an assignment on thin layer chromatography

an assignment on thin layer chromatography

1. An assignment on Thin layer chromatography (TLC)
Introduction: TLC is one of the simplest, fastest, easiest and least expensive of several
chromatographic techniques used in qualitative and quantitative analysis to separate organic
compounds and to test the purity of compounds.
Definition: Thin layer chromatography can be defined as a method of separation or
identification of a mixture of components into individual components by using finely divided
adsorbent solid/liquid spread over glass plate and liquid as a mobile phase.
TLC is a form of liquid chromatography consisting of:
 A mobile phase (developing solvent)
 A stationary phase (a plate or strip coated with a form of silica gel)
 Analysis is performed on a flat surface under atmospheric pressure and room
temperature.
Classes: The most common classes of TLC are:
 Normal phase
 Reversed phase
Normal phase: Normal phase is the terminology used when the stationary phase is polar; as for
example silica gel and the mobile phase is an organic solvent or a mixture of organic solvents
which is less polar than the stationary phase.
Reversed phase: Reversed phase is the technology used when the stationary phase is silica
bonded with an organic substance such as a long chain aliphatic acid like C-18 and the mobile
phase is a mixture of water and organic solvent which is more polar than the stationary phase.

2. Procedure of TLC :

Step 1: Prepare the developing
container
The developing container for TLC can be a
specially designed chamber, a jar with a lid, or
a beaker with a watch glass on the top. Pour
solvent into the chamber to a depth of just less
than 0.5 cm. To aid in the saturation of the
TLC chamber with solvent vapors. Cover the
beaker with a watch glass.

Step 2: Prepare the TLC plate
TLC plates are purchased as 5 cm x 20 cm
sheets. Each large sheet is cut horizontally into
plates which are 5 cm tall by various widths.

Step 3: Spot the TLC plate
If the sample is not already in solution,
dissolve about 1 mg in 1 mL of a volatile
solvent such as hexanes, ethyl acetate, or
methylene chloride. As a rule of thumb, a
concentration of 1% usually works well for
TLC analysis.

3. Step 4: Develop the plate
Place the prepared TLC plate in the developing
beaker, cover the beaker with the watch glass,
and leave it undisturbed on bench top. The
solvent will rise up the TLC plate by capillary
action. Make sure the solvent does not cover
the spot.

Allow the plate to develop until the solvent is
about half a centimeter below the top of the
plate. Remove the plate from the beaker and
immediately mark the solvent front with a
pencil. Allow the plate to dry.

Step 5: Visualize the spots
If there are any colored spots, circle them
lightly with a pencil. Most samples are not
colored and need to be visualized with a UV
lamp. Hold a UV lamp over the plate and
circle any spots you see. Beware! UV light is
damaging both to your eyes and to your skin!
Make sure you are wearing your goggles and
do not look directly into the lamp. Protect your
skin by wearing gloves.
Application:
 Identification and purity testing of drugs
 Clinical chemistry, Forensic chemistry and Biochemistry
 Cosmology
 Food analysis
 Environmental analysis
 Analysis of Inorganic substance

4. Advantages of TLC:
 Simple method and cost of the equipment is low
 Rapid technique
 Any type of compound can be analyzed
 Corrosive spray reagents can be used without damaging the plate and needs less solvent
 Requires minimal training
 Adoptable to most pharmaceuticals.

Conclusion: Thin layer chromatography is done exactly as it says - using a thin, uniform layer
of silica gel or alumina coated onto a piece of glass, metal or rigid plastic. It is most common and
easiest method.



References:
1) Touchstone, Joseph C. Practice of thin layer chromatography. 2nd edition. New York:
Wiley, 1983.Print.
2) Pharmaceutical drug analysis, by Ashutosh Kar, 2001 edition, page no-515
3) A text book of pharmaceutical analysis by Dr.Swadodkar, A.V.Kasture, Dr.H.N.More,
Volume-II, 19
th
edition, June 2010, page no: 18-27.