | GROUP 3

 PROCEDURE:

05 Add 3 drops of concentrated sulfuric acid and mix carefully.

06 Watch for color changes.

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RESULT AND OBSERVATION
- After a few minutes, The formation of a green or green-blue color
is observed and that indicates the presence of cholesterol in a sample.

- If no blue-green color appears immediately, let the mixture stand up

10-15 minutes before discarding.

- Other color may appear earlier but they must revert back to green to
be positive for cholesterol.

- If a fat is rancid, it may produce muddy colors that interfere with

cholesterol test.

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 ACTIVITY 12: LIPIDS
REVIEW QUESTION #1

RQ1. What is the principle of the Liebermann-

Burchard and Salkowski Colorimetric assay? Explain
its principle and include their respective reagents used,
the individual procedures, and the results.

ACTIVITY 12: LIPIDS | GROUP 3

 ACTIVITY 12: LIPIDS | GROUP 3

LIEBERMANN- BURCHARD TEST PRINCIPLE:

- Liebermann – Burchard test is a chemical estimation of cholesterol. The cholesterol react as a typical
alcohol with a strong concentrated acids to give colored substances. In this test, acetic anhydride are used as
solvent and dehydrating agents, and the sulfuric acid is used as dehydrating and oxidizing agent. After
adding these acids to the cholesterol solution, a result is observed when the solution becomes red or pink,
then purple, blue and finally bluish – green color.
 REAGENTS:
- Acetic Anhydride - Sulfuric Acid -Chloroform -Cholesterol

PROCEDURE: RESULTS:
-In this test, dissolve a 0.5 mL of cholesterol
crystals in a 5 mL chloroform. Then measure a 2 Production of
mL cholesterol solution in chloroform in a dry test bluish-green color is a
tube.
positive result.
- Add 12 drops of Acetic anhydride
- Add 5 drops of concentrated sulfuric acid and
mix.
- Observe for possible reactions.
- The reaction progressed from pink to purple
to blue and eventually to bluish-green.
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 A C T I V I T Y 1 2 : L I P I D S | G R O UP 3

SALKOWSKI COLORIMETRIC ASSAY PRINCIPLE:

- Salkowski test is used to detect cholesterol in a solution. It is an important test used to detect
cholesterol depending on the colors (distinct and clear colors) that yield from the reaction of
cholesterol with concentrated sulfuric acid. In Salkowski reaction, red color is due to the formation
of bi-cholestadiene. In this reaction, concentrated sulfuric acid removes two molecules of water from
two molecules of cholesterol and bicholestadien is formed. Sulfuric acid sulphonates due to the
formation of bi-sulphonic acid of bi-cholestadiene color is produced when the result is positive.
 REAGENTS:
- Cholesterol - Chloroform -Concentrated sulfuric acid

RESULTS:
PROCEDURE:
The tube is allowed to stand
-Dissolve a small amount of cholesterol in 2 until the mixture separates into
mL of chloroform in a dry test tube two layers: The chloroform
- Add 2 mL of concentrated sulfuric acid and (top layer) is red and sulfuric
mix.
acid (bottom layer) is yellow.

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CONCLUSION:
- Both Liebermann- Burchard Test and
Salkowksi Test are Chemical Tests for Steroids.

- In Liebermann- Burchard Test, when chloroform solution of

steroid is treated with acetic anhydride and concentrated sulfuric
acid, green color is formed.

- In Salkowski test, when chloroform solution of steroid is

treated with concentrated sulfuric acid, red color is formed.

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REFERENCE:

Supriya, N. (n.d.) Qualitative Analysis of Lipids. Retrieved from https://www. Biology-

reader.com/qualitative-analysis-of-lipids.html#BurchardTest

Mad Hav University (n.d.) Identification Test for Steroids, Lipids and Biological Importance
of Lipids. Retrieved from https://www.madhavuniversity.edu.in/identification-test.html

(n.a.) (2020). 3-Qualitative tests of cholesterol. Retrieved from https://www. Uomustansiriyah

.edu.iq/media/lectures/6/6_2020_03_15!02_22_40_PM.pdf

Duaa, Z. et al. (n.d.) Qualitative Analysis for Carbohydrates. Retrieve from https://ww
w.kau.edu.sa/Files/0017586/Subjects/Bioc-371.pdf

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THANK YOU!
BY SHERWIN CARL R. DIMACUTAC

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