Thin Layer Chromatography

THIN LAYER CHROMATOGRAPHY
( TLC )
VIGNAN PHARMACY COLLEGE

(APPROVED BY AICTE,PCI-NEW DELHI & AFFILIATED TO JNTUK)
VADLAMUDI,GUNTUR DISTRICT 522213
BY
BHUKYA.NOM KUMAR NAIK
12-08-2019 Vignan pharmacy college , Vadlamudi 1

 Introduction
1.History
2.Principle
 Practical requirements
1.S.P ( Stationary phase ).
2.Glass plates .
3.M.P ( Mobile phase ).
 Methodology
1.Preparation of slurry.
2.Coating of plates.
3.Activation of TLC plate.
4.Application of sample.
5.Development tank.
6.Development techniques.
7.Detecting / Visualizing agents
 Application

History E
 The origin of thin layer chromatography is a little obscure but was probably first
developed and utilized by Schraiber in 1939.
 Schraiber working with Izmailov at the Khar'kov Chemistry and Pharmacy
Research Institute developed the techniques for use in the analysis of
pharmaceuticals. Quoting from her publication,
 " It occurred to us that a thin layer of the sorbent could be used in lieu of a strip
of paper; also we felt that the flat bed could be considered as a cut-out of the
adsorbent column. We believed that in carrying out the separation process in such
a layer, the process would be accelerated significantly. In our work, we deposited
a drop of the solution being investigated on the flat adsorbent layer and observed
the separation into concentric circular zones which could become visible because
of their fluorescence in the light of a UV lamp."

Advantages of thin layer chromatography : -
1. Corrosive spray reagents can be used with out damaging the plates.
2. Simple method and low cost.
3. Any type of compound can be separated even it is in g level.
4. Less quantity of M.P & S.P re required when compared with column
chromatography.
5. Detection is very easy and fast.
6. Very less equipment are used.
7. It is very simple method.
8. It is also sensitive method.
9. The components present in the sample can be separated and recovered out
easily by scratching the powdery coating on the plate and hence quantitative
separation of spots or zone are possible

Disadvantages Of Thin Layer Chromatography : -
1. Thin Layer Chromatography plates do not have longer stationary phase.
2. The separation takes place in an open.
3. Results obtained from TLC are difficult to reproduce.
4. Only soluble components of the mixtures are possible.
5. This is the only qualitative analysis possible, not quantitative.
6. Usually, it is not automatic.
7. TCL works in the open system, therefore, due to temperature and humidity can
affect the results.

PRINCIPLE
1) The major principle involved in the Thin Layer Chromatography is

“Adsorption”.
2) The compound which has more affinity towards S.P travels slowly.
3) The compound which has less affinity towards S.P travel faster.
4) The compounds are separated based up on Adsorption towards Stationary

phase.
M.P : - Solid phase ( Silica gel )
S.P : - Liquid

PRACTICAL REQUIREMENTS
 S.P ( Stationary phase ).
 Glass plates .
 M.P ( Mobile phase ).
Methodology / Procedure
 Preparation of slurry.
 Coating of plates.
 Activation of TLC plate.
 Application of sample.
 Development tank.
 Development techniques.
 Detecting / Visualizing agents.
STATIONARY PHASE
The stationary phase should be solid & it is an adsorbent material.

eg : - silica gel, aluminium oxide
Factors to be considered for choosing an adsorbent : -
1. Characteristic of the compound to be separated.
2. Solubility of the compound.
3. Nature of the compound ( acidic,basic,Amphoteric ).
4. Whether the compound is liable to react chemically with adsorbent.
Note : - The adsorbents contain binders like gypsum & starch,Hydrated silicone
dioxide.

Sl.no Name of the Adsorbents Composition Adsorbent : Water
1 Silica gel H Silica with out binder 1 : 1.5
2 Silica gel G Silica with out Calcium sulphate 1:2
Silica + binder & Fluorescent

3 Silica gel GF254 indicator 1: 2
4 Cellulose powder Cellulose powder with out binder 1:5
 Generally in Silica gel GF – G stands for gypsum,F stands for Fluorescent indicator (zinc
silicate ).
 Silica gel PF 254+366 it is meant for preparative TLC.
 Silica gel HF 254 it contain binder and flourescent indicator.

