INDEX

1) Abstract……………………………………………….……………………………..…………[1]
2) Introduction
 Chromatography…………………………………………………………..…….[2]
 Principles of Chromatography……………………………………….…..….[3]
 Types of Chromatography…………………………………………..…..…….[6]
3) Introduction To Thin Layer Chromatography (TLC)
 Background and Significance of Thin Layer Chromatography
(TLC)……………………………………………………………………….….….[7]
 Research Objectives and Scope…………………………………….......[8]
4) Literature Review-
 History of TLC: -………………………………………………………………….….[10]
 PRINCIPLE………………………………………………………………..……….….[12]
 Applications of Thin Layer Chromatography……………………………....[14]
5) Classification of THIN LAYER CHROMATOGRAPHY………………..……………..[17]
6) INSTRUMENTATION OF TLC …………………………………………………..………….[19]
7) Sample Preparation and Application in Thin Layer Chromatography (TLC)
 Sample preparation …………………………………………………………….…[30]
 Sample application method ……………………………………………...….…[36]
8) Development and detection methods
 Development Methods …………………………………………………..…….…[37]
 Detection methods ………………………………………………………..…….…[47]
9) Data analysis and interpretation …………………………………………………………[49]

10) Result and discussion

 Result …………….…………………………………………………………………..…[53]
 Discussion ………/……………………………………………………………………[55]
11) Application and future dir.ection …………………………………………………………[57]
12) Conclusion ……………………/……………………………………………………………..…[63]

13) References ……………………/…………………………………………………..……………[64]

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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.

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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.

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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.

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:

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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.

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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.

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Types of Chromatography

There are several types of chromatography, including:

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

2. Thin Layer Chromatography (TLC): Uses a thin layer of adsorbent material
as the stationary phase.
3. Gas Chromatography (GC): Uses a gas as the mobile phase.
4. Liquid Chromatography (LC): Uses a liquid as the mobile phase.
5. High-Performance Liquid Chromatography (HPLC): A type of LC that
uses high pressure to separate components.

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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

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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 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.

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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

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.

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.

3. Environmental monitoring: This study will examine the application of TLC in

detecting and quantifying environmental pollutants, such as pesticides and
heavy metals.

4. Biotechnology: This research will explore the use of TLC in analysing and
purifying biomolecules, such as proteins and DNA.

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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.

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Key Figures

1. Mikhail Tsvet: Considered the father of chromatography, Tsvet introduced

the concept of TLC.

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

1837-1919
3. Erich Heftmann: A German-American chemist, Heftmann made significant
contributions to the development of modern TLC.

1922- 1993

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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 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
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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.

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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:

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

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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.

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.

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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

1. Cosmetics and personal care products: TLC is used to analyse cosmetics and
personal care products for contaminants and impurities.

2. Agricultural analysis: TLC is used to analyse agricultural products for

contaminants and impurities.

3. Industrial analysis: TLC is used to analyse industrial products for contaminants

and impurities.

4. Research and development: TLC is used in research and development,

including the analysis of new compounds and materials.

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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.

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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

2. 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

3. 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
Application: Separation of compounds with varying polarities, such as
pharmaceuticals, agrochemicals, and environmental pollutants

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INSTRUMENTATION OF TLC

Instrumentation used in Thin Layer Chromatography (TLC):

1. TLC Plates:

TLC plate

Material: Glass, aluminium, or plastic

Size: 20 x 20 cm or 10 x 10 cm

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.
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4. Purification: TLC plates can be used to purify compounds by separating them

from impurities or other contaminants.

2. Spotting Device:

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.

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3. Development Chamber:

Type: Glass or plastic tank

Size: 20 x 20 x 10 cm

 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.

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2. Plastic chambers: These are lightweight and portable development

chambers made of plastic.

3. Automated development chambers: These are electronic devices that can

control the development process, including temperature, humidity, and solvent
flow.

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4. Mobile Phase:

Type: Various solvents (e.g., hexane, ethyl acetate, methanol)

 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.

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5. Detection Methods:

UV Light: Detects compounds that absorb UV light

Iodine Vapor: Detects compounds that react with iodine

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.

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6. Densitometer:

Type: 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.

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7. TLC Scanner:

Type: 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.

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8. Chromatography Software:

Cambag software

Type: 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.

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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:

Type: 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.

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

10. TLC Plate Cooler:

Type: 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.

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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.

3. Stratified sampling: A stratified sample is collected from different subgroups

or strata within the material of interest.

 Extraction:
1. 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.
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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.
Step 1: Solvent Selection
1. Select a non-polar solvent: Select a non-polar solvent, such as hexane,
to separate the non-polar compounds.
2. Select a polar solvent: Select a polar solvent, such as methanol, to
separate the polar compounds.
3. Select a binary solvent system: Select a binary solvent system, such as
hexane-methanol, to separate both non-polar and polar compounds.

Step 2: Optimization
1. Optimize the solvent ratio: Optimize the ratio of the solvents in the binary
solvent system.
2. Optimize the solvent strength: Optimize the strength of the solvent
system.

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2. 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.
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3. Extraction time selection:

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

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.

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4. 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

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,
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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:

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1. Detection limits: Improving sensitivity.

2. Quantitation accuracy: Increasing analyte concentration.

Common applications include:

1. Environmental analysis: Concentrating pollutants.

2. Biological samples: Concentrating biomarkers.

 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

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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.

