Proteomics/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

Introduction

Chromatography separation consists of a stationary phase and a mobile phase. In a mixture of proteins, the target molecule interacts with the stationary while the rest of the proteins in the mixture remain in the mobile phase. As the name indicates, the mobile phase moves and flows out of the system. The target protein has been successfully isolated. The next step is to remove the target protein from the stationary phase and get it into the mobile phase. This is done by eluting the target molecule.

The mobile phase flows out of the system while molecules in the stationary phase remain immobile

In chromatography, the target protein is desorbed from the stationary phase to the mobile phase. This illustrates a desorption curve. During this time the sample is transferred from the stationary phase to the mobile phase due to altered buffer conditions

State of Mobile Phase

The mobile phase can be either liquid or gas. Gas chromatography is very widely used in analytical chemistry. It has less application to proteomics because the technique requires high temperatures which are often unsuitable for the large polymers involved in proteomics. Gas chromatography depends on the partition equilibrium between a solid stationary phase and a gaseous mobile phase. Gas chromatography is almost always performed in a tube. The stationary phase usually consists of solid beads packed into a column adhered to a capillary tube.

A more useful mobile phase in proteomics is one that is in a liquid state. All of the techniques discussed in this chapter involve liquid chromatography. In this technique, there is traditionally a partition equilibrium between a solid stationary phase and a liquid mobile phase. Liquid chromatography is either carried out in a column or a plane.

Shape of Stationary Phase

The stationary phase is almost always solid, however, there are examples of chromatography experiments in which the stationary phase is in another state. For example, in counter current chromatography (CCC), both the mobile and stationary phase are liquid.

The shape of the stationary phase usually can take two forms:

1. planar chromatography
2. column chromatography

Planar Chromatography

This type of chromatography involves a stationary phase that is planar. The plane can consist of a layer of paper or a substance such as a gel that is supported by a glass plate. The mobile phase diffuses and is allowed to interact with the plane.

Figure 2. Paper Chromatography. The components of the sample move up the paper at different rates.

In paper chromatography, the target molecule is placed on chromatography paper. The mobile phase is a solvent. The solvent rises up the paper. When it reaches the sample, the solvent carries it with it. Different components of the sample travel at different rates thus separating the sample into strips. In thin layer chromatography, a matrix of gel or silica is used instead of paper. This tend to provide faster runs and better resolution than paper chromatography.

Column Chromatography

Column chromatography is probably the most common form of chromatography used in proteomics. The stationary phase is in a column. The column usually consists of a tightly packed bead matrix. The mobile phase enters the column and flows out at a constant rate. The column is usually coupled to a detection device such as a mass spectometry device. the absorption of the sample can be measured as it flows out of the column. This way, any protein that has been eluted in the mobile phase can be detected.

References

1. Chromatography on Wikipedia
2. The affinity chromatography separation mechanism. GE Healthcare Life Sciences