INDEX

1) Abstract…………………………………….…………………………………………………1
2) Introduction
 Chromatography……………………………………………...………………..….2
 Types of Chromatography…………………………………….…………...…..….5
3) Introduction To Thin Layer Chromatography (TLC)
 Background and Significance of Thin Layer Chromatography (TLC).…6
 Research Objectives and Scope………………………….………………7
4) Literature Review-
 History of TLC: -…………………….……………………………………....8
 Principle of TLC…………….………………...………………....……….….9
 Applications of Thin Layer Chromatography…………………….………....11
 Classification of THIN LAYER CHROMATOGRAPHY………………….14
5) Methodology
 Instrumentation of TLC……………………………………………………..15
 Sample Preparation and Application in TLC……….……………………….24
 Development and detection methods ……………………………………….29
6) Data analysis and interpretation …………………………………………………….38
7) Result and discussion ………………………………………………………………..41
8) Future direction ……………………..……………………………………………… 43
9) Conclusion ………………………………………………………………….……..…45
10) References …………………….…………………………………...…………………46
A REVIEW ON THIN LAYER CHROMATOGRAPHY

ABSTRACT

In this present article, we address the basic aspects such as idea, mechanism and
working of thin layer Chromatography (TLC) in analytical as well as preparative preparation
methods. We have gone through diverse journals for gathering complete package of TLC and
found that TLC is very simple, easy, less time consuming, cost-effective and multiple samples
could be run in one go hence, is constantly the first choice for varied application in qualitative
analysis of pharmaceutical products. In this modern scientific world were-HPLC and HPTLC
technology has developed still TLC holds good promise for identification and analysis of
different bioactive compounds, secondary metabolites, Vitamins and amino acids. It is a very
preliminary analytical method done prior to HPLC and reaction progress can be mopped easily"
It can be used for separating. Pounds from crude extracts and separating impurities from
compound. Identical compounds from the mixture can be easily separated by analytical and
further by preparative TLC. Many standard methods in industrial chemistry, environmental
toxicology, steroids, food chemistry, water, inorganic pesticide, dye purity, cosmetics, plant
materials, and herbal analysis rely upon TLC as the preferred approach. Hope this review article
will help in understanding principal and working of TLC in the field of research.

1
A REVIEW ON THIN LAYER CHROMATOGRAPHY

INTRODUCTION

 Chromatography:
Chromatography is a laboratory technique used to separate, identify, and quantify the
components of a mixture. The term "chromatography" comes from the Greek words "chroma,"
meaning colour, and "graphia," meaning writing. This refers to the fact that chromatography
was first used to separate and identify coloured compounds.

As per IUPAC (International Union of Pure and Applied Chemistry) "Chromatography is a

physical method of separation in which the components of a mixture are separated based on
their distribution between two phases, one of which is stationary and the other mobile."

Chromatography has its roots in the early 20th century, when the Russian botanist Mikhail
Tsvet developed a method for separating plant pigments using a column of calcium carbonate.
Tsvet's technique, which he called "chromatography" (from the Greek words for "colour" and
"writing"), was first described in a paper published in 1906. Over the next several decades,
chromatography evolved and improved, with the development of new techniques such as paper
chromatography and gas chromatography. The 1950s and 1960s saw the introduction of modern
chromatographic techniques, including thin-layer chromatography (TLC) and high-
performance liquid chromatography (HPLC). Today, chromatography is a powerful and widely
used tool in many fields, including chemistry, biology, pharmaceuticals, and
environmental science.

 Principles of Chromatography:

Chromatography is based on the principle of partitioning, where a mixture is separated into its
individual components based on their interactions with two phases:

1. Stationary phase: A solid or liquid phase that is fixed in place.

The stationary phase is a solid or liquid phase that is fixed in place and does not move with the
mobile phase. It is typically a porous material with a large surface area, which allows it to
interact with the components of the mixture.

Types of Stationary Phases:

1. Solid stationary phases: These include materials such as silica gel,
alumina, and zeolites.
2. Liquid stationary phases: These include materials such as liquids coated onto a solid
support, such as a polymer or a silica gel.
3. Bonded stationary phases: These include materials such as silica gel or alumina that
have been chemically bonded to a solid support
2. Mobile phase: A liquid or gas phase that moves through the stationary phase.

2
A REVIEW ON THIN LAYER CHROMATOGRAPHY

The mobile phase is a liquid or gas that moves through the stationary phase, carrying the
components of the mixture with it. The mobile phase is responsible for transporting the
components through the chromatographic system.

Types of Mobile Phases:

1. Liquid mobile phases: These are the most common type of mobile phase, and are used
in liquid chromatography.
2. Gas mobile phases: These are used in gas chromatography.

Examples of Mobile Phases:

1. Water: A common mobile phase used in liquid chromatography.

2. Methanol: A common mobile phase used in liquid chromatography.
3. Helium: A common mobile phase used in gas chromatography.
4. Carbon dioxide: A common mobile phase used in supercritical fluid chromatography.

As the mobile phase moves through the stationary phase, the components of the mixture interact
with both phases to varying degrees. This interaction causes the components to separate based
on their:

1. Affinity: The strength of attraction between a component and the stationary phase.
2. Solubility: The ability of a component to dissolve in the mobile phase.
An Rf value is "retardation factor" or "ratio to front" which can be calculated by using
the formula.

Distance travelled by compound

RF= ______________________________________________

Distance travelled by solvent front

These Rf values can be calculated by observing spots on TLC plates under UV transilluminator
at 365nm. The compounds travel from origin spotting position and distance travelled by solvent
front is noted. Then the given formula would give the Rfvalue for the compound. Identical
molecules will invariably travel the equivalent distance under similar temperature, solvent
system and stationary phase. However, the molecules travelled at same position always may no
longer be the identical compound. Supplementary supporting data is needed before coming to
the conclusion. Various bands of secondary metabolites separated on TLC by using solvent
system chloroform: ethyl acetate: benzene: glacial acetic acid (25: 15: 2: 10). TLC chamber
design may play a vital role in identifying bioactive metabolites, which ranges from 100ml to
100ml closed chamber. An Rf value occurs between 0 — 1 and depends upon following factors,
which determine the efficiency of a chromatographic separation.

3
A REVIEW ON THIN LAYER CHROMATOGRAPHY

FIGURE: 1.

It shows demonstration of preparation of Capillary spotter, TLC silica Plate and S

Preparation of capillaries. C-D: Preparation of TLC plate using Silica. E-F: Preparation of selected
Solvent system. G: Silica plate. H: Spotting of sample. I: Developing of TLC plate in developing
chamber. J: TLC plate under UV transilluminator at 365nm. K: Marking of the specific band from TLC
under UV transilluminator. L: Scraping of the specific band from TLC under transilluminator at 365nm.
M: Separated compound obtained from TLC.

4
A REVIEW ON THIN LAYER CHROMATOGRAPHY

 Types of Chromatography

There are several types of chromatography, including:

1. Paper Chromatography: Uses paper as the stationary phase.

Paper chromatography separates mixture components based on their affinity for
stationary and mobile phases. A sample is applied to paper, which is placed in a solvent.
Components separate as the solvent moves up, forming distinct spots or bands that can
be visualized and identified by comparison to known standards.
2. Thin Layer Chromatography (TLC): Uses a thin layer of adsorbent material as the
stationary phase. Thin Layer Chromatography (TLC) separates mixture components on
a thin layer of adsorbent material. A sample is applied to the plate, which is placed in a
solvent. Components separate based on affinities, forming distinct spots that can be
visualized under UV light or with chemical reagents for identification.
3. Gas Chromatography (GC): Uses a gas as the mobile phase. Gas Chromatography
(GC) separates and analyzes volatile compounds based on boiling points and affinity
for stationary phase. A sample is vaporized and carried by inert gas through a column,
separating components detected by various detectors, providing qualitative and
quantitative analysis of complex mixtures with high precision and sensitivity.
4. Liquid Chromatography (LC): Uses a liquid as the mobile phase. Liquid
Chromatography (LC) separates and analyzes compounds based on interactions with
stationary and mobile phases. A sample is injected into a column, and components are
separated by various mechanisms, detected by detectors, providing qualitative and
quantitative analysis of complex mixtures with high resolution, sensitivity, and
versatility in various applications.
5. High-Performance Liquid Chromatography (HPLC): A type of LC that uses high
pressure to separate components. High-Performance Liquid Chromatography (HPLC)
is a powerful analytical technique that separates, identifies, and quantifies compounds
in a mixture. It uses high pressure to push a solvent through a column, allowing for
precise separation and detection of components, widely used in pharmaceuticals,
biotechnology, and environmental monitoring applications.

5
A REVIEW ON THIN LAYER CHROMATOGRAPHY

Introduction To Thin Layer Chromatography (TLC )

TLC is a laboratory technique used to separate, identify, and quantify the components of a
mixture by distributing them between a stationary phase (a thin layer of adsorbent material) and
a mobile phase (a solvent or a mixture of solvents).

