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Thin Layer Chromatography (TLC): Principle and Procedure

Updated on December 28, 2015

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.

Principle

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.

Procedure

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

Advantages

  • 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.

Applications

  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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      sai 5 months ago

      Thanks this was soo helpful for my pg exam.

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      Shweta singh 6 months ago

      Thanks...it was very helpful

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      saima 10 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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      pradeep c londhe 16 months ago

      Lot of thank tou......

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      Aliya 17 months ago

      Thankuuu

      Its useful

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      Adan Iman 17 months ago

      its very useful article.thank u so much

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      mahi 19 months ago

      its very use full thnks

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      Sajina 20 months ago

      Tq so much

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      Ruchika 22 months ago

      Thanks...it helped a lot...just to the point and we summarized..

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      kashaf 23 months ago

      Good

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      KUSHAL DOLEY 23 months ago

      Thanks ,it is very useful note book

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      surbhib268@gmail.com 24 months ago

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

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      indira Gawariya 2 years ago

      Thanks for the help

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      narendra waje 2 years ago

      its very simple languge....thnx

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      kajal 2 years ago

      itz lot useful

      ty

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      maya 2 years ago

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

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      Gayathrisyam 2 years ago

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

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      HASIB QURESHI 2 years ago

      Thank you sir. really its was easy to understand.

    • bheem profile image
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      bheem 2 years ago

      @Aishwarya Murali ! Welcome and glad with you complements.

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      Aishwarya Murali 2 years 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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      shahida 2 years ago

      informative & easy to understand

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      shahida 2 years ago

      informative & easy to understand

    • bheem profile image
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      bheem 2 years ago

      @Tejraj Rajput ! Welcome again.

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      Tejraj Rajput 2 years ago

      Thanks for imformation

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      yogita thakur 2 years ago

      Exellent...thank u

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      Syeda Farhath Unnisa 2 years ago

      Very useful and Informative...

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      FORTUNATE 2 years ago

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

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      bheem 2 years ago

      @Sharad Devkatte ! welcome...

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      Sharad Devkatte 2 years ago

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

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      Namrata vyas 2 years ago

      this book is very use full so thanx very much

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      bheem 2 years ago

      Hi sonali, you can mail me at bheemnr@gmail.com..

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      Sonali nimbalkar 2 years ago

      Hi bheem,

      This is very gud material of chromatography. Well done

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

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      Monika Mohanty 2 years 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...

    • profile image

      Monika Mohanty 2 years 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...

    • bheem profile image
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      bheem 2 years ago

      @roshani! You are welcome...

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      roshani 2 years ago

      thanks

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      bheem 3 years ago

      lobsang! You are welcome...:-)

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      lobsang 3 years ago

      Awesome ...thanks

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      manoj 3 years ago

      nice

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      Himadri 4 years ago

      very helpful and very easy to read.

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      bheem 4 years ago

      Hi rinku! welcome....

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      rinku 4 years ago

      thank u for help

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      bheem 4 years ago

      NAVV DEEP KAUR! welcome again..

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      NAVV DEEP KAUR 4 years ago

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      bheem 4 years ago

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

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      Amit Sharma 4 years ago

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      thank you.......

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      bheem 5 years ago

      Happy for your complements Dilip and Feranmi..

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      bheem 5 years ago

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

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

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      bheem 5 years ago

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      bheem 5 years ago

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      bheem 5 years ago

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      B.Idris 5 years ago

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      bheem 5 years ago

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

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      dk 5 years ago

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      bheem 5 years ago

      Thanks Novera......

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      bheem 5 years ago

      Yassir! you are welcome.....

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      bheem 5 years ago

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

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