CHROMATOGRAPHY

INTRODUCTION:
Chromatography (from Greek chroma "color" and graphein "to write") is the collective term for a set of laboratory techniques for the separation of mixtures. The mixture is dissolved in a fluid called the "mobile phase", which carries it through a structure holding another material called the "stationary phase". The various constituents of the mixture travel at different speeds, causing them to separate. The separation is based on differential partitioning between the mobile and stationary phases. Subtle differences in a compound's partition coefficient result in differential retention on the stationary phase and thus changing the separation. Chromatography may be preparative or analytical. The purpose of preparative chromatography is to separate the components of a mixture for further use (and is thus a form of purification). Analytical chromatography is done normally with smaller amounts of material and is for measuring the relative proportions of analytes in a mixture. The two are not mutually exclusive.

History:
Chromatography, literally means "color writing", was first employed by Russian scientist Michael Tsvet in 1900. He continued to work with chromatography in the first decade of the 20th century, primarily for the separation of plant pigments such as chlorophyll, carotenes, and xanthophylls. Since these components have different colors (green, orange, and yellow, respectively) they gave the technique its name. New types of chromatography developed during the 1930s and 1940s made the technique useful for many separation processes. Chromatography technique developed substantially as a result of the work of Archer John Porter Martin and Richard Laurence Millington Synge during the 1940s and 1950s. They established the principles and basic techniques of partition chromatography, and their work encouraged the rapid development of several chromatographic methods: paper chromatography, gas chromatography, and what would become known as high performance liquid chromatography. Since then, the technology has advanced rapidly. Researchers found that the main principles of Tsvet's chromatography could be applied in many different ways. Advances are continually improving the technical performance of chromatography, allowing the separation of increasingly similar molecules.

Steps involved in chromatography:

1. Adsorption or retention of a substance or substances on the stationary phase. 2. Separation of the adsorbed substances by mobile phase.
3. Recovery of the separated substances by a continuous flow of the mobile phase, the

method being called Elution. 4. Qualitative and quantitative analysis of the eluted substances . THEORIES OF CHROMATOGRAPHY:

The Theoretical Plate Model of Chromatography

The plate model supposes that the chromatographic column is contains a large number of separate layers, called theoretical plates. Separate equilibrations of the sample between the stationary and mobile phase occur in these "plates". The analyte moves down the column by transfer of equilibrated mobile phase from one plate to the next.

It is important to remember that the plates do not really exist; they are a figment of the imagination that helps us understand the processes at work in the column.They also serve as a way of measuring column efficiency, either by stating the number of theoretical plates in a column, N (the more plates the better), or by stating the plate height; the Height Equivalent to a Theoretical Plate (the smaller the better). If the length of the column is L, then the HETP is HETP = L / N The number of theoretical plates that a real column possesses can be found by examining a chromatographic peak after elution;

Where w1/2 is the peak width at half-height.

As can be seen from this equation, columns behave as if they have different numbers of plates for different solutes in a mixture.

The Rate Theory of Chromatography

A more realistic description of the processes at work inside a column takes account of the time taken for the solute to equilibrate between the stationary and mobile phase (unlike the plate model, which assumes that equilibration is infinitely fast). The resulting band shape of a chromatographic peak is therefore affected by the rate of elution. It is also affected by the different paths available to solute molecules as they travel between particles of stationary phase. If we consider the various mechanisms which contribute to band broadening, we arrive at the Van Deemter equation for plate height; HETP = A + B / u + C u where u is the average velocity of the mobile phase. A, B, and C are factors which contribute to band broadening.

A - Eddy diffusion
The mobile phase moves through the column which is packed with stationary phase. Solute molecules will take different paths through the stationary phase at random. This will cause broadening of the solute band, because different paths are of different lengths.

B - Longitudinal diffusion
The concentration of analyte is less at the edges of the band than at the centre. Analyte diffuses out from the centre to the edges. This causes band broadening. If the velocity of the mobile phase is high then the analyte spends less time on the column, which decreases the effects of longitudinal diffusion.

C - Resistance to mass transfer

The analyte takes a certain amount of time to equilibrate between the stationary and mobile phase. If the velocity of the mobile phase is high, and the analyte has a strong affinity for the stationary phase, then the analyte in the mobile phase will move ahead of the analyte in the stationary phase. The band of analyte is broadened. The higher the velocity of mobile phase, the worse the broadening becomes.

Van Deemter plots Plate height versus average linear velocity of mobile phase

Such plots are of considerable use in determining the optimum mobile phase flow rate.

Types of chromatography:
Chromatography characterized as a separation method based on the differential migration of solute through a system of two phases, one is mobile phase another one is stationary phase. Chromatography is a technique by which the components in a sample, carried by the liquid or gaseous phase, are resolved by desorption steps on the stationary phase. Chromatography is mainly divided into two categories:
1. Adsorption chromatography: separation is mainly due to the interaction between

solute and surface on the adsorbent. In this, stationary phase is solid and mobile phase is liquid. E.g.; TLC, HPTLC, and GC.
2. Partition chromatography: separation is based on the partition between two phases.

In this mode, both stationary phase and mobile phase are liquids. E.g.; GLC.

CLASSIFICATION OF CHROMATOGRAPHY Chromatography

Gas chromatography
Gas-solid gas-liquid partition

Liquid chromatography

Adsorption Ion exchange

Affinity Chiral

Size exclusion

Normal phase

reverse phase

Selection of LC modes based on solubility and molecular mass.