Bio Instrumentation

What Is Thin Layer Chromatography?
Thin Layer Chromatography is a technique used to isolate non-volatile mixtures. The experiment
is conducted on a sheet of aluminium foil, plastic, or glass which is coated with a thin layer of
adsorbent material. The material usually used is aluminium oxide, cellulose, or silica gel.
On completion of the separation, each component appears as spots separated vertically. Each
spot has a retention factor (Rf) expressed as:
Rf = dist. Travelled by sample / dist. Travelled by solvent
The factors affecting retardation factor are the solvent system, amount of material spotted,
adsorbent and temperature. TLC is one of the fastest, least expensive, simplest and easiest
chromatography technique.
Thin Layer Chromatography Principle

Like other chromatographic techniques, thin-layer chromatography (TLC) depends on the
separation principle. The separation relies on the relative affinity of compounds towards both the
phases. The compounds in the mobile phase move over the surface of the stationary phase. The
movement occurs in such a way that the compounds which have a higher affinity to the
stationary phase move slowly while the other compounds travel fast. Therefore, the separation of
the mixture is attained. On completion of the separation process, the individual components from
the mixture appear as spots at respective levels on the plates. Their character and nature are
identified by suitable detection techniques.
Thin Layer Chromatography Procedure

Before starting with the Thin Layer Chromatography Experiment, let us understand the different
components required to conduct the procedure along with the phases involved.
Thin Layer Chromatography Plates – ready-made plates are used which are chemically inert and
stable. The stationary phase is applied on its surface in the form of a thin layer. The stationary
phase on the plate has a fine particle size and also has a uniform thickness.
Thin Layer Chromatography Chamber – Chamber is used to develop plates. It is responsible to
keep a steady environment inside which will help in developing spots. Also, it prevents the
solvent evaporation and keeps the entire process dust-free.
Thin Layer Chromatography Mobile phase – Mobile phase is the one that moves and consists of
a solvent mixture or a solvent. This phase should be particulate-free. The higher the quality of
purity the development of spots is better.
Thin Layer Chromatography Filter Paper – It has to be placed inside the chamber. It is moistened
in the mobile phase.
Thin Layer Chromatography Experiment
The stationary phase that is applied to the plate is made to dry and stabilize.
To apply sample spots, thin marks are made at the bottom of the plate with the help of a pencil.
Apply sample solutions to the marked spots.
Pour the mobile phase into the TLC chamber and to maintain equal humidity, place a moistened
filter paper in the mobile phase.
Place the plate in the TLC chamber and close it with a lid. It is kept in such a way that the sample
faces the mobile phase.
Immerse the plate for development. Remember to keep the sample spots well above the level of
the mobile phase. Do not immerse it in the solvent.
Wait till the development of spots. Once the spots are developed, take out the plates and dry
them. The sample spots can be observed under a UV light chamber.
Thin Layer Chromatography Applications

The qualitative testing of Various medicines such as sedatives, local anaesthetics,
anticonvulsant tranquilisers, analgesics, antihistamines, steroids, hypnotics is done by TLC.
TLC is extremely useful in Biochemical analysis such as separation or isolation of biochemical
metabolites from its blood plasma, urine, body fluids, serum, etc.
Thin layer chromatography can be used to identify natural products like essential oils or volatile
oil, fixed oil, glycosides, waxes, alkaloids, etc.
It Is widely used in separating multicomponent pharmaceutical formulations.
It Is used for the purification of samples and direct comparison is done between the sample and
the authentic sample.
It Is used in the food industry, to separate and identify colours, sweetening agent, and
preservatives
It is used in the cosmetic industry.
It is used to study if a reaction is complete.
Disadvantages Of Thin Layer Chromatography:
Thin Layer Chromatography plates do not have longer stationary phase.
When compared to other chromatographic techniques the length of separation is limited.
The results generated from TLC are difficult to reproduce.
Since TLC operates as an open system, some factors such as humidity and temperature can be
can affect the final outcome of the chromatogram.
The detection limit Is high and therefore if you want a lower detection limit, you cannot use
TLC.
It is only a qualitative analysis technique and not quantitative
Affinity chromatography is a powerful technique for isolating and purifying specific

