BENGAL SCHOOL OF TECHNOLOGY

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REPORT WRITING
ON

TITLE OF THE TOPIC: Steps Involve In Thin Layer Chromatography.

Limitation Of TLC. Application Of TLC.
SUBMITTED BY:

NAME: SUSMITA GHOSH

COURSE: B.PHARM YEAR/SEMESTER: 4th YEAR / 7th SEM

ROLL NO: 19301920115 REGISTRATION NO: 201930201920001

SUBJECT: Instrumental Method Of Analysis. SUBJECT CODE: PT 711

ACADEMIC SESSION: 2022-23 DATE OF SUBMISSION: 24.08.2022

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 INTRODUCTION

Thin layer chromatography was first discovered by Izmailov and Schreiber in 1938.Further Stahl
(1958) perfected the method and developed equipment and standardized adsorbents for the preparation
of uniform layers on glass plates. This technique , chromatography using thin layers of an adsorbent
held on a glass plate or other supporting medium is called as thin layer chromatography.

Chromatography is an important biophysical technique that enables the separation , identification , and
purification of the components of a mixture for qualitative and quantitative analysis.

Thin layer chromatography (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.

THIN LAYER CHROMATOGRAPHY

Thin layer chromatography is a method of analysis in which the stationary phase , a finely divided
solid , is spread as a thin layer on a rigid supporting plate ; and the mobile phase , a liquid is allowed to
migrate across the surface of the plate by means of capillary action.

The principle for separation is adsorption. The component move according to their affinities towards
the adsorbent.

More affinity towards the stationary phase – travels slowly.

More affinity towards the stationary phase – travels faster.

Thus the components are separated on thin layer chromatographic plate based on the affinity of the
components towards the stationary phase.

http://chromatographyscience.blogspot.com/2012/08/thin-layer-chromatography.

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 Different Steps Involve In Thin Layer Chromatography

 Selection of adsorbent and Preparation of Stationary Phase.

 Selection of glass plate.
 Preparation of the TLC plate.
 Activation of TLC plates.
 Preparation & Application of sample in TLC Plates.
 Development.
 Visualization or Detection of compounds on TLC.
1. Selection Of Adsorbent and Preparation Of Stationary Phase :

A large no. of coating materials are available. The Adsorbents classified in various types.

Acidic : silica. Basic : Alumina. Neutral : keisleguhr , cellulose powder.

There are so many several adsorbents which can be used as stationary phases. Some of the stationary
phases , their composition and the ratio in which they have to be mixed with water or other solvents to
form a slurry for preparing thin layer chromatographic plates. E.g. Silicagel GF : Silica gel + Binder+
Flourescent indicator.

2. Selection Of Glass Plates :

Glass plates which are specific dimension like 20cm × 20cm (fullplate). 20cm × 10cm (half plate).
20cm ×5cm (quarter plate) can be used. These dimensions are used since the width of the
commercially available TLC spreader is 20cm.

Microscopic slides can also be usede when the TLC plates are prepared without the use of TLC
spreader. In general , the glass plates should be of good quality and should withstand temperatures
used for drying the plates.

3. Preparation Of The Plates :

TLC plates made by mixing silica gel + inert binder calcium sulphate (gypsum) + distilled water in a
beaker & then shake well for two minutes and slurry is prepared.

Plates are washed with soap & water (and then washed with acetone for removal of fatty materials)
and dried oven. After preparing the slurry , the TLC plates can be prepared by using any one of the
following technique : Pouring , Dipping , Spraying , Spreading.

Cont……

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Pouring : In pouring technique , the slurry is prepared and poured on to glass plate which is
maintained on a leveled surface. The slurry is spread uniformly on the surface of the glass plate. After
setting , the plates are dried in an oven. The disadvantage is that uniformly in thickness can not be
ensured.

Dipping : In dipping technique , two plates (either of standard dimensions microscopic slides) are
dipped in to the slurry and are separated a removing from slurry and later dried. The disadvantage is
that a large quantity of slurry is required even for preparing fewer plates.

Spraying : Spraying technique resembles that of using a perfume spray on cloth. The suspension of
adsorbent or slurry is spread on a glass plate using a sprayer. The disadvantage is that the layer
thickness cannot be maintained uniformly all over the plate.

Spreading : Spreading technique is best technique where a TLC spreader are stacked on a base plate.
The slurry after preparation is poured inside the reservoir of TLC spreader. The thickness of the
adsorbent layer is adjusted by using a knob in the spreader. Normally a thickness of 0.25mm is used
for analytical purpose and 2mm thickness for preparative purpose. Then the spreader is rolled only
once on the plates. The plates are allowed for setting (air drying). This is done to avoid cracks on the
surface of adsorbent after setting , the plates are activated by keeping in an oven at 100° c to 120° c for
1hour.

http://www.expertsmind.com/topic/thin-layer-chromatography/stationary-phases-912769.aspx

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 4. Activation Of TLC Plates :
Activation of TLC plates is nothing but removing water / moisture and other adsorbed substances
from the surface of any adsorbent by heating at high temperature so that adsorbent activity is retained.
The activated plates can be stored in thermostatically controlled oven or in desiccators and can be used
whenever required.
5. Preparation & Application of sample in TLC Plates :
A straight line is ruled along one side of the plate about 1 inch from the edge. The sample which may
range from a few micrograms to milligram dissolved in 10-1000 µl of a volatile solvent. Usually to get
good spots , the concentration of the sample or standard solution has to be minimum. 2-5µl of a 1%
solution of either standard or test sample is spotted usually with a capillary tube or a micro syringe or
micro-pipette. Samples may be applied as spots or as thin bands. These applied spots should be about
¾ inch apart and 1inch from the bottom of the plate. The spotting area should not be immersed in the
mobile phase in the development tank. At least 4 spots can be spotted conveniently on a quarter plate.