Sl.no Name of the Adsorbents Composition Adsorbent : Water
5 Cellulose powder Cellulose powder with binder 1:6
6 Keiselguhr G Diatomaceous earth + binder 1:2
7 Polyamide powder Polyamide 1:9
8 Al 2O 3 G Al 2O 3 + Binder 1:2
9 Silica gel N Silica with out binder 1 : 1.5

Adsorbents
Organic Adsorbents Inorganic Adsorbents
a) The organic adsorbent like like Cellulose,acetylate

cellulose,Manitol,Sucrose,Starch.
b) The inorganic adsorbent like aluminium hydroxide & oxide,
silicate,bentonite,calcium hydroxide & oxide,silicate,carbonate

SELECTION - GLASS PLATES
 In TLC the glass plates are used.
 They are available in different sizes.
 Based up on size of the plate they are they are devided into 3 types
They are : -
1.20 cm  20 cm ( full plate )
2.20 cm  10 cm ( Half plate )
3. 20 cm  5 cm ( Quater plate )

MOBILE PHASE
It should be more polar than Stationary phase ( S.P ).

It may be a single or mixture of the solvents.
It should not react with Compound & spraying reagents.
There are two types of mobile phases
1.Hydrophilic mobile phase.
Eg : - 1.n-butanol : Glacial acetic acid : water (4 : 1: 5 ).
2.Isoproponal : Ammonia : water ( 9: 1: 2 ).
2.Hydrophobic mobile phase.
Eg : - Cyclo-hexane and di-ethyl ether.

If one know the chemical nature of the solute in the mixture ,it is
possible to know the suitable solvent by using “ Stahl`s triangle “
It gives an interaction between adsorptive activity , nature of the solute

and solvent.

Preparation of slurry
1. Powder and solvent ( water ) is mixed and slurry is prepared.
2. The optimum thickness of slurry is prepared.
Note : -
1. If the slurry is too-thick there will be no separation of compound
will occur .
2. If the compound if too-thin during spotting compounds will spread

and no exact or accurate spots can be detected.

Coating of plates /Spraying techniques
Techniques
Pouring Spraying Dipping Spreading

Pouring : - The prepared slurry is poured and spraded over the plate.
It is less time consuming technique.
No uniform thickness is observed.
Spraying : - The prepared slurry is sprayed over the plate.

No uniform thickness is observed

Dipping : - In this technique the two glass slides are taken together and
dipped in prepares slurry.
No uniform thickness is observed.
Large amount of slurry is required.
Spreading : - In this specific instance , The slurry or suspension is

carefully introduced into an application, which in turn is duly moved
over the stationary glass – plate or vice versa .This method is known
as spreading and It gives quite uniform thin layer on the glass plates.

Kirchner (1951) evolved another spreading
methodology that predominantly compress of
the following important steps : -
1. Selection of absolutely uniform
surface glass plate.
2.Holding them in position between
glass or metal that happen to be Thicker than the
glass plate by quantum which is desired for the
adsorbent layer to form , and
3. Spreading the slurry uniformly on
the glass plate with the help of a clean glass rod
gently.

Activation of plates
After completion of coating let air dry the plate at room temperature
for 30min.
And then place the plate at 110 in Hot air oven .
Note : - Silica gel G is impure in nature which contains Iron as
impurities Which can bee removed by 9 : 1 v/v Methanol and HCL.
Spotting of sample
spotting of sample can be done with the help of Micropippettes or
Capillary tube .
Note : - Spot diameter - 2-3 mm.
Distance between two diameters – 2cm.
Base line should be drawn from below - 2 cm.
Developing chambers
1. After spotting of sample the plate has to keep in the developing

chamber because to develop colour spots.
2. There are different types of developing chambers .
1.Old type chamber

2.New type : -
In this type of chamber the filter paper
is chilled with solvent and placed inside
the chamber to avoid edge effect.
In this type of chamber we can place two TLC plates

at a time

Developing Techniques
Techniques
Multiple Horizontal
One dimensional Two dimensional
Dimensional dimensional
Descending
Ascending

Descending TLC
Development of the chromatogram is done
by allowing the solvent to travel down the
paper. Here, mobile phase is placed in
solvent holder at the top. The spot is kept at
the top and solvent flows down the TLC
from above.
Ascending TLC
Here the solvent travels up the
chromatographic paper. Both descending and
ascending TLC are used for the separation of
organic and inorganic substances. The sample
and solvent move upward
( Against gravitational force ).

Two - dimensional TLC
In two-dimensional TLC development, the sample is applied to a starting point in a corner of
the TLC plate. The plate is placed in a normal chamber and developed once from bottom to
top. After drying, the plate is turned 90° and placed in another chamber with a different
solvent and developed again. The chromatogram track from the first development is used as
the starting line for the second development. Two-dimensional TLC offers the advantage of
running a standard with either development. However, the standard cannot be developed in
two dimensions on the same chromatogram since it would mix with the sample.

