It is a modified version of high-performance liquid chromatography (HPLC) and is based on the phenomenon of attraction between opposite charges. In other words, it’s based on the theory that opposites attract!
The method combines both chromatographic and ion equilibrium theories into one technique in order to separate ionic species. It is commonly used in biomolecule purification, and there are several specialized modes used in laboratories today. Scientists and researchers rely on ion chromatography for its usefulness at all stages and levels of purification, it’s concentrating and controllable capabilities, as well as its high selectivity, capacity, and recovery.
Furthermore, it is able to look at major anions such as fluoride, chloride, nitrate, nitrite, and sulfates, as well as major cations such as lithium, sodium, ammonium, potassium, calcium, and magnesium. However, this chromatography method must be performed in an environment that is one unit away from the isoelectric point (pl) of a protein.
The principle of ion exchange chromatography is as follows:
“A crude sample containing charged molecules is used as the liquid phase. When it passes through the chromatographic column, molecules bind to oppositely charged sites in the stationary phase.The molecules separated on the basis of their charge are eluted using a solution of varying ionic strength. By passing such a solution through the column, highly selective separation of molecules according to their different charges takes place.”
The stationary phase is typically a polymeric resin. It’s selected so that it has a particular charge and attracts the sample molecules with the opposite charge. This phase is also termed as the ion exchanger. The ion exchanger can be two types, either a cation exchanger or an anion exchanger. Cation exchangers have a negative charge and attract positively charged cations, while anion exchangers are positively charged and attract negatively charged anions.
The mobile phase, or eluent, is a typically a solution—an inorganic salt dissolved in a suitable solvent—which introduces the ions that need to be separated into the system. Aqueous acid and base solutions, as well as organic, non-aqueous solutions can also be used as the mobile phase. The solution acts as the carrier portion of the mobile phase, and carries the analytes through the ion chromatograph.
There are a number of ion chromatography analyses performed in laboratories today, some of which include ammonia, cyanide, transition metals, and pharmaceuticals analysis. Yet, despite each method’s distinct uses, ion chromatography instrumentation is composed of several standard components and is similar to that of HPLC: a pumping system, chromatography columns, and a detecting system.
IEX, which is synonymous with ion chromatography, is also known as high-pressure ion chromatography (HPIC), and, as mentioned, is a form of liquid chromatography that divides ionic species so that their concentrations may be measured. To expand on our definition above, we will go over the basics of IEX in further detail.
The solution that the material is being held in is put through a chromatographic column and the ions are separated and absorbed. The sizes and types of electrovalent molecules will determine how they separate themselves.
Ions that are less attracted to the specific resin, or stationary phase used, will move faster through the column and be filtered out, or eluted, first while ions that are more attracted to the resin will move slower and be eluted later. Once they have left the column, the results are analyzed and the data can be plotted on a chromatographic readout. In ion-exchange chromatography, there are two chromatographic approaches: anion-exchange, and cation-exchange.
In ion-exchange chromatography, there are two chromatographic approaches, which require different columns: anion-exchange and cation-exchange. (Different ion exchange resins are used in IEX, depending on the material being separated.)
Anion Exchange (AIEC):
For anion exchange chromatography, an anion-exchange column and resin is used, both of which have positively charged groups like a diethylaminoethyl group, or DEAE, and are able to filter out more negatively charged molecules. This is done by coating the resin with positively charged counter-ions, which will attract the negatively charged ions in the solution.
In more chemically technical terms, this means that the pI is less than the pH for the chromatography. This technique is commonly used to purify negatively charged acidic substances at higher pH levels such as amino acid, proteins, sugars, and carbohydrates.
Cation Exchange (CIEC):
This technique, referred to as cation exchange chromatography, is used when the molecule that is to be observed is positively charged. This positive charge is due to the fact that the pI is more than the pH for chromatography.
Similar to AIEC, CIEC uses a cation-exchange columns and resin that have negative charges in order to attract positively charged ions. This technique is used to measure the concentration of positively charged cations like sodium potassium, calcium, and magnesium.
Ion-exclusion chromatography, or high-performance ion chromatography exclusion (HPICE), is a useful method to separate weak inorganic and organic acids. The method relies on either a cation or anion charged exchange resin to separate ionic solutes from weakly ionic or neutral solutes.