Pencil is always used to mark a TLC plate since the graphite carbon is inert. If organic ink is used to
mark the plate , it will chromatograph just as any other organic compound and give incorrect results.

6. Development Of The Chromatogram :

 The majority of the TLC development is done by allowing the mobile phase to migrate up
(ascend) the plate , which is standing in a suitable sized chambers . For the purpose of
development Developing chambers are used , it may glass , plastic or metal ; glass chambers
are the most common.
 At first the mobile phase is taken in the development chamber and saturated completely with
the mobile phase. The development tank or chamber should be lined inside with filter paper
moistened with the mobile phase so as to saturate the atmosphere. If this kind of saturation of
the atmosphere is not done “edge effect” occurs where the solvent front in the middle of TLC
plate moves faster than that of the edge. Therefore the spots are distorted and not regulated.
 Then the plate is inserted into the chamber keeping the origin of spot downwards. Solvent level
should be below the starting line of TLC , else spots will dissolve in the solvent.
 The solvent will gradually climb up and past the spot. Once the solvent is within 1-2cm of the
top of the TLC sheet , the TLC is removed from the developing chamber and the farthest extent
of the solvent (the solvent front) is marked with a pencil.
 Thus a thin layer chromatogram is developed. The solvent is allowed to evaporate from the
TLC sheet in the hood.

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 7. Detection or Visualization Of Compounds On TLC :
After he development of the chromatogram , colored compounds can be easily detected on the TLC
plate.
But if the compounds is colorless , it can be detected by various methods. Usually the compounds are
not colored. Then –
If any fluorescent or quenching compound is present , the plates can be visualized under UV lamp and
marked. The UV light at 254nm (short) and 366nm (long) is usually used to detect the spot or band of
fluorescent compounds. Usually the commercially available pre-coated TLC plates itself contain a
fluorescing substance (Zinc silicate and Zinc Cadmium sulfide, 0.5% each) which fluoresces
everywhere except where a quenching compound is present on the plate. These compounds appear as
black spot on the TLC plate.
Compounds which do not contain any chromophore are not visualized under the UV light.

http://chemicalinstrumentation.weebly.com/uploads/6/8/0/9/6809778/5344418.jpg?1300183570

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 LIMITATION OF THIN LAYER CHROMATOGRAPHY
 It cannot tell the difference between enantiomers and some isomers.
 In order to identify specific compounds , the Rf values for the compounds if interest must be
known beforehand.
 TLC plates do not have long stationary phases. Therefore , the length of separation is limited
compound to other chromatographic techniques.

APPLICATION OF TLC

1. Qualitative Analysis :
If the separated compounds are colored then identification is very easy. All the visualizing
agents used in paper chromatography (detecting agents or indicators) can be used in TLC.
From Rf value qualitative analysis can be performed.
2. Quantitative Analysis :

The size of the spot increases with the amount. Square root of the spot area is find out from
that amount of solute can be found out.

Flurometry or emission Spectroscopy is also used.

3. Other Application Of TLC :

 TLC can be applicable in the field of medicinal preparation , Pharmaceutical preparation ,

natural product extract and related compounds.
 Separation of mixtures of drugs of chemical or biological origin , plant extracts , etc.
 Separation of carbohydrates , vitamins , antibiotics , proteins , alkaloids , glycosides , etc.
 Identification of certain drugs like Amoxycilline trihydrate , Chlorpromazine HCl , Digitoxin ,
Levodopa , Methyl dopa , Allopurinol , Diazepam etc.
 Assaying the radiochemical purity of radiopharmaceuticals.
 Determination of the pigments in plants.
 In forensic science Laboratory detection of pesticides and insecticides in food , poison etc.

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CONCLUSION :

Here I have been discussed about the THIN LAYER CHROMATOGRAPHY , various steps
involve in TLC , and its limitation and its application. It is concluded that due to simplicity , sharpness
of separation , high sensitivity , speed of separation and ease of recovery of separated compounds ,
TLC has found increasing application in all branches and this technique has been proved especially
versatile process for the analytical investigation of substances that available in very small quantities.

ACKNOWLEDGEMENT :

I would like to express my special thanks of gratitude to my respected teachers Mr. Biswanath
Ghosh , Mr. Dipra Dastidar. For gave me the golden opportunity to do this report on “Steps Involve In
TLC , Limitation of TLC , Application of TLC “.

REFERENCES :

1. Gurdeep R. Chatwal , Sham K Anand , 2017 , Fifth revised edition , Thin Layer
Chromatography : Instrumental methods of Chemical Analysis , Himalaya Publishing House.
PP - (2.599-2.615)
2. Mendham J , Denney R. C , Barnes J. D , Thomas M , Sivasankar B , 6 th edition , Thin Layer
Chromatography : Vogel’s Textbook of Quantitative Chemical Analysis , Pearson Education
Ltd. PP- (234-242)
3. MERCK (n.d) , Thin-Layer Chromatography Process ,
https://www.sigmaaldrich.com/IN/en/technical-documents/protocol/analytical-chemistry/thin-
layer-chromatography/tlc-process , 23/08/2022 , 11.00 a.m.
4. Chemistry Libre Texts , (n.d) , Thin Layer Chromatography ,
https://chem.libretexts.org/Ancillary_Materials/Demos_Techniques_and_Experiments/General
_Lab_Techniques/Thin_Layer_Chromatography. 23/08/2022 , 11.20 a.m.

5. Sharma B.K., 2007 5thedition , Instrumental methods of chemical analysis, Goel publishing
house, Meerut, PP - 241-264.