2021

Pharmaceutical
analysis
Thin Layer Chromatography
Muhammad Wajid
Assistant Professor
Gulab Devi Institute of Pharmacy, GDEC, Lahore
 THIN LAYER CHROMATOGRAPHY

INTRODUCTION
In 1949, Mein hard and Hall first proposed Thin Layer Chromatography (TLC) method by
using the starch as binder to separate the inorganic ions. Kirchner proposed the conventional
ascending TLC method. TLC is a solid–liquid technique in which two phases are there: one is
stationary phase and other is mobile phase. The TLC principle was first introduced by the
Izmailov and Shraiber. Stahl was first designed the standard equipment for the TLC. It is
similar to that of the paper chromatography.

PRINCIPLE
The main principle involved in the TLC is the mobile phase flows through the stationary
phase where a solid or liquid is supported on the solid and carries the components of mixture
which will be separated into individual analytes. The separation is based upon the migration
of the analytes between the stationary phase and mobile phase. The components which have
more affinity towards the stationary phase elute later and which have less affinity elutes first.
There are mainly two types of TLC methods. They are as follows:

Normal phase TLC: Where the stationary phase is polar and the mobile phase is non-polar.

Reverse phase TLC: Where the stationary phase is non-polar and the mobile phase is the
polar.

THEORY

The theory of the TLC is as follows: the plastic or glass or aluminium sheet is coated with a
layer of silica gel or alumina powder. The silica gel used is in the form of silicon dioxide.

Silicon atoms are joined through the oxygen atoms in a giant covalent structure.
A very small amount of a sample solution of the substance to be analysed is applied as small
spot with a capillary tube on TLC plate 1 cm from the bottom. Then this TLC plate is
developed in a chamber containing mobile phase where the different types of solvents are
used. Then the mobile phase moves up through the TLC plate by capillary action which
dissolves the sample and moves up based on the inter molecular forces between the stationary
phase and mobile phase with sample components. The free particles are completely dissolved
in the liquid or gaseous mobile phase. Then the adsorbed particles are bound to the stationary
phase. This can be attained by the equilibrium between the mobile phase and stationary
phase. The equilibrium between the mobile phase and the stationary phase depends on the
following:

o The polarity and size of the sample.

o The polarity of the stationary phase molecules.
o The polarity of the mobile phase.

THE PROCESS OF TLC

Preparation of the plate: The preparations of the TLC plates are done by the slurry of the
adsorbent for example, silica gel, cellulose powder, etc., is spread uniformly over the plate by
means of commercially available spreaders. The adsorbent layer should have the thickness of
150–250 μm. Then these plates are dried at 80–90 °C for 30 min in hot air oven or air drying
over-night. There are different methods for applying the slurry of adsorbent on the plate.
They are as follows:
(a) Pouring: This is nothing but the direct pouring of the slurry on the plate and then
uniformly spreader by revolving the plate.

(b) Spreading: In this, the slurry is applied with the help of the applicator and then
moved over the plate and uniformly spread.

(c) Dipping: This method was first proposed by the PEIFER. In this method, the plates
are dipped in the slurry.
 (d) Spraying: This method was first proposed by the REITSEMA. This is carried out by
the spraying with the help of sprayer.
(e) Precoated plates: In this method, the adsorbents are pre-coated on glass or plastic
sheets. The main advantage is ready to use. The main disadvantage is these are very
expensive.

Activation of the plates: These plates are activated by placing the adsorbent-coated plates in
the oven at 50–60 °C for 15–20 min. This step is mainly to remove the water molecules
present in the slurry.

Marking the plate: The plate is marked with the help of pencil leaving 2–4 cm from the
bottom. The marking is also done by the marker pen instead of pencil to get accurate results.

Spotting of the sample: The sample applied with the help of micro syringe or with the help
of micro capillary to get the minute sample is spotted properly. The capillary is filled with the
sample solution where the sample is dissolved in the appropriate solvent and then allows the
solvent to evaporate from the spot completely.

Preparing the development tank: The plates of the TLC are generally developed by the
ascending technique where the plates are placed in the developing solvent that is mobile
phase. The tank is saturated with the solvent vapours. The care should be taken for the
complete closing of the chamber with the help of the lid. The development of the plates is
done by the immersing in the tank which contains the mobile phase. The plates are immersed
in such a way that the 0.5 cm of the plate is dipped in the solvent.

Solvents (mobile phase): The selection of solvent system is mainly based on the polarity and
nature of the sample to be analysed. Generally used solvents in the TLC are the following:

Very polar solvents: methanol, ethanol and isopropanol.

