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 thin layer chromatography 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.

For more details go through the book on thin layer chromatography by James.

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Comments 60 comments

3ola 3laeldin 3 years ago

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

vandana shenoy 3 years ago

thanx! it ws of grt use ! :)

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bheem 3 years ago Hub Author

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

Yassir rather 3 years ago

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

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bheem 3 years ago Hub Author

Yassir! you are welcome.....

novera sadaqat 3 years ago

good effert

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bheem 3 years ago Hub Author

Thanks Novera......

dk 3 years ago

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

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bheem 3 years ago Hub Author

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

hygx 919 3 years ago

sweet and useful

B.Idris 3 years ago

Thanks so much 4 dis. Peace be unto you

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bheem 3 years ago Hub Author

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

Gideon Ngotho 3 years ago

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

hellen mugodo 3 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 3 years ago Hub Author

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

nethu 3 years ago

really was brief and informative....

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bheem 3 years ago Hub Author

Nethu!happy to know u liked the article..

NIMISHA VIRANI 3 years ago


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bheem 3 years ago Hub Author

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

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bheem 3 years ago Hub Author

Happy for your complements Dilip and Feranmi..

chandra nath 3 years ago

thank you.......

anju thakur 3 years ago is very interesting topic.thank you sir it is great

Amit Sharma 2 years ago

Thanks for the help.

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bheem 2 years ago Hub Author

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

Iytee 2 years 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 2 years ago


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bheem 2 years ago Hub Author

NAVV DEEP KAUR! welcome again..

rinku 2 years ago

thank u for help

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bheem 2 years ago Hub Author

Hi rinku! welcome....

Himadri 2 years ago

very helpful and very easy to read.

manoj 16 months ago


lobsang 14 months ago

Awesome ...thanks

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bheem 14 months ago Hub Author

lobsang! You are welcome...:-)

roshani 13 months ago


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bheem 13 months ago Hub Author

@roshani! You are welcome...

Monika Mohanty 11 months ago

Thanku vry much for dis article....dis help me out on my xams...but d there is vry less points for its application...plzz add some more points...

Monika Mohanty 11 months ago

Thanku vry much for dis article....dis help me out on my xams...but d there is vry less points for its application...plzz add some more points...

Sonali nimbalkar 10 months ago

Hi bheem,

This is very gud material of chromatography. Well done

But want your email id any difficulty in chromatography then I mail send me matrial as soon as possible

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bheem 10 months ago Hub Author

Hi sonali, you can mail me at

Namrata vyas 10 months ago

this book is very use full so thanx very much

Sharad Devkatte 9 months ago

Thanxx...this is excellent information of chromatography. ..simple language. ..

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bheem 9 months ago Hub Author

@Sharad Devkatte ! welcome...

FORTUNATE 9 months ago

Thank you so much it was of really helped me to draft my prac report

Syeda Farhath Unnisa 8 months ago

Very useful and Informative...

yogita thakur 8 months ago

Exellent...thank u

Tejraj Rajput 8 months ago

Thanks for imformation

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bheem 8 months ago Hub Author

@Tejraj Rajput ! Welcome again.

shahida 8 months ago

informative & easy to understand

shahida 8 months ago

informative & easy to understand

Aishwarya Murali 7 months ago

Thank u sir.. Am so thankful to u. The infrmtn abt various chrmtgrphy had helpd me to stdy for my labrtry exams.thanx a lot.

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bheem 7 months ago Hub Author

@Aishwarya Murali ! Welcome and glad with you complements.

HASIB QURESHI 6 months ago

Thank you sir. really its was easy to understand.

Gayathrisyam 4 months ago

Thank You sir.. It is very very simple to Read and understand. Really improved my knowledge..

maya 4 months ago

It is so useful for everyone....thank you sir....

kajal 3 months ago

itz lot useful


narendra waje 3 months ago

its very simple languge....thnx

indira Gawariya 2 months ago

Thanks for the help 5 weeks ago

thank you was so helpful for understanding the basic principle of practical as well as theoratical knowledge...

KUSHAL DOLEY 4 weeks ago

Thanks ,it is very useful note book

kashaf 13 days ago


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