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TLC thin layer chromatography: Principle, Procedure

TLC chromatography or Thin layer chromatography

is a type of planar chromatography. TLC is routinely used by researcher in the field of phyto-chemicals, biochemistry etc. to identify the components in a compound mixture like alkaloids, phospholipids, amino acids etc..

It is a semi quantitative method of analysis and its sophisticated version or highly precise quantitative version is High performance thin layer chromatography (HPTLC).

Thin layer chromatography principle:

Similar to other chromatographic methods TLC is also based on the principle of separation. The separation depends on the relative affinity of compounds towards stationary and mobile phase. The compounds under the influence of mobile phase (driven by capillary action) travel over the surface of stationary phase. During this movement the compounds with higher affinity to stationary phase travel slowly while the others travel faster. Thus separation of components in the mixture is achieved.

Once separation occurs individual components are visualized as spots at respective level of travel on the plate. Their nature or character are identified by means of suitable detection techniques.

TLC chromatography System components:

TLC System consists of

a) TLC plates preferably ready made with stationary phase: These are stable and chemically inert plates on to whose surface a thin layer of stationary phase is applied. The stationary phase on the plates is of uniform thickness and consists of fine particle size.

b) TLC chamber: This is used for the development of TLC plate. The chamber maintains uniform environment inside for proper development of spots. It also prevents the evaporation of solvents and keep the process dust free.

c) Mobile phase: This comprises of a solvent or solvent mixture recommended for the purpose. The mobile phase used should be particulate free and of highest purity for proper development of TLC spots. The solvents recommended are chemically inert with the sample, stationary phase.

d) A filter paper moistened in the mobile phase, to be placed inside the chamber. This helps uniform rise in mobile phase over the length stationary phase.

TLC chromatography

Thin layer chromatography procedure:

The stationary phase is applied onto the plate uniformly and then allowed to dry and stabilize. But now a days ready made plates are preferred.


A thin mark is made at the bottom of the plate with a pencil to apply the sample spots.

Then samples solutions are applied on the spots marked on the line at equal distances.

The mobile phase is poured into the TLC chamber to a level few centimeters above the chamber bottom. A filter paper moistened in mobile phase is placed on the inner wall of the chamber to maintain equal humidity in the entire chamber and there by avoid edge effect.

Then the plate prepared with sample spotting is placed in TLC chamber such that the side of the plate with sample line is towards the mobile phase. Then the chamber is closed with a lid.

The plate is immersed such that sample spots are well above the level of mobile phase but not immersed in the solvent as shown in the picture for development.

Allow sufficient time for development of spots. Then the plates are removed and allowed to dry. The sample spots are visualized in suitable UV light chamber or any other methods as recommended for the said sample.

TLC chromatography video demo

Advantages of TLC

The Thin layer chromatography advantages include:

It is simple process with short development time.

It helps in visualization of separated compound spots easily.

The method helps to identify the individual compounds.

It helps in isolation of most of the compounds.

The separation process is faster and the selectivity for compounds is higher (even small differences in chemistry is enough for clear separation.

The purity standards of the given sample can be assessed easily.

It is a cheaper chromatographic technique.

Applications of Thin layer chromatography

1. To check purity of given samples.

2. Identification of compounds like acids, alcohols, proteins, alkaloids, amines, antibiotics etc.

3. To evaluate reaction process by assessment of intermediates, reaction course etc.

4. To purify samples i.e for purification process.

5. To keep a check on the performance of other separation processes.

Being a semi quantitative technique, tlc chromatography is used for rapid qualitative measurements than for quantitative purposes. But due its rapidity of results, easy handling and inexpensive procedure, it finds its application as one of the most widely used chromatography techniques


 Last updated on December 7, 2012

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Comments 33 comments

3ola 3laeldin 2 years ago

Thank You Very Much, This has been very helpful in my assignment


vandana shenoy 2 years ago

thanx! it ws of grt use ! :)


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bheem 2 years ago Hub Author

Glad to know you found the article useful..Thanks....


Yassir rather 2 years ago

Glad to find so much detail.Its of great importance to me.thanks


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bheem 2 years ago Hub Author

Yassir! you are welcome.....


novera sadaqat 2 years ago

good effert


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bheem 2 years ago Hub Author

Thanks Novera......


dk 2 years ago

very good...............................................


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bheem 2 years ago Hub Author

dk! glad that you liked the article......


hygx 919 2 years ago

sweet and useful


B.Idris 2 years ago

Thanks so much 4 dis. Peace be unto you


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bheem 2 years ago Hub Author

@ hygx & idris! glad you found the article useful.....


Gideon Ngotho 2 years ago

Has helped me to write my practical report in my campus.Thank you


hellen mugodo 2 years ago

its a good document very helpful my project is now a star project because of ur good work


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bheem 2 years ago Hub Author

@Gideon & Hellen, your comments are encouraging.........Thanks..


nethu 2 years ago

really superb....it was brief and informative....


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bheem 2 years ago Hub Author

Nethu!happy to know u liked the article..


NIMISHA VIRANI 2 years ago

ITS TOO EASY AND UNDERSTANDABLE.ITS A WELL FOR PRESENTATION...


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bheem 2 years ago Hub Author

NIMISHA VIRANI! glad with your complement...Welcome again..


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bheem 2 years ago Hub Author

Happy for your complements Dilip and Feranmi..


chandra nath 2 years ago

thank you.......


anju thakur 24 months ago

wow.it is very interesting topic.thank you sir it is great


Amit Sharma 22 months ago

Thanks for the help.


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bheem 22 months ago Hub Author

Welcome guys.......glad with your compliments.


Iytee 18 months ago

Very useful and informative material, would like to get the full name of the author as I intend making proper references where necessary in my term paper.


NAVV DEEP KAUR 17 months ago

THNK U, REALLY HELPED ME LOT


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bheem 17 months ago Hub Author

NAVV DEEP KAUR! welcome again..


rinku 16 months ago

thank u for help


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bheem 15 months ago Hub Author

Hi rinku! welcome....


Himadri 14 months ago

very helpfull and very easy to read.


manoj 2 months ago

nice


lobsang 4 weeks ago

Awesome ...thanks


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bheem 3 weeks ago Hub Author

lobsang! You are welcome...:-)

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