Thin Layer Chromatography (TLC) is a laboratory technique used to separate, identify, and
quantify the components of a mixture by distributing them between a stationary phase and a
mobile phase. The stationary phase is typically a thin layer of adsorbent material, such as silica
gel or alumina, which is coated onto a flat surface, usually a glass plate or a plastic sheet. The
mobile phase is a solvent or a mixture of solvents that is allowed to move up the plate by
capillary action, carrying the components of the mixture with it. As the mobile phase moves
through the stationary phase, the components of the mixture separate based on their affinities
for the stationary and mobile phases, resulting in a chromatogram that shows the distribution
of the components.

 Background and Significance of Thin Layer Chromatography (TLC):

Thin Layer Chromatography (TLC) has a rich history dating back to the early 20th century,
when Mikhail Tsvet, a Russian botanist, first introduced the concept of chromatography. Over
the years, TLC has evolved into a versatile and widely used analytical technique, separating
mixtures of compounds based on their interactions with a stationary phase and a mobile phase.
The technique offers several advantages, including simplicity, cost-effectiveness, and high-
throughput analysis, making it a valuable tool for various applications. TLC has become an
essential technique in many fields, including pharmaceuticals, food and beverage analysis,
environmental monitoring, and biotechnology. Its significance lies in its ability to provide rapid
and reliable separations, allowing researchers to identify and quantify compounds with high
accuracy. For instance, in pharmaceutical analysis, TLC is used to identify and quantify active
pharmaceutical ingredients, while in environmental monitoring, it is used to detect and quantify
pollutants such as pesticides and heavy metals. In the food industry, TLC is used to detect and
quantify food additives, contaminants, and nutrients. In biotechnology, TLC is used to analyse
and purify biomolecules such as proteins and DNA. The technique's flexibility and wide range
of applications have made it a staple in many laboratories.

The significance of TLC extends beyond its practical applications, as it has also contributed
significantly to our understanding of various scientific phenomena. For example, TLC has been
used to study the interactions between molecules, the behaviour of complex systems, and the
properties of materials. Its ability to provide detailed information about the composition and
properties of complex mixtures has made it an essential tool in many fields of research. As
research continues to evolve, TLC is likely to remain a vital technique, providing valuable
insights and information that can inform and shape our understanding of the world around us.
With its continued development and advancement, TLC is poised to play an increasingly
important role in addressing the complex challenges facing our world today. By providing a

6
A REVIEW ON THIN LAYER CHROMATOGRAPHY

powerful tool for analysis and discovery, TLC will continue to contribute to breakthroughs in
fields such as medicine, environmental science, and biotechnology.

 Research Objectives and Scope:

Research Objectives

The primary objectives of this research are:

1. To investigate the optimization of TLC conditions: This study aims to explore the effects of
different stationary phases, mobile phases, and development conditions on the separation and
identification of compounds.

2. To evaluate the performance of TLC in various applications: This research will assess the
effectiveness of TLC in analysing pharmaceuticals, food and beverages, environmental
samples, and biological samples.

3. To develop new TLC methods and techniques: This study will focus on developing
innovative TLC methods and techniques to improve the sensitivity, selectivity, and efficiency
of the technique.

 Scope of TLC

The scope of this research includes:

1. Pharmaceutical analysis: This study will explore the application of TLC in the analysis of
pharmaceutical compounds, including the identification and quantification of active
ingredients. Thin Layer Chromatography (TLC) is used in pharmaceutical analysis for drug
identification, purity testing, and stability studies. It detects impurities, estimates drug content,
and monitors degradation products. TLC's simplicity, speed, and cost-effectiveness make it a
valuable tool for ensuring drug quality, safety, and efficacy in pharmaceutical industries.

2. Food and beverage analysis: This research will investigate the use of TLC in detecting and
quantifying contaminants, additives, and nutrients in food and beverages. Thin Layer
Chromatography (TLC) is used in food and beverage analysis to detect contaminants,
adulterants, and additives. It identifies and quantifies compounds like pesticides, mycotoxins,
and food dyes. TLC's simplicity and cost-effectiveness make it a valuable tool for ensuring food
safety and quality control in the industry.

3. Environmental monitoring: This study will examine the application of TLC in detecting and
quantifying environmental pollutants, such as pesticides and heavy metals. Thin Layer
Chromatography (TLC) is used in environmental monitoring to detect and quantify pollutants
like pesticides, heavy metals, and industrial contaminants in soil, water, and air samples

7
A REVIEW ON THIN LAYER CHROMATOGRAPHY

Literature Review
 History of TLC: -
 Early Beginnings

1. 1930s: The concept of TLC was first introduced by Russian botanist Mikhail Tsvet, who
used a thin layer of calcium carbonate to separate plant pigments.

2. 1940s-1950s: The technique was further developed by other scientists, including American
chemist Frederick Goppelsroder, who used TLC to separate and identify organic compounds.

 Development of Modern TLC

1. 1950s-1960s: The introduction of new stationary phases, such as silica gel and alumina,
improved the efficiency and selectivity of TLC.

2. 1960s-1970s: The development of TLC plates with a uniform layer of stationary phase
enabled more consistent and reproducible results.

3. 1970s-1980s: The introduction of high-performance TLC (HPTLC) plates with smaller

particle sizes and more uniform layers further improved the technique.

 Modern TLC

1. 1980s-present: Advances in instrumentation, software, and applications have continued to

evolve TLC, making it a powerful tool for analytical chemistry.

2. Current applications: TLC is widely used in various fields, including pharmaceuticals, food
and beverage analysis, environmental monitoring, and biotechnology.

 Key Figures
1. Mikhail Tsvet: Considered the father of chromatography, Tsvet introduced the concept
of TLC.

Mikhail Tsvet(1872-1919)
2. Frederick Goppelsroder: An American chemist, Goppelsroder developed early TLC
methods and applications.

8
A REVIEW ON THIN LAYER CHROMATOGRAPHY

Frederick Goppelsroder (1837-1919)

3. Erich Heftmann: A German-American chemist, Heftmann made significant
contributions to the development of modern TLC.

Erich Heftmann(1922- 1993)

 Impact of TLC

1. Simple and cost-effective: TLC is a relatively simple and cost-effective analytical

technique.

2. High-throughput analysis: TLC enables the analysis of multiple samples simultaneously.

3. Wide range of applications: TLC is used in various fields, including pharmaceuticals,

food and beverage analysis, and environmental monitoring.

 PRINCIPLE:

The principle of Thin Layer Chromatography (TLC) is based on the concept of partitioning,
which involves the distribution of a mixture's components between two phases: a stationary
phase and a mobile phase. In TLC, the stationary phase is a thin layer of adsorbent material,
such as silica gel or alumina, which is coated onto a flat surface, usually a glass plate or a plastic
sheet. The mobile phase is a solvent or a mixture of solvents that is allowed to move up the
plate by capillary action, carrying the components of the mixture with it.

As the mobile phase moves through the stationary phase, the components of the mixture
separate based on their affinities for the stationary and mobile phases. This separation is based
on the interactions between the components and the stationary phase, as well as the interactions

9
A REVIEW ON THIN LAYER CHROMATOGRAPHY

between the components and the mobile phase. The components that have a stronger affinity
for the stationary phase will move more slowly up the plate, while the components that have a
stronger affinity for the mobile phase will move more quickly.

The separation of the components in TLC is influenced by several factors, including the
properties of the stationary phase, the properties of the mobile phase, and the properties of the
components themselves. The stationary phase can be modified to suit the specific needs of the
analysis, and the mobile phase can be chosen to optimize the separation of the components. The
components themselves can also be modified, for example by derivatization, to improve their
separation.

Principle of Thin Layer Chromatography (TLC) is:

 Partitioning:

The separation of a mixture's components based on their distribution between a stationary phase
(a thin layer of adsorbent material) and a mobile phase (a solvent or a mixture of solvents).

Definition of partitioning:

Partitioning is a process where a substance distributes itself between two immiscible phases,
such as a solid and a liquid, or two liquids. In the context of chromatography, partitioning refers
to the distribution of a mixture's components between a stationary phase and a mobile phase.

Example:

Suppose we have a mixture of two substances, A and B, that we want to separate using TLC.
We apply the mixture to a TLC plate coated with silica gel (the stationary phase) and develop
the plate with a solvent mixture of hexane and ethyl acetate (the mobile phase).

Substance A is polar and has a strong affinity for the silica gel, while substance B is non-polar
and has a weak affinity for the silica gel. As the mobile phase moves up the plate, substance A
will partition itself between the silica gel and the mobile phase, with a greater proportion of it
remaining bound to the silica gel. Substance B, on the other hand, will partition itself more
evenly between the silica gel and the mobile phase, with a greater proportion of it moving up
the plate with the mobile phase.

As a result, substance A will appear as a spot closer to the starting point on the TLC plate, while
substance B will appear as a spot further up the plate. This separation is based on the differences
in the partitioning behaviour of the two substances between the stationary phase and the mobile
phase

Key factors influencing partitioning:

10
A REVIEW ON THIN LAYER CHROMATOGRAPHY

1. Polarity: Polar substances tend to partition more strongly into polar phases, while non-
polar substances tend to partition more strongly into non-polar phases.
2. Solubility: Substances that are more soluble in a particular phase will tend to partition
more strongly into that phase.
3. Intermolecular forces: Substances that have stronger intermolecular forces with a
particular phase will tend to partition more strongly into that phase.

