BCM 112 BIOCHEMISTRY I “Thin-Layer chromatography (TLC)”

BCM 112
BIOCHEMISTRY I
Week 7 Practical 5
“Thin-Layer chromatography (TLC)”
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Chromatography is an analytical procedure by which a multi-component
mixture is separated into its constituents with a differential migration
phenomenon. The constituents are separated by continuous
redistribution between two phases.
All chromatography systems consist of two phases: the first is solid
named stationary phase or immobilized phase may be solid, liquid or
solid/liquid and the second is the mobile phase, which flows over or
through the stationary phase may be liquid or gas.
There are different types of chromarography e.g. TLC
Definition of TLC :
Is a method of separating two or more chemical compounds in a solution
through their differential migrations across a thin layer of adsorbent
spread over a glass or plastic plate. that in which the stationary phase is
a thin layer of an adsorbent such as silica gel coated on a flat plate such
as glass, plastic plate or aluminium foil.
Principle :
Thin layer chromatography (TLC) is based on the separation of a mixture
of compounds as they migrate (with the help of a solvent system) through
a thin layer of adsorbent material. When using a nonpolar solvent system
(mobile phase), polar compounds are held back by the adsorbent while
non-polar compounds advance further.
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Procedure :
1. With your pencil, draw a line about 1 cm from the bottom edge of a
silica gel chromatography sheet.
2. On this line, apply 2 spots: the first for the lipid mixture and the second
for cholesterol as a reference. N.B. mark with your pencil “M” for the
mixture and “C” for cholesterol.
3. Put the TLC sheet vertically in a beaker containing 10 ml of a nonpolar
solvent system composed of petroleum ether : diethylether : glacial
acetic acid (70:30:1).
4. Cover the beaker immediately with aluminium foil.
5. Allow the solvent to rise to the appropriate level (about 1 cm from the
upper end of the sheet).
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6. Remove the TLC sheet from the beaker and immediately draw a line
with your pencil at the front line (level reached by the solvent).
7. Allow the solvent to evaporate and place the TLC sheet vertically in
another beaker containing few
crystals of iodine; cover the beaker
with a watch glass, iodine will
sublime and colorize the lipid spots
with a yellow color.
8. Remove the TLC sheet and
encircle spots with a pencil.
9. Record the Retention Factor (R f )
value for each spot. R f values are
numbers between zero and one, representing the position of the spot on
the TLC plate.
Calculating Retention Factors (R f ) for Thin Layer Chromatography:
R f values are numbers between zero and one, representing the position
of the spot on the TLC plate.
distance traveled by the spot
Rf = _______________________________________
distance traveled by the solvent
• Note that if the lipid mixture contains cholesterol, a spot with the
same Rf value of reference cholesterol is obtained.
• Comment on the polarity of compounds in the separated spots.
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Application and importance of Thin-Layer Chromatography In
Clinical Chemistry :
1. Lipid analysis, separation of different lipid classes from animal and
plant tissues;
a- simple lipids i.e. cholesterol esters, triglycerides, free fatty
acids, cholesterol and diacylglycerols.
b- Complex lipids such as phospholipids e.g. lecithin and
sphingomyelin (a semiquantitative ratio of these compounds in
amniotic fluid is widely used as an indicator of foetal lung maturity).
2. Analytical TLC is used in drug analysis, in cases of acute poisoning
3. Thin layer chromatography (TLC) is very useful to chemists as an
analytical technique to separate and identify the compounds in a
mixture.
4. Detection of abnormal components in inborn errors of metabolism in
the neonatal population e.g. inborn errors of carbohydrate metabolism.
TLC has been used to detect the presence of abnormal sugars in
blood, urine or faeces.
5. Amino-aciduria can be investigated in urine to detect amino acids by
using two dimensional TLC system.
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BCM 112
BIOCHEMISTRY I
Week 7 Practical 5
“Thin-Layer Chromatography (TLC)”
Student Name: …………………………………… Student number: ……………
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Detection of cholesterol in a lipid mixture by TLC:
Rf value of cholesterol spot = ………………………………………………
Does the mixture contain cholesterol ? ……………………………………
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