The ion exchange chromatography is an analytical technique that relies on the principles of chromatography to produce the separation of ionic and molecular species that exhibit polarity. This is based on the premise of how related these substances are in relation to another called ion exchanger..
In this sense, substances that have an electric charge are secreted thanks to ionic displacement, in which one or more ionic species are transferred from a fluid to a solid through exchange, due to the fact of having equal charges..
These ionic species bind to functional groups located on the surface through electrostatic interactions that facilitate ion exchange. Furthermore, the effectiveness of ion separation depends on the rapidity of matter exchange and the equilibrium between both phases; that is, it is based on this transfer.
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Before starting the ion exchange chromatography process, certain important factors must be taken into account, which allow optimizing the separation and obtaining better results..
These elements include the amount of analyte, the molar mass or molecular weight of the sample, and the charge of the species that make up the analyte..
These factors are essential to determine chromatography parameters, such as stationary phase, column size and matrix pore dimensions, among others..
There are two types of ion exchange chromatography: one that involves cation displacement and one that involves anion displacement..
In the first, the mobile phase (which constitutes the sample to be separated) has ions with a positive charge, while the stationary phase has ions with a negative charge..
In this case, the positively charged species are attracted to the stationary phase depending on their ionic strength and this is reflected in the retention time shown in the chromatogram..
Similarly, in chromatography involving anion shift, the mobile phase has negatively charged ions, while the stationary phase has positively charged ions..
In other words, when the stationary phase has a positive charge it is used in the separation of the anionic species, and when this phase is anionic in nature it is used in the segregation of the cationic species present in the sample..
In the case of compounds that present an electrical charge and exhibit solubility in water (such as amino acids, small nucleotides, peptides and large proteins), these combine with fragments that present the opposite charge, producing ionic bonds with the phase. stationary that is not soluble.
When the stationary phase is in equilibrium, there is a functional group that is susceptible to ionization, in which the substances of interest in the sample are segregated and quantified, being able to combine while moving along the column. chromatographic.
Subsequently, the species that have been combined can be eluted and then collected using an eluting substance. This substance is made up of cationic and anionic elements, giving rise to a higher concentration of ions throughout the column or modifying its pH characteristics..
In summary, first a species capable of exchanging ions is surface charged in a positive way with counterions, and then the combination of the ions that will be secreted takes place. When the elution process starts, the weakly bound ionic species are desorbed..
After this, the ionic species with stronger bonds also become desorbed. Finally, regeneration occurs, in which it is possible that the initial state is reconstituted by washing the column with the buffered species that initially intervenes..
Ion exchange chromatography is based on the fact that the species that manifest an electrical charge present in the analyte are segregated thanks to the electrostatic-type attractive forces, when they move through an ionic-type resinous substance in specific temperature and pH conditions.
This segregation is caused by the reversible exchange of ionic species between the ions found in the solution and those found in the displacement resinous substance that has ionic nature..
In this way, the process used for the segregation of compounds in the sample is subject to the type of resin used, following the principle of anion and cation exchangers described above..
Since the ions of interest are trapped in the resinous substance, it is possible for the chromatographic column to flow until the rest of the ionic species is eluted..
Subsequently, the ionic species that are trapped in the resin are allowed to flow, while they are transferred by a mobile phase with greater reactivity along the column..
As in this type of chromatography the separation of substances is carried out due to ionic exchange, it has a large number of uses and applications, among which are the following:
- Separation and purification of samples that contain combinations of compounds of an organic nature, consisting of substances such as nucleotides, carbohydrates and proteins.
- Quality control in water treatment and in deionization and solution softening processes (used in the textile industry), as well as the segregation of magnesium and calcium.
- Separation and purification of drugs, enzymes, metabolites present in blood and urine, and other substances with alkaline or acid behavior, in the pharmaceutical industry.
- Demineralization of solutions and substances, where it is desired to obtain high purity compounds.
- Isolation of a specific compound in a sample to be separated, in order to obtain a preparatory separation of it to later be the object of other analyzes.
Likewise, this analytical method is widely used in the petrochemical, hydrometallurgical, pharmaceutical, textile, food and beverage, and semiconductor industries, among other areas..
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