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troubleshooting thin layer chromatography

Troubleshooting Thin Layer Chromatography: Some TLC for Your TLC

The whole TLC technique sounds easy to do, but it can be difficult and tricky during interpretation or give unexpected results, especially when working with biomolecules. For this reason, it is important to be familiar with troubleshooting thin layer chromatography.

Some of the common problems faced during TLC and their solutions are listed below:

Solvent Front Runs Unevenly/Crookedly

  • The thickness of the TLC slurry is uneven (usually seen when the TLC plate is prepared on a glass slide). The plate touches the sides of the container/chamber/filter paper.
  • The slurry/silica on the slide has fallen off.

No Spots Seen on the Plate

  • The concentration/quantity of the sample is too low. You can overcome this problem by spotting the sample multiple times in the same place on the slide (allowing the solvent to dry in between applications).
  • The development method needs to be changed.
  • There is no compound in the sample due to experimental failure.
  • The level of the solvent system is too high. If the level of the solvent system in the chamber is above the spot when the plate is inserted, the compound from the spot will dissolve in the solvent instead of migrating up the plate.
  • You may see irreproducible results if you reuse the solvent system for several TLCs. Always make it fresh.

Smeared Blue Spots after TLC

  • Using a pen instead of a pencil to draw a start line might cause this as the ink can travel with the solvent system on the TLC plate.

Several Unexpected Spots Seen

  • Dropping organic compounds accidentally on the TLC plate or touching the surface of the plate while handling it might result in unexpected spots upon developing the plate.

The Compound Runs as a Streak Rather than a Spot

  • Sample was overloaded (applied in excess).
  • The polarity of solvent system is inappropriate for the compound. Try a new solvent system.
  • The sample contains two or more compounds with slight differences in their polarity (try changing the solvent system).

As has been noted, you might face several problems while performing TLC for any given sample, but with repetition you will move towards perfecting your TLC experiment. Best luck with your TLC.

5 Comments

  1. Eitel MISSE on April 11, 2019 at 10:47 pm

    is it possible to perform an TLC experiment with a solvent system known immiscible? eg hexane-methanol

  2. kashif on October 9, 2018 at 8:44 am

    My sample give spot on staining, It contain 2 amino group expected is 2ndary and other is 3rd. and may b tertiary. I tried with many solvent system but dont travel along solvent phase. i checked with high polarity to low polarity solvent.

  3. kim on December 12, 2017 at 11:14 am

    What is the cause if The rf is higher than specify eg expected rf .7 for one component and another at rf .3 actual rf .8 and rf .5 solvent is a mixture of ammonia chloroform n methanol. Is it the homogeneity of the solvent or saturation of the mobile phase
    Ps Can one conclude it is still the same component for id purpose

  4. williana on January 29, 2017 at 6:32 pm

    do you know what causes this problem. my sample give positive result using mobile phase A,, but give negative result using mobile phase B. please answer,,thank you

    • Sandesh Marathe on April 27, 2017 at 1:55 pm

      It depends on your sample and mobile phase. Will you please tell what sample and mobile phases are you working with?

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