This document presents an overview of thin layer chromatography (TLC), a technique for isolating non-volatile mixtures, developed in 1938. It details the procedure, key components, and applications of TLC, emphasizing its use in analytical and preparative capacities in various industries, including pharmaceuticals and food. The document also mentions the limitations of TLC, such as challenges in reproducibility and its qualitative nature.

1. Thin Layer
Chromatography
Mehran University of Engineering &
Technology
Jamshoro, Pakistan

2. HELL
O!
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I’m Mujeeb UR
Rahman
Chemical Engineering student @Mehran University of
Engineering & Technology Jamshoro, Pakistan.
“I’m here to present you how to isolate non-volatile mixture by
using Thin Layer Chromatography technique.”
You can find me at SlideShare @MujeebURRahman38
• ResearchGate @Mujeeb UR Rahman, Academia @Mujeeb UR Rahman

3. CHROMATOGRAPHY
 Chromatography is the technique for the separation, purification, and
testing of compounds..
 Works by allowing the molecules present in the mixture to distribute
themselves between a stationary and a mobile medium.
 Molecules that spend most of their time in the mobile phase are carried
along faster.
 Chroma -"color" and graphein - "to write”.
 Color bands - separation of individual compounds
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4. Thin layer chromatography
is the technique to isolate
non-volatile mixtures.
It was developed by
Izmailov in the year 1938
considering Mikhail Tswett’s
description of column
chromatography.
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WHAT IS THIN LAYER
CHROMATOGRAPHY
'
”
TLC
chamber
Mobile
phase
TLC
plate
Spotting
line

5. THIN LAYER CHROMATOGRAPHY
(TLC)
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 The experiment is conducted on a sheet of aluminum foil, plastic or glass
which is coated with a thin layer of adsorbent material. The material
usually used is aluminum oxide, cellulose or silica gel.
 On completion of the separation, each component appears as spots
separated vertically. Each spot has a retention factor (RF) expressed as:
𝑹𝑭 =
𝒅𝒔𝒊𝒕. 𝒕𝒓𝒂𝒗𝒆𝒍𝒍𝒆𝒅 𝒃𝒚 𝒔𝒂𝒎𝒑𝒍𝒆
𝒅𝒔𝒊𝒕. 𝒕𝒓𝒂𝒗𝒆𝒍𝒍𝒆𝒅 𝒃𝒚 𝒔𝒐𝒍𝒗𝒆𝒏𝒕
 The factor affecting retardation factor are the solvent system, amount of material
spotted, absorbent and temperature TLC is one of the fastest, least expensive, simple
and easiest chromatography.

6. PRICNIPLES OF CHROMATOGRAPGHY
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 Physical method of separation that distributes components
to separate between two phases moves in a definite
direction.
 Substances are separated based on their differential
distribution between two phases.
 Substances will move with the mobile phase at different
rate depending upon their Partition or Distribution
co- efficients.

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CHROMATOGRAPGHY PROCEDURE
 Before stating with the Thin Layer Chromatography Experiment let us
understand the different components required to conduct the procedure
along with the phase involved:
 Thin layer chromatography Plates
 Ready-made plates are used which are chemically inert and stable. The
stationary phase is applied on its surface in the form of a thin layers.
The stationary phase on the plate has a fine particle size and has a
uniform thickness.

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CHROMATOGRAPGHY PROCEDURE
 Thin layer chromatography chamber
 Chamber is it used to develop plates. It is responsible to keep a steady
environment inside which will help in developing spots. Also, it prevents the
solvent evaporation and keeps the entire process dust-free.
 Thin layer chromatography mobile phase
 Mobile phases the one that moves and consists of solvent mixture or a
solvent. This phase should be a particulat-free. The higher the quality of
purity the development of spots is better.
 Thin layer chromatography filter paper
 It must be glass inside the chamber it is my stand in the mobile face phase.

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 To apply sample spots, thin marks are made at the bottom of the plate with the help of a
pencil.
 Apply sample solutions to the marked spots.
 Pour the mobile phase into the TLC chamber and to maintain equal humidity, place a
moistened filter paper in the mobile phase.
 Place the plate in the TLC chamber and close it with a lid. It is kept in such a way that the
sample faces the mobile phase.
 Immerse the plate for development. Remember to keep the sample spots well above the
level of the mobile phase. Do not immerse in the solvent.
 Wait till the development of spots. Once the spots are developed, take out the plates and
dry them. The sample spots can be observed under a UV light chamber.
Thin Layer Chromatography Experiment

10. CHROMATOGRAPGHY PROCEDURE
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11. PURPOSE OF CHROMATOGRAPHY
 Analytical
• Determine Chemical composition of a sample
 Preparative
• Used to purify sufficient quantities of a substance
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12.  Chromatograph - equipment that enables a sophisticated separation
EX. Gas chromatography or Liquid chromatography
 Eluent - Fluid entering column/ solvent that carries the analyte.
 Eluate - Mobile phase leaving the column.
 Stationary phase - Immobilized phase
• Immobilized on the support particles or on the inner wall of the
column tubing.
• Examples : Silica layer - Thin Layer Chromatography
CHROMATOGRAPHY TERMS
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13.  Mobile phase - Moves in a definite direction. Liquid (LC),
▪ Gas (GC). The mobile phase moves through the chromatography
column (the stationary phase) where the sample interacts with the
stationary phase and is separated.
 Retention time: Time takes for a particular analyte to pass through the
system (from the column inlet to the detector) under set conditions.
 Sample (Analyte) : Substance analyzed in chromatography.
 Solvent: Any substance capable of solubilizing another substance.
CHROMATOGRAPHY TERMS
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14. THIN LAYER CHROMATOGRAPHY
APPLICATIONS
 The qualitative testing of various medicines such s sedatives, local anesthetics,
anticonvulsant tranquilizers, analgesics, antihistamines, steroids, hypnotics is done by TLC.
 Thin layer chromatography can be used to identify natural products like essential oils or
volatile oil, fixed oil, glycosides, waxes, alkaloids, etc.
 It is widely used in separating multicomponent pharmaceutical formulations.
 It is used in the cosmetic industry.
 It is used to purify of any sample and direct comparison is done between the sample and the
authentic sample.
 It is used in the food industry, to separate and identify colors, sweetening agent, and
preservatives.
 It is used to study if a reaction is complete.
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15.  Thin layer chromatography plates do not have longer stationary phase.
 When compared to other chromatographic techniques the length of separation
is limited.
 The result generated from TLC are difficult to reproduce.
 Since TLC operates as an open system some factors such as humidity and
temperature can be consequences the outcome of the chromatogram.
 The detection limit is high and therefore if you want a lower detection limit,
you cannot use TLC.
 It is only a qualitative analysis technique and not quantitative.
DISADVANTAGES OF THIN LAYER
CHROMATOGRAPHY
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16. Thank
s!
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