Unlike ion-exchange, in ion-exclusion, the ions that have a partial charge similar to the charged resin used are separated. This means if a cation exchange resin is used, partially negatively charged ions are divided. This is done by creating a Donna membrane that attracts smaller, similarly charged particles to them, meaning that they will elute later than larger charged particles. Because of the size-dependent factor of this method, it is also called size-exclusion chromatography.
The suppressed ion chromatography utilizes a suppressor to reduce background conductivity and enhance anion conductivity. It can be used with universal detectors to act as a desalting device, thereby removing the interference resulting from the presence of ionic salts in the eluent. A sodium carbonate aqueous solution is used during this method. The suppressor removes sodium ions in the eluent, changing the sodium carbonate to carbonic acid (which has lower conductivity). This reduces background conductivity levels.
In addition, the suppressor changes sodium ions (cations) to hydrogen ions with higher conductivity. This increases peak response and allows for a highly sensitive analysis of anions.
The non-suppressed ion chromatography method does not utilize a suppressor. Instead, it relies on a conductivity detector and chromatographic columns. Although simpler in design, this method produces higher background conductivity and lower sensitivity. Furthermore, the usable eluent is limited to low-conductivity solvents like phthalic acid.
The pump is an essential component of this instrument. It is used to provide a constant flow of eluent through the sample injector, column, and detector. The flow rate used during ion exchange chromatography is controlled by the pump, and is especially important.
There are a number of pump types used, some of which include: constant flow pumps, which include two types, reciprocating piston pumps or positive displacement pumps, and dual piston pumps, which reduce pulsation downstream of the pump.
Sample injection is possible a number of different ways, the simplest of which involves an injection valve. The valve has two positions: Load and Inject. While in the Load position, the injector can be filled with the exact sample volume required. This is done while the eluent passes past the injector into the column. When the injector valve is turned to Inject, the eluent will pass through the injector and carry the sample into the column.
More advanced instrumentation typically features an autosampler or microprocessor, where the sample is introduced into the column automatically.
In IEX, there are few different materials used for columns. In general, these materials include either stainless steel, titanium, glass, or an inert plastic. Capillary columns are becoming common as well, specifically in high-pressure ion chromatography. These are made of fused silica glass.
The column diameter can vary depending on whether it will be used for normal analytical purposes, high speed analyses, preparative work, or microanalysis.
Columns can either be used for anion exchange or cation exchange, a choice that depends on whether the material is anionic or cationic. When anion exchange columns are used, the packing is positively charged and therefore retains negatively charged molecules. When cation exchange columns are used, the media inside the column is negatively charged, binding positively charged molecules.
As mentioned, the suppressor reduces the background conductivity of the chemicals used to elute samples from the ion-exchange column. This improves conductivity measurement of the ions being tested.
In general, IEX suppressors are membrane-based devices that are designed to convert the ionic eluent to water in order to enhance the sensitivity. It can be used with universal detectors to act as a desalting device, removing the interference resulting from the presence of ionic salts in the eluent.
However, these devices are normally used with purely aqueous eluents. Because of this, establishing whether the device can be used with an aqueous or organic eluent is important.
After the sample ions have been separated, they need to be identified and quantified. This will be accomplished by using a detector, which will analyze the output at the end of the column. By doing so, each time analyte molecules emerge from the column, the detector will generate a measurable signal which prints out as a peak on the chromatogram.
The most commonly used device is the electrical conductivity detector. Another commonly used device includes the electrochemical detector, which is flow- and volume-optimized for high-performance amperometric detection in both capillary and analytical ion chromatography, and provide low backgrounds.
Other detectors available include photodiode array detectors, multiple wavelength UV/Vis detectors, and mass spectrometers.
The most important marker that we look for in the search for life beyond our planet is the presence of water. Carbon-based life cannot exist without it, so the detection of water on other planets or celestial bodies is the first step towards determining if life is present there.
NASA has been developing a series of instruments and tests to perform in space to determine if water is present on a planet or not. Ion chromatography is one of these important tests. They are working to develop an ion chromatography chip that will be both lightweight and effective for water analysis and desalting.
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