CHOLESTEROL

LIEBERMANN BURCHARD REACTION

Making of L-B reagent

Before preparing the reagent, operator must wear rubber gloves for use. The reagent preparation made
use of a round-bottomed flask. The flask is supported in a bath of ice cubes and water to chill 20
volumes of acetic anhydride. When the chilled acetic acids temperature is about 5C, slowly add 1
volume of sulfuric acid with gentle swirling to mix. Allow mixture to remain chilled for 9 minutes, then
add 10 volumes of glacial acetic acid, swirl well to mix, and allow mixture to come up to room
temperature. Then transfer mixture to small propylene screw-capped bottles for usage.

There were two routinely used methods namely Liebermann-Burchard and Zak Reactions. The processes
are using oxidizing agents to oxidize the reactions. The oxidizing agent used Liebermann-Burchard is SO3
and in the Zak reaction Ferric ion. The final product of the Liebermann-Burchard is green while Zak
reaction is reddish violet.

STEPS:

The experiment uses four set-ups of 25-ml glass-stoppered centrifuge tube, one for the test, one for the
low standard, one for the high standard and one for the control. Pipette 0.5ml of specimen, add 5ml of
alcoholic potassium hydroxide to test and control tubes; stoppered and mix.

To the high standard, pipette 5ml of working standard and add 0.3ml of 33% potassium hydroxide. To
the low standard pipette 2.5ml of working standard, add 2.5ml of ethanol and 0.3ml of 33% potassium
hydroxide to it. Stopper both tubes and mix well. All four tubes are then heated in a 37C water bath for
55 minutes. Then cooled down to room temperature. Pipette 10ml of petroleum spirit or petroleum
ether to each of the four tubes and mixed. Then, add 5ml of distilled water to each four tubes,
stoppered and shake well for 1 minute. Then subject to centrifugation at low speed 1000-1500rpm for
five minutes.

For tests and control, pipette 4ml of petroleum spirit layer or the upper layer into 150 x 19mm tubes or
similar. From both the standards, pipette 3 ml aliquots into similar tubes. Place all tubes in water bath in
the fume hood at 60C. The petroleum spirit is evaporated off. The tubes are then allowed to cool.

Place all tubes plus an empty tube for reagent blank into a 25C water bath. At 1- minute intervals add 6
ml of Liebermann-Burchard reagent, mix on a vortex, seal with parafilm and placed back in water bath.
Wait and observe for a green-colored reaction. The green colored reaction is indicative of positive result.

R: Raphael, S.(1983) Lynchs Medical Laboratory Technology 4th ed. Philadelphia: W. B. Saunders

The experiment utilized a 25ml glass stoppered centrifuge tube. To this tube, 0.50 ml of specimen, and
5ml alcoholic potassium hydroxide were added, stoppered and mixed.
To make a standard, mix two 5 ml aliquots of cholesterol standard with 0.3ml 33% potassium hydroxide
in a 25-ml stoppered tube. The two tubes are incubated at 37C for 55 minutes. After 55 minutes, cool
the tubes to room temperature, the add 10 ml of petroleum ether to each tube and mix. Add 5ml water
to tubes, mix and shake for 1 minute. Then centrifuge at about 100rpm for 5 minutes or until emulsion
breaks.

For the standards, transfer 3 ml aliquots of petroleum ether layers, equaling to .6mg of cholesterol, to a
new test tube. For the unknowns, transfer duplicate 4 ml aliquots of petroleum ether layer to test tubes.
The petroleum is then evaporated with the aid of stream of dry Nitrogen. The tubes are cooled to room
temperature and stopper with dry, clean corks. Place all tubes in 25C water bath. add 6ml of
Liebermann-Burchard reagent to each tube at staggered intervals, mix and return to bath. A positive
result will give a green colored reaction.

R: Sonnenwirth, A. & Jarett, L. (1983) Gradwohls Clinical Laboratory Methods and Diagnosis 8th ed.
Missouri: C.V. Mosby Company