Chromatography:Chromatography is an analytic technique developed in 1940s to 50s.
Its procedure relies on the stationary phase and mobile phase which separate molecules under test.
In early stages its applications and efficiency was much limited and the system was rudimentary.
We assume you know what is chromatography, If not see “What is Chromatography? definition, principle & uses“.
In the initial stages, the time for analysis was too long like few days or hours. Also the range of compounds that could be detected was minimum.
Therefore the techniques of chromatography were modified and developed to meet the demands of modern analytical chemistry.
For better separation, enhanced stationary phases and mobile phases were incorporated. Due to this modification large molecules like proteins, fats and small molecules like monoamines can be separated efficiently.
Technologies like spectroscopy and electrochemical methods were added to enhance detection of separated components.
To reduce the time of run, pressure was applied by use of pumps.
With the latest developments, there is an improved efficiency in the separation and also the quantification.
Further the range and type of substances which could be analyzed has been greatly increased.
Different Types of chromatography:
The differentiation is based on techniques of chromatography or principle or physical changes used
- The physical states of stationary phase and mobile phases.
- The on principle of separation used.
- The chemical nature of stationary phase and mobile phases used (polarity).
- Based on the shape of stationary phase employed.
- Based on purpose of chromatography experiment.
- Based on physical or chemical character of the stationary phase.
♠ Homogeneous techniques have both stationary phase and mobile phase as liquid. Ex: Liquid-liquid chromatography.
♠ Heterogeneous techniques employ different stationary and mobile phases. Ex: Solid-liquid chromatography, Solid-Gas chromatography, Liquid-Gas chromatography etc.
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 mobile phase. This principle works when stationary phase is a solid and mobile phase is liquid solvent.
♠ Partition chromatography: Here the samples molecules get separated due to relative differences of dissolution and partition into different phases/layers. The molecules with greater partition or dissolution into mobile phase is separated faster while that with partition into solid phase liquid moves slower or latter. Here both stationary phase and mobile phase are liquid in nature or liquid as stationary phase and gas as mobile phase. The liquid on stationary phase exists as a thin layer on a solid background.
3. Based on the chemical nature of stationary phase and mobile phase:
This differentiation is based on chromatography column i.e. the nature of stationary phase inside the column. The chromatography affinity for the sample is decided by both stationary phase and mobile phase combination.
♠ Normal phase chromatography: Here the stationary phase is polar in nature and mobile phase is in non-polar nature. Hence on elution, non-polar compounds are eluted first and polar compounds later as they have greater affinity to stationary phase. Mostly used in column chromatography technique
Ex: Normal phase column chromatography.
♠ Reverse phase chromatography: This is reverse to the above method. The stationary phase is non-polar and mobile phase is polar in nature. In practice this reversed phase chromatography is highly used in routine analysis as most of the substances like drugs etc. used in daily life are polar in nature.
Ex: Reverse phase HPLC systems.
4. Based on the shape of stationary phase: The shape of stationary phases depends on the support used to place the stationary phase. Hence based on the shape of stationary phase, there are two types like
Column chromatography and planar chromatography.
♠ Columnar chromatography is one where stationary phase is column shape. It is widely used in types like High pressure liquid chromatography (also medium pressure liquid chromatography), Column chromatography, Gas chromatography etc. Development of chromatogram occurs in volume aspect.
♠ Planar chromatography is one wherein a stationary phase is flat. The development occurs on the planar surface (only area).
5. Based on 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 sample is not disposed off. 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 off.
6. Based on physical or chemical character of the stationary phase:
This is especially followed in columnar chromatography where in the stationary phase used has specific character like being porous or charged.
♠ Size exclusion chromatography: Here the stationary phase has pores in its matrix. When molecules are allowed to pass through, those with large size travel a short path under mobile phase influence and pass out of column first and vice-verse.
♠ Ion exchange chromatography: Here the stationary phase has a definite charged ions. When the sample is passed through, it retains all the molecules with opposite charge and leaves off molecules with same charge. So to elute the bound molecules, you need to pass another mobile phase with similar charge to 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 11 types of chromatography
So over all the available chromatography techniques for regular analysis include.
- Column chormatography: It is similar to the pic shown above where you pour mobile phase from top of the column to flow through the sample present on stationary phase in the column to get separated.
- High performance liquid chromatography (HPLC): Here mobile phase is pumped into the column at a defined pressure and further the column particles are very small so the surface area is high and better separation takes place. See HPLC applications
- Gas chromatography (GC): Here gas is used as mobile phase. For details see gas chromatography principle.
- Ion-exchange chromatography: Here the mobile phase is charged and sample molecules with similar charge present on the charged stationary phase get eluted out as the mobile phase molecules with charge displace them.
- Size exclusion chromatography. Here the column is loaded with charged some gel having pores. Sample particles when poured along with mobile phase has to pass through the sieve like net work of the stationary phase. In doing so, the larger particles elute out first and smaller ones last. The reason is the smaller ones take longer path in the column stationary phase while larger particles take short path to elute out.
- Thin layer chromatography (TLC): Here the stationary phase is a thin layer and plate like.
- High performance thin layer chromatography (HTLC): Similar to TLC but more efficient.
- Paper chromatography.Here column is paper either rectangular or circular.
- Affinity chromatography.
- LC-MS: Liquid chromatography combine with Mass spectroscopy (detector)
- GC-MS: Gas chromatography combined with Mass spectroscopy (detector)
- Ultra high performance chromatography
The chromatography is still under improvement with regards to one or other part of it. But the basic concept remains same in all.