Horizontal development of TLC : -

Multiple Dimensional TLC
In this method, the TLC plate undergoes multiple
developments with drying between each cycle. The
solvent travels repeatedly through the layer, re
concentrating and deforming the spots, often
producing elliptical shapes or narrow bands. This
significantly improves resolution for substances
with Rf values below 0.5. Multiple development
can be performed over different separation
distances, using the same solvent or different
solvents of varying polarity.

Spraying agents or detecting agents or visualizing agents
 Base on physiochemical nature

Detection
Physical Chemical
Specific
Non-specific
UV chamber / Fluroscence Radioactive method
Geiger-muller Auto-radioactive
method

Physical method
1.Radioactive method
A.Geiger-muller method : -
Chromatogram was placed in GMC ( geiger-muller chamber ). And it is used

for the identification of radioactivity.
B.Autoradioactive method : -
Chromatogram is pressed between glass slide and x – ray slide , from these we
can identify the quantity of a compound.

2.UV chamber method : -
 This method is used for the detection of “Florescent and Non – florescent
compounds.
 Non – florescent compounds can be identified at 256 rpm .
 Florescent compounds produce different colours like “ Green , Blue , Violet “

with a background of Black colour.
 Non – florescent compounds produce “ Black colour “ with a background of

different colours like Blue, Green , Violet .

Chemical method
Specific method : -
Sl.no Name of the compounds Spraying reagent Colour obtained

1
Phenols & Tannins Ferric chloride Blue , green , Violet
2
Amino acids Ninhydrine Pink /Purple
3
sugars Aniline-hydrogenpthalate Black grey
4
Cardiac glycosides Di-nitrobenzoic acid Orange
5
Anthraquinones Ammonia Pink

 All these are selective and specific reagent in chemical method of
detection.
 Universal spraying reagent is “Sulphuric acid”( 4 ml sulphuric acid +

96 ml ethanol ).
 Modified plates should be taken to spray Sulphuric acid (Universal

sprayer) and
After staining the reagent ( Sulphuric acid ) the plates will appear as
Black colour

Non specific or Non selective method : -
1.Iodine chamber method
2.Uv method
3.Universal sprayer reagent ( sulphuric acid )
Note : -
1. All the selective methods are destructive methods including universal sprayer because
they destroy the original compound and gives colour when we spray reagent.
2. All the non selective methods are non destructive except universal sprayer reagent.

Applications : -
1.To detect both qualitative and quantitative analysis.
Qualitative analysis : - Qualitative analysis is done based on
Migration parameters. That are Rf , Rx , Rm values
Rf Value (Retardation Factor)

In the TLC , the results are represented by Rf value which
represents the movement or migration of solute relative to the solvent
front.
Rf = Distance travelled by the solute
Distance travelled by the solvent front
The Rf value range from 0 – 1 , but ideal value range between 0.3 –
0.8.

Rx Value : -
a. In many cases , it has been observed that the solvent front is run off the
end of the paper .Rx value is thus used.
b. Rx value is nothing the ratio of distance travelled by the sample and the
distance travelled by the Standard Rx value is always closer to 1.
Rm value : -
a. Rm value is used in qualitative analysis to find out whether the
compound belongs to a homologous series .
b. If they belongs to Homologous series .
c. The Rm value are constant.
Rm = log  1/Rf - 1

Quantitative analysis : -
Quantitative analysis is done in two ways
1.Direct method.
2.Indirect method.
1.Direct method : -
a) The quantity of the individual spot can be determined by using
densitometric method ( densitometer ).
b) This technique is also called as in- situ method and
c) It is described earlier in detecting technique.

2.In direct method : -
a) Quantitative analysis can be done after eluting the individual spot
with solvent and filtering off the stationary phase.
b) The solution can be concentrated and the exact quantity of the
compound can be determined by using conventional methods like
colorimetry , UV Spectrophotometry ,Flourescence method , etc....
2. Presence of foreign alkaloids in certain alkaloid formulations(Drug)

e.g : - Codeine , Atropine sulphate etc....
3. Presence of ninhydrin positive compounds in Official aminoacids
e.g : - Leucine , Glutamic acid etc....
4. Critical occurrence of Foreign steroids in Steroidal drugs
e.g : - Betamethasone valerate.etc.....
5. Presence of closely related substances in abroad spectrum of Potent
Pharmaceutical substances
e.g : - Aminophylline , Carbamazepine and Baclofen etc......
6.Presence of undiserable compounds as Impurities in a number of
pharmaceutical products .
e.g : - Hydrazine in Carbidopa , Morphine in Apomorphine
hydrochloride etc....

