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Chromatography: Principle, Types, and Techniques

Updated on August 18, 2018

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.

Paper chromatography
Paper chromatography

Types

Based on the technique employed in separation of components it is broadly classified as

Adsorptionbased

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.

Partition-based

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:

  1. 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.
  2. 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.

Planar Type

Thin layer chromatography
Thin layer chromatography

Techniques

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.

Planar:

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

  1. High performance thin layer chromatography (HPTLC).

Columnar:

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:

  1. Column chromatography
  2. Gas chromatography.
  3. HPLC.
  4. Size exclusion
  5. Ion exchange

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).

Comments

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    • profile image

      sunil nagap 

      2 years ago

      nice one...

    • profile image

      madhavi 

      2 years ago

      its great

    • profile image

      Ateeb 

      2 years ago

      good work

    • profile image

      Rajitha 

      3 years ago

      AWESOME

    • profile image

      bunty 

      3 years ago

      good

    • profile image

      Hasib 

      3 years ago

      Thanks for this beautiful knowledge.

    • profile image

      nishantgupta 

      3 years ago

      superb for knowledge

    • profile image

      muralikrishna 

      3 years ago

      thank for giving velubel information

    • profile image

      RK SINGH 

      3 years ago

      Very nice for study and knowledge.

    • profile image

      Arti yadav 

      3 years ago

      thanks four knowledge

    • profile image

      Syeda Farhath Unnisa 

      3 years ago

      This is quite easy and simple....

    • profile image

      vishwjeet jadhav 

      3 years ago

      thank you so much....make it very easy

    • profile image

      upendrasinh 

      3 years ago

      Thanks four knowledge

    • profile image

      mallikarjun huggi 

      3 years ago

      Good...! it is helpful for me.

    • bheem profile imageAUTHOR

      bheem 

      4 years ago

      Ananthi! Inspiring comment to write even better articles. :-)

    • profile image

      ananthi 

      4 years ago

      It is very good article and then very useful for my preparation of the chromatography. thanking u

    • bheem profile imageAUTHOR

      bheem 

      4 years ago

      Nabeel! a new pic on column chromatography used in a lab will be updated shortly for easy understanding..

    • profile image

      Nabeel 

      4 years ago

      good but there should be more diagrams of column chromatography

    • profile image

      Mahalakshmi 

      4 years ago

      thank you for your explanation

    • profile image

      Anjali 

      4 years ago

      This is very educational and also interesting .

    • bheem profile imageAUTHOR

      bheem 

      5 years ago

      dr mudassar ! welcome..

    • profile image

      dr mudassar 

      5 years ago

      thanks

    • profile image

      vijay 

      5 years ago

      i have learnt thank you still give informations

    • profile image

      saami khan 

      5 years ago

      very nice n informative

    • bheem profile imageAUTHOR

      bheem 

      6 years ago

      Rahul refer to rajaha.com for more info on HPLc and GC.

      Regards...

    • profile image

      rahul 

      6 years ago

      hi, bheem sir i m rahul n i m also intersted to study HPLC,GC so plz give me hub addres

    • bheem profile imageAUTHOR

      bheem 

      6 years ago

      Amy & ademilokun!Happy to know you liked the article...

    • profile image

      Ademilokun Ayodele 

      6 years ago

      This is quite nice and pin-pointing.

    • profile image

      amy 

      6 years ago

      very informative

    • bheem profile imageAUTHOR

      bheem 

      6 years ago

      Glad to know you found the article useful sunshinecloudyday.

    • profile image

      Sunshinecloudyday  

      7 years ago

      Very interesting and educational, I enjoyed reading this hub.

    • bheem profile imageAUTHOR

      bheem 

      7 years ago

      Thanks for your complement kafsoa.

    • kafsoa profile image

      kafsoa 

      7 years ago

      Simple, nice and easy way to explain chromatography method in separation. very useful hub!

    • bheem profile imageAUTHOR

      bheem 

      7 years ago

      Ya rudra! that is one of the important tools we use in my lab on regular basis.

    • Rudra profile image

      Rudra 

      7 years ago

      Very good indeed about chromatography. Have you tried HPLC.

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