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What is column chromatography?

The column chromatography is a technique that allows separation of the components of a mixture of substances, in order to achieve the purification of a substance for identification, characterization or use.

Column chromatography has two phases: a stationary phase and a mobile phase. The stationary phase can be in a solid or liquid phase and the substances to be separated interact with it, prior to or simultaneously with the action of the mobile phase.

The degradation of the colors indicates that the substances elute in different ways due to their variable affinities for the stationary phase: the bluish substance, being higher up, is the one that suffers the strongest retention. Source: Максим Фомич, CC BY 2.0 <https://creativecommons.org/licenses/by/2.0>, via Wikimedia Commons

The mobile phase, on the other hand, moves during the chromatography and allows the separation of the components of the mixture. The mobile phase can be in a liquid or gaseous state , and interacts with substances in chromatography by similarity in their polarities.

Column chromatography is a versatile technique that has numerous applications in various industries, as well as in medicine. Therefore, chromatographic techniques have constantly evolved: such is the case of so-called high-performance liquid chromatography (HPLC).

The HPLC allows to analyze, in a very short time, numerous substances, since all the stages of the process are automated. For example, it is very useful in endless drug quality testing, where many samples must be tested on a daily basis.

We also have size exclusion chromatography, which, as its name indicates, is based on the size of the molecules and not so much on their polarities. This technique is used when it is required to separate mixtures of pigments, resins, asphaltenes, biomolecules , etc., which do not differ too much in their chemical nature.

Types of column chromatography

There are several types of chromatography that are carried out on a column, glass structure or other material, generally in the form of straight tubes. Among the types of column chromatography are the following:

  • Absorption or column.
  • Liquid-liquid partition.
  • Ion exchange.
  • Exclusion by size.
  • Gas.
  • High Resolution Liquid (HPLC).
  • Affinity.

Fundamentals and materials

Column chromatography in laboratory

Adsorption chromatography or column chromatography

This chromatography has the same principles as thin layer chromatography, based on the use of a solid stationary phase of a polar nature (alumina or silica gel), and a mobile phase made up of a non-polar solvent in liquid state.

The polar substances present in a mixture of substances, interact with the polar stationary phase and are adsorbed by it. Furthermore, they have low affinity for the non-polar solvent of the mobile phase.

Liquid-liquid partition chromatography

This term is used to refer to a type of chromatography in which the stationary phase and the mobile phase are in a liquid state. The stationary phase is a polar liquid that permeates a solid support, usually inert silica. And the mobile phase corresponds to a non-polar liquid.

This arrangement of the phases in partition chromatography is called normal phase, whereas if the non-polar liquid permeates the silica, constituting the stationary phase, then it will be called reverse phase partition chromatography.

Ion exchange chromatography

In this type of chromatography the stationary phase is electrically charged. When its charge is positive, it retains the anions (-); and if the charge is negative, to the cations (+).

Amines, sulfonic acid, diatomaceous earth, cellulose and sephadex (obtained from dextran) are often used as the stationary phase. A positive charge is added to these latter materials, for example, diethylaminoethyl (DEAE) to obtain DEAE-cellulose or DEAE-sephadex, to thus interact with the anions.

A negative charge can also be added to them through the union of the carboxymethyl group (CM), thus forming CM-cellulose or CM-sephadex, which function as cation exchangers.

The existing electrostatic interactions in this type of chromatography can be regulated by means of modifications of the pH and the ionic strength of the liquid that forms the mobile phase.

At neutral or basic pH, proteins are usually negatively charged and interact with the positive charge of DEAE-cellulose and DEAE-sephadex; but by decreasing the pH of the mobile phase, the proteins can become positive and stop interacting with the positive charge of the stationary phase.

Using this experimental strategy, the different proteins present in a mixture can be separated.

Also, inorganic compounds such as aluminosilicates (zeolites) are used in ion exchange chromatography .

Size Exclusion Chromatography

This type of chromatography is based on the existence of particles that have channels inside them, which allow the circulation of a substance of small size and low molecular weight through them. Meanwhile, larger substances cannot cross the channels and are excluded from them.

