THIN LAYER

CHROMATOGRAPH

PRESENTED BY:
1)Ashwin 22D207
2)Dharanish 22D211
3)Harshad 22D219
4)Arun 23D433
 TABLE OF CONTENTS

01 Introduction What is TLC

The principle on which TLC is

02 principle based

03 working Working of TLC

Factors The important factors affecting in

04 affecting making of a TLC

Application of TLC in
05 Application biomedical field
INTRODUCTION
What is chromatography ?
Chromatography is a physical method of
separation in which the components to be separated are distributed between two
phases, one of which is stationary (stationary phase) while the other (the mobile
phase) moves in a definite direction
● Types of chromatograph
1) Column/Adsorption Chromatography

2) Partition Chromatography

3) paper Chromatography

4)Thin layer Chromatography (TLC)

5) Gas-Liquid Chromatography(GLC)

7) Gas- solid Chromatography (GSC)

Advantages of TLC over other type of Chromatography

● Cost effectiveness
● Speed
● Ease of use
● Flexibility
● Sample size
● Visualization
● Scalability
● portability
TLC is one of the simplest, fastest, easiest and least expensive
compared to other chromatographic techniques used in qualitative and
quantitative analysis to separate organic compounds and to test the
purity of compounds. TLC is a form of liquid chromatography consisting
of:

1)A mobile phase

2) A stationary phase
Principle of TLC
It is based on the principle of adsorption
chromatography or partition chromatography or combination of both, it
depends on the differential affinities of compounds towards stationary
phase and mobile phase.
Working of TLC
● Preparation of the TLC Plate:
A thin layer of adsorbent material (like silica gel or
alumina) is coated onto a glass, plastic, or aluminum plate. This layer is
typically about 0.1 to 0.25 mm thick.

● Sample Application:
A small amount of the mixture to be analyzed is spotted
onto the base line of the TLC plate. This is usually done with a micro-pipette
or capillary tube.
● Development:
The TLC plate is then placed in a shallow container (like a
developing chamber) containing a solvent mixture. The solvent moves up the
plate through capillary action, carrying the sample components along with it.

● Separation:
As the solvent moves up the plate, it interacts differently with the
components of the sample mixture. Some components will be more strongly
adsorbed to the stationary phase (the adsorbent material on the TLC plate)
and will move more slowly, while others will be less strongly adsorbed and
will move faster.
● Visualization:
Once the solvent front reaches near the top of the plate, the
plate is removed from the developing chamber and allowed to dry. The
separated components are then visualized using various methods, such as
UV light, iodine vapor, or chemical staining.

● Analysis:
The distance traveled by each component (Rf value) can be
calculated and compared to known standards to identify the components in
the mixture
RETARDATION FACTOR
Factors affecting Rf value:
● Nature adsorbent
● Mobile phase
● Activity
● Thickness of layer
● Temperature
● Equilibrium
● Loading
● Dipping zone
● Chromatographic techniques
Application of TLC in biomedical field

● 1. Drug Analysis and Pharmacokinetics:

- TLC is used to analyze the composition and purity of pharmaceuticals.
It helps in identifying and quantifying active ingredients and impurities.
- It assists in studying the pharmacokinetics of drugs by monitoring their
metabolism and distribution in biological fluids.

● 2. Clinical Diagnostics:
- TLC is employed to detect metabolic disorders by analyzing urine or
blood samples. For example, it can help diagnose phenylketonuria by identifying
abnormal metabolites.
- It is used in the screening of neonatal jaundice by measuring bilirubin
levels in blood samples.
● 3. Detection of Pathoge
● 4. Forensic Toxicology
● 5. Biomolecule Separation and Identification
● 6. Hormone and Steroid Analysis
● 7. Nutritional and Metabolic Studies
● 8. Environmental Monitoring

These applications demonstrate the critical role of TLC in advancing

biomedical research, clinical diagnostics, and therapeutic monitoring.
CONCLUSION
THANK YOU
REFERENCES
● "Thin-Layer Chromatography: A Modern Practical Approach" by Peter E. Wall and Sabine E. König.
This book provides a comprehensive introduction to thin-layer chromatography, covering theory,
instrumentation, methods, and applications.
● "Thin Layer Chromatography in Phytochemistry" edited by Monika, Joseph Sherma, and Teresa
Kowalska. This book focuses on the application of TLC in phytochemistry, particularly for the
analysis of plant constituents.
● "Handbook of Thin-Layer Chromatography" edited by Joseph Sherma and Bernard Fried. This
comprehensive handbook covers all aspects of thin-layer chromatography, including theory,
techniques, method development, and applications in various fields.
● "Modern Thin-Layer Chromatography" by Salvatore Fanali, Joseph Sherma, and Stig Pedersen-
Bjergaard. This book provides an up-to-date overview of thin-layer chromatography, including
recent advances in instrumentation and method development.