Thin layer Chromatography
Thin
layer Chromatography (TLC) :-
TLC is method of
analysis in which the stationary phase is spread as a thin layer on a rigid
supporting plate.
The mobile phase a
liquid is allowed to migrate across the stationary of plate.
TLC is a solid- liquid
form of Chromatography, where the s.p. is polar and m.p. is non polar.
Principle
:-
The principle of
separation is adsorption.When
a mixture containing one or more components are spotted on a TLC of absorbant
coated on a chromatographic plate and introduce into a m.p. which are
continuing distributing.
The m.p. is flow by
capillary action.
The components with
more affinity travel slow and that of lesser affinity travel faster.
Advantage
of TLC :-
- Simple method.
- Rapid technique.
- Low cost technique and
less time consuming.
- Applied for all types of
mixture.
- Detection is easy and not
tedious.
- Efficiency of separation :-
very small particle size can be ised which increase the efficiency of
separation. Flow rate is not altered because of the particle size since it
is not a closed column. It is planar type having thin layer of absorbant.
- Corrosive spray reagents can be
used with out damage plate.
Practical
Requirements :-
- Stationary phase
- Glass plate
- Preparation and activation of
TLC plates
- Application of sample
- Development of tank
- Mobile phase
- Development of technique
- Detecting agent or visualising
agent
1.
Stationary phase :-
A large number of
adsorbent can be used as stationary phase.
Choice of adsorbent:-
- Solubility of the substance :-
like hydrophilic or lipophilic
- Nature of the compound :- acidic,basic
or natural
- Chemically reactivity of
compound with the binder example is
- For inorganic absorbant :-
alumina, calcium carbonate
- For organic adsorbent :-
charcoal, dextrane etc
Mobile Phase, Activation of TLC Plates, Development, Detecting agent
Practical
Requirements :-
- Stationary phase
- Glass plate
- Preparation and activation of
TLC plates
- Application of sample
- Development of tank
- Mobile phase
- Development of technique
- Detecting agent or visualizing agent
Now discuss further
2.
Glass plates :-
Glass plate which are
specific dimensional like 20 × 20 cm ( full plate), 20×10 cm ( half plate), 20
× 5 cm (Quarter plate) can be used.
3.
Preparation and activation of TLC plates:-
Preparation of the TLC
plate :- there are mainly four method
1. Pouring :- The
adsorbent is made into an slurry and is poured into a glass plate which is kept
on a level surface.
The slurry is spread
uniformly over the surface and is dried in on oven.
2. Dipping :- whole
plate is dipped in slurry.
3. Spraying :- The
adsorbent is made into a suspension and is sprayed on a glass plate using a
sprayer.
4. Spreading :- The
adsorbent is made into a slurry and is placed in an applicator and is spreaded
on a plate by means of moving the applicator or plate.
4.
Activation of adsorbent layer :-
Since water or other
polar solvent greatly affect the development by adsorption chromatography they
should be removed from the adsorbent layer. plate can be activated by placing
in an oven at 120°-130° C° for about 30 minutes.
5.
Mobile phase :-
Ideal requirements of
mobile phase:-
- It should be safe.
- It should be non toxic.
- It should be easily available.
- It is not destory sample.
- Chemically inert.
As per ICH guide lines
mainly three types of the solvent
Class-I
Class -II
Class – III
In normal phase separation
non polar solvent like hexane, toluene, cyclohexane are useful and for reverse
phase, polar solvent give effective separation.
6.
Sample Application:-
Sample solution
containing analyte mixture is dissolved in suitable solvent and applied 1-2 mm
from the edge of the plate.
Sample is applied with
the help of fixed volume disposable capillaries or micropipette. For best
separation efficiency the spot diameter should be not more than 5 mm.
7.Development
of tank :-
By development of the
tank we are using less amount of mobile phase and use more number of TLC
plates.
When anew Method is
developed,it is better to develop in glass beakers, specimen jar, etc to avoid
more wastage of solvent.
New type of
development of the chamber in one chamber we have put two TLC plates.
Other types of new
development we have more than two plates are placed in chamber.
Development
of technique :-
Ascending method :
Development of chromatogram is usually carried out by ascending method,by using
special TLC chamber.
Chamber is allowed to
saturate with solvent vapour in the atmosphere. Plate is also allowed to
saturate with these solvent vapour if required by exposing the plate to solvent
vapour is closed chamber. Then the plate is dipped in developing to effect
separation of analyte mixture.
Horizontal,
sandwich,two dimensional technique are also use in TLC.
Visualisation
:-
After the development
of TLC plates, the spot should be visualized.
Detection
of spot can be done by two Method
A. Non specific method
:- It is used when the number of spot can be detected but not the exact nature
or type of compound.
Example
1. Iodine chamber :– where brown or Amber
spots are observed when the TLC plates are kept in a tank with few iodine
crystal at the bottom.
2. UV chamber for
flourescent compound :- when compound are view under uv chamber at 254nm or at
365 nm fluroscence can be detected.
3. Using fluroscence
stationary phase
4. Sulphuric acid
spray reagents.
B.
Specific method :- specific spray
reagents or detecting agent or visualising agent are used to find out the
nature of compound or for identification purpose.
Example
are
- Ferric chloride – for phenolic
compound
- Ninhydrin in Acetone – Amino
acid
- 3,5 – Dinitro benzoic acid –
for cardiac glycoside.
Identification
of solute :-
Similar to paper
Chromatography Rf value can be Calculated. Sometime Rf value are preferred to
be express as hrf. It is obtained by multiplying RF value by 100 e.g.0.15 RF
multiple by 100 give hrf value 15 which is convenient to report.
These observation can
be useful for qualitative or quantitative.
Application
of TLC :-
- TLC can be successful used for
isolation of vitamin like A,D,andE using silica gel.
- Tetracycline has been separated
in silica gel.
- Neomycin sulphate can be
isolating on activated compound.
- Amino acid protein and peptide
can be isolated and recovery by using silica gel as an adsorbent.
- Identification of drug.
- To detect decomposition product
in drug.