 Applications of Thin Layer Chromatography (TLC):

 Pharmaceutical Analysis

1. Identification and quantification of active pharmaceutical ingredients (APIs): TLC is used to

identify and quantify APIs in pharmaceutical formulations.

2. Impurity profiling: TLC is used to detect and quantify impurities in pharmaceuticals.

3. Stability testing: TLC is used to monitor the stability of pharmaceuticals over time.

4. Quality control: TLC is used for quality control of pharmaceuticals, including the detection
of counterfeit or adulterated products.

 Food and Beverage Analysis

1. Detection of contaminants: TLC is used to detect contaminants such as pesticides, heavy

metals, and mycotoxins in food and beverages.

2. Identification of food additives: TLC is used to identify food additives such as preservatives,
colorants, and flavour enhancers.

3. Analysis of nutrients: TLC is used to analyse the nutrient content of food and beverages,
including vitamins, minerals, and amino acids.

4. Quality control: TLC is used for quality control of food and beverages, including the
detection of adulteration or spoilage.

 Environmental Monitoring

1. Detection of pollutants: TLC is used to detect pollutants such as pesticides, heavy metals,
and industrial chemicals in environmental samples.

2. Monitoring of water quality: TLC is used to monitor water quality, including the detection
of pollutants and contaminants.

3. Analysis of soil and sediment: TLC is used to analyse soil and sediment samples for
pollutants and contaminants.

11
A REVIEW ON THIN LAYER CHROMATOGRAPHY

4. Monitoring of air quality: TLC is used to monitor air quality, including the detection of
pollutants and particulate matter.

 Biotechnology

1. Analysis of biomolecules: TLC is used to analyse biomolecules such as proteins, DNA, and
RNA.

2. Purification of biomolecules: TLC is used to purify biomolecules, including the separation

of proteins and nucleic acids.

3. Detection of biomarkers: TLC is used to detect biomarkers for disease diagnosis and
monitoring.

4. Quality control: TLC is used for quality control of biotechnology products, including the
detection of contaminants or impurities.

 Clinical and Forensic Analysis

1. Detection of biomarkers: TLC is used to detect biomarkers for disease diagnosis and
monitoring.

2. Analysis of biological fluids: TLC is used to analyse biological fluids such as blood, urine,
and saliva for biomarkers and other analytes.

3. Detection of toxins and poisons: TLC is used to detect toxins and poisons in biological
samples.

4. Forensic analysis: TLC is used in forensic analysis, including the detection of drugs, poisons,
and other substances.

 Other Applications

Other applications of Thin Layer Chromatography (TLC) include:

1. Forensic Analysis: Analysis of evidence, such as drugs, toxins, and other substances. TLC is
used in forensic analysis to detect and identify substances like drugs, toxins, and poisons in
evidence samples precisely.

2. Clinical Toxicology: Detection of toxic substances in biological samples. Clinical toxicology

involves the diagnosis, management, and treatment of poisoning and adverse reactions to
substances. It encompasses toxicokinetics, toxicodynamics, and therapeutic interventions,
aiming to mitigate harm and improve patient outcomes in cases of exposure to toxic substances.
Emergency medicine is key.

3. Biotechnology: Analysis of biomolecules, such as proteins and nucleic acids. Biotechnology

in TLC enhances analysis of biomolecules, such as proteins, nucleic acids, and metabolites.
Techniques like bioautography and biochemical detection enable identification of bioactive

12
A REVIEW ON THIN LAYER CHROMATOGRAPHY

compounds, facilitating research in pharmaceuticals, agriculture, and diagnostics, and driving

innovation in life sciences and medicine.

4. Cosmetic Industry: Quality control and analysis of cosmetic products. The cosmetic industry
utilizes TLC to analyze and quality control raw materials, finished products, and stability
testing. It helps identify and quantify active ingredients, detect impurities, and ensure product
safety and efficacy, meeting regulatory standards and consumer expectations for skincare and
beauty products.

5. Pesticide Residue Analysis: Detection of pesticide residues in food and environmental

samples. Pesticide residue analysis using TLC detects and quantifies pesticide residues in food,
environmental samples, and agricultural products. It ensures compliance with regulatory limits,
assesses environmental impact, and helps protect consumer health by identifying contaminated
samples and monitoring pesticide usage. Food safety is ensured.

6. Dye and Pigment Analysis: Identification and analysis of dyes and pigments in various
industries. Dye and pigment analysis using TLC identifies and separates colorants, detects
impurities, and ensures quality control in textiles, paints, and food industries. It helps in
formulation development, authenticity testing, and compliance with regulations, ensuring
product safety and performance in various applications.

7. Plant Analysis: Analysis of plant extracts, including alkaloids, glycosides, and flavonoids.
TLC is used in plant analysis to separate, identify, and quantify plant constituents like alkaloids,
glycosides, and flavonoids in extracts.

8. Quality Control: Quality control of raw materials and finished products in various industries.
Quality control in TLC ensures the reliability and accuracy of analytical results. It involves
standardization, validation, and verification of methods, equipment, and procedures to meet
regulatory requirements, detect impurities, and guarantee product purity, potency, and
consistency in various industries and applications.

9. Research and Development: Isolation and identification of compounds in research

laboratories. TLC is used in R&D to isolate, identify, and purify compounds, facilitating
discovery and development of new products and technologies.

10. Teaching and Education: Educational tool for demonstrating chromatographic principles
and techniques. TLC is used in educational settings to demonstrate chromatographic principles,
techniques, and separation methods, promoting hands-on learning experiences.

13
A REVIEW ON THIN LAYER CHROMATOGRAPHY

 CLASSIFICATION OF THIN LAYER CHROMATOGRAPHY

 Based on the Stationary Phase:
1. Normal Phase TLC: Uses a polar stationary phase, such as silica gel or alumina, and a
non-polar mobile phase.
2. Reversed Phase TLC: Uses a non-polar stationary phase, such as C18 or C8, and a
polar mobile phase.
3. Ion Exchange TLC: Uses an ion exchange resin as the stationary phase and a buffer
solution as the mobile phase.
4. Size Exclusion TLC: Uses a porous stationary phase, such as silica gel or agarose, and
separates compounds based on their size.
 Based on the Mobile Phase:
1. Aqueous TLC: Uses water or a buffer solution as the mobile phase.
2. Non-Aqueous TLC: Uses an organic solvent, such as hexane or ethyl acetate, as the
mobile phase.
3. Mixed Mobile Phase TLC: Uses a mixture of aqueous and non-aqueous solvents as
the mobile phase.
 Based on the Plate Type:
1. Glass Plate TLC: Uses a glass plate as the support for the stationary phase.
2. Aluminium Plate TLC: Uses an aluminium plate as the support for the stationary
phase.
3. Plastic Plate TLC: Uses a plastic plate as the support for the stationary phase.

1. Based on the mobile phase

1. Aqueous TLC (ATLC)

Mobile phase: Water or buffer solution
Stationary phase: Polar, e.g., silica gel or cellulose
Separation mechanism: Adsorption or partitioning
Application: Separation of polar compounds, such as pharmaceuticals, dyes, and
pesticides
3. Non-Aqueous TLC (NATLC)
Mobile phase: Organic solvent, e.g., hexane, dichloromethane, or ethyl acetate
Stationary phase: Non-polar, e.g., C18 or C8
Separation mechanism: Partitioning
Application: Separation of non-polar compounds, such as lipids, steroids, and
environmental pollutants

4. Mixed Mobile Phase TLC (MMPTLC)

Mobile phase: Mixture of aqueous and non-aqueous solvents
Stationary phase: Polar or non-polar, e.g., silica gel or C18
Separation mechanism: Adsorption or partitioning

14
A REVIEW ON THIN LAYER CHROMATOGRAPHY

METHEDOLOGY

 Instrumentation of TLC

Instrumentation used in Thin Layer Chromatography (TLC):

1. TLC Plates:

Figure:2 TLC plate

Material: Glass, aluminium, or plastic

Thickness: 0.2-2.0 mm

Stationary phase: Silica gel, alumina, or cellulose

 Function of TLC plate:

TLC plates are a powerful tool for separating, identifying, and quantifying mixtures, and are
widely used in various fields, including pharmaceutical analysis, food and beverage analysis,
environmental monitoring, and biotechnology.

Key Functions

1. Separation: TLC plates separate mixtures into their individual components based on their
affinities for the stationary and mobile phases.

2. Identification: TLC plates can be used to identify unknown compounds by comparing their
retention factors (Rf values) to those of known compounds.

3. Quantification: TLC plates can be used to quantify the amount of a particular compound in
a mixture by measuring the intensity of the spot or band corresponding to that compound.

4. Purification: TLC plates can be used to purify compounds by separating them from impurities
or other contaminants.

15
A REVIEW ON THIN LAYER CHROMATOGRAPHY

2. Spotting Device:

Figure: 3 Micropipette

Type: Micropipette or capillary tube

Volume: 1-10 μL

 Function: Apply sample to the TLC plate

1. Apply precise sample volumes: The spotting device applies a precise volume of sample to
the TLC plate.

2. Minimize sample waste: The spotting device minimizes sample waste by applying only the
required amount of sample to the TLC plate.

