Chromatography: An Overview

Did you know that green is a mixture of yellow and blue? This theory can be demonstrated by the separation technique, chromatography which means colored writing. It is one of the newest techniques for separating, identifying, and purifying the mixture of components. 

The IUPAC defines it as a “physical method of separation in which the components to be separated are distributed between two phases; one of which is the stationary phase while the other is the mobile phase that moves in a definite direction.

Brief History

Early Discovery

  • The first use of the chromatography method was purely by dye chemists. In 1861, Friedrich Goppelsroder described a method of obtaining a colored ring of drops of inorganic compounds in the center of filter paper. He gave it the name “capillary analysis.”
  • However, Russian botanist Mikhail Semyonovich Tsvet was the first documented inventor of the chromatography technique. He discovered this technique while researching chlorophyll in 1900. He mentioned the details of the process and named it chromatography.

Further Modification

  • In 1941, Archer J.P Martin and Richard L.M Synge for the separation of amino acids in wool. They were awarded the Nobel Prize in Chemistry in 1952 for discovering partition-chromatography. The technique was similar in principle to Tsvet’s approach.
  • Due to different defects in the silica used in his technique, Martin and his team used filter paper as the stationary medium This modification gave the technique more reproducibility. It also replaced the column used by Tsvet to a large extent. The method was termed “paper chromatography.”
  • Likewise, two Soviet pharmacists, Nikolay A. Izmaylov and Maria S. Shrayber distributed a thin layer of support material on a glass plate. The German Chemist Egon Stahl further studied the spectrum of its application. And the system was termed “thin layer chromatography.”
  • Similarly, chemist Erika Cremer defined a new chromatographic technique where the stationary phase was solid instead of liquid. The technique is termed gas chromatography.

Principle of Chromatography

The basic principle of the chromatography technique is the same even though it has various types. This technique consists of the two-phase; stationary and mobile phase. The stationary phase does not move. The desired mixture of compounds (analytes) is present in the mobile phase. The analytes and the mobile phase move around the stationary phase.

The different components of analytes react at different times with the stationary phase while navigating through the stationary phase. It is because of their polarity with the stationary phase. This reaction time is termed retention time. Since the various components have different retention times, separation of the components occurs at different levels. The separated components pass through a detector. Afterward, they are plotted in a chromatogram.

Chromatography techniques
Chromatography techniques

Types of Chromatography

There are eight common types of chromatography which are; paper, thin-layer, gas, column, ion exchange, affinity, high-performance liquid, and immunochromatography.

  1. Paper chromatography is a technique that uses paper sheets that are absorbent as the stationary phase through which the liquid sample passes. This type commonly applies to check the purities of chemicals in various laboratories, determine the level of drugs in humans and animals, etc.
  2. Thin-layer chromatography is the process that uses aluminum foils or glass coated with a thin layer of absorbent material like cellulose, silica, or gel as a stationary phase through which liquid mobile moves. This movement and affinity of components in the mixture with the stationery phase helps separate the components. It is helpful for various qualitative and quantitative analyses of different compounds.
  3. Gas chromatography: It is the separation process that uses gas (carrier gas) as a stationary phase where the liquid or gas sample gets injected/inserted. It is useful in the separation of volatile substances.
  4. Column chromatography: It is the technique that uses a column with absorbent material as a stationary phase where the mobile phase along with sample travel. The movement depends on the difference in the affinity of components of the sample with the stationary phase leading to the separation of compounds at different times.
  5. Ion exchange chromatography is the technique that separates charged ions or polar compounds based on ionic interactions between the stationary phase and the mixture present in the mobile phase. It is applicable in various fields and works for any compounds that are capable of ionization.
  6. Affinity chromatography: It is the method based on the specific binding reaction between an immobilized stationary phase and mobile phase with the mixture.
  7. High-performance liquid chromatography (HPLC): It is the method in which pumps help liquid solvent to pass in high pressure through the column with solid absorbent material (stationary phase).
  8. Immunochromatography or Lateral flow Immunoassay: It is the combination of immunoassay and chromatography where immobilized antigen/antibody in the nitrocellulose membrane captures antibody/antigen present in the sample when allowed to pass through it.

Applications/ Uses of Chromatography

This method has wide applications in various fields of science. Some of the applications of chromatography are as follows:

  1. Chromatographic techniques apply in quality control testing in various industries.
  2. The food industry uses different chromatographic techniques to detect the presence of chemical additives in food. This technique also helps in assessing the nutritional value of food and the shelf life.
  3. Pharmaceutical industries use these techniques to detect the chemicals and traces of foreign substances in the drugs. This method applies to developing new drugs too. 
  4. The chemical industries detect the quality of air, water, pesticides, and impurities in petroleum products and oils using this technique.
  5. Forensic science also uses this technique to analyze various evidence.
  6. This technique also applies in molecular science to separate various proteins and nucleic acid components.

Limitations

Although chromatography has a wide spectrum of applications, it has few limitations. Some of its limitations are as follows:

  1. Chromatographic instruments and materials are expensive.
  2. Trained manpower is necessary for operating this technique.
  3. Maintenance and care of the equipment are necessary from time to time.
  4. Some chromatographic techniques may require a high load of electricity whereas others may require a high amount of solvents.

Reference

  • MacNaught, A., & Wilkinson, A. (1997). Compendium of chemical terminology. Blackwell Science.
  • Chromatography – New World Encyclopedia. Newworldencyclopedia.org. (2022). Retrieved 11 May 2022, from https://www.newworldencyclopedia.org/entry/Chromatography.
  • Chromatography | Definition, Types, & Facts. Encyclopedia Britannica. (2022). Retrieved 11 May 2022, from https://www.britannica.com/science/chromatography.
  • Coskun, O. (2016). Separation Techniques: CHROMATOGRAPHY. Northern Clinics Of Istanbul. https://doi.org/10.14744/nci.2016.32757.

Ashma Shrestha

Hello, I am Ashma Shrestha. I am currently pursuing my Master's Degree in Microbiology. Passionate about writing and blogging. Key interest in virology and molecular biology

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