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.

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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Comments 66 comments

3ola 3laeldin 4 years ago

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vandana shenoy 4 years ago

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bheem 4 years ago Author

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


Yassir rather 4 years ago

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bheem 4 years ago Author

Yassir! you are welcome.....


novera sadaqat 4 years ago

good effert


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bheem 4 years ago Author

Thanks Novera......


dk 4 years ago

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


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bheem 4 years ago Author

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


hygx 919 4 years ago

sweet and useful


B.Idris 4 years ago

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bheem 4 years ago Author

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


Gideon Ngotho 4 years ago

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hellen mugodo 4 years ago

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bheem 4 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

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


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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 3 years ago

thank you.......


anju thakur 3 years ago

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Amit Sharma 3 years ago

Thanks for the help.


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bheem 3 years ago Author

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


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

THNK U, REALLY HELPED ME LOT


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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

nice


lobsang 23 months ago

Awesome ...thanks


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bheem 23 months ago Author

lobsang! You are welcome...:-)


roshani 22 months ago

thanks


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bheem 22 months ago Author

@roshani! You are welcome...


Monika Mohanty 20 months ago

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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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bheem 20 months ago Author

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


Namrata vyas 19 months ago

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Sharad Devkatte 18 months ago

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


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bheem 18 months ago Author

@Sharad Devkatte ! welcome...


FORTUNATE 18 months ago

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Syeda Farhath Unnisa 17 months ago

Very useful and Informative...


yogita thakur 17 months ago

Exellent...thank u


Tejraj Rajput 17 months ago

Thanks for imformation


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bheem 17 months ago Author

@Tejraj Rajput ! Welcome again.


shahida 17 months ago

informative & easy to understand


shahida 17 months ago

informative & easy to understand


Aishwarya Murali 16 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 16 months ago Author

@Aishwarya Murali ! Welcome and glad with you complements.


HASIB QURESHI 15 months ago

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Gayathrisyam 13 months ago

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maya 13 months ago

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kajal 12 months ago

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narendra waje 12 months ago

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indira Gawariya 12 months ago

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surbhib268@gmail.com 10 months ago

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kashaf 9 months ago

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Sajina 6 months ago

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Adan Iman 4 months ago

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Aliya 4 months ago

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pradeep c londhe 2 months ago

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