References
1. A text book of pharmaceutical analysis by Dr.S.Ravisankar
2. Instrumental methods of chemical analysis by G.R.Chatwal.
3. Pharmaceutical analysis Vol – 2 by A.V.Kasture.
4. Pharmaceutical analysis by Ashutoskar , VOLUME – 2
5. Pharmaceutical analysis , A text book for pharmacy students and pharmaceutical
chemists by David G.Watson.
6. https://en.wikipedia.org/wiki/Thin-layer_chromatography
7. http://classes.kvcc.edu/chm220/TLC%20Lab/prelab/introduction.htm
8. https://byjus.com/chemistry/thin-layer-chromatography/
9. https://chem.libretexts.org/Ancillary_Materials/Demos%2C_Techniques%2C_and_Experi
ments/General_Lab_Techniques/Thin_Layer_Chromatography

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THIN LAYER CHROMATOGRAPHY

VIGNAN PHARMACY COLLEGE

12-08-2019 Vignan pharmacy college , Vadlamudi 1

12-08-2019 Vignan pharmacy college , Vadlamudi 2

12-08-2019 Vignan pharmacy college , Vadlamudi 4

12-08-2019 Vignan pharmacy college , Vadlamudi 5

12-08-2019 Vignan pharmacy college , Vadlamudi 6

1) The major principle involved in the Thin Layer Chromatography is

4) The compounds are separated based up on Adsorption towards Stationary

M.P : - Solid phase ( Silica gel )

12-08-2019 Vignan pharmacy college , Vadlamudi 7

The stationary phase should be solid & it is an adsorbent material.

12-08-2019 Vignan pharmacy college , Vadlamudi 10

1 Silica gel H Silica with out binder 1 : 1.5

2 Silica gel G Silica with out Calcium sulphate 1:2

Silica + binder & Fluorescent

4 Cellulose powder Cellulose powder with out binder 1:5

12-08-2019 Vignan pharmacy college , Vadlamudi 11

5 Cellulose powder Cellulose powder with binder 1:6

6 Keiselguhr G Diatomaceous earth + binder 1:2

7 Polyamide powder Polyamide 1:9

9 Silica gel N Silica with out binder 1 : 1.5

12-08-2019 Vignan pharmacy college , Vadlamudi 12

Organic Adsorbents Inorganic Adsorbents

a) The organic adsorbent like like Cellulose,acetylate

b) The inorganic adsorbent like aluminium hydroxide & oxide,

silicate,bentonite,calcium hydroxide & oxide,silicate,carbonate

12-08-2019 Vignan pharmacy college , Vadlamudi 13

 In TLC the glass plates are used.

 They are available in different sizes.

1.20 cm  20 cm ( full plate )

2.20 cm  10 cm ( Half plate )

12-08-2019 Vignan pharmacy college , Vadlamudi 14

It should be more polar than Stationary phase ( S.P ).

12-08-2019 Vignan pharmacy college , Vadlamudi 15

It gives an interaction between adsorptive activity , nature of the solute

12-08-2019 Vignan pharmacy college , Vadlamudi 16

1. Powder and solvent ( water ) is mixed and slurry is prepared.

2. The optimum thickness of slurry is prepared.

2. If the compound if too-thin during spotting compounds will spread

12-08-2019 Vignan pharmacy college , Vadlamudi 18

Pouring Spraying Dipping Spreading

12-08-2019 Vignan pharmacy college , Vadlamudi 19

Spraying : - The prepared slurry is sprayed over the plate.

12-08-2019 Vignan pharmacy college , Vadlamudi 20

Spreading : - In this specific instance , The slurry or suspension is

12-08-2019 Vignan pharmacy college , Vadlamudi 21

12-08-2019 Vignan pharmacy college , Vadlamudi 22

1. After spotting of sample the plate has to keep in the developing

12-08-2019 Vignan pharmacy college , Vadlamudi 24

In this type of chamber we can place two TLC plates

12-08-2019 Vignan pharmacy college , Vadlamudi 25

12-08-2019 Vignan pharmacy college , Vadlamudi 26

12-08-2019 Vignan pharmacy college , Vadlamudi 27

12-08-2019 Vignan pharmacy college , Vadlamudi 28

12-08-2019 Vignan pharmacy college , Vadlamudi 29

12-08-2019 Vignan pharmacy college , Vadlamudi 30

 Base on physiochemical nature