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1. Ascending development:

Ascending development is a technique used in TLC where the solvent moves up

the plate by capillary action.

 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

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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.
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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.

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 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.

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 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.

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

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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.

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3. Horizontal Development:

Horizontal development is a technique used in TLC where the solvent moves

horizontally across the plate.

Direction of mobile phase

paper

support

mobile phase

 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.

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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. 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.

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 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.

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.

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 Detection methods

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

 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.

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 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.

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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.

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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
Rf 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

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.
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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.

Using the stated approach and the above experiment, the

Rf (retardation/retention factor) values can be computed.

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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

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Results and Discussion

 Results:

Results in TLC

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.
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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.

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 Discussion:

. Compound Identification

Rf values are interpreted to identify compounds based on their retention

properties. This involves:

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.

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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.

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Applications and Future Directions

 Applications:
 Pharmaceutical Analysis:

Pharmaceutical analysis is a critical application of Thin Layer Chromatography

(TLC). TLC is widely used in the pharmaceutical industry for the identification,
purity testing, and quantification of active pharmaceutical ingredients (APIs) and
impurities. The technique provides rapid analysis and detection of compounds,
making it an essential tool in drug development, quality control, and stability
testing. TLC is used to identify APIs, detect impurities, and determine the purity
of pharmaceutical products. It is also used to study the stability of APIs and
detect degradation products.

The advantages of TLC in pharmaceutical analysis include its simplicity, cost-

effectiveness, and robustness. TLC is a relatively simple technique that requires
minimal equipment and expertise, making it accessible to a wide range of
laboratories. Additionally, TLC is a cost-effective technique compared to other
chromatographic methods, such as High-Performance Liquid Chromatography
(HPLC). The technique is also robust, allowing for the analysis of complex
pharmaceutical samples. Overall, TLC is a valuable tool in pharmaceutical
analysis, providing critical information about the identity, purity, and stability of
pharmaceutical products. Its applications in drug development, quality control,
and stability testing make it an essential technique in the pharmaceutical
industry. By leveraging TLC, researchers and manufacturers can ensure the
quality, safety, and efficacy of pharmaceutical products.

 Food and Beverage Analysis:

Food and beverage analysis is another significant application of Thin Layer

Chromatography (TLC). TLC is used to detect contaminants, adulterants, and
residues in food and beverages, ensuring their safety and quality. The technique
is applied to identify and quantify various compounds, such as pesticides, heavy
metals, and food additives. TLC is also used to detect foodborne pathogens and
toxins, helping to prevent foodborne illnesses. Additionally, TLC is used in the
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analysis of food and beverage components, such as flavourings, colourings, and

preservatives.

The advantages of TLC in food and beverage analysis include its simplicity, cost-
effectiveness, and ability to analyse complex samples. TLC is a valuable tool for
food manufacturers, regulatory agencies, and research institutions. Its
applications in food safety testing, quality control, and research make it an
essential technique in the food and beverage industry. By leveraging TLC,
researchers and manufacturers can ensure the safety and quality of food and
beverages, protecting public health and preventing economic losses. TLC's role
in detecting contaminants and adulterants helps to maintain consumer trust and
confidence in the food supply.

Food and beverage analysis is a significant application of Thin Layer

Chromatography (TLC). TLC detects contaminants, adulterants, and residues,
ensuring safety and quality. It's used to identify and quantify compounds like
pesticides, heavy metals, and food additives. TLC also detects foodborne
pathogens and toxins, preventing foodborne illnesses. Its simplicity, cost-
effectiveness, and ability to analyse complex samples make it valuable for food
manufacturers, regulatory agencies, and research institutions.

 Environmental Monitoring:

Environmental monitoring is a vital application of Thin Layer Chromatography

(TLC). TLC detects and quantifies pollutants in environmental samples, including
pesticides, heavy metals, and industrial pollutants. The technique provides rapid
analysis and detection of contaminants, enabling researchers to assess
pollution levels and identify contamination sources. TLC's simplicity, cost-
effectiveness, and ability to analyse complex samples make it a valuable tool for
environmental monitoring.

TLC's applications in environmental monitoring are diverse. It detects pesticide

residues in soil, water, and air, and identifies heavy metal contaminants in
environmental samples. TLC also analyses industrial pollutants, such as
polycyclic aromatic hydrocarbons (PAHs), and detects other organic pollutants.
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By leveraging TLC, researchers and environmental agencies can inform

remediation efforts, ensure public health, and protect the environment. TLC's
role in environmental monitoring supports sustainable development and
environmental stewardship.

The benefits of TLC in environmental monitoring include its ability to provide rapid
and cost-effective analysis, making it an ideal technique for large-scale
environmental monitoring programs. Additionally, TLC's high sensitivity and
selectivity enable researchers to detect and quantify pollutants at low
concentrations, which is critical for environmental protection. Overall, TLC is a
valuable tool in environmental monitoring, providing critical information on
pollution levels and contamination sources, and supporting efforts to mitigate
the impact of human activities on the environment. By using TLC, researchers can
contribute to a safer and healthier environment.

 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:

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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:

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1. Improved detection: Enhanced sensitivity and specificity.

2. Identification: Structural information for compound identification.

3. Complex mixture analysis: Analysis of complex mixtures.

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.

3. Isomer distinction: Distinction between isomers.

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:

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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, biotechnology, and food analysis.

 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.

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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.

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