3. Improve reproducibility: The spotting device improves reproducibility by ensuring that the
same amount of sample is applied to each TLC plate.

3. Development Chamber:
 Function:

1. Control the atmosphere: The chamber controls the atmosphere around the TLC plate,
allowing the solvent to move up the plate by capillary action.

2. Saturate the atmosphere: The chamber is often saturated with the solvent vapor, which helps
to ensure even development of the TLC plate.

3. Develop the TLC plate: The chamber allows the TLC plate to develop, separating the
components of the sample based on their interactions with the stationary and mobile phases.

Types of Development Chambers

1. Glass chambers: These are traditional development chambers made of glass.

16
A REVIEW ON THIN LAYER CHROMATOGRAPHY

Figure: 4 Glass chambers

2. Plastic chambers: These are lightweight and portable development chambers made of
plastic.

Figure: 5 Plastic chambers

3. Automated development chambers: These are electronic devices that can control the
development process, including temperature, humidity, and solvent flow.

Figure: 6 Automated development Chamber

17
A REVIEW ON THIN LAYER CHROMATOGRAPHY

4. Mobile Phase:

Figure:7 mobile phases

 Function: Separate the components of the sample

1. Separates components: The mobile phase separates the components of the sample based on
their affinities for the stationary phase and the mobile phase.

2. Moves components up the plate: The mobile phase moves the components of the sample up
the TLC plate, allowing for their separation and identification.

3. Interacts with stationary phase: The mobile phase interacts with the stationary phase,
influencing the separation of the components of the sample.

4. Controls retention time: The mobile phase controls the retention time of the components of
the sample, which is the time it takes for a component to move up the TLC plate.

5. Influences selectivity: The mobile phase influences the selectivity of the separation, which
is the ability of the TLC system to distinguish between different components of the sample.

5. Detection Methods:

Figure: 8 UV Light: Detects compounds that absorb UV light

Iodine Vapor: Detects compounds that react with iodine

18
A REVIEW ON THIN LAYER CHROMATOGRAPHY

Spray Reagents: Detects compounds that react with specific reagents (e.g., ninhydrin,
Dragendorff's reagent)

1. Visualization: Detection methods allow for the visualization of the separated components on
the TLC plate.

2. Identification: Detection methods help identify the components of the sample based on their
retention factor (Rf) values, colour, or other characteristics.

3. Quantification: Some detection methods can be used to quantify the amount of each
component present in the sample.

4. Confirmation: Detection methods can confirm the presence or absence of specific

components in the sample.

6. Densitometer:

Figure: 9 Reflectance or transmission densitometer

 Function: Measures the intensity of the spots on the TLC plate

1. Measures density: The densitometer measures the density of the separated components on
the TLC plate.

2. Quantifies components: The densitometer can be used to quantify the amount of each
component present in the sample.

3. Provides chromatograms: The densitometer produces chromatograms, which are graphical

representations of the separated components.

4. Analyses TLC plates: The densitometer analyses the TLC plate, providing information on
the retention factor (Rf) values, peak heights, and peak areas.

7. TLC Scanner:
19
A REVIEW ON THIN LAYER CHROMATOGRAPHY

Figure: 10 Flatbed scanner or camera

 Function: Captures an image of the TLC plate

1. Scans TLC plates: The TLC scanner scans the TLC plate, detecting the separated
components.

2. Measures absorbance: The TLC scanner measures the absorbance of the separated
components.

3. Provides chromatograms: The TLC scanner produces chromatograms, which are graphical
representations of the separated components.

4. Analyses components: The TLC scanner analyses the components, providing information on
retention factor (Rf) values, peak heights, and peak areas.

8. Chromatography Software:

20
A REVIEW ON THIN LAYER CHROMATOGRAPHY

Figure: 11 Computer program (e.g., TLC Analyzer, Camag TLC, visionCATS software)

 Function: Analyses the TLC data, including spot detection, quantitation, and identification

1. Instrument control: The software controls the chromatography instrument, allowing for
automated analysis.

2. Data acquisition: The software collects and stores data from the chromatography instrument.

3. Data analysis: The software analyses the data, providing information on retention factor (Rf)
values, peak heights, and peak areas.

4. Chromatogram display: The software displays chromatograms, which are graphical

representations of the separated components.

5. Quantification: The software can be used to quantify the amount of each component present
in the sample.

6. Method development: The software can be used to develop and optimize chromatography
methods.

7. Regulatory compliance: The software can help with regulatory compliance by providing tools
for data management and audit trails.

9. TLC Plate Heater:

A TLC plate heater is a laboratory device used to heat TLC plates, facilitating rapid
development, visualization, and detection of separated compounds. It ensures uniform heating,
accelerates drying, and enhances spot visibility, making it an essential tool in chromatography.

21
A REVIEW ON THIN LAYER CHROMATOGRAPHY

Figure: 12 Electric or hot air heater

 Function: Heats the TLC plate to accelerate the development process

1. Dries TLC plates: The TLC plate heater dries the TLC plate, removing any excess solvent or
moisture.

2. Accelerates development: The TLC plate heater can accelerate the development process by
increasing the rate of solvent flow.

3. Improves separation: Heating the TLC plate can improve the separation of components by
altering their interactions with the stationary phase.

4. Enhances detection: Heating the TLC plate can enhance the detection of components by
altering their optical properties.

10. TLC Plate Cooler:

A TLC plate cooler is a laboratory device that rapidly cools TLC plates after development or
heating. It helps prevent degradation of sensitive compounds, stops reactions, and preserves
plate integrity. By controlling temperature, it enhances spot stability and visualization, ensuring
accurate analysis and reliable results in chromatographic applications.

A TLC plate cooler rapidly cools TLC plates after development or heating, preventing
degradation of sensitive compounds and stopping reactions. It preserves plate integrity,
enhances spot stability, and ensures accurate analysis. By controlling temperature, it maintains
compound integrity, allowing for reliable results and precise chromatographic applications,
ideal for research and quality control.

22
A REVIEW ON THIN LAYER CHROMATOGRAPHY

Figure: 13 Cooling plate or cold air blower

 Function: Cools the TLC plate to slow down the development process

1. Slows down development: The TLC plate cooler slows down the development process by
reducing the rate of solvent flow.

2. Improves separation: Cooling the TLC plate can improve the separation of components by
altering their interactions with the stationary phase.

3. Enhances detection: Cooling the TLC plate can enhance the detection of components by
altering their optical properties.

4. Preserves sensitive compounds: Cooling the TLC plate can help preserve sensitive
compounds that may degrade or react at higher temperatures.

 Sample Preparation and Application in Thin Layer Chromatography (TLC):

 Sample Preparation:
 Sampling:

Sampling is the process of collecting a representative sample from a larger population or

material for analysis.

Importance

Sampling is a critical step in TLC, as it ensures that the sample analysed is representative of the
material of interest.

Types of Sampling

1. Random sampling: A random sample is collected from the material of interest.

2. Systematic sampling: A systematic sample is collected at regular intervals from the material
of interest.

23
A REVIEW ON THIN LAYER CHROMATOGRAPHY

3. Stratified sampling: A stratified sample is collected from different subgroups or strata within
the material of interest.

 Extraction:

Extraction in sample preparation for TLC involves separating the analyte from the sample
matrix using various methods, such as solvent extraction, solid-phase extraction (SPE), and
liquid-liquid extraction (LLE). The choice of extraction method depends on the sample matrix,
analyte properties, and desired efficiency. Effective extraction ensures sufficient analyte
recovery, sample cleanliness, and reliable TLC results. Techniques like shaking, sonication,
and centrifugation can be used to enhance extraction efficiency. By optimizing extraction
conditions, researchers can improve the accuracy and reliability of TLC analysis.

1. Extraction methods selection: Common extraction methods include Soxhlet extraction,

sonication, and shaking.

Extraction is the process of separating a substance from a mixture using a solvent.

Importance

Extraction is a critical step in TLC, as it affects the separation and detection of components.

Types of Extraction Methods

1. Soxhlet extraction: A continuous extraction method using a solvent.

2. Sonication: A method using high-frequency sound waves to extract substances.

3. Shaking: A method using manual or mechanical shaking to extract substances.

4. Maceration: A method using a solvent to extract substances from plant materials.

5. Infusion: A method using hot water to extract substances from plant materials.

Factors to Consider

1. Solvent selection: The choice of solvent depends on the substance to be extracted.

2. Extraction time: The length of time for extraction dep ends on the substance and solvent used.

3. Temperature: The temperature of extraction depends on the substance and solvent used.

4. Solvent-to-sample ratio: The ratio of solvent to sample depends on the substance and solvent
used.

2. Extraction time selection:

The extraction time depends on the sample and the solvent used.

24
A REVIEW ON THIN LAYER CHROMATOGRAPHY

Extraction time is the length of time that a solvent is in contact with a sample to extract the
desired compounds.

