What is Chromatography?
Chromatography is a physical process of separation in which the components to be separated are distributed between two phases; one phase is stationary phase while the other is the mobile phase moves through it in a definite direction.
The chromatographic method happens due to variations in the distribution constant of the specific sample elements.
Chromatography is a process used to isolate and recognize the elements of a mixture.
Works by providing the molecules present in the compound to categorize themselves between a stationary and a mobile medium.
Those molecules that spend most of their time within the mobile phase are carried along faster.
The chromatographic technique is principally based on the differences in the rate by which the components in a mixture moves or flow through the porous medium (also known as the stationary phase) under the influence of some gas or solvent (also known as the mobile phase).
In general, the chromatographic method consists of the subsequent steps:
1. Retention or adsorption of substances on the stationary phase.
2. Separation of the adsorbed substances by the mobile phase.
3. Elution is the process in which the separated elements are recovered from the constant flow of the mobile phase.
4. Qualitative and quantitative examination of the eluted materials.
• This technique was discovered by Mikhail Tswett, Russian Botanist, in 1872-1919.
• In 1906 Tswett used the chromatography method to separate the plant pigments.
• He called the new method chromatography because the result of the analysis was ‘written in color’ along the length of the adsorbent column.
• In chromatography word, Chroma means “color” and graphein means to “write”.
• Chromatography has importance in all branches of the physical and biological sciences.
• Twelve Nobel prizes were awarded between 1937 and 1972 alone for work in which chromatography performed an important role in identification.
Classification of chromatography
Chromatographic techniques are classified based on the type of stationary and mobile phase used or chosen. The stationary phase could also be a solid or a liquid and the mobile phase could also be liquid or a gas. Separation occurs due to a mixture of two or more factors like rates of migration, capillary action, and extent of adsorption, etc.
• Classification of chromatography according to mobile phase:
1- Liquid chromatography: mobile phase is a liquid. (LLC, LSC).
2- Gas chromatography: mobile phase is a gas. (GSC, GLC)
• Classification according to the packing of the stationary phase:
1- Thin-layer chromatography (TLC): the stationary phase is a thin layer supported on glass, plastic, or aluminum plates.
2- Paper chromatography (PC): the stationary phase is a thin film of liquid supported on an inert support.
3- Column chromatography (CC): stationary phase is packed in a glass column
• Classification according to the force of separation:
1- Adsorption chromatography.
2- Partition chromatography.
3- Ion exchange chromatography.
4- Gel filtration chromatography.
5- Affinity chromatography
Theory of Chromatography
Two theories are suggested regarding the rate of migration of solute and the development of peaks within the chromatogram.
They are the plate theory and the rate theory or the kinetic theory.
(a) Plate Theory:
According to the plate theory developed by Martin and Synge, a chromatographic column consists of a series of imaginary, discrete yet connected horizontal layers that are termed the general plates. Equilibration of the solute among the stationary and the mobile phases takes place at each of these plates. Migration of the solute is then expected to occur by a series of stepwise transfers between one plate to the other directly below. The efficiency of partition in a chromatographic column gets increases as the number of theoretical plates increases. This is because the number of equilibrations will also correspondingly develop.
(b) Rate Theory:
Rate theory describes the effect of variables, for example, the process of diffusion and mobile phase velocity to decide the width of an elution band. It relates to the effects of these variables on the time taken by a solute to make its appearance at the end of the column. A molecule alternates rapidly between adsorbed and desorbed states.
The time which the molecule spends in any one phase depends on the accidental energy gained by the molecule from its environment and is highly variable. A particle can migrate only if it is present in the mobile phase and as a result, the migration down the column is also highly irregular. Consequently, some solute molecules may migrate rapidly whereas others may lag.
The net result of all these random and unique processes is the asymmetric distribution of velocities around the mean value, which describes the behavior of the most common distribution of velocities or average particles. The width of the zone gets increased as it migrates down the column because more time is needed for migration to take place. Hence, the zone width is directly related to the residence or retention time on the column and inversely proportional to the mobile phase velocity.
• The technique of chromatography is non-destructive in nature for resolving multicomponent mixtures of minor, major, and trace elements into their fractions.
• Significant improvements have been made and the method is used to distribute colored as well as colorless substances.
• Chromatography is probably the most important single analytical method used today and is likely to continue to be so in the foreseeable future.
• Chromatography columns are considered to consist of several closest zones.
• Chromatographic peaks are generally by three kinetically controlled processes,
▪️ Eddy diffusion,
▪️ Longitudinal diffusion,
▪️ Non-equilibrium mass transfer.