biomolecules, particularly proteins, from a complex mixture. It utilizes the highly specific
interaction between a ligand and its binding partner to achieve separation. Imagine a lock-and-
key mechanism, where the ligand is the lock and the target biomolecule is the key. Only
molecules with the complementary structure can bind to the ligand and be retained.
This technique offers several advantages, including:
* High selectivity: Affinity chromatography targets specific biomolecules with minimal
interference from other components in the mixture.
* High resolution: It enables the isolation of closely related molecules based on subtle
differences in their binding affinities.
* Efficiency: Affinity chromatography can often achieve significant purification in a single step,
saving time and resources.
PROCEDURE
Affinity chromatography relies on two key components: a stationary phase and a mobile phase.
* The stationary phase consists of a solid support matrix with a ligand attached to its surface.
The ligand is a molecule that has a high affinity for the target biomolecule you want to purify.
* The mobile phase is a solution containing the mixture of biomolecules, including your target
molecule.
The process works like this:
* The mixture is loaded onto the chromatography column containing the immobilized ligand.
* As the mobile phase flows through the column, the target biomolecules bind specifically to the
ligand, while other non-binding molecules pass through unhindered.
* After washing away unbound components, the target molecule is eluted from the ligand using
a specific elution buffer that disrupts the binding interaction.
This technique offers a highly specific and efficient way to isolate and purify desired
biomolecules from complex mixtures.
Affinity chromatography can be visualized through the following steps:
 Sample Loading: A complex mixture containing the target biomolecule is loaded onto the
chromatography column.
 Specific Binding: The target biomolecules bind specifically to the ligand immobilized on the
stationary phase.
 Washing: Unbound components are washed away with a buffer.
 Elution: The target molecule is eluted from the ligand using a specific elution buffer,
allowing it to be collected.
 Affinity chromatography is a versatile technique with numerous applications in

biochemistry, molecular biology, and biotechnology. Here are some of its key applications:
 * Protein purification: This is the most common application of affinity chromatography. It
allows for the isolation of specific proteins from complex mixtures, such as cell lysates or
biological fluids. This is crucial for downstream applications like studying protein function,
enzyme assays, or protein crystallization.
 * Nucleic acid purification: Affinity chromatography can be used to purify DNA or RNA
from biological samples. This is useful for various molecular biology techniques like PCR,
sequencing, or cloning.
 * Removal of contaminants: Affinity chromatography can be used to remove unwanted

components from a sample, such as endotoxins, toxins, or other impurities. This can be
particularly important for therapeutic protein production or for research purposes where a
pure sample is essential.
 * Studying biomolecular interactions: Affinity chromatography can be used to investigate

the interactions between biomolecules, such as protein-protein or protein-DNA interactions.
This can provide valuable insights into cellular processes and signaling pathways.
 * Drug discovery and development: Affinity chromatography can be used to identify and
purify drug targets, as well as to screen for potential drug candidates.
 Affinity chromatography relies on several key instruments to achieve efficient purification:
 * Chromatography Column: A cylindrical container packed with the affinity matrix, where
the ligand is immobilized.
 * Liquid Chromatography Pump: Delivers the mobile phase containing the sample mixture
through the column at a controlled flow rate.
 * Detector: Monitors the components eluting from the column, allowing for identification
and collection of the purified target molecule. Common detectors include UV-Vis
spectrophotometers for proteins and refractive index detectors for general use.
 * Fraction Collector: Collects eluents exiting the column at specific intervals, enabling the
isolation of the desired purified fraction.

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What Is Thin Layer Chromatography?

Rf = dist. Travelled by sample / dist. Travelled by solvent

Thin Layer Chromatography Principle

Thin Layer Chromatography Procedure

Thin Layer Chromatography Applications

Affinity chromatography is a powerful technique for isolating and purifying specific

Affinity chromatography can be visualized through the following steps:

 Washing: Unbound components are washed away with a buffer.

 Affinity chromatography is a versatile technique with numerous applications in

 * Removal of contaminants: Affinity chromatography can be used to remove unwanted

 * Studying biomolecular interactions: Affinity chromatography can be used to investigate

 Affinity chromatography relies on several key instruments to achieve efficient purification:

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