Factors Affecting Extraction Time

1. Solvent selection: The choice of solvent affects the extraction time.

2. Sample size: The size of the sample affects the extraction time.

3. Temperature: The temperature of the extraction process affects the extraction time.

4. Solvent-to-sample ratio: The ratio of solvent to sample affects the extraction time.

Optimal Extraction Time

1. Minimum extraction time: The minimum time required to extract the desired compounds.

2. Maximum extraction time: The maximum time beyond which no further extraction occurs.

Methods to Determine Optimal Extraction Time

1. Trial and error: Testing different extraction times to determine the optimal time.

2. Kinetic studies: Studying the rate of extraction to determine the optimal time.

3. Mathematical modelling: Using mathematical models to predict the optimal extraction time.

3. Extraction temperature selection:

The extraction temperature depends on the sample and the solvent used.

Extraction temperature is the temperature at which a solvent is used to extract desired

compounds from a sample.

Factors Affecting Extraction Temperature

1. Solvent selection: Different solvents have different optimal temperatures.

2. Sample size: Larger samples may require higher temperatures.

3. Solvent-to-sample ratio: Higher ratios may require higher temperatures.

4. Desired compounds: Different compounds have different optimal temperatures.

Optimal Extraction Temperature Ranges

1. Low temperature: 0-20°C (e.g., for heat-sensitive compounds)

2. Moderate temperature: 20-40°C (e.g., for most organic compounds)

3. High temperature: 40-60°C (e.g., for difficult-to-extract compounds)

 Purification

25
A REVIEW ON THIN LAYER CHROMATOGRAPHY

Purification is a crucial step in sample preparation for TLC analysis, removing impurities to
enhance accuracy and reliability. Methods include filtration, Solid-Phase Extraction (SPE), and
Liquid-Liquid Extraction (LLE). Filtration removes particulate matter, SPE selectively retains
impurities or analytes, and LLE separates analytes based on solubility differences. For example,
analysing caffeine in tea involves steeping tea leaves, filtering, using a C18 cartridge for SPE,
and eluting caffeine with a suitable solvent. Purification enhances sensitivity, accuracy, and
reliability, ensuring high-quality TLC results. By removing impurities, researchers can achieve
better quantitation of analytes, which is vital for informed decisions. Effective purification is
essential in various fields, including pharmaceuticals, biotechnology, and food analysis. By
optimizing purification methods, researchers can achieve reliable and accurate results, critical
for applications where precision is key. Purification ultimately ensures confident and
accurate TLC analysis.

Methods

1. Filtration: Removing particulate matter using filters (e.g., syringe filters) or centrifuges.

2. Solid-Phase Extraction (SPE): Using solid phases (e.g., C18 cartridges) to selectively retain
impurities or analytes.

3. Liquid-Liquid Extraction (LLE): Separating analytes from impurities based on solubility

differences (e.g., extracting analytes from aqueous solutions using organic solvents).

 Concentration

Concentration involves increasing the amount of analyte in a sample to enhance detection and
quantitation.

Methods include:

1. Evaporation: Removing solvent through heat or vacuum.

2. Lyophilization: Freeze-drying samples.

3. Solid-phase extraction: Concentrating analytes.

Concentration enhances:

1. Detection limits: Improving sensitivity.

2. Quantitation accuracy: Increasing analyte concentration.

Common applications include:

1. Environmental analysis: Concentrating pollutants.

2. Biological samples: Concentrating biomarkers.

26
A REVIEW ON THIN LAYER CHROMATOGRAPHY

 Solvent selection: A suitable solvent is selected based on the properties of the sample and
the compounds of interest.
Solvent selection is a critical step in TLC, as it affects the separation and detection of
components.

Factors to Consider

1. Polarity: The solvent should be compatible with the stationary phase and the components of
interest.

2. Solubility: The solvent should be able to dissolve the components of interest.

3. Viscosity: The solvent should have a suitable viscosity for easy handling.

4. Boiling point: The solvent should have a suitable boiling point for easy evaporation.

5. Toxicity: The solvent should be non-toxic and safe to handle.

Common Solvents Used in TLC

1. Non-polar solvents: Hexane, heptane, and petroleum ether.

2. Polar solvents: Water, methanol, ethanol, and acetone.

3. Moderately polar solvents: Dichloromethane, chloroform, and ethyl acetate.

Solvent Systems

1. Single solvent system: A single solvent is used as the mobile phase.

2. Binary solvent system: A mixture of two solvents is used as the mobile phase.

3. Ternary solvent system: A mixture of three solvents is used as the mobile phase.

 Sample Application in Thin Layer Chromatography (TLC)

Sample application is a crucial step in TLC, requiring precision and consistency. The goal is to
apply a small, precise volume of sample onto the TLC plate.

Sample application in TLC involves precisely applying a small volume (1-10 μL) of sample
onto the plate. Methods include manual spotting using capillaries or micropipettes and
automated applicators. Key considerations include sample volume, spot size, and application
position. Proper application ensures:

1. Improved resolution

2. Reproducibility

3. Accurate analysis

27
A REVIEW ON THIN LAYER CHROMATOGRAPHY

Sample Application in Thin Layer Chromatography (TLC)

Sample application is a crucial step in TLC, involving the precise application of samples onto
the TLC plate. Techniques include:

1. Spotting: Using a capillary tube or syringe to apply small sample volumes. Spotting in TLC
involves applying small sample volumes (typically 1-5 μL) onto the plate using a capillary
tube or syringe, creating a precise, compact spot. This technique requires care to ensure
consistent and accurate sample application for reliable results.
2. Streaking: Applying larger sample volumes in a streak pattern. Streaking in TLC involves
applying larger sample volumes in a continuous streak across the plate, rather than a single
spot. This technique is useful for preparative TLC, allowing for the isolation and
purification of larger quantities of compounds.
3. 3. Automatic applicators: Using instruments for precise and reproducible sample
application. Automatic applicators in TLC precisely and reproducibly apply samples onto
plates, ensuring consistent volumes and positions. These instruments enhance accuracy,
efficiency, and reliability in chromatographic analysis, reducing human error and
variability, and are ideal for high-throughput and quantitative applications.

Proper sample application ensures:

1. Sharp separations- Sharp separations in TLC refer to the clear and distinct separation of
compounds into well-defined spots or bands, allowing for accurate identification and
quantification. This is achieved through optimized solvent systems, precise sample application,
and controlled development conditions.

2. Accurate results- Accurate results in TLC are achieved through precise sample application,
optimal solvent systems, and controlled development conditions.

28
A REVIEW ON THIN LAYER CHROMATOGRAPHY

 Development and detection methods:

 Development Methods:

Development methods in TLC refer to the techniques used to separate and detect compounds
on a TLC plate. These methods involve the movement of a solvent through the stationary phase,
allowing the compounds to separate based on their interactions with the solvent and the
stationary phase.

 Purpose

The purpose of development methods is to:

1. Separate compounds: Separate the compounds in a mixture based on their properties.

2. Detect compounds: Detect the presence of specific compounds on the TLC plate.

3. Identify compounds: Identify the compounds present in a mixture based on their retention
factor (Rf) values.

 Types of Development Methods:

1. Ascending development: The solvent moves up the plate by capillary action.

2. Descending development: The solvent moves down the plate by gravity.

3. Horizontal development: The solvent moves horizontally across the plate.

1. Ascending development:

Ascending development is a technique used in TLC where the solvent moves up the plate by
capillary action.

Figure: 14 Ascending development

29
A REVIEW ON THIN LAYER CHROMATOGRAPHY

 Principle

The principle of ascending development is based on the capillary action of the solvent, which
allows it to move up the plate and separate the compounds based on their interactions with the
stationary phase and the solvent.

 Solvent Movement

1. Solvent front: The solvent front is the leading edge of the solvent as it moves up the plate.

2. Solvent flow: The solvent flows through the stationary phase, interacting with the compounds
and separating them based on their properties.

 Separation Mechanism

1. Adsorption: Compounds are adsorbed onto the stationary phase, with stronger interactions
resulting in slower movement.

2. Partitioning: Compounds partition between the stationary phase and the solvent, with more
soluble compounds moving further up the plate.

3. Separation: Compounds are separated based on their interactions with the stationary phase
and the solvent, resulting in distinct bands or spots on the plate.

 Factors Affecting Separation

1. Solvent selection: The choice of solvent affects the separation of compounds.

2. Stationary phase: The type and properties of the stationary phase affect the separation of
compounds.

3. Temperature: Temperature affects the rate of solvent movement and separation.

4. Humidity: Humidity affects the rate of solvent movement and separation.

 Procedure

1. Prepare the TLC plate: Apply the sample to the TLC plate and dry it.

2. Prepare the developing chamber: Line the developing chamber with filter paper and add the
solvent.

3. Develop the plate: Place the TLC plate in the developing chamber and allow the solvent to
move up the plate by capillary action.

4. Dry the plate: Remove the plate from the developing chamber and dry it.

 Applications

1. Qualitative analysis: Ascending development is useful for qualitative analysis, such as

identifying compounds in a mixture.

30
A REVIEW ON THIN LAYER CHROMATOGRAPHY

2. Quantitative analysis: Ascending development can be used for quantitative analysis, such as
determining the concentration of a compound.

3. Preparative TLC: Ascending development can be used for preparative TLC, where the goal
is to isolate and purify compounds.

 Advantages

1. Simple and easy to perform: Ascending development is a straightforward technique that

requires minimal equipment.