Although it seems strange, substances of greater size move through the mobile phase more easily than those of smaller size, since they are not retained in the channels of the particles used in chromatography. Among these particles are zeolite and sephadex.

The so-called sephadex was created by the Pharmacia company from the polysaccharide dextran. There are many types of sephadex whose use is selected based on the size or molecular weight of the substances to be separated.

Gas chromatography

In this chromatography, the stationary phase covers the wall of the columns or fills their interior, while the mobile phase is made up of an inert gas: nitrogen or helium. The chromatography columns are made of glass or metal; although at the moment they have narrow forms of capillaries, whose walls are lined with silica.

The chromatography columns have a length between 1 meter and 100 meters, the columns being inside an oven capable of supplying temperatures higher than 300 ºC. Substances used in chromatography must be volatile, as they must evaporate during chromatography.

Substances evaporate from the surface of the stationary phase according to their boiling point . The evaporation of the substances occurs as a function of the increase in the temperature of the furnace, which allows the substances to reach their boiling point sequentially.

Once they have reached their boiling point, the substances evaporate and are carried by the inert gas stream to the detection and analysis system.

The gas chromatography technique is basically analytical and not preparative. That is, it is not usually used to obtain a certain substance.

High Performance Chromatography (HPLC)

The fundamentals of HPLC are similar to those of so-called column or adsorption chromatography, but the difference is that the solvent (mobile phase) passes through the stationary phase, driven by a pressure of around 400 atmospheres.

The HPLC columns have a length between 15 and 25 cm, and an internal diameter of 0.46 cm. They are usually made of stainless steel. The stationary phase is made up of very small silica particles that have a polar characteristic.

Normal phase

In the normal phase of HPLC, a non-polar solvent is usually used as the mobile phase, usually hexane. Polar substances interact with silica and emerge late from chromatography columns. Meanwhile, nonpolar substances have a higher affinity for nonpolar solvents, they do not interact with silica particles and therefore emerge rapidly from the columns.

Reverse phase

In the so-called reverse phase, the polar silica particles are rendered nonpolar by the addition of a hydrocarbon chain of 8 to 18 carbons. In the mobile phase, a polar solvent made up of a mixture of methanol and water is used.

The polar substances do not interact with the modified (non-polar) silica particles but they do interact with the polar solvent, allowing them to quickly exit the chromatography column, being taken to a detection system in the presence of ultraviolet light.

Affinity chromatography

This chromatography is based on the interaction of a particular substance with a ligand for it, fixed to a support that can be agarose, dextran, cellulose, etc. Affinity chromatography is a technique used for the separation and purification of molecules with a biological function.

The affinity chromatography technique allows the purification of a protein, using a specific antibody fixed to a support, which constitutes the stationary phase. Copper, cobalt and nickel are used as ligands to obtain proteins that contain the amino acid histidine.

This technique is also used for the purification of DNA and RNA, using as a ligand a nucleic acid with a complementary base sequence. The substance bound to its ligand can be separated by modifying the pH, or the ionic strength of the solution that is used as the mobile phase.

Applications

Column chromatography is a technique used for the separation of a substance from a mixture of them, with different purposes or objectives. For example: diagnosing a problem, identifying drugs, obtaining a substance, etc.

Adsorption chromatography is used in the removal of impurities from substances, in the isolation of metabolites from biological fluids, in the determination of harmful organic acids in food, etc.

High-performance liquid chromatography is a technique that has numerous uses, including: in the detection of antibiotics, sedatives, steroids, or others of pharmacological interest; in the identification of pollutants such as pesticides, herbicides, phenols, etc.

Gas chromatography is used in the study of many volatile substances. It is used in the pharmaceutical industry in the analysis of drugs, such as aspirin, ibuprofen, and paracetamol. In addition to the identification in the urine of doping agents, such as amphetamines, cocaine, LSD, cannabis, etc.

Size exclusion chromatography and affinity chromatography are mainly used in the isolation and purification of biological macromolecules, such as proteins and nucleic acids.

And finally, ion exchange chromatography is used in the purification of proteins and polypeptides.

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