In water analysis, quality control, and protein purification, ion exchange chromatography is usually used. Ion exchange chromatography is defined as the process of separating polar molecules and ions based on charge. This process may be used for any charged molecule like amino acids, large proteins, and small nucleotides.
There are different methods used in chromatography, and the term ‘ion exchange chromatography’ is applicable to the separation of analytes’ components for analytical applications.
Ion Exchange Chromatography – The Principle
The analyte molecules are retained in their ionic or coulumbic interactions. When it comes to purification of charged molecules and proteins, the ion exchange chromatography is the most common.
In ion exchange chromatography, the conductivity solution is low to medium (e.g. salt concentration). Ionic interaction drives molecule absorption between ionic groups that are oppositely charged. It is the location and number of charges on the functional group and molecules that determines the interaction’s strength.
The buffer’s pH should be between the charged group’s pKa on solid support and the charged molecules pKa or pl. For instance, if the pKa is 1.2 for the functional group, and pl of 8.2 for sample molecule, the buffer during the mobile phase can be pH 8.0 – this is a good example for cation exchange chromatography.
Protein samples are usually used in different ion exchange chromatography methods wherein it is injected into the column, provided that the condition will ensure the former’s strong retention. A step or linear gradient is used to elute sample components. By using the step gradient, less complex equipment can be used and it is also very effective in eluting different fractions when salt concentrations are known.
In ion exchange chromatography, pH changes are used in order to affect separation. When the mobile phase buffer’s pH is raised in ion exchange chromatography, the molecules less protonated and positively charged. Because of this, the protein can’t interact with the solid support which is negatively charged, and the molecules elute. When the pH is lowered in (anion) ion exchange chromatography, the molecules become more positively charged or protonated. Ionic interactions between the solid support and proteins are no longer possible.
Ion Exchange Chromatography – Important Techniques
In ion exchange chromatography, the analysis can determine the analytes presence or concentration in the sample. Separating mixtures is usually required in laboratories, and this is made possible through ion exchange chromatography. A set of techniques and lab methods are used to achieve this.
In the mobile phase, a mixture is dissolved and it is passed through the stationary phase. After this, the analytes separate from other mixture components. Once isolated, it is easier to measure the analytes. Such technique is analytical and preparatory in nature. In preparatory ion exchange chromatography, a mixture’s components are separated for analysis, purification applications, and cleansing.
In analytical ion exchange chromatography, a smaller amount is required to measure analytes in relative proportions. For instance, a chemical substance is poured into a glass tube that contains adsorbent material. You will notice color bands while the substance components are absorbed into the material.
There are several categories for the analysis techniques in ion exchange chromatography, and it includes the following – gas chromatography, liquid chromatography, and affinity ion exchange chromatography.
The said categories above determine the mobile phase. For instance, in gas
exchange chromatography, gas is the mobile phase while for liquid exchange chromatography, it is liquid.
There are still other separation mechanisms, and the ion exchange chromatography is one. Here, analytes are separated through ion exchange. Columns are usually required to perform ion exchange chromatography, but some use the planar mode. The charged stationary phase is used to split charged compounds like proteins, peptides, and amino acids.
The typical technique for the ion exchange chromatography is as follows –
Aan autosampler introduces the sample, or it can also be done manually, into the sample loop. The column houses the material in stationary phase and the aqueous solution (mobile phase) carries the sample to it. The material in the column is in gel matrix or resin form, consisting of cellulose or agarose beads with charged functional groups that bonded covalently. The stationary phase retains the analytes (cation or anion), though it can be eluted. To displace analyte ions, all you have to do is increase the concentration of species with similar charges.
In cation chromatography, you can add sodium ions that are positively charged to displace positively charged analytes. You can detect the analytes that you are going to examine/analyze through visible light absorbence, UV, or conductivity. The CDS or chromatography data system is required to control the IC system. The CDS can also control HPLC and gas chromatography.
The proteins are separated in ion exchange chromatography depending on the net charge. The latter is dependent on the mobile phase’s composition. It is a known fact that proteins can have positive and negative functional groups. You can separate protein molecules by adjusting the mobile phase’s ionic concentration or pH.
The ion exchange chromatography is also used in the measurement of water purification, porphyrin, and HbA1c. It isn’t hard to separate the various elements of a substance through chromatography. The naked eye can’t see the specific compounds, but experts can easily decipher odorless and colorless substances.
There are many situations where chromatography can be helpful. Just imagine how much time you can save if you are able to analyze the compounds that make up a certain material. You will have more time for other important matters of whatever it is that you are investigating. This can be very beneficial in forensics. The evidence gathered can be examined and analyzed quickly, thereby allowing the investigator to focus on other important tasks.
As you can see, ion exchange chromatography is not that complicated. Regardless of the field you are in, you may find this process very useful. The major types of chromatography are gas, liquid, paper, and thin layer chromatography. Each of these types has its own merits. For instance, paper chromatography is used in RNA fingerprinting while forensics focus more on the thin layer chromatography. Despite the modern advancements, ion exchange chromatography is still being used widely in different situations and industries.