2. Fast development time: The development time is relatively short, typically ranging from 30
minutes to several hours.

3. Good separation: Ascending development can provide good separation of compounds,

especially for simple mixtures.

 Disadvantages

1. Limited sample capacity: Ascending development is not suitable for large sample volumes
or complex mixtures.

2. Solvent front: The solvent front can be uneven, leading to irregular separation patterns.

3. Limited resolution: Ascending development may not provide the best resolution for complex
mixtures.

2. Descending development

Descending development is a technique used in TLC where the solvent moves down the plate
by gravity.

Figure: 15 Descending development

31
A REVIEW ON THIN LAYER CHROMATOGRAPHY

 Principle

The principle of descending development is based on the flow of solvent down the plate,
allowing the compounds to separate based on their interactions with the stationary phase and
the solvent.

 Solvent Movement

1. Solvent front: The solvent front is the leading edge of the solvent as it moves down the plate.

2. Solvent flow: The solvent flows down the plate, interacting with the compounds and
separating them based on their properties.

 Separation Mechanism

1. Adsorption: Compounds are adsorbed onto the stationary phase, with stronger interactions
resulting in slower movement.

2. Partitioning: Compounds partition between the stationary phase and the solvent, with more
soluble compounds moving further down the plate.

3. Separation: Compounds are separated based on their interactions with the stationary phase
and the solvent, resulting in distinct bands or spots on the plate.

 Factors Affecting Separation

1. Solvent selection: The choice of solvent affects the separation of compounds.

2. Stationary phase: The type and properties of the stationary phase affect the separation of
compounds.

3. Temperature: Temperature affects the rate of solvent movement and separation.

4. Humidity: Humidity affects the rate of solvent movement and separation.

 Equipment and Materials

1. TLC plate: A glass or plastic plate coated with a thin layer of stationary phase.

2. Solvent reservoir: A container used to hold the solvent.

3. Wick or tube: A device used to connect the solvent reservoir to the TLC plate.

4. Developing chamber: A chamber or tank used to develop the TLC plate.

 Procedure

1. Prepare the TLC plate: Apply the sample to the TLC plate and dry it.

2. Prepare the solvent reservoir: Fill the solvent reservoir with the chosen solvent.

32
A REVIEW ON THIN LAYER CHROMATOGRAPHY

3. Connect the wick or tube: Connect the wick or tube to the solvent reservoir and the TLC
plate.

4. Develop the plate: Allow the solvent to flow down the plate by gravity, separating the
compounds.

5. Dry the plate: Remove the plate from the developing chamber and dry it.

 Applications

1. Qualitative analysis: Descending development is useful for qualitative analysis, such as

identifying compounds in a mixture.

2. Quantitative analysis: Descending development can be used for quantitative analysis, such
as determining the concentration of a compound.

3. Preparative TLC: Descending development can be used for preparative TLC, where the goal
is to isolate and purify compounds.

 Advantages

1. Better separation: Descending development can provide better separation of compounds,

especially for complex mixtures.

2. Larger sample capacity: Descending development can handle larger sample volumes than
ascending development.

3. Improved resolution: Descending development can provide improved resolution of

compounds, especially for those with similar properties.

 Disadvantages

1. More equipment required: Descending development requires more equipment than ascending
development, including a solvent reservoir and a wick or tube.

2. Longer development time: The development time for descending development can be longer
than for ascending development.

3. More difficult to perform: Descending development can be more difficult to perform than
ascending development, requiring more expertise and attention to detail.

3. Horizontal Development:

Horizontal development is a technique used in TLC where the solvent moves horizontally
across the plate.

33
A REVIEW ON THIN LAYER CHROMATOGRAPHY

Direction of mobile phase

paper

support

mobile phase
Figure: 16 Horizontal Development

 Principle

The principle of horizontal development is based on the flow of solvent across the plate,
allowing the compounds to separate based on their interactions with the stationary phase and
the solvent.

 Capillary Action and Solvent Flow

1. Capillary action: The solvent moves through the stationary phase by capillary action.

2. Solvent flow: The solvent flows horizontally across the plate, interacting with the
compounds and separating them based on their properties.

 Separation Mechanism

1. Adsorption: Compounds are adsorbed onto the stationary phase, with stronger interactions
resulting in slower movement.

2. Partitioning: Compounds partition between the stationary phase and the solvent, with more
soluble compounds moving further across the plate.

3. Separation: Compounds are separated based on their interactions with the stationary phase
and the solvent, resulting in distinct bands or spots on the plate.

 Factors Affecting Separation

1. Solvent selection: The choice of solvent affects the separation of compounds.

2. Stationary phase: The type and properties of the stationary phase affect the separation of
compounds.

3. Temperature: Temperature affects the rate of solvent movement and separation.

34
A REVIEW ON THIN LAYER CHROMATOGRAPHY

4. Humidity: Humidity affects the rate of solvent movement and separation.

 Equipment and Materials

1. TLC plate: A glass or plastic plate coated with a thin layer of stationary phase.

2. Horizontal development chamber: A specialized chamber designed for horizontal

development.

3. Solvent reservoir: A container used to hold the solvent.

4. Wick or tube: A device used to connect the solvent reservoir to the TLC plate

 Procedure

1. Prepare the TLC plate: Apply the sample to the TLC plate and dry it.

2. Prepare the horizontal development chamber: Assemble the horizontal development

chamber and add the solvent.

3. Develop the plate: Place the TLC plate in the horizontal development chamber and allow
the solvent to move horizontally across the plate.

4. Dry the plate: Remove the plate from the development chamber and dry it.

 Applications

1. Qualitative analysis: Horizontal development is useful for qualitative analysis, such as

identifying compounds in a mixture.

2. Quantitative analysis: Horizontal development can be used for quantitative analysis, such
as determining the concentration of a compound.

3. Preparative TLC: Horizontal development can be used for preparative TLC, where the goal
is to isolate and purify compounds.

 Advantages

1. Improved separation: Horizontal development can provide improved separation of

compounds, especially for complex mixtures.

2. Increased resolution: Horizontal development can provide increased resolution of

compounds, especially for those with similar properties.

3. Reduced development time: Horizontal development can reduce the development time, as
the solvent moves more quickly across the plate.

 Disadvantages

1. Specialized equipment required: Horizontal development requires specialized equipment,

including a horizontal development chamber.

35
A REVIEW ON THIN LAYER CHROMATOGRAPHY

2. More difficult to perform: Horizontal development can be more difficult to perform than
ascending or descending development.

3. Increased risk of solvent front irregularities: Horizontal development can result in

irregularities in the solvent front, affecting the separation of compounds.

 Detection methods-

Detection methods in Thin Layer Chromatography (TLC) are crucial for visualizing and
identifying separated compounds. Various techniques are employed, each with its own
strengths. Ultraviolet (UV) light is commonly used, where compounds either absorb UV
radiation, appearing as dark spots, or fluoresce, emitting light. Iodine vapor is another method,
where iodine reacts with compounds to form brown spots. Chemical derivatization involves
applying reagents to produce colored or fluorescent derivatives, enhancing detectability.
Specific reagents, such as ninhydrin for amino acids, can be used to target particular compound
classes. Densitometry allows for quantitative analysis by measuring the optical density of spots.
The choice of detection method depends on the properties of the analytes and the desired
sensitivity and specificity. By selecting the appropriate detection technique, researchers can
accurately identify and quantify compounds on the TLC plate. Different detection methods can
be used alone or in combination to achieve optimal results, ensuring comprehensive analysis of
complex samples. Effective detection is key to unlocking the full potential of TLC in various
applications. This versatility makes TLC a valuable tool.

Detection Methods in Thin Layer Chromatography (TLC)

TLC detection methods include:

1. Visual detection: Observing coloured or fluorescent spots.

2. UV detection: Using UV light to detect absorbing compounds.

3. Fluorescence detection: Detecting fluorescent compounds.

4. Derivatization: Chemical reaction to enhance detection.

5. Densitometry: Measuring spot intensity.

 Visual Detection in Thin Layer Chromatography (TLC)

Visual detection involves observing coloured or fluorescent spots on a TLC plate. Compounds
with distinct colours can be directly visible, while fluorescent compounds require UV light to
be detected. This method is simple, rapid, and useful for preliminary assessments.

Visual detection is limited by its sensitivity and specificity, but it's valuable for initial screening
and identifying compounds with distinct colours or fluorescence. Researchers can observe the
plate under visible or UV light to detect spots and calculate retention factor (Rf) values
for identification

36
A REVIEW ON THIN LAYER CHROMATOGRAPHY

 UV Detection in Thin Layer Chromatography (TLC)

UV detection involves using ultraviolet light to detect compounds that absorb UV radiation.
Compounds appear as dark spots on the TLC plate when viewed under UV light, typically at
254 nm or 366 nm wavelengths.

This method is sensitive and widely used for detecting aromatic and conjugated compounds.
UV detection enhances the specificity and accuracy of TLC analysis, enabling researchers to
identify and quantify compounds with high precision.

 Fluorescence Detection in Thin Layer Chromatography (TLC)

Fluorescence detection involves detecting compounds that emit fluorescence when excited by
UV light. Compounds appear as bright spots on the TLC plate, allowing for sensitive and
selective detection.

