Chromatography is the technique for separating the components, or solutes, of a mixture on the basis of the relative amounts of each solute distributed between stationary phase and mobile phase.
It works on the principle of distribution coefficient (Kd)
separation in phase A
Kd = ---------------------------------
seperation in phase B
History : Tswett separate plant pigments by the chromatography
Word meaning : "chroma" - color and graphein - "to write"
Chromatography: Principles, History, and Types
Chromatography is a powerful technique used for separating the components of a mixture based on their relative affinities between two phases: the stationary phase and the mobile phase. It is widely employed in chemical analysis, biochemistry, and other scientific fields to purify, identify, and quantify compounds.
Principle of Chromatography
At the heart of chromatography is the concept of the distribution coefficient (Kd), which is a ratio of a compound's concentration between two phases:
The two phases include:
Stationary Phase: A solid or liquid substance that remains fixed in place.
Mobile Phase: A liquid or gas that moves through the stationary phase, carrying the mixture’s components along with it.
The separation occurs because different substances in a mixture have varying degrees of affinity for the stationary phase and mobile phase. Components that interact more strongly with the stationary phase will move more slowly, while those with higher affinity for the mobile phase will travel faster.
History of Chromatography
The concept of chromatography was first introduced in 1906 by Russian botanist Mikhail Tswett, who used it to separate plant pigments. Tswett applied this technique to analyze chlorophyll, the green pigment in plants, by passing a solution through a column packed with solid adsorbent. The pigments separated into distinct colored bands, coining the term "chromatography" from the Greek words "chroma" (meaning "color") and "graphein" (meaning "to write").
Though initially focused on plant pigments, chromatography evolved into a highly versatile tool that can separate and analyze a wide range of chemical substances, even colorless ones.
Key Terms in Chromatography
Several essential terms are associated with chromatography, which are critical to understanding how it works:
Mobile Phase: The solvent or gas that flows through the chromatography system, carrying the solutes of the mixture.
Stationary Phase: The phase that stays immobile inside the column or on the chromatographic medium, interacting with the solutes and causing separation.
Elution and Elution Volume: Elution refers to the process of extracting one substance from another by washing it out with a solvent. The elution volume is the amount of mobile phase needed to elute a particular solute from the column.
Void Volume: The volume of mobile phase required to pass through the column without any interaction with the stationary phase, essentially representing the space not occupied by the stationary phase.
Column: A cylindrical container filled with a stationary phase through which the mobile phase passes, facilitating the separation of the mixture’s components.
Types of Chromatography
Chromatography comes in many forms, each designed to target specific types of mixtures and molecules. Below are the main types:
1. Adsorption Chromatography: In this method, the stationary phase is a solid, and the separation occurs because solutes adsorb onto the surface of the stationary phase. Components with stronger adsorption remain in the stationary phase longer, separating from others more quickly carried by the mobile phase.
2. Partition Chromatography: This technique relies on a liquid stationary phase that forms a thin film on a solid support. The solutes partition themselves between the liquid stationary phase and the mobile phase, leading to their separation based on solubility differences.
3. Size Exclusion Chromatography (SEC): Also known as gel filtration or molecular sieve chromatography, SEC separates molecules based on their size. The stationary phase contains porous beads, and smaller molecules enter the pores, slowing them down, while larger molecules pass through more quickly.
4. Affinity Chromatography: This type exploits the specific interactions between a molecule and a particular ligand bound to the stationary phase. It’s commonly used in biochemistry to isolate proteins, enzymes, or antibodies by exploiting their binding affinity to specific molecules.
5. Ion Exchange Chromatography: This method separates molecules based on their charge. The stationary phase contains charged groups that interact with oppositely charged ions in the mobile phase. By varying the pH or ionic strength of the mobile phase, the ions of interest can be selectively eluted.
Conclusion
Chromatography is a versatile and essential tool in modern science, with applications in fields as diverse as chemistry, biochemistry, medicine, and environmental analysis. Its ability to separate complex mixtures into individual components allows scientists to study and manipulate compounds with precision, driving progress in research and development. Whether isolating plant pigments or analyzing biomolecules, chromatography continues to be a foundational technique in laboratories worldwide.
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