The technique of column chromatography can be defined in two ways in accordance with the field of science in which it is being utilized – Chemistry and Biology. When applied in chemistry, it is used to separate one or sometimes more chemical compounds from a mixture dissolved in an appropriate solvent.
Whereas in biology, it most often is employed to separate a biological compound such as nucleic acids or Proteins from a mixture (also dissolved in a suitable solvent).
Since a column is used in this separation technique, hence the name column chromatography. This column is filled or packed with a solid adsorbent also called the stationary phase (e.g., silica gel or beads).
While the mobile phase consists of a solvent (in which the mixture from which a compound’s separation is required is dissolved), is passed through the stationary phase which allows the adsorbent to capture the compound to be separated.
After this, the compound attached to the stationary phase is eluted using elusion solvents. A typical column is shown in figure 1.
Figure 1. a typical column used in chromatographic separations and its different parts.
Column chromatography is used to carry out all the major types of chromatographic techniques. These are:
- Adsorption chromatography
- Ion exchange chromatography
- Gel filtration chromatography
- Affinity chromatography
- Gas chromatography, and
- High-performance liquid chromatography
Let us now take a brief look at each of these with understandable examples where possible.
It is a type of liquid chromatography in which compounds to be separated from the mixture are retained or adsorbed on the surface of the stationary phase.
Usually, the forces involved in the binding of the compound with the solid support are van der Waals forces and steric interactions. Adsorption chromatography is also known as solid-liquid chromatography. Adsorption chromatography was first invented by the Russian botanist Mikhail Tsvet in 1901. Figure 2 illustrates how molecules might become adsorbed on the surface of the solid support.
Figure 2. Adsorption of solute molecules on the surface of the adsorbent.
The invention of adsorption chromatography is also associated with its very first example. In this, the chlorophyll and carotenoid pigments were separated using calcium carbonate, sucrose, and alumina as a stationary phase with petroleum and ether/ethanol mixtures as eluents.
Ion exchange chromatography
Ion chromatography or ion-exchange chromatography is another type of column chromatography in which mostly polar and ion compounds are separated. The basic principle lies in the charge of the molecules and they are separated by attaching to the oppositely charged stationary phase.
Then finally in the elusion step, the molecules of the eluent compete for the ionic compound being purified, take their places in the stationary phase and thus the ionic compound is purified (see Figure 3).
Figure 3. Ion exchange column chromatography. This particular type is anion exchange (negative ions are being exchanged)
As an example, let us look at an anionic resin quaternary amine (-N+(CH3)3). It is a strong anion exchanger known by its commercial name of Q resin. As it is an anion exchanger (having a positive charge on itself), it can be used for the purification of a variety of negatively charged compounds.
Gel filtration chromatography
Gel filtration chromatography is also known as size exclusion chromatography and molecular sieve chromatography. It is a type of chromatographic separation technique in which molecules in a mixture can be separated on the basis of their sizes or molecular weights.
Its most common applications are in the separation of macromolecular protein complexes and polymers of industrial importance.
The resins used for gel filtration chromatography consist of spherical particles or beads which form a porous matrix upon the packing of the column. These beads are non-reactive and non-adsorptive. Since the pores formed by different resins are of a particular size, the molecules in a sample larger than the pore size of the column would be eluted first.
The molecules having sizes in accordance with the sizes of the pores (or smaller than them) would penetrate the pores and elute in a way that smallest of the molecules would come last since they would enter the pores completely (Figure 4).
Figure 4. The figurative representation of the principal of gel filtration chromatography. In red are the molecules of large size which do not enter the pores of the resin and thus are eluted early. While blue particles, which are smaller in size, become trapped inside the pores of the resin and this takes longer to elute than the larger molecules.
It is a type of column chromatography in which biomolecules are separated from a mixture based on their specific interactions with the resin. It utilizes in its principle the high specificity and interaction of two biological molecules.
The purification and separation of the desired compound from the mixture is achieved when the molecule of interest (called the ligand) interacts with its target attached on the surface of the stationary phase. This is then eluted with the help of a solvent which competes with it for binding sites on the stationary phase (Figure 5)
Figure 5. the principle of affinity chromatography.
The most commonly used application of the affinity chromatography is the separation of a protein from a mixture. This is usually done by using the column or resin containing the antibodies for that protein attached.
Therefore, when the protein solution will be passed through the column, only the protein of interest will be attached with its antibodies and the rest of them will be washed out. Finally, in the elusion step, this protein will be collected in fractions (as illustrated in Figure 5).
It is the most commonly used type of chromatography used in analytical chemistry and biochemistry for the detection and analysis of compounds in their vapor form (must not decompose upon vaporization).
The mobile phase consists of an inert carrier gas containing the sample to be analyzed and the stationary phase is a thin microscopic layer of a polymer or liquid attached on the inside of a column. It is also called vapor-phase chromatography or gas-liquid partition chromatography.
Figure 6. the diagrammatic representation of gas chromatography. The sample in the gaseous form is passed through the column and the components are then detected and recorded in the form of peaks. The data gathered is then further analyzed.
High-performance liquid chromatography (HPLC)
It is a separation technique extensively being utilized in analytical chemistry and biochemistry. Also known formerly as high-pressure liquid chromatography, it differs from typical liquid column chromatographic techniques in that it involves the application for high pressures (50-35- bars) for its operation.
The basic principles are quite similar to traditional column chromatography. The adsorbent is attached to the walls of the column through which the solvent containing the compound to be separated is passed with pressure. The attached compound can then be eluted with an appropriate elusion buffer.
HPLC strengthens a wide variety of fields by being applicable to them. For example, it is used extensively in the manufacturing industry for the production of biological and pharmaceutical products. Most of the recombinant proteins commercially available for research purposes are purified using HPLC. Besides, it is also broadly used in conventional and forensic research and other medical purposes.