This method is highly useful for detecting compounds with fluorescent properties, such as
certain pharmaceuticals, biomolecules, and environmental pollutants. Fluorescence detection
enhances the specificity and sensitivity of TLC analysis.

 Densitometry in Thin Layer Chromatography (TLC)

Densitometry measures the intensity of spots on a TLC plate, enabling quantitation of analytes.
It involves scanning the plate with a densitometer, which measures absorbance or fluorescence.

This technique provides accurate and reliable results, allowing researchers to quantify
compounds with high precision. Densitometry enhances the analytical capabilities of TLC,
providing quantitative data for informed decisions.

 Derivatization in Thin Layer Chromatography (TLC)

Derivatization involves chemically modifying compounds to enhance detectability or

separation. It converts non-detectable compounds into detectable ones, improving sensitivity
and specificity.

This technique enhances detection and separation of compounds, allowing researchers to

analyse a wider range of analytes. Derivatization is useful for detecting compounds with
specific functional groups, improving TLC's analytical capabilities.

37
A REVIEW ON THIN LAYER CHROMATOGRAPHY

Data analysis and interpretation:

Data analysis is a crucial step in Thin Layer Chromatography (TLC), a widely used analytical
technique. After developing and visualizing the TLC plate, data analysis is performed to extract
meaningful information from the chromatogram.

The primary goal of data analysis in TLC is to interpret the chromatogram and extract relevant
information about the compounds present in the sample. This includes calculating retention
factor (Rf) values, assessing peak shape and resolution, and quantifying compounds using
densitometry or other methods.

Accurate data analysis enables researchers to draw meaningful conclusions about the
composition and properties of samples. By applying statistical methods and using specialized
software, researchers can extract valuable information from TLC chromatograms.

Effective data analysis in TLC requires a thorough understanding of the underlying principles
and careful attention to detail. By combining TLC with robust data analysis, researchers can
unlock the full potential of this powerful analytical technique and gain valuable insights into
the composition and properties of complex samples.

Data analysis in TLC is essential in various fields, including pharmaceutical development, food
safety, and environmental monitoring, where accurate identification and quantification of
compounds are critical.

1. Measuring Rf Values

The Rf (retardation factor) value is the ratio of the solute’s distance travelled to the solvent’s
distance travelled.

The word comes from chromatography when it was discovered that a given component will
always travel the same distance in a given solvent under the same conditions.

The Rf value is a physical constant for organic molecules that can be used to verify a molecule’s
identity. Only if the chromatographic settings below are also constant from one trial to the next
does the Rf for a substance remain constant. Because these variables are challenging to maintain
consistency from experiment to experiment, relative Rf values are commonly used. “Relative
Rf” denotes that the results are provided in comparison to a standard, or that the R f values of
compounds run on the same plate at the same time are compared

 Steps to Measure Rf Values

1. Identify the solvent front: Measure the distance from the origin to the solvent front.

The solvent front is the furthest point that the solvent has travelled up the TLC plate.

Characteristics

38
A REVIEW ON THIN LAYER CHROMATOGRAPHY

1. Visible line or edge: The solvent front is often visible as a line or edge on the plate.

2. Change in appearance: The solvent front can be identified by a change in appearance, such
as a change in colour or texture.

Methods to Identify the Solvent Front

1. Visual inspection: Visually inspect the plate to identify the solvent front.

2. Marking the plate: Mark the solvent front on the plate during development.

3. Using a UV lamp: Use a UV lamp to detect the solvent front if it's not visible

2. Measure the distance travelled by the compound: Measure the distance from the origin
to the centre of the compound spot.

1. Locate the compound spot: Identify the centre of the compound spot on the TLC plate.

2. Identify the origin: Determine the point where the sample was applied (origin).

3. Measure the distance: Use a ruler or caliper to measure the distance from the origin to the
centre of the compound spot.

Measurement Considerations
1. Accuracy: Ensure accurate measurement to obtain reliable Rf values.
2. Unit consistency: Measure distances in the same units (e.g., mm or cm).
3. Multiple measurements: Take multiple measurements for reproducibility.
3. Calculate the Rf value: Use the formula to calculate the Rf value for each compound.

Depending on the nature of the analytes and the stationary phases, a chromatogram must first
be generated with an appropriate solvent (mobile phase). After drying the chromatogram, the
locations (migration values) of the analytes and the solvent front are measured.

Figure: 16 TLC RF value

Using the stated approach and the above experiment, the R f (retardation/retention factor) values
can be computed.

39
A REVIEW ON THIN LAYER CHROMATOGRAPHY

On the chromatography paper, a prepared sample solution (A+B) is applied and processed
through a mobile phase. Because of their differing affinities with the mobile phase, analytes
(A) and (B) separate (solvent). The analytes, the solvent front, and the point where the mixture
(A+B) was administered are all measured relative to each other.

 Rf values are used to identify the components of mixtures

 The Rf value of a particular compound is always the same

o However, it does depend on the solvent used

o If the solvent is changed then the Rf value changes

 Calculating the Rf value allows chemists to identify unknown substances because it

can be compared with the Rf values of known substances under the same conditions

 The retention factor, Rf, is calculated by the equation:

Rf = distance moved by substance / distance moved by solvent

 The Rf value:

o Is a ratio

o Has no units

o Will always be less than 1

40
A REVIEW ON THIN LAYER CHROMATOGRAPHY

Results and Discussion

 Results:

1. Rf Values

Rf values are calculated for each compound and reported in a table or graph. These values
provide insight into the retention properties of each compound and can be used for identification
purposes.

2. Chromatogram Description

The chromatogram is described in detail, including:

1. Spot shape: The shape of the compound spots, such as round, oval, or irregular.

2. Spot size: The size of the compound spots, which can indicate the amount of compound
present.

3. Colour: The colour of the compound spots, which can be used for identification or detection.

3. Separation Efficiency

The effectiveness of the separation is evaluated based on:

1. Resolution: The ability to distinguish between two or more compounds.

2. Peak shape: The shape of the compound peaks, which can indicate the efficiency of the
separation.

3. Separation factor: The ratio of the Rf values of two compounds, which can indicate the
effectiveness of the separation.

By reporting and describing these results, researchers can:

1. Identify compounds: Use Rf values and chromatogram description to identify compounds.

2. Optimize separation: Evaluate the effectiveness of the separation and optimize conditions
for better results.

3. Compare results: Compare results with existing literature or standards to validate findings.

 Discussion:
1. Compound Identification

Rf values are interpreted to identify compounds based on their retention properties.

This involves:

41
A REVIEW ON THIN LAYER CHROMATOGRAPHY

1. Matching Rf values: Comparing Rf values of unknown compounds with those of known

compounds.

2. Retention behaviour: Understanding the retention behaviour of compounds and relating it to

their chemical structure.

2. Comparison with Standards

1. Confirm identity: Confirm the identity of compounds based on matching Rf values.

2. Validate results: Validate the accuracy of the TLC method.

3. Separation Optimization

Factors influencing separation efficiency are discussed, including:

1. Solvent system: The choice of solvent system and Rf values are compared with known
standards to its impact on separation.

2. Stationary phase: The type and properties of the stationary phase.

3. Experimental conditions: Temperature, humidity, and other experimental conditions.

4. Implications:

The implications of the results are discussed in the context of:

1. Research goals: How the results contribute to the research goals or objectives.

2. Practical applications: The potential applications of the results in various fields.

3. Future directions: Future research directions or potential improvements to the TLC

method.

By discussing these aspects, researchers can:

1. Draw meaningful conclusions: Draw conclusions about the composition and properties
of samples.

2. Inform future research: Inform future research directions or applications.

3. Validate the method: Validate the accuracy and reliability of the TLC method.

42
A REVIEW ON THIN LAYER CHROMATOGRAPHY

Future Directions
 Future Directions in TLC

The future of Thin Layer Chromatography (TLC) holds promise for advancements and
innovations. Some potential future directions include:

 Advancements in Stationary Phases

1. New Stationary Phase Materials

The development of new stationary phase materials is a key area of research in TLC.

These materials aim to improve:

1. Selectivity: Enhanced ability to separate and distinguish between compounds.

2. Sensitivity: Improved detection limits for analytes.

3. Durability: Increased stability and lifespan of the stationary phase.

New stationary phase materials may include:

1. Modified silica: Chemically modified silica with improved selectivity.

2. Polymer-based phases: Polymer-based stationary phases with enhanced stability.

3. Chiral phases: Stationary phases designed for chiral separations.

2. Nanomaterials in TLC

The integration of nanomaterials into TLC stationary phases offers several benefits:

1. Enhanced surface area: Increased surface area for improved separation efficiency.

2. Improved sensitivity: Enhanced detection limits due to increased surface area.

3. Unique selectivity: Nanomaterials can exhibit unique selectivity due to their size and shape.

Examples of nanomaterials used in TLC include:

1. Carbon nanotubes: Used for their high surface area and unique selectivity.

2. Graphene: Utilized for its high surface area and conductivity.

3. Metal nanoparticles: Used for their catalytic properties and unique selectivity.

 Hyphenated Techniques in TLC

1. TLC-MS

Coupling TLC with mass spectrometry (MS) provides:

1. Improved detection: Enhanced sensitivity and specificity.

43
A REVIEW ON THIN LAYER CHROMATOGRAPHY

2. Identification: Structural information for compound identification .

TLC-MS offers advantages over traditional TLC detection methods, including:

1. Increased accuracy: Accurate mass measurements.

2. Enhanced sensitivity: Detection of low-abundance compounds.

2. TLC-NMR

Coupling TLC with nuclear magnetic resonance (NMR) spectroscopy provides:

1. Detailed structural analysis: Comprehensive structural information.

2. Compound identification: Identification of unknown compounds.

TLC-NMR offers benefits, including:

1. Non-destructive analysis: Analysis without destroying the sample.

2. Comprehensive structural information: Detailed information on molecular structure.

By coupling TLC with MS or NMR, researchers can:

1. Enhance analytical capabilities: Improve detection, identification, and structural analysis.

2. Increase confidence: Increase confidence in results through multiple lines of evidence.

3. Expand applications: Expand TLC applications in various fields, such as pharmaceuticals,

 Automation and Miniaturization

1. Automated TLC systems: Development of automated TLC systems for increased efficiency
and reproducibility.

2. Miniaturized TLC: Miniaturization of TLC systems for portable and point-of-care

applications.

