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Principle of Chromatography

Chromatography Principles

Nowadays, several scientific laboratories utilize the chromatography technique, an effective method of separating complex compounds. This term initially came into existence in the early 1900s from the Greek. Chromatography arrives from two words, called “Chrome” and “Graphy,” meaning color and writing, respectively. Thus, the word chromatography means color writing. A proper definition for chromatography would be:

An analytical technique that helps segregate a mixture into individual compounds. This phenomenon starts by separating, identifying, and purifying the mixture for further qualitative and quantitative analysis.

Who Introduced Chromatography?

AA Russian botanist, Mikhail Tswett, worked on the plant pigments, including chlorophyll a and b, xanthophyll, and carotenoids, in 1906.

He held these pigments and applied different biological techniques to purify the substances. This way, the concept was introduced, and many people these days access this skill to filter objects. Several types of chromatography are present, for instance, types of column chromatography, gas chromatography, thin-layer chromatography, etc.

Phases of Chromatography

It is a segregating technique, applied in all branches of science, and has two essential phases. These are the stationary and the mobile phase.

Stationary Phase

This phase of chromatography will always be either liquid or solid-phase connected with the surface where components, to be separated, are absorbed. It is named because of is static, while other portions are mobile. Numerous compounds used as stationary phases are usually porous, making it easy to run the process. The type of stationary phase used depends on the nature and type of chromatography.


Some general examples are gases, silica, uniform paper, glass, etc.

Mobile Phase

A mobile phase can exist in a liquid or gas state that will further travel in the chromatographic tank from where components separate. This phase is the solvent compared to the stationary phase, which helps to transfer the mixture towards the other side. The term indicates that this state is a continuous state whose components are picked up, depending on the type and nature of chromatography.


Several mobile phase examples are present, including acetic acid, alcohol, acetone, water, etc.

How Does Chromatography Work?

This technique works on a basic principle, involving the above two main phases. The process starts when molecules from one phase pass on to the other phase, separating the individual components from one another. The initial phase, as discussed above, is called stationary, while the second phase is termed as mobile phase. Both exist in the three states; solid, liquid, and gas.

As chromatography is used to separate the mixture, it demands a sample called the solute. This solute is separated due to its relative affinity after distributing itself between the stationary and the mobile phase. The distribution of components of a mixture between the stationary phase and the mobile phase is governed by the distribution coefficient, K. A mixture with a smaller K value remains in the stationary phase as the moving phase flows over it. Similarly, the compound with a greater K value remains in the mobile phase. This entire segregation process involves some compelling factors, such as

  • Adsorption – liquid-solid
  • Partition – liquid-solid
  • Affinity or differences between the component weights

These factors help a mixture process rapidly and leave the chromatographic tank, leaving the two phases behind.

Types of Chromatography

ion exchange chromatography

There are numerous types of chromatography that hold much significance. All of them are used in our daily life experiments and to separate biological molecules from one another. We will soon come up with an article enlisting all the chromatography types in detail. Till then, here is the list of prototypes of chromatography:

  • Gas Chromatography
  • Flash Chromatography
  • Paper Chromatography
  • Liquid Chromatography
  • Affinity Chromatography
  • Column Chromatography
  • Ion Exchange Chromatography
  • Size Exclusion Chromatography
  • Reverse-Phase Chromatography
  • Thin Layer Chromatography (TLC)
  • Hydrophobic Interaction Chromatography
  • High-Performance Liquid Chromatography (HPLC)

Applications of Chromatography

This procedure has been universally utilized in various sectors, including chemical corporations, pharmaceutical sectors, molecular biology, biochemistry, forensic laboratories, etc. All the chromatography types are equally important to analyze a substance qualitatively and quantitatively. Experts use this phenomenon in:

Food Sector

  • To determine the high-grade food quality.
  • To detect the additives and toxins in food.

Chemical Companies

  • To test the air quality and samples used.
  • Gas Chromatography is extensively utilized to identify different contaminants that help forms the chemicals.

Pharmaceutical Companies

  • To determine the samples of all minor elements
  • Chromatography is used in the identification and formation of drugs.

Molecular Biology

  • Chromatography helps in several hyphenated techniques for research purposes.
  • We use HPLC to separate essential proteins and vitamins for human use, such as plasma fractionation and insulin purification.

Forensic Laboratories

  • To test the blood or hair sample of the criminal or the suspect that will help identify the culprit.

In a Verdict

Chromatography has become an indispensable technique across many fields, with prevalent applications in the food, chemical, pharmaceutical, molecular biology, and forensic industries. Its ability to separate complex substances and mixtures into individual components has revolutionized analytical chemistry and our understanding of biological systems. Continued improvements in chromatographic columns, detectors, and data analysis will ensure chromatography remains a vital tool for scientific discovery into the foreseeable future. With each new advance, we gain deeper insights into the composition and interactions of the natural world at the molecular level.

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