Chromatography is the process of breaking down the mixture of compounds into the components that make up it dependent on their interaction to an inert matrix. The process of ion exchange chromatography (or Ion Chromatography) is a method that permits the separation of polar molecules and ions in accordance with their affinity to the ion exchangers. The idea behind separation is therefore based on the reversible exchange of ions from the targets ions within the samples solution and the ions that are present on ion exchangers. In this procedure, two kinds of exchangers i.e. the cationic as well as anionic exchangers are available.
- Cationic exchangers are positively charged groups and they draw positively charged Cations that are positively charged. They are also referred to as “Acidic ion exchange” materials due to their negative charges. are the result of the ionization process of acidic groups.
- Anionic exchangers possess positively charged groups that draw negatively charged ions. These are also known as “Basic ion exchange” materials.
Ion exchange chromatography can be commonly performed as a method that of column chromatography. But, there are thin-layer chromatographic methods which are in a fundamental way based on the concept of Ion exchange.
Working Principle of ion exchange chromatography
This type of chromatography is based on the interaction between an oppositely charged stationary phases, referred to as an ion exchanger and analyte. The ion exchangers generally contain charged groups that are covalently connected onto the top of the matrix. The charged elements of the matrix may be charged either negatively or positively. If suspended in an aqueous solution the charged elements that make up the matrix can be covered by ions with opposite charge. This “ion cloud”, ions are able to be exchanged in a reverse manner without altering the structure and nature of the structure that the matrix.
Instrumentation of ion exchange chromatography
This type of chromatography is based on the interaction between an oppositely charged stationary phases, referred to as an ion exchanger and analyte. The ion exchangers generally contain charged groups that are covalently connected onto the top of the matrix. The charged elements of the matrix may be charged either negatively or positively. If suspended in an aqueous solution the charged elements that make up the matrix can be covered by ions with opposite charge. This “ion cloud”, ions are able to be exchanged in a reverse manner without altering the structure and nature of the structure that the matrix.
Ion exchange chromatography instrumentation
A typical IC instrumentation comprises pump and injector, column detector, suppressor and recorder, also known as a data system.
1. Pump
It is the IC pump is believed to one of the most vital components of the system. It is required to ensure a uninterrupted flow of fluid through the IC column, injector and detector.
2. Injector
The introduction of samples can be done using a variety of ways. The most straightforward technique is to utilize injectors. Liquid samples can be injected straight and solid samples should be dissolved in a suitable solvent. Injectors should offer the possibility of injecting liquid samples within the limit between 0.1 to 100 ml volume that is reproducible and at extreme pressure (up to four thousand psi).
3. Columns
Depending on the end-user and the area of use The column’s materials could be titanium, stainless steel glass, or an inert plastic like PEEK. The column could range in size from 2 millimeters to 5 centimeters and in length ranging from 3 cm to 50 centimeters, based on whether it’s to be used for standard analysis microanalysis, high-speed analysis or even for preparation work.
The Guard column is located prior to the column which is separating. This is a safeguarding feature that increases the lifespan and efficiency of the column that separates. These are solid columns made to remove or filter out particles that block the separation column.
4. Suppressor
The suppressor lowers the background conductivity of chemicals used to extract samples from the ion exchange column. This improves the measurement of conductivity of the samples being examined. IC suppressors comprise membranes that are specifically designed for conversion of the liquid into water in the hopes to increase the sensitiveness.
5. Detectors
The electrical conductivity detector is in common employed.
6. Data system
In the case of routine analysis, in which there is no need for automation A pre-programmed computing integrator could be enough. To achieve higher levels of control an intelligent device is required for example, minicomputers or data stations.
Procedure of ion exchange chromatography
- Separations of ions are typically carried out in columns containing an ion exchanger.
- Ionic exchangers are readily accessible. They are composed of divinylbenzene and styrene. Example. DeAE-cellulose can be described as an anionic exchanging. CMC-cellulose is a cationic switcher.
- The choice of exchanger is based on the charge of the particle that needs which is being separated. To separate anion “Anionic exchanger” is used for separating cations “Cationic exchanger” is used.
- The column is first filled with an ion exchanger, Then the sample is placed to the buffer. The tris-buffer and pyridine buffers and acetate buffers, as well as citrate as well as phosphate buffers can be all widely utilized.
- The particles that have an affinity that is high for an ion exchanger will be deposited in the column, along with buffers.
- The next step is to use the corresponding buffers, the particles that are tightly bound.
- Then, these particles are examined using spectroscopic analysis.
Resin Selection in Ion Exchange Chromatography
Ion exchange resins possess either negatively or positively charged functional groups that are connected to a solid matrix. Matrices are generally made from polystyrene, cellulose, Polyacrylamide, and agarose. A few of the elements that affect the selection of a resin include anion or cation exchangers, flow rate, strength or weak Ion Exchanger, the dimension of the material and the binding capacity. Stability of the protein in question determines whether to use either an anion or a cation exchanger. Either can be employed if stability is not an issue.
Applications of ion exchange chromatography
- A major use for ion exchange Chromatography is for regular analysis of mixtures of amino acids.
- The 20 amino acids that are the principal ones in blood serum or the hydrolysis process of proteins are identified and utilized to make clinical diagnoses.
- This is the most effective method of purifying water. Deionization complete of the water (or) an electrolyte-free solution is accomplished by exchanging solute cations with hydrogen ions and solute anions with hydroxyl ions. This typically happens using process used to soften the PH the drinking water.
- Analyzing the substances from the hydrolysis of nucleic acid. This way, information can be gathered regarding the structure of these molecules and how they relate to their biological functions as carriers of information from hereditary sources.
- Chelating resins can be used to extract small amounts of trace metals in the sea.
- To examine lunar rocks and trace elements that are found on Earth.
Advantages of ion exchange chromatography
- It is among the most effective methods to separate charged particles.
- It is suitable to treat almost any charged molecule such as large proteins smaller nucleotides as well as amino acids.
- Ion exchange can be used in both analytical and preparative purpose in the laboratory The analytical applications being the most common.
- Inorganic ions are also separated with an ion-exchange chromatography
Limitations of ion exchange chromatography
- Only charged molecules can be separated.
- Buffer Requirement
