You are on page 1of 11

PAPER AND THIN LAYER CHROMATOGRAPHY (TLC)

Chromatography Is the collective term for a set of laboratory techniques for the
separation of mixtures. Chromatography is a method by which a mixture is
separated by distributing its components between two phases. The stationary
phase remains fixed in place while the mobile phase carries the components of
the mixture through the medium being used The separation of materials is based
on differential partitioning [retardation]between the mobile and stationary phases.

 The mobile phase (also known as solvent) may be either liquid or gas.
 The stationary phase (also known as sorbent) can be either a solid or liquid,
a liquid stationary phase is held stationary by a solid(support or matrix).
 The molecules in the mixture to be separated are the
Classification of chromatography according to mobile phase:
1. Liquid chromatography: mobile phase is a liquid. (LLC, LSC).
2. Gas chromatography : mobile phase is a gas. (GSC, GLC)

Classification according to the packing of the stationary phase:


1. Thin layer chromatography (TLC): the stationary phase is a thin layer
supported on glass, plastic or aluminium plates.
2. Paper chromatography (PC): the stationary phase is a thin film of liquid
supported on an inert support.
3. Column chromatography (CC): stationary phase is packed in a glass column

Classification according to the force of separation:


1. Adsorption chromatography.
2. Partition chromatography.
3. Ion exchange chromatography.
4. Gel filtration chromatography.
5. Affinity chromatography

Different Types of chromatography


Thin layer chromatography (TLC)

Thin-layer chromatography (TLC) has developed into a very sophisticated


technique for identification of compounds and for determination of the presence of
trace impurities. Since it was one of the earliest chromatographic techniques, a
huge array of TLC-based tests is available and pharmacopoeial monographs reflect
the extent to which this technique has been developed as a fundamental quality
control technique for trace impurities.

It is a method for identifying substances and testing the purity of compounds. TLC
is a useful technique because it is relatively quick and requires small quantities of
material.
The stationary phase:
is a thin layer of adsorbent (usually silica gel or alumina) coated on a plate.

The mobile phase:


is a developing liquid which travels up the stationary phase, carrying the samples
with it.
Components of the samples will separate on the stationary phase according to
how much they adsorb on the stationary phase versus how much they dissolve in
the mobile phase
Samples Analysis
Visualization
Spots in paper and TLC chromatograms can be detected in 4 different ways:
1.By their natural color
2.By their fluorescence
3.By their chemical reactions that take place after the paper has been sprayed with
various reagents for example: during paper chromatography of amino acids, the
chromatograms are sprayed with ninhydrin.
4.By radioactivity
Expression of the results.
The spots are usually identified by comparing of standards of known(relative flow)
Rf values.
Is the ratio of the distance moved by a solute to the distance moved by the solvent
Under similar conditions, a component should always travel at the same speed.
Its identity can be found by comparing The distance it moves relative to the
solvent.

[Rf] Value:
-If Rƒvalue of a solute is closer to zero, the solute has more attraction to stationary
phase.
-If Rƒvalue of a solute is closer to 1,then the solute has more affinity for the
mobile phase and travels further.
-The final chromatogram can be compared with other knownmixture
chromatograms to identify sample mixes, using the Rf value in an experiment

Some factors that may affect separation


1. Temperature: Since temperature can effect the solubility of the solute in a
given solvent. often a chromatography laboratory has a fixed temperature for
optimum results
2. Composition of the solvent: Since some compounds are more soluble in one
solvent than in the other the mixture of solvents used affect separation of the
compounds.
3. Any ionized impurities in the support medium will tend to bind or attract
oppositely charged ions and will therefore reduce the mobility of these solutes .

Principles

An analyte migrates up or across a layer of stationary phase (most commonly


silica gel), under the influence of a mobile phase (usually a mixture of organic
solvents), which moves through the stationary phase by capillary action. The
distance moved by the analyte is determined by its relative affinity for the
stationary vs the mobile phase.

Partition of a solute between a moving solvent phase and a stationary aqueous


phase. The solute moves in the direction of a solvent flow at a rate determined by
the solubility of the solute in the moving phase. Thus a compound with high
mobility [less polarity] is more attracted to the moving solvent [mobile phase]than
to the stationary phase.

Applications
• Used to determine impurities in pharmaceutical raw materials and formulated
products.
• Often used as a basic identity check on pharmaceutical raw materials.
• Potentially useful in cleaning validation, which is part of the manufacture of
pharmaceuticals.
 determine the number of components in a mixture
• verify a substance’s identity
• monitor the progress of a reaction
• determine appropriate conditions for column chromatography
• analyze the fractions obtained from column chromatogrpahy
Paper Chromatography

A method of partition chromatography using filter paper strips as carrier or inert


support.
The factor governing separation of mixtures of solutes on filter paper is the
partition between two immiscible phases.
One is usually water adsorbed on cellulose fibres in the paper (stationary phase).
The second is the organic solvent flows past the sample on the paper (stationary
phase).

one of thetypes of chromatographyprocedures which runs on a piece of specialized


paper.
Two types of paper chromatography:

1. Descending
-In this method, the solvent is kept in a trough at the top of the chamber and is
allowed to flow down the paper.
-The liquid moves down by capillary actionas well as by the gravitational force.
-In this case, the flow is more rapid as compared to the ascending method.
2. Ascending
 In this method, the solvent moves upward against gravitational force.
 The only force that cause the motion[of solvent and the componds] is
capillary force. So the speed of the process is slow.

solutes are separated according to their solubility in mobile phase, or their


attraction to the stationary phase Depend in the polarity
TLC vs PC

References:

1. owlcation.com/stem/tlc-thin-layer-chromatography-Principle-Procedure
2. [David_G_Watson]_Pharmaceutical_analysis__a_textb(BookZZ.org)
3. paper_and_thin_layer_chromatography_tlc
4. http://cdn.biologydiscussion.com/wp-
content/uploads/2015/10/clip_image028.jpg

Md.Nazmul Islam (ABIR)


#151330 (2ND Batch)
Contact: 01766388247
Abir140044@gmail.com
Department of Pharmacy
Pabna University of Science and Technology

You might also like