Chromatography is an analytic technique that is based on the separation of molecules of a sample over two phases. These phases are called the stationary and the mobile phase. Based on the technique, efficiency, and applications they are diverse types.
Types Of Chromatography
- Column chromatography
- High-performance liquid chromatography
- Gas chromatography
- Paper chromatography
- Thin-layer chromatography
- Ion exchange chromatography
- Size exclusion chromatography
- Supercritical fluid chromatography
- High-performance thin-layer chromatography (HPTLC)
- Liquid chromatography-Mass spectroscopy
- Gas chromatography – Mass spectroscopy
- HPLC – atomic absorption spectroscopy
- Gas chromatography – atomic absorption spectroscopy
Initially, chromatography was developed as column chromatography. But its applications and efficiency were much limited.
To overcome this limitation chromatography was modified with the integration of advanced techniques for separation and detection of the sample components.
This lead to the advancement of multiple types of chromatography.
However, in this article, we will discuss all the instrumental methods and theoretical classifications. So read the article till the end.
Details on Different Types of chromatography
Based on Instrumentation and procedure we have 10 types as
It is the most widely used chromatography which finds daily use in research labs and industries. It is easy to operate and less expensive technique. As the name indicates, there is a long column which is suspended in the air with the help of a stand.
Into the column, the stationary phase is packed and the mobile phase is allowed to flow taking the sample through the solid column.
Similar to the picture shown below, you pour the mobile phase from top of the column to flow through the sample present on the stationary phase in the column to get separated.
High-performance liquid chromatography (HPLC)
This is a modification of column chromatography wherein a high pressure is employed for effective separation of compounds.
Additionally, the stationary phase used is made up of small size particles to increase the surface area for effective separation. So it is called as a high performance instead of high-pressure liquid chromatography.
The principle is similar to column chromatography. The components of a mixture in the mobile phase travel in the column with different speeds. This rate of travel depends on the relative affinity of the components towards the mobile phase and stationary phase.
The mobile phase is pumped into the column at a defined pressure along with the sample. The sample components coming out of the column are detected by the use of either UV-Visible spectroscopic detector or potentiometric or any other suitable detector.
The application of pressure reduces the time of the run in the column. For better separation, enhanced stationary phases and mobile phases are incorporated.
Due to this modification large molecules like proteins, fats, and small molecules like monoamines can be separated efficiently.
Those components with relatively similar chemistry like monoamines, steroids, etc. can be analyzed well by HPLC.
It is very versatile and is used in forensic, drug, food, clinical, environmental and biotechnological applications. See the HPLC principle and method for more details.
Gas chromatography (GC)
In this type, a gas is used as a mobile phase and a liquid layer coated on a solid support is used as the stationary phase. The volatile components of a mixture based on their relative affinities to the mobile phase are separated.
This technique is not widely used due to the limited number of stationary phases and also expensive instrumentation and operation procedures.
It is quite useful for the analysis of a few drugs. For details see the gas chromatography principle.
As the name indicates, here a specially designed filter paper is used as a stationary phase. The cellulose layers in the filter paper contain moisture which acts as a stationary phase. Buffers and other solvents are used as mobile phases.
The principle of separation is partition but even and adsorption method can also be performed.
Very wide range of samples can be analyzed by using paper chromatography
The sample dissolved in the mobile phase is marked on the Whatman’s paper with a capillary tube or micropipette. Then the paper placed in the solvent vessel to allow it to percolate through the paper.
In doing so, it separates the sample over the paper surface which is then subjected to color formation for identification.
Samples like the carbohydrates, proteins, amino acids, vitamins, drugs, glycosides, alkaloids, metabolites in blood, and urine can be analyzed.
Thin-layer chromatography (TLC)
This method was first used for the separation of plant extract on a 2mm layer of alumina loaded on a glass plate. The principle of separation is adsorption. One or more samples are spotted on to the adsorbent coated over a chromatographic plate.
This plate is partially dipped into a mobile phase as shown in the picture above. The solvent moves along the adsorbent. The sample components move along with the mobile phase on with different rates based on their affinity to the adsorbent. This leads to the separation of components which are detected with visualizing agents.
This thin layer chromatography similar to paper chromatography can be used to analyze any type of sample. But it is routinely used to analyze plant extracts, drugs, carbohydrates, proteins, antibiotics, vitamins, etc.
Here the mixture of charged ions is separated using an ion exchange resin. There occurs a reversible exchange of similar ions between those present in the mobile phase and the ion exchange resin. This exchange is based on the relative affinities. For a sample to be separated by this method, it should have charged particles as anions or cations. As seen in the equation below,
Resin-H+ + M+ (mobile solution) ————–> Resin-M+ + H+ (in mobile solution)
The mobile phase is charged and sample molecules with similar charges present on the charged stationary phase (resin) get exchanged.
These ions are displaced from resin along with oppositely charged ion in the mobile phase get eluted out of column first. Then a buffer solution is used to elute those molecules bound to the resin.
This ion-exchange chromatography is used for softening, deionization of water, purification of solutions, etc.
You can read more about ion-exchange chromatography.
Size exclusion chromatography
Here the column is loaded with charged some gel having pores. Sample particles when poured along with the mobile phase have to pass through the sieve-like network of the stationary phase. In doing so, the larger particles elute out first and smaller ones last. The reason is the smaller ones take a longer path in the column stationary phase while larger particles take a short path to elute out. Find more about gel permeation chromatography.
