Column chromatography exists in two ways under the field of science in which it is being utilized, Chemistry and Biology. When applied in chemistry, it separates one or sometimes more chemical compounds from a mixture dissolved in an appropriate solvent.
Laboratory experts most often employ this technique in biology to separate a biological compound such as nucleic acids (DNA or RNA) or Proteins from a mixture (also dissolved in a suitable solvent).
Since a column is used in this separation technique, hence named column chromatography. This column comes 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, it passes through the stationary phase, allowing the adsorbent to capture the separated compound. After this, the compound attached to the stationary phase elutes using elution solvents.
Figure 1. a regular column used in chromatographic separations and their different parts.
What are the Different Types of Column Chromatography?
This procedure helps in carrying 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 these different types of column chromatography with understandable examples where possible.
Adsorption Chromatography
It is a type of liquid chromatography which retains the compounds from the mixture, adsorbed on the surface of the stationary phase.
Usually, the forces involved in binding the compound with the solid support are Van Der Waals forces and steric interactions. Besides, adsorption chromatography refers to as solid-liquid chromatography. This 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 associated with its very first example. The carotenoid and chlorophyll a and b pigments were formerly separated using calcium carbonate, sucrose, and alumina as a stationary phase, with petroleum and ether/ethanol mixtures as eluents.
Ion Exchange Chromatography
Ion Exchange Chromatography
Ion chromatography or ion-exchange chromatography is another type of column chromatography that mostly separates polar and ionic compounds. The basic principle lies in the molecules’ charge that separates by attaching to the oppositely charged stationary phase.
Then finally, in the elution 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)
For example, let us look at an anionic resin quaternary amine (-N+(CH3)3). It is a strong anion exchanger with a commercial name of Q resin. As it is an anion exchanger (having a positive charge on itself), it helps purify various negatively charged compounds.
Gel Filtration Chromatography
Gel filtration chromatography is also known as size exclusion chromatography and molecular sieve chromatography. It is a separation technique, where molecules in a mixture separate based on their sizes or molecular weights. Its most common applications are in the separation of macromolecular complexes, such as proteins, and polymers of industrial importance.
The resins used for gel filtration chromatography consist of spherical particles or beads that form a porous matrix upon the column’s packing. These beads are non-reactive and non-adsorptive. The molecules larger than the pore size of the column elutes first since these are of different resins and particular sizes.
The molecules having sizes following the magnitudes of the pores (or smaller than them) would penetrate them. Furthermore, they will elute so that the smallest of the molecules would come last since they are completely entering the pores (Figure 4).
Figure 4. The symbolic representation of the principle of gel filtration chromatography. In red are the molecules of large size which do not enter the resin’s pores and thus elute early. While blue particles, smaller in size, trap inside the resin’s pores and take longer to elute than the larger molecules.
Affinity Chromatography
It is a type of column chromatography where biomolecules disintegrate 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 accomplishes when the molecule of interest (called the ligand) interacts with its target attached to the surface of the stationary phase. It 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 affinity chromatography is the separation of a protein from a mixture. It is usually processed by using the column or resin containing the antibodies for that protein attached. Therefore, when the protein solution passes through the column, only the protein of interest attaches with its antibodies, and the rest washes out. However, affinity chromatography has some disadvantages too.
Gas Chromatography
It is another type of chromatography that helps to detect and analyze compounds in their vapor form.
The mobile phase consists of an inert carrier gas containing the sample of examination. The stationary phase is a thin microscopic layer of a polymer or liquid attached to the inside of a column. Experts usually term it vapor-phase chromatography or gas-liquid partition chromatography.
Figure 6. the diagrammatic representation of gas chromatography. The sample in the gaseous form passes through the column, where the components detect and further record in the form of peaks. The data gathered is then analyzed.
High-Performance Liquid Chromatography (HPLC)
It is also a separation technique extensively utilized in analytical chemistry and biochemistry. Also known formerly as high-pressure liquid chromatography, it differs from regular 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 initially attaches to the column walls through which the solvent containing the compound passes with pressure. The attached compound further elutes with an appropriate elution buffer.
HPLC strengthens a wide variety of fields by applying them. For example, it is broadly used in the manufacturing industry to produce biological and pharmaceutical products. Most of the recombinant proteins commercially available for research purposes are purified using HPLC. Besides, it also helps in conventional and forensic research and other medical purposes.
Column Chromatography – A Versatile Approach
We cannot deny how column chromatography is the best way to purify and separate different phases of matter. It actively plays a role in isolating major constituents and helps disintegrate complex mixtures. Moreover, it also assists in the estimation of drugs in drugs formulation, etc.
Jeannie has achieved her Master’s degree in science and technology and is further pursuing a Ph.D. She desires to provide you the validated knowledge about science, technology, and the environment through writing articles.
