Till now, we have discussed about plane chromatography technique. Now, coming to column chromatography. The five major types of column chromatography are as:
1. Adsorption chromatography in which the components of mixture are selectively adsorbed on the surface of packing column material i.e. adsorbent.
2. Partition chromatography in which component is partitioned between the mobile phase and their stationary phase is held stationary on inert solid support.
3. Ion exchange chromatography in which the constituent of sample is selectively retained by exchange resin by replacing ion/s on packing material.
4. Gel chromatography is the method in which column is packed with a permeable gel which brings out separation by sieving (gel permeation )
5. Affinity chromatography utilizes the specificity of a enzyme for its substrate or substrate analogue for the enzyme’s separation.
We will discuss in details all these types of chromatogrpahic techniques. But for now, lets understand that all these chromatographic techniques have a lot of features in common. So, lets discuss what all they share in common.
The columns are usually made up of glass or polyacrylate plastic. They all are different in dimensions and range from 2 mm in diameter to 70 mm in diameter and lengths vary from 15 cm to 150 cm. The choice of the column is determined by the amount or the volume of the sample.
The commonly used glass column shave a sintered glass disc at the bottom to support the stationary phase. Another affordable alternative is the use of a plug of glass wool with a small amount of quartz sand and glass beads.
Some columns might get affected by the temperature and hence, for such techniques, the columns with thermostat jackets are used. All the columns are provided with an inlet and an outlet.
Packing of the column
The column is held in the upright position and as already mentioned above, the bottom is sealed with glass wool or other such supports and the outlet is closed. Now, the mobile phase is added into the column so that 1/3rd is filled with it. Next, the stationary phase in the form of slurry i.e., degassed gel or adsorbent or resin is gently poured in the column. The slurry is added such that the 3/4th of the column is full. The outlet is now opened and the column is stabilized by adding the mobile phase. Note that a nylon disk is placed on the top of the column so as to prevent the disturbance of the column by the addition of the mobile phase or by the addition of the sample.
Introducing the sample
The addition of the sample is a critical step as it should not disturb the column. Also, the sample should be added in a less volume and the sample should be desalted so as to avoid anomalous adsorption effects.
The sample is mixed with sucrose or ficoll to 1% concentration so as to increase the density of the sample. This sample now sinks below the top layer of the solvent to the surface of the column. Bromophenol blue dye can also be added in place of sucrose which will help in tracking as well. Alternate method of sample addition is to reach the column surface directly with the help of the syringe or capillary tubing.
The continuous flow of mobile phase (called the "eluant" – mobile phase exiting the column is the eluant) through the packed column separates the components of the sample which is applied to the column. This process is called column development. There are two main techniques of elution:
Isocratic elution: When there is a single solvent acting as an eluant during development, then, this type of process is called isocratic elution.
Gradient elution: However, sometimes, single solvent elution may not be sufficient as the resolution may not be satisfactory. So, in such cases, pH or ionic strength or polarity of the eluant is changed w.r.t. time. This leads to the formation of gradient in the column which leads to a better resolution of the sample components. So, this process where the composition of the mobile phase is changed thereby giving rise to the gradient is called gradient elution.
This was about the basics of the column chromatography. From next posts on, we will discuss the types of column chromatography in details.