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Chromatography- Definition, Principle, Types, Applications

September 10, 2022 by Sagar Aryal

Edited By: Sagar Aryal

Table of Contents
What is chromatography?
Principle of Chromatography (how does chromatography work)
Types of Chromatography
Commonly employed chromatography techniques include:
Applications of Chromatography
References
Chromatography- definition, principle, types, applications

What is chromatography?
Chromatography is an important biophysical technique that enables the
separation, identification, and purification of the components of a mixture for
qualitative and quantitative analysis.
The Russian botanist Mikhail Tswett coined the term chromatography in 1906.
 The first analytical use of chromatography was described by James and Martin in
1952, for the use of gas chromatography for the analysis of fatty acid mixtures.
A wide range of chromatographic procedures makes use of differences in size,
binding affinities, charge, and other properties to separate materials.
It is a powerful separation tool that is used in all branches of science and is often
the only means of separating components from complex mixtures.

Principle of Chromatography (how does chromatography work)

Image Source: Khan Academy

Chromatography is based on the principle where molecules in mixture applied

onto the surface or into the solid, and fluid stationary phase (stable phase) is
separating from each other while moving with the aid of a mobile phase.
The factors effective on this separation process include molecular characteristics
related to adsorption (liquid-solid), partition (liquid-solid), and affinity or
differences among their molecular weights.
Because of these differences, some components of the mixture stay longer in the
stationary phase, and they move slowly in the chromatography system, while
others pass rapidly into the mobile phase, and leave the system faster.

Three components thus form the basis of the chromatography technique.

1. Stationary phase: This phase is always composed of a “solid” phase or “a layer of
a liquid adsorbed on the surface solid support”.
2. Mobile phase: This phase is always composed of “liquid” or a “gaseous
component.”
3. Separated molecules

The type of interaction between the stationary phase, mobile phase, and substances
contained in the mixture is the basic component effective on the separation of
molecules from each other.

Image Source: Khan Academy

Types of Chromatography
 Substances can be separated on the basis of a variety of methods and the
presence of characteristics such as size and shape, total charge, hydrophobic
groups present on the surface, and binding capacity with the stationary phase.
This leads to different types of chromatography techniques, each with their own
instrumentation and working principle.
For instance, four separation techniques based on molecular characteristics and
interaction type use mechanisms of ion exchange, surface adsorption, partition,
and size exclusion.
Other chromatography techniques are based on the stationary bed, including
column, thin layer, and paper chromatography.

Commonly employed chromatography techniques include:

1. Column chromatography
2. Ion-exchange chromatography
3. Gel-permeation (molecular sieve) chromatography
4. Affinity chromatography
5. Paper chromatography
6. Thin-layer chromatography
7. Gas chromatography (GS)
8. Dye-ligand chromatography
9. Hydrophobic interaction chromatography
10. Pseudoaffinity chromatography
11. High-pressure liquid chromatography (HPLC)
Applications of Chromatography
Pharmaceutical sector

To identify and analyze samples for the presence of trace elements or chemicals.
Separation of compounds based on their molecular weight and element
composition.
Detects the unknown compounds and purity of mixture.
In drug development.

Chemical industry

In testing water samples and also checks air quality.

HPLC and GC are very much used for detecting various contaminants such as
polychlorinated biphenyl (PCBs) in pesticides and oils.
In various life sciences applications

Food Industry

In food spoilage and additive detection

Determining the nutritional quality of food

Forensic Science

In forensic pathology and crime scene testing like analyzing blood and hair
samples of crime place.
Molecular Biology Studies

Various hyphenated techniques in chromatography such as EC-LC-MS are applied

in the study of metabolomics and proteomics along with nucleic acid research.
HPLC is used in Protein Separation like Insulin Purification, Plasma Fractionation,
and Enzyme Purification and also in various departments like Fuel Industry,
biotechnology, and biochemical processes.

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References
1. https://chromatography.conferenceseries.com/events-list/applications-of-
chromatography
2. http://www.biologydiscussion.com/biochemistry/chromatography-
techniques/top-12-types-of-chromatographic-techniques-biochemistry/12730
3. http://library.umac.mo/ebooks/b28050630.pdf
4. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5206469/
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Chromatography- definition, principle, types, applications

About Author

Sagar Aryal

Sagar Aryal is a microbiologist and a scientific blogger. He attended St. Xavier’s

College, Maitighar, Kathmandu, Nepal, to complete his Master of Science in
Microbiology. He worked as a Lecturer at St. Xavier’s College, Maitighar,
Kathmandu, Nepal, from Feb 2015 to June 2019. After teaching microbiology for
more than four years, he joined the Central Department of Microbiology, Tribhuvan
University, to pursue his Ph.D. in collaboration with Helmholtz-Institute for
Pharmaceutical Research Saarland (HIPS), Saarbrucken, Germany. He is
interested in research on actinobacteria, myxobacteria, and natural products. He
 has published more than 15 research articles and book chapters in international
journals and well-renowned publishers.

12 thoughts on “Chromatography- Definition, Principle, Types,

Applications”

Bharti
December 25, 2022 at 8:59 AM

Because mobile phase travels against gravity so the gravitational force on it

causes it to slow down

Reply

Priyanshi
April 23, 2022 at 6:56 AM

Osm article …. Thanks

Reply

Nikita hembade
November 29, 2021 at 2:04 PM

Thanks for this article…. It was excellent..,..

Reply
 Poonam HARINKHEDE
March 27, 2021 at 11:09 PM

Thanks you

Reply

ƝΔƬΣ ƧӇƛШ
October 16, 2020 at 2:14 PM

Thank You So Much. Needed Info For A Project And Got It All Here!

Reply

Irtaza Gillani PM
June 15, 2020 at 12:08 PM

Very good , i like it very much

Reply

Manmohan Singh
June 1, 2020 at 6:08 PM
Why is the rate of movement of mobile phase less in ascending Paper
Chromatography?

Reply

Bakhtawar
August 10, 2020 at 8:29 AM

It is because of the molecular weight. Components with large molecular

weight rise up slowly.

Reply

Amidu Rafat
May 20, 2020 at 6:17 PM

Excellent article really like it

Reply

AMAR CHAND MALAKAR

April 24, 2020 at 12:38 PM

excellence article related to chromatography.brief revision of

chromatography. thanks all team.

Reply
 Dr.Satyendra Singh
September 25, 2018 at 5:36 PM

Excellent article.

Reply

Manmohan Singh
June 1, 2020 at 6:15 PM

Why is the rate of movement of mobile phase less in ascending Paper

Chromatography?
What is Chromatogram?

Reply

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