Chromatography: Principle, Types, and Techniques
Chromatography is a non-destructive procedure for resolving a complex mixture into its individual fractions or compounds.
- It is a separation procedure, and the separated entities are identified by other analytical techniques like UV-visible, Infrared, NMR (nuclear magnetic resonance), Mass spectroscopy, and so forth.
- For a quantitative analysis, measurements of the area under the curve in the chromatogram are taken.
- Its name is derived from two words: "chromo" meaning colour, and "graphy" meaning writing. In other words, colour bands are formed in the procedure, which are then measured or analysed. These bands are due to separation of individual compounds at different lengths on the column, as seen in column chromatography and on paper in paper chromatography.
- However, in modern methods like HPLC or gas chromatograph, colour bands can't be seen.
The basic principle of chromatography has advanced a lot to cater to the growing needs of the industry and for research purposes.
Definition and Principle
It is defined as the process of separation of the individual components of a mixture based on their relative affinities towards stationary and mobile phases.
Principle: The samples are subjected to flow by mobile liquid onto or through the stable stationary phase. The sample components are separated into fractions based on their relative affinity towards the two phases during their travel.
The fraction with a greater affinity to stationary layer travels slower and at a shorter distance, while that with a lesser affinity travels faster and longer.
Based on the technique employed in separation of components it is broadly classified as
Here, the stationary layer is a solid while the mobile phase is liquid. The compounds travel on the solid surface under the influence of mobile liquid. The separation depends on the extent of physical adsorption to the solid surface.
In this mode, both the stationary and mobile phase are liquids. So the compounds have an affinity based on their partition coefficients into the individual liquid layer. The one with the greater partition to mobile liquid has a higher affinity to it, so it travels faster, and vice versa.
Based on the type of stationary material used for separation, there are two types:
- Normal phase: Here, the stationary material is polar in nature and hence the compounds with a higher polarity elute out last while non polars come out first.
- Reverse phase: Here, the stationary material is non-polar in nature and hence the compounds with a lower polarity elute out last, and vice versa.
In most HPLC analyses, the type used is the reverse one, as many of the biological, phytochemical compounds, and drugs estimated by HPLA are polar in nature.
There are many developments which have occurred over years based on the requirements and also technology employed in evaluation of mixtures.
The techniques can be broadly divided into planar and columnar techniques.
In this type, the stationary phase is a plane surface (two dimensions surface where only length and breadth are taken as area), on which chromatograms are formed.
This method is adopted in techniques like
In this type, there is use of a column wherein on its walls lies a stationary phase, while the mobile phase is flushed through the column.
The techniques which employ this method are:
Pros and Cons of Both Techniques:
Both techniques have their pros and their cons.
- Planar techniques have the advantages of faster separation, visualization of formed chromatograms, or spots, and they are less expensive. However they are not useful for preparative purposes.
- Columnar techniques have the advantages of having better or more effective separations of even complex mixtures — possibility to yield to a large amount of compounds by separation of mixtures, i.e in preparative mode. However they have the disadvantage of being expensive, time consuming, and cumbersome (heavy).