Moderately polar solvents: acetonitrile, ethyl acetate, chloroform and diethyl ether.
Non-polar solvents: cyclohexane, petroleum ether, hexane and pentane. The solvent
selection depends on the following factors:

o Nature of the sample.

o Nature of the stationary phase.
o Mode of chromatography.
o Mode of separation.

SELECTION OF MOBILE PHASE

Trial & Error: If one does not know about the nature of component of mix to be separated
the best elute is found by trial & error method.

Stahl’s triangle:

If one knows the chemical nature of solute to be separated it is possible to know a solvent by
using Stahl’s triangle.

Drying of the plate: The plate developed in the development tank is dried by means of
exposing to the air or placing it in the oven at 50–60 °C for 30 min.

Detection of components: The spots dried are detected by the exposure to the UV light or by
treating the spots with the chemical reagents. First the spots formed are lined with the help of
the pencil. The boundary of the spot is marked with pencil. Then the spot is detected with the
different methods. They are the following:

Iodination: The plates are placed in the chamber containing the iodine crystals.

Ninhydrin technique: Where the spots are sprayed with the ninhydrin reagent. This method
is mainly used for the determination of the compounds containing amine as a functional
group.
 o By using the fluorescent radiation.
o By using the UV radiation.

Rf measurement: The Rf is defined as the ratio of the distance of the centre of the spot
moved to that of the solvent front moved.

Rf value ranges from 0-1. Ideal values are 0.3-0.8. If Rf = 1 then analyte move along with m/p
without separating and if Rf = 0.1 then analyte is adsorbed to adsorbent and not moving.

Rx value:

Distance travelled by sample/distance travelled by standard. It is always closer to 1. M/P

allowed traveling throughout the plate.

RS Values:

Resolution is defined as the separation of the two analytes on TLC chromatogram which is
known as resolution commonly denoted as Rs.
Flow constant is determined by taking the migration rate of the solvent front which is denoted
by the K

Where K is the flow constant; Z is the distance between the solute front and solvent front; t is
the development time.

Rm values:

To find whether compounds belong to a homologous series. It is a combined value.

Delta Rm = log (1/Rf – 1)

Recovery of the components: The recovery of the sample is done by using the Craig tube
for the extraction of the solvent from the powder and to remove the adsorbent. Then the other
method used is the removing of the zones by means of the spatula and followed by the
extraction with solvent.
DEVELOPMENT TECHNIQUES

One dimensional development or vertical: Conventional method. Solvent flows against

gravity, because of capillary action. (Bottom to top).

Horizontal TLC: Plate is kept in horizontal manner. Spotting is done in middle of the plate
and M/P is added slowly through sides.

Descending TLC: Flow of solvent is assisted by gravity and hence development is faster.
Solvent holder is on top.

Multiple Development TLC: Similar to vertical TLC. After developing once, the plate
is dried and then again kept in same mobile phase (same composition) and in same direction.
Without detection multiple developments are done. It is done to separate some complex
mixture (more no. of compounds).

Step wise TLC: Plate size is bigger 30cm plate. Usually done when development
distance is long

ADVANTAGES OF TLC OVER OTHER TECHNIQUES

o Simple equipment compared to other methods.

o The sample preparation is simple.
o Mobile phase required is less.
o Less time consuming.
o There is wide choice of selection when compared to the other methods.
o Detected easily by using different methods.
o Low cost.
o Reliability is high.
o High sensitivity.
o High resolution.
o Inert stationary phase.

DISADVANTAGES OF TLC

o Over large spots formation.

o Uneven migration of the solvent front.
o Some spots are appeared as the streaked spots.
TROUBLE SHOOTING IN TLC

o Over migration of the spots: decrease the polarity of the solvent.

o Tailing of the peaks: reduce the concentration of the sample or decrease the acidity
or basicity.
o Distorted solvent front: equilibrate the mobile phase.
o Edge effect: Where the solvent front moves faster in middle of the plate than that of
the edges. Therefore spots are distorted and not regular. If chamber saturation is not
done edge effect occurs.

APPLICATIONS

o Used in the determination of the impurities from which the purity of the compounds
are determined.
o Used in the identification of the compounds:
 Stramonium leaf constituents are identified.
 Fixed oils are identified.
 Steroids are identified.
 Phenothiazines are identified.
o Used in the determination of the analgesics. Example: Paracetamol, analgin.
o Used in the determination of the biologically active substances.
o Used in the determination of preservatives, oxidants, surfactants and fixed oils.
o Used in the determination of pesticides in the water.
o Used in the determination of inorganic ions.
o Used in the determination of the reaction rate.
o Used in the determination of amino acids and peptides.
o Used in the inks analysis