Chromatography

Chromatography is defined as a procedure by which solutes are separated by a dynamic differential migration process in a system consisting of two or more phases. One of which moves continuously in a given direction and in which the individual substances exhibit different mobilities by reason of differences in adsorption, partition, solubility,vapor pressure, molecular size, or ionic charge density.

Two Phases of Chromatographic Technique

A chromatographic technique requires solute to distribute between two phases, the stationary phase which is fixed and mobile phase in which is moving.

1. Mobile phase – it carries the solute through the medium until emerges separately from other solutes that are eluted already or later. The solute is transferred in a flowing stream of liquid or a gaseous solvent known as eluant through the separation medium.
2. Stationary phase – acts through adsorption. Adsorbents such as activated alumina, silica gel, and ion-exchange resins, or it may act by dissolving the solute, thus partitioning the latter between the stationary and mobile phases.

Types of Chromatography

1. Column Chromatography
2. Gas Chromatography
3. Paper Chromatography
4. Thin-layer Chromatography
5. High Performance Liquid Chromatography

Use of References Substances in Identity Test

R_f is the ratio of distance traveled on the medium by a given compound to the distance traveled by the front of the mobile phase, from the point of application of the test substance. This distance measured to the point of maximum intensity of the spot.

R_r is the ratio between the distances traveled by a compound and a reference substance.

Location of Components

Spots created by paper or thin-layer chromatography can be located by:

1. Direct inspection. Executed under white or either short-wavelength (254nm), or long-wavelength (360nm) ultraviolet light.
2. Inspection in white or ultraviolet light after treatment with reagents that will make the spots visible such as reagents used in atomizer.
3. Use of a Geiger-Muller counter or autoradiographic techniques in the case of the presence of radioactive substances.
4. Evidence resulting from stimulation or inhibition of bacterial growth by the placing of removed potions of the adsorbent and substance on inoculated media.

For open-column, pressurized liquid and gas chromatography the retention time, t, is defined as the time elapsed between sample injection and appearance of the peak concentration of the eluted sample zone, may used as a parameter of identification. The ration of the retention times of the test substance, the reference compound, and a mixture of these, to the retention time of an internal standard is call the relative retention time, R_r and is also used frequently as a parameter of identification.