# Proteomics/Protein Separations - Chromatography/Chromatography Theory

 « Protein Separations - Chromatography Chromatography Theory » Protein Separations - Chromatography Stationary and Mobile Phases

Chapter written by: Laura Grell and Alexander Butarbutar
Contact llg3875@rit.edu or nbb3924@rit.edu for contributions
Chapter modified by Kai Burnett and Dalia Ghoneim
Contact kab9783@rit.edu or dxg6098@rit.edu

## Chromatography Theory

Chromatography is a method of separating molecule. The method takes advantage of differences between a mobile phase and a stationary phase to separate the different components in a mixture. The target molecules can interact with the stationary phase based on characteristics such as charge, size, and hydrophobicity.

There are two theories of Chromatography:

1. Plate theory
2. Rate theory

### Plate Theory of Chromatography

Archer John Porter Martin and Richard Laurence Millington Synge created the plate theory of chromatography. The plate theory describes the stationary phase and the mobile phase as being in equilibrium. The partition coefficient k between these two plates can be defined as:

${\displaystyle K={\frac {Concentration\ of\ solute\ in\ stationary\ phase}{Concentration\ of\ solute\ in\ mobile\ phase}}}$

As k increases, the time it takes for the solutes to separate increases. If chromatography is being performed in a column of fixed length and flow rate, the retention time ${\displaystyle (t_{R})}$ and retention volume ${\displaystyle (V_{r})}$ can be measured and used to determine the value of K.

### Rate Theory of Chromatography

Rate theory was introduced by van Deemter to account for chromatographic behavior that could not be explained by plate theory. Rate theory is based on three terms: path-dependent diffusion (A),longitudinal diffusion (B) and mass transfer (C).

A. Path-dependent diffusion occurs when the packing in a chromatography column is not uniform, so that two identical analytes may migrate differently because one had further to travel than the other.

B. Longitudinal diffusion is the result of materials moving from an area of high concentration (the center of a band on a chromatography column) to an area of low concentration (the outside edges of the same band). Longitudinal diffusion increases with increasing temperature.

C. Mass transfer effects result from the fact that materials take time to equilibrate between the stationary and mobile phases. While that time is elapsing, the mobile phase is still moving. This leads to band broadening.