Supercritical fluid chromatography
It is a normal phase chromatography with instrumentation similar to HPLC. Here mobile phase is mostly supercritical fluid-like carbon dioxide.
High-performance thin-layer chromatography (HPTLC)
As the name indicates, this is similar to TLC but more efficient. Its efficiency is increased due to the use of small particles in the stationary phase, more choices of stationary phase material, auto-sampling technique, etc. Read more about HPTLC.
This liquid chromatography equipment (HPLC) which connected with Mass spectroscopy (MS) as a detector
Here Gas chromatography equipment column is connected with Mass spectroscopy as a detector.
HPLC instrument column is coupled with an atomic absorption spectroscopy instrument.
Gas chromatography AAS
If the chemical sample is volatile and has metal ions to be analyzed, the sample is separated with gas chromatography and passed into atomic absorption spectroscopy.
The types of chromatography mentioned are instrumental based. But theoretically, the classification can be done based on other facts.
Different types of chromatography based on
- The physical states of the stationary phase and mobile phases.
- Based on the principle of separation used.
- The chemical nature of the stationary phase and mobile phases used (polarity).
- Based on the shape of the stationary phase employed.
- Based on the purpose of the chromatography experiment.
- Based on the physical or chemical character of the stationary phase.
Based on Physical state of both phases
These are broadly classified as homogeneous or heterogeneous. The chromatography systems differ based on the physical states of the phases used.
Homogeneous techniques have both the stationary phase and the mobile phase as a liquid. Ex: Liquid-liquid chromatography.
Heterogeneous techniques employ different stationery and mobile phases. Ex: Solid-liquid chromatography, Solid-Gas chromatography, Liquid-Gas chromatography, etc.
Based on the principle of separation used
Here the principle used in separation is considered i.e adsorption method or partition method.
♠ Adsorption chromatography: Here the sample molecules get separated due to greater affinity to adsorb the solid stationary phase compared to that of the mobile phase. This principle works when the stationary phase is a solid and the mobile phase is a liquid solvent.
♠ Partition chromatography: Here the molecules of the sample get separated due to relative differences of dissolution and partition into different phases/layers. The molecules with greater partition or dissolution into the mobile phase are separated faster while that with a partition into solid-phase liquid moves slower or latter. Here both stationary phase and mobile phase are liquid in nature or liquid as the stationary phase and gas as mobile phase. The liquid on the stationary phase exists as a thin layer on a solid background.
Based on the chemical nature of the stationary phase and mobile phase:
This differentiation is based on chromatography column i.e. the nature of the stationary phase inside the column. The chromatography affinity for the sample is decided by both the stationary phase and mobile phase combination.
♠ Normal phase chromatography: Here the stationary phase is polar in nature and the mobile phase is in non-polar nature. Hence on elution, non-polar compounds are eluted first and polar compounds later as they have a greater affinity to the stationary phase. Mostly used in column chromatography technique. Read the details on Normal Phase Chromatography.
Ex: Normal phase column chromatography.
♠ Reverse-phase chromatography: This is reverse to the above method. The stationary phase is non-polar and the mobile phase is polar in nature. In practice, this reversed-phase chromatography is highly used in the routine analysis as most of the substances like drugs, etc. used in daily life are polar in nature.
Ex: Reverse phase HPLC systems.
Based on the shape of the stationary phase: The shape of stationary phases depends on the support used to place the stationary phase. Hence based on the shape of the stationary phase, there are two types like
Column chromatography and planar chromatography.
♠ Columnar chromatography is one where the stationary phase is a column shape. It is widely used in types like High-pressure liquid chromatography (also medium pressure liquid chromatography), Column chromatography, Gas chromatography, etc. The development of chromatograms occurs in the volume aspect.
♠ Planar chromatography is one wherein a stationary phase is flat. The development occurs on the planar surface (only area).
This type of chromatography is used in Thin-layer chromatography (TLC), High-Pressure thin layer chromatography (HPTLC), and Paper chromatography.
Based on the purpose of chromatography experiment: This is one of the types of chromatography Here the idea of experiment different. This can be done on both planar type and columnar type of chromatography. The types are
♠ Preparative chromatography: The amount of sample injected or applied is very large and the separated and pure component is collected for use. The desired component of the sample is not disposed of. This is also exclusively applied in column types as preparative column chromatography.
♠ Analytic chromatography: Here the sample size applied or injected is very small and the intention is aimed to identify the components in the sample and also their individual concentrations in the sample. The eluted sample from the outlet is disposed of.
Based on the physical or chemical character of the stationary phase:
This is especially followed in columnar chromatography wherein the stationary phase used has a specific character like being porous or charged.
♠ Size exclusion chromatography: Here the stationary phase has pores in its matrix. When the molecules pass through, those with large size travel a short path under mobile phase influence and pass out of column first and vice-versa.
♠ Ion exchange chromatography: Here the stationary phase has a definitely charged ions. When the sample is passed through, it retains all the molecules with the opposite charge and leaves off molecules with the same charge. So to elute the bound molecules, you need to pass another mobile phase with a similar charge to the stationary phase to recover the bound molecules. (like molecule displacement method).
So the above-mentioned types of chromatography are theoretically classified. But practically we have only 10 types of chromatography.