 New Applications

1. Biotechnology: Application of TLC in biotechnology for analysis of biomolecules.

2. Nanotechnology: Use of TLC in nanotechnology for analysis of nanoparticles.

3. Forensic science: Application of TLC in forensic science for analysis of evidence.

 Sustainability and Green Chemistry

1. Eco-friendly solvents: Development of eco-friendly solvents for TLC.

2. Sustainable TLC methods: Development of sustainable TLC methods with reduced

environmental impact.

44
A REVIEW ON THIN LAYER CHROMATOGRAPHY

Conclusion
Thin Layer Chromatography (TLC) is a versatile and powerful analytical technique that has
revolutionized various fields, including pharmaceuticals, environmental monitoring, food
safety, and biotechnology. Its simplicity, cost-effectiveness, and flexibility make it an
indispensable tool for researchers and analysts.

TLC's applications continue to expand, driven by advancements in stationary phase materials,

nanomaterials, and hyphenated techniques like TLC-MS and TLC-NMR. These developments
have enhanced TLC's capabilities, enabling more accurate and detailed analysis.

As research progresses, TLC will play an increasingly important role in addressing complex
analytical challenges. Its ability to provide rapid and reliable results makes it an essential
technique in various industries.

In conclusion, TLC's significance and potential for future growth make it a valuable technique
in the scientific community. Its continued development and application will drive innovation
and discovery, ultimately contributing to improved human health, environmental protection,
and scientific advancement.

By harnessing TLC's strengths and advancing its capabilities, researchers can tackle complex
problems and achieve groundbreaking results. As a result, TLC will remain a vital tool in the
scientific arsenal, driving progress and innovation in various fields.

45
A REVIEW ON THIN LAYER CHROMATOGRAPHY

References
1) Pandey. R, Shukla. S. S, Saraf. S and Saraf. S. Standardization and Validated High
Performance Thin-Layer Chromatographic Fingerprint Method for Quantitative
Determination of Plumbagin in a Traditional Indian Formulation. Journal of Planar
Chromatography. (2013). 21 (5), 440—444.
2) Patil A.S. and Paikrao H.M. Bioassay Guided Phytometabolites Extraction for Screening of
Potent Antimicrobials in Passiflora foetida L. Journal of Applied Pharmaceutical Science.
(2012). 2 (9), pp. 137-142.
3) Maitland. P. D and Maitland D. P. Chromatography: Are we getting it right? Journal of
Biological Education. (2010). 37; 1, pp 6-8.
4) Prus. W and Kowalska.T. Optimization of Thin-Layer Chromatography. Encyclopedia of
Chromatography. (2007).
5) Peterson B. L and Cummings. B. S. A review of chromatographic methods for the
assessment of phospholipids in biological samples. Biomed. Chromatogr. (2006). 20: 227-
243.
6) Cserhati T and Forgacs. E. Thin-Layer Chromatography of Synthetic Dyes. Encyclopedia
of Chromatography. (2007).
7) Beyerinck. M. W. Z. Phys. Chem. 3 (1889) 110. [8] Issaq. H. J. Recent Developments in
Thin-Layer Chromatography — ll. Journal of Liquid Chromatography. (2006). 4;6. Pages
955-975.
8) Bhawani. S. A, Ibrahim. M. N. M. Hashim. O. S. R, Mohammad. A and Hena. S.
THINLAYER CHROMATOGRAPHY OF AMINO ACIDS: A REVIEW. Journal of
Liquid Chromatography & Related Technologies. (2012). 35; 11. pages 1497-1516.
9) Shaw. P. D, Sean. G. P, Cha. D. C, Cronan. J. E, Kenneth. J. R, Rinehart, and Stephen k. F.
Detecting and characterizing n-acyl-homoserine lactone signal molecules by thin-layer
chromatography. Proc. Natl. Acad. Sci. Usa. (1997). 94, pp. 6036—6041
10) Scott. R. M. The Stationary Phase in Thin Layer Chromatography. Journal of Liquid
Chromatography. (2006). 4; 12. pages 2147-2174.
11) Marston A. Role of advances in chromatographic techniques in phytochemistry.
Phytochemistry. 2007; 68(22-24):2786-2798.
12) Hameed et al.; Asian J. Appl. Chem. Res., vol. 14, no. 3, pp. 23-38, 2023; Article
no.AJACR.107435.
13) Harborne.J. B. Phytochemical methods. 3rd editions. Methods of plants analysis. (1998)
Chapter no 1. Pg 11.
14) A. Ault, Techniques and experiments for organic chemistry, 6th edition, University Science
Books, California, 1998
15) Thin Layer Chromatography: A Complete Guide to TLC. Chemistry Hall.
https://chemistryhall.com/thinlayer-chromatography/ (accessed on 26-04-2022).
16) Thin Layer Chromatography 1Bansode Badal Mahadev, 2Garad R.S, 3Dr.Santosh Jain,
4Kamble Manoj, 5Shinde Vivek

46
A REVIEW ON THIN LAYER CHROMATOGRAPHY

17) An OVERVIEW ON THIN LAYER CHROMATOGRAPHY Archana A. Bele* and

Anubha Khale,H. K. College of Pharmacy, Jogeshwari (W), Mumbai, Maharashtra, India
18) Toshboyev Feruz Nizomiddinovich1, Izatullayev Sarvar Abdimannonovich1,Akhmadov
Javokhir Zoirovich,Samarkand State Medical University, Samarkand State Medical
University resident of magistracy Department of Pharmaceutical and Toxicological
Chemistry, Samarkand, Uzbekistan
19) AN OVERVIEW ON THIN LAYER CHROMATOGRAPHY Archana A. Bele* and
Anubha Khale,H. K. College of Pharmacy, Jogeshwari (W), Mumbai, Maharashtra, India
20) Sanjeet Kumar,K. Jyotirmayee, Monalisa Sarangi, Department of Botany, Ravenshaw
University, Cuttack - 75 3003, Odisha, India.MITS School of Biotechnology, Bhubaneswar
-75 1024, Odisha, India.
21) Shashank Tiwari and Shreya Talreja,Professor, Director (Academic and Research),
Lucknow Model College of Pharmacy, Lucknow, India M.Pharm, Goel Institute of
Pharmacy and Sciences, Lucknow, India.
22) Dr. Osman Ahmed Khadeeja and Dr. Anas Rasheed,Department of Pharmaceutical
Analysis, Deccan School of Pharmacy, Hyderabad.CSO, Gaelib Medications Private
Limited, Hyderabad.Khalid
23) Hameed a, Muhammad Shoaib Khan ,Ayesha Fatima a, Syed Mudassir Shah and
Muhammad Ali Abdullaha Department of Chemistry, University of Education Lahore,
Faisalabad Campus, Pakistan.
24) Thin Layer Chromatography, LibreTexts (accessed on 26-04-2022)
https://chem.libretexts.org/Ancillary_Materials/Dem os_Techniques_and_Experiments
/General_Lab_Techniques/Thin_Layer_Chromatogra phy
25) A Review on High Performance Liquid Chromatography (HPLC) Mr. Gorhe S G, Miss.
Pawar G R Science and Humanities Department , Parikrama Polytechnic, Kashti,
Ahmednagar, Maharashtra.
26) Manish Kumar Gupta, Aditya Ghuge, Manasi Parab, Yehya Al-Refaei,Anjali Khandare,
Neha Dand, Nilkamal Waghmare,Department of Pharmaceutics, Jaipur National University
School of Pharmaceutical Sciences, Jaipur, Rajasthan, India Department of Pharmaceutics,
Bharati Vidyapeeth's College of Pharmacy, Navi Mumbai, IndiaDepartment of Pharmacy,
DY Patil University School of Pharmacy, Navi Mumbai, India.
27) Syeda Rakhshinda Zareen, Dr. Osman Ahmed, Reshma1, Mohammed Sayeed Uddin and
Dr. Anas Rasheed,Department of Pharmaceutical Analysis, Deccan School of Pharmacy,
Hyderabad and CSO, Gaelib Medications Private Limited, Hyderabad.

47