Chromatography: Rayaan Ahmed 9-Yellow

CHROMATOGRAPHY
Rayaan Ahmed
9-Yellow
This Method is used for separation of mixtures
of compounds between two phases
• Stationary ( solid or liquid located on neutral medium
• Mobile (gas or liquid)
This method is used for separation of mixture of

compounds with different speed of migration a given
compounds in porous medium with a different ability of
absorption or ion exchange or different solubility in
solvents
Chromatography is used for ;
 Separation of compounds from mixtures
Purification
Identification
Quantity and Quality analysis
Choice of method depend on ;

 Amount of analyzed mixtures
Kind of compounds
Complexity of seperation
Methods of
Chromatography
• Gas Chromatography
• Liquid chromatography
• Paper Chromatography
• High pressure liquid chromatography
• Thin layer chromatography
But we are only going to cover Paper and Thin layer

Chromatography
The principle of separation is mainly partition rather than adsorption. Substances are
distributed between a stationary phase and mobile phase. Cellulose layers in filter paper
contain moisture which acts as stationary phase. Organic solvents/buffers are used as mobile
phase. The developing solution travels up the stationary phase carrying the sample with it.
Components of the sample will separate readily according to how strongly they adsorb onto
the stationary phase versus how readily they dissolve in the mobile phase.
5.Development of the Chromatogram
Sample loaded filter paper is dipped carefully into the solvent not more than a height of 1 cm and waited until the solvent front
reaches near the edge of the paper.
Different types of development techniques can be used:
ASCENDING DEVELOPMENT
•Like conventional type, the solvent flows against gravity.
•The spots are kept at the bottom portion of paper and kept in a chamber with mobile phase solvent at the bottom.
DESCENDING TYPE
•This is carried out in a special chamber where the solvent holder is at the top.
•The spot is kept at the top and the solvent flows down the paper.
•In this method solvent moves from top to bottom so it is called descending chromatography
ASCENDING – DESCENDING DEVELOPMENT
•A hybrid of above two techniques is called ascending-descending chromatography.
•Only length of separation increased, first ascending takes place followed by descending.
CIRCULAR / RADIAL DEVELOPMENT
•Spot is kept at the centre of a circular paper.
•The solvent flows through a wick at the centre & spreads in all directions uniformly.
6.Drying of Chromatogram
After the development, the solvent front is marked and the left to dry in a dry cabinet or oven.
7.Detection
Colourless analytes detected by staining with reagents such as iodine vapour, ninhydrin etc.
Radiolabeled and fluorescently labeled analytes detected by measuring radioactivity and florescence respectively.
Rf values
Some compounds in a mixture travel almost as far as the solvent does; some stay
much closer to the base line. The distance travelled relative to the solvent is a
constant for a particular compound as long as other parameters such as the type
of paper and the exact composition of the solvent are constant. The distance
travelled relative to the solvent is called the Rf value.
Thus, in order to obtain a measure of the extent of movement of a

component in a paper chromatography experiment, “Rf value” is
calculated for each separated component in the developed chromatogram.
An Rf value is a number that is defined as distance traveled by the
component from application point.
Applications of Paper Chromatography
•To check the control of purity of pharmaceuticals,
•For detection of adulterants,
•Detect the contaminants in foods and drinks,
•In the study of ripening and fermentation,
•For the detection of drugs and dopes in animals & humans
•In analysis of cosmetics
•Analysis of the reaction mixtures in biochemical labs
Advantages of Paper Chromatography
•Simple
•Rapid
•Paper Chromatography requires very less quantitative material.
•Paper Chromatography is cheaper compared to other chromatography methods.
•Both unknown inorganic as well as organic compounds can be identified by paper
chromatography method.
•Paper chromatography does not occupy much space compared to other analytical
methods or equipment's.
•Excellent resolving power
Limitations of Paper Chromatography
•Large quantity of sample cannot be applied on paper chromatography.
•In quantitative analysis paper chromatography is not effective.
•Complex mixture cannot be separated by paper chromatography.
•Less Accurate compared to HPLC or HPTLC
References
1.http://frndzzz.com/Advantages-and-Disadvantages-of-Paper-Chromatography
2.https://www.slideshare.net/shaisejacob/paper-chromatography-pptnew?next_slideshow=1
3.https://www.slideshare.net/shaisejacob/paper-chromatography-ppt-new
4.https://www.biochemden.com/paper-chromatography/
5.https://pubs.acs.org/doi/abs/10.1021/ac60051a002

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CHROMATOGRAPHY

This method is used for separation of mixture of

Choice of method depend on ;

But we are only going to cover Paper and Thin layer

Thus, in order to obtain a measure of the extent of movement of a

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