32.8: Principles Of Column Chromatography

The chromatography technique was first invented in 1901 by Michael S. Tswett, a Russian botanist, to separate plant pigments using organic solvents. Further, in 1941, Archer John Porter Martin and R. L. M. Synge modified the technique by packing silica gel into a column. A mixture of amino acids was then separated on the packed column using chloroform and water mixture as the mobile phase. This was the first report on column chromatography. At present, column chromatography is a widely used technique for separating various types of compounds from a sample mixture.

Factors Influencing Efficient Separation of Proteins

Various parameters like column material, packing, and operational conditions such as flow rates and temperature determine the efficiency of separation by column chromatography.

The choice of column material or matrix determines the extent of interaction with the sample. The matrix material must be tightly and uniformly packed in the column. Air bubbles, debris, large particles, and precipitates interfere with the uniform flow of the solvent through the column, affecting its separation efficiency. The column should also be free from particulate matter.

The sample injected into the column should be clear and free from aggregates that might clog the column, hindering solvent flow. The flow rate of solvent also affects the separation. Very high or very low flow rates of solvents result in inefficient separation of compounds and impure preparations. Very high rates may also disturb the column packing, affecting process efficiency. In addition, the composition of elution buffer is also an important factor. It should be non-corrosive and compatible with the sample and also the column material to prevent in situ precipitation or dissolution.

Another operational parameter, temperature, also plays an important role in the process. It decides the stability of the sample, column material, and solvent buffer. Also, a constant temperature throughout the column efficiently resolves compounds. After completing the separation process, the columns must be washed thoroughly by repeatedly passing a suitable solvent to avoid sample contamination in further runs. Occasionally, the solvent is passed in a column in the reverse direction to remove any clogged material.

Limitations

Though a very widely used technique, the method still has some limitations. It is a very time-consuming method as the flow rates need to be slower for better resolution of the compounds. Also, large quantities of highly pure solvents required in the mobile phase make the process expensive. This also raises the scaling-up cost when higher yields of pure compounds are needed.

Column chromatography is a biochemical technique used to separate compounds based on their physical and chemical properties.

It has two main components — the solid stationary phase or the matrix and the liquid mobile phase or the solvent.

The matrix-packed column is loaded with a sample, such as a protein mixture. The solvent is then used to carry the sample through the column.

Inside the column, the matrix acts as a molecular mesh filtering the proteins based on their size or interaction with the matrix. The larger proteins tend to travel faster than the smaller proteins, resulting in a size-based separation.

Additionally, proteins in a sample may interact with the matrix. Weak interactions allow proteins to pass quickly, while strong interactions retain proteins within the column.

A gradual change in the pH or ionic strength of the solvent alters the proteins’ interactions with the matrix and helps to elute proteins into separate fractions.