Tuesday, October 4, 2016

Chromatography - Principles


What is chromatography?
Chromatography is a technique of separating or analyzing complex mixtures. Now, the question asked is, what is so special about chromatography?  Well, chromatography can separate the components from the mixture with acute precision. For example, some proteins, which vary by a single amino acid, can also be separated by the chromatography technique. Also, the conditions employed in chromatography are not severe and for this reason, chromatography can be used when one needs to separate delicate products.

The inventor of chromatography is Michel Tswett who was the Russian biochemist. In 1906, he separated chlorophyll from the plant pigments mixture. In this mixture of pigments, each band had a distinct color and so the procedure was named ‘chromatography’ where ‘chromo’ means colour while ‘graphy’ means to write.

Before understanding the principle, first lets get familiarized with frequently used terms in chromatography.
  • Analyte – Mixture whose components needs to be separated
  • Mobile phase (carrier) – Solvent moving through the column
  • Stationary phase (adsorbent) – Substance that stays fixed inside the column
  • Eluent – Fluid that enters the column
  • Eluate – Fluid that exits the column

In all chromatography, there are two phases – a stationary phase and a mobile phase.  The mobile phase can be either a liquid or a gas while the stationary phase can be either a solid or a liquid. The analyte is composed of different components that need to be separated. So, the mobile phase (solvent) is made to flow through the stationary phase (like, silica bed). The components of analyte show different degrees of adhesion to silica. The components that adhere to the stationary phase more strongly, travel slowly as compared to those components who have a weaker adhesion. Thus, we can say that, when an analyte or mixture of components is introduced into the column (narrow zone), then, the different components move at different rates in the direction of solvent (mobile phase) flow.

Now, lets ask ourselves certain questions. 
(a) What decides the separation of the the components in the analyte?
The basic principle behind the separation is that the affinities of the components of the analyte is different towards the mobile phase and the stationary phase. The smaller the affinity the component has for the stationary phase, the shorter the time it will spend in the column.

(b) What decides the affinity?
The affinity, in turn, is decided by two properties:
  • Adsorption: Adsorption is the property as to how firmly a component sticks to the stationary phase. So, the higher the adsorption, the slower the molecule will move through the column.
  • Solubility: Solubility is the property as to how well a component of the analyte can get dissolved in the mobile phase. The higher the solubility in the mobile phase, the faster the molecule will move through the column. Note: Adsorption and solubility of a molecule can be changed by choosing the appropriate stationary and mobile phases.

(c) Why different components have different affinity towards stationary and mobile phase? 
The answer to this question lies in ‘polarity’. The component that is polar in nature, will adsorb on the polar stationary phase (say silica), while the components which are non-polar in nature, will readily dissolve in the non-polar mobile phase without adhering to the stationary phase and will elute from the column as can be seen in the adjacent diagram.

There are two techniques of chromatography as:
A. Plane chromatography which includes:

  1. Paper chromatography 
  2. Thin layer chromatography

B. Column chromatography which  includes:

  1. Adsorption chromatography
  2. Partition chromatography
  3. Gel permeation chromatography
  4. Ion Exchange chromatography
  5. Affinity chromatography

Keeping all these basics in mind, we will discuss in the next few posts, the above mentioned different types of chromatography.