Thin layer Chromatography (TLC)

Definition
Principle
Steps in TLC Analysis
Development Techniques
Detection
Application of TLC

1. S.GOKULAKRISHNAN
M.Pharm (Pharmaceutics) – I Year,
Department of Pharmaceutics,
College of Pharmacy,
Mother Theresa Post Graduate and Research Institute of Health Sciences,
(A Government of Puducherry Institution)
Puducherry.

THIN LAYER CHROMATOGRAPHY
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INTRODUCTION
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.

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Michael Tswett is credited as being the father
of liquid chromatography. Tswett developed
his ideas in the early 1900’s.

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TLC



•The two most common classes of TLC
are:

– Normal phase

– Reversed phase

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Normal Phase




Normal phase is the terminology used when the
stationary phase is polar; 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.

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Reversed Phase




Reversed phase is the terminology used when the
stationary phase is a silica bonded with an organic
substrate 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.

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THIN LAYER
CHROMATOGRAPHY

Thin layer chromatography is used to separate mixtures of substances into
their components.

Similar to P.C, except that a thin layer of some inert material,
i.e. Aluminium oxide, mag.oxid. , sili.oxide is used instead of
paper.

A layer of any one of these oxide is made from a slurry of power in a
suitable inert solvent.
Slurry is spread over a flat surface ( glass, metal or rigid plastic ) &
dried.

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PRINCIPLE

ADSORPTION

The component with more affinity towards the S.P travels slower

The component with lesser affinity towards the S.P travels faster

ADVANTAGES OF TLC
Simple mtd. & cost of the equipment is low
Rapid technique & not time consuming like C.C
Separation of µg of the substances can be achieved
Any type of compound can be analyzed
Corrosive spray reagents can be used without damaging the plate &
needs less solvent

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STEPS IN TLC ANALYSIS



The following are the important components of a typical
TLC system:

 Apparatus (developing chamber)

 Stationary phase layer and mobile phase
 Application of sample

 Development of the plate

 Detection of analyte

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GENERAL PROCEDURE


 Decide if you are going to do Normal or Reversed
phase chromatography.

 Prepare a plate or select a plate with the proper sorbent
material.

 Prepare the mobile phase

 Mark the plate

 Apply the sample
 Develop the plate

 Detect the analytes

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PRACTICAL REQUIREMENTS

STATIONARY PHASE

Adsorbents mixed with water or other solvents→ slurry

Silica gel H ( Silica gel with out binder )

Silica gel G ( Silica gel + CaSO4 )

Silica GF (Silica gel + binder + fluorescent indicator)

Alumina, Cellulose powder.

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COATER, HAND OPERATED

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2. GLASS PLATE

Specific dimensions- 20cm Х 20cm, 20cm Х 10cm, 20cm Х 5cm Microscopic slides can also be used
Plates should be of good quality & withstand high temperatures

PREPARATION & ACTIVATION OF TLC PLATES

1.Pouring ( simplest methods )

2.Dipping (used for small plates )
3.Spraying ( difficult to get uniform layers )

4.Spreading ( best technique ) TLC Spreader

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ACTIVATION OF PLATES
After spreading → Air dry (5 to 10 minutes)

Activated by heating at about 100˚C for 30 min. Then plates may
be kept in desiccators

4.APPLICATION OF SAMPLE

Using capillary tube or micropipette

Spotting area should not be immersed in the mobile phase
5.DEVELOPMENT TANK

Better to develop in glass beakers, jars to avoid more wastage of
solvents
When standard method is used, use twin trough tanks

Do chamber saturation to avoid “edge effect”

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6. MOBILE PHASE
M.P used depends upon various factors
Nature of the substance
Nature of the S.P
Mode of Chromatography
Separation to be achieved, Analytical/Preparative

e.g. → pyridine, pet. ether, carbon tetrachloride, acetone,
water, glycerol, ethanol, benzene….

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DEVELOPMENT TECHNIQUE

One dimensional development

Two dimensional development

Horizontal development

Multiple development

19. THIN LAYER CHROMATOGRAPHY
DRYING OF CHROMATOGRAM
After the solvent has moved a certain distance for certain
time the chromatogram is taken out from the tank & position
of the solvent front is marked with a pencil.
They are dried by cold or hot air depending on volatility of
solvents. A simple hair dryer is a convenient device to dry
chromatograms.


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20. THIN LAYER CHROMATOGRAPHY
DETECTING / VISUALISING AGENTS
If the substance are colored they are visually detected easily.
But for colorless substance, Physical and chemical methods are used
to detect the spot.
(a)Non specific methods ( Physical methods)
E.g. iodine chamber method,
UV chamber for fluorescent compounds – at 254 or at 365nm.

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21. (b) Specific methods (Chemical methods) or Spraying
method
EXAMPLES
Ferric chloride-Phenolic comp. & tannins
Ninhydrin in acetone-Amino acids
Dragendroff’s reagent-Alkaloids
3,5 dinitro benzoic acid-Cardiac glycosides





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22. Following detecting tech. can also be categorized as
1) DESTRUCTIVE TECHNIQUES
Specific spray reagents, samples destroyed before detection e.g. – ninhydrin
reagent
2) NON-DESTRUCTIVE TECHNIQUES
For radio active materials - Geiger Muller counter
uv chamber, iodine chamber
QUANTITATIVE ESTIMATIONS
The method can be divided into two main groups
1.Direct techniques-
2.Indirect techniques-

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23. THIN LAYER CHROMATOGRAPHY
Direct Measurement Method
(i) Comparison of visible spots
A rough quantitative measurements
Component in a mixture can be carried out by comparing the intensity
and size of the spot with a standard substance.
(ii) Photo densitometry
The method is used with the chromatograms of colored
compound, instrument which measures quantitatively the density of the
spots.

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24. THIN LAYER CHROMATOGRAPHY
(iii) Fluorimetry
The compound to be determined by fluorimetry must be fluorescent or
convertible into fluorescent derivatives.
(iv) Radiotracer Method
The compound containing radioactive element is labeled and treated with
locating reagent. Using Geiger Muller counter.
(v) Polarographic & Conductometric methods
Used to measure the amount of material in the spot
INDIRECT MEASUREMENT METHOD
In this technique, the spots are cut into portions and eluted with solvents.
This solution can be analyzed by any techniques of analysis like
spectrophotometry, electrochemical methods, etc.


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25. Rf VALUE (Retardation Factor)
In Thin layer chromatography the results are represented by Rf
value which represent the movement or migration of solute relative to
the solvent front.
Rf value=
the ratio of the distance traveled by the substance
the distance traveled by the solvent

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26. Factors affecting Rf VALUE
 The temperature
 The purity of the solvents used
 The quality of the paper, adsorbents & impurities
present in the adsorbents
 Chamber saturation techniques, method of drying & development
 The distance travelled by the solute & solvent
 Chemical reaction between the substances.
 pH of the solution

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27. APPLICATIONS
Purity of sample
Examination of reaction
Identification of compounds
Biochemical analysis

In pharmaceutical industry

Separation of multicomponent pharmaceutical formulations

In food and cosmetic industry

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28. REFERENCE
S.RAVI SHANKAR, Text book of pharmaceutical analysis,
Rx publications, 2001.Pg no : 13-4 to 13-13.
WWW.Google.com

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