Thin Layer Chromatography (TLC): Principle and Procedure

TLC or Thin Layer Chromatography

TLC is a type of planar chromatography.

  • It is routinely used by researchers in the field of phyto-chemicals, biochemistry, and so forth, to identify the components in a compound mixture, like alkaloids, phospholipids, and amino acids.

  • It is a semi quantitative method consisting of analysis.

  • High performance thin layer chromatography (HPTLC) is the more sophisticated or more precise quantitative version.


Similar to other chromatographic methods, thin layer chromatography is also based on the principle of separation.

  1. The separation depends on the relative affinity of compounds towards stationary and the mobile phase.
  2. The compounds under the influence of the mobile phase (driven by capillary action) travel over the surface of the 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.
  3. Once separation occurs, the individual components are visualized as spots at a respective level of travel on the plate. Their nature or character are identified by means of suitable detection techniques.

System Components

TLC system components consists of

  1. TLC plates, preferably ready made with a stationary phase: These are stable and chemically inert plates, where a thin layer of stationary phase is applied on its whole surface layer. The stationary phase on the plates is of uniform thickness and is in a fine particle size.
  2. TLC chamber. This is used for the development of TLC plate. The chamber maintains a uniform environment inside for proper development of spots. It also prevents the evaporation of solvents, and keeps the process dust free.
  3. Mobile phase. This comprises of a solvent or solvent mixture The mobile phase used should be particulate-free and of the highest purity for proper development of TLC spots. The solvents recommended are chemically inert with the sample, a stationary phase.
  4. A filter paper. This is moistened in the mobile phase, to be placed inside the chamber. This helps develop a uniform rise in a mobile phase over the length of the stationary phase.


The stationary phase is applied onto the plate uniformly and then allowed to dry and stabilize. These days, however, ready-made plates are preferred.

  1. With a pencil, a thin mark is made at the bottom of the plate to apply the sample spots.
  2. Then, samples solutions are applied on the spots marked on the line in equal distances.
  3. The mobile phase is poured into the TLC chamber to a leveled few centimeters above the chamber bottom. A moistened filter paper in mobile phase is placed on the inner wall of the chamber to maintain equal humidity (and also thereby avoids edge effect this way).
  4. Now, the plate prepared with sample spotting is placed in TLC chamber so that the side of the plate with the sample line is facing the mobile phase. Then the chamber is closed with a lid.
  5. The plate is then immersed, such that the sample spots are well above the level of mobile phase (but not immersed in the solvent — as shown in the picture) for development.
  6. Allow sufficient time for the development of spots. Then remove the plates and allow them to dry. The sample spots can now be seen in a suitable UV light chamber, or any other methods as recommended for the said sample.

Video Demo


  • It is a simple process with a short development time.
  • It helps with the visualization of separated compound spots easily.
  • The method helps to identify the individual compounds.
  • It helps in isolating 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.


  1. To check the purity of given samples.
  2. Identification of compounds like acids, alcohols, proteins, alkaloids, amines, antibiotics, and more.
  3. To evaluate the reaction process by assessment of intermediates, reaction course, and so forth.
  4. To purify samples, i.e for the purification process.
  5. To keep a check on the performance of other separation processes.

Being a semi quantitative technique, TLC is used more 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.

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Comments 67 comments

3ola 3laeldin 5 years ago

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

vandana shenoy 5 years ago

thanx! it ws of grt use ! :)

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bheem 5 years ago Author

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

Yassir rather 5 years ago

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bheem 5 years ago Author

Yassir! you are welcome.....

novera sadaqat 5 years ago

good effert

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bheem 5 years ago Author

Thanks Novera......

dk 5 years ago

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

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bheem 5 years ago Author

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

hygx 919 5 years ago

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B.Idris 5 years ago

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bheem 5 years ago Author

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

Gideon Ngotho 5 years ago

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hellen mugodo 5 years ago

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bheem 5 years ago Author

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

nethu 4 years ago

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bheem 4 years ago Author

Nethu!happy to know u liked the article..

NIMISHA VIRANI 4 years ago


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bheem 4 years ago Author

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

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bheem 4 years ago Author

Happy for your complements Dilip and Feranmi..

chandra nath 4 years ago

thank you.......

anju thakur 4 years ago is very interesting topic.thank you sir it is great

Amit Sharma 4 years ago

Thanks for the help.

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bheem 4 years ago Author

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

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Iytee 3 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 3 years ago


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bheem 3 years ago Author

NAVV DEEP KAUR! welcome again..

rinku 3 years ago

thank u for help

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bheem 3 years ago Author

Hi rinku! welcome....

Himadri 3 years ago

very helpful and very easy to read.

manoj 2 years ago


lobsang 2 years ago

Awesome ...thanks

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bheem 2 years ago Author

lobsang! You are welcome...:-)

roshani 2 years ago


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bheem 2 years ago Author

@roshani! You are welcome...

Monika Mohanty 2 years ago

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Monika Mohanty 2 years ago

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Hi bheem,

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bheem 2 years ago Author

Hi sonali, you can mail me at

Namrata vyas 2 years ago

this book is very use full so thanx very much

Sharad Devkatte 2 years ago

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

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bheem 2 years ago Author

@Sharad Devkatte ! welcome...

FORTUNATE 24 months ago

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

Syeda Farhath Unnisa 23 months ago

Very useful and Informative...

yogita thakur 23 months ago

Exellent...thank u

Tejraj Rajput 23 months ago

Thanks for imformation

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bheem 23 months ago Author

@Tejraj Rajput ! Welcome again.

shahida 23 months ago

informative & easy to understand

shahida 23 months ago

informative & easy to understand

Aishwarya Murali 22 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 22 months ago Author

@Aishwarya Murali ! Welcome and glad with you complements.

HASIB QURESHI 21 months ago

Thank you sir. really its was easy to understand.

Gayathrisyam 19 months ago

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

maya 19 months ago

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kajal 18 months ago

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narendra waje 18 months ago

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indira Gawariya 18 months ago

Thanks for the help 16 months ago

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

KUSHAL DOLEY 16 months ago

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kashaf 15 months ago


Ruchika 14 months ago helped a lot...just to the point and we summarized..

Sajina 12 months ago

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mahi 11 months ago

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Adan Iman 10 months ago

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Aliya 10 months ago


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pradeep c londhe 8 months ago

Lot of thank tou......

saima 2 months ago

It is in very much detail and very helpful but can you pls tell me how tlc helps in separating a polar and non polar substance and what is the difference in their movement and why?

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