Nov 15, 2009

RBF Teaches: Thin Layer Chromatography (TLC)

Welcome Kiddies,

To the first ever RBF teaches blog post. In these posts we will be uploading a number of videos that go through the various techniques used in an organic chemistry lab and perhaps outside of one [MSG EDIT: Please don't try experiments at home kiddies] also.

Today we will be learning all about Thin Layer Chromatography (TLC). This is perhaps one of the most useful widely used techniques in the organic chemistry lab as it provides a fast, reliable and reproducible qualitative analysis of most reactions in the lab. YAY!

So, what does that mean? Well, for instance, let us assume we are performing a reaction in the lab. WOOOO! Now, how do we know when the reaction is complete? If your compounds are all colourless and no colour change occurs upon completion (as is usually the case) how can you know whether to stop the reaction? The answer my young friends is TLC! And Here is How we do it!

By taking a tiny sample of the reaction mixture (and I mean tiny, usually a drop is plenty) and comparing that to your starting material(s) you can determine whether the reaction is complete or not.
So, you may now be thinking "OK cool its magic or something, but I must know the secret Oh Great One!" Well its just chemistry these secrets will be revealed.
Here's how it works and how to run a TLC all of your very own and on the cheap from the very beginning:
  1. TLC plates can usually be purchased as large square (20x20cm) sheets or as a large roll of silica (SiO4) (or aluminium oxide AlO4) adsorbed onto an aluminium backing sheet. You can also get TLC sheets with plastic or glass backing.
  2. From these large square sheets you can cut them using a sharp knife (mark the whole plate or a guillotine (cut the plates silver side up). I usually aim for my TLC plates to be 5cm long and 2cm wide, this gives me 40 plates per sheet. To cut the plates it is possible to mark with a pencil on the silica side (i.e. the white side) HOWEVER you must press very gently so that the silica is not etched with your pencil you only want to lightly glide the pencil over the silica.
  3. Once you have cut your plate it can be used for a TLC analysis. To begin with a straight baseline must be drawn across the bottom of the plate usually 0.5-1cm from the bottom and parallel to the base. This is your baseline and all of your spots will begin from this point so that there is no error. It should look something like this:

  4. Once you have this base line you can begin to plan your TLC. Usually, you spot at least 3 spots depending on the reaction. If for example, you have one starting material and a reagent which does not run on TLC the TLC would be spotted as follows 1. Starting material (SM) 2. Reaction mixture (R) 3. Co-spot (C, a spot on which both starting material and reaction mixture is spotted. This reduces the chance of a false positive due to a poor solvent front etc.). [MSG EDIT: The use of a co-spot lane is also helpful when your product and starting material spots have very similar Rfs. You will read about Rfs later] If you have more than one material or a reagent that runs then this spot should also be added as well as being spotted to the co-spot. Also, if you have a pure sample of the desired product then it is also a good idea to spot this too to ensure that your reaction mixture has the correct product being formed (this is also the case in which a known by-product that can be produced from the reaction). This should also be spotted on the co-spot.
  5. Having the plan in place you can now gently mark the TLC plate with a pencil. Remember, lightly so as not to scratch the silica. Mark the plate so that you can understand it and not be confused as well as marking it so that all the spots are evenly spaced from each other and not too close to the edge of the plate. A good example can be seen below:
  6. Once you have a sample that is reasonably well diluted (5mg/mL usually suffices) you can begin to spot the plate. [MSG EDIT: In general, aim for higher dilution, that is, lower concentration. Using high concentration spotting solutions leads to larger spots, which means poor resolution. If the concentration is too low, then you can simply spot more] Using a TLC spotter (these can be purchased or made simply by blunting the end of a small 21-23G needle) gently spot a small spot on the plate. This may take some practice. It is easiest to use a quick motion to avoid leeching of the solvent from the TLC spotter onto the plate this avoids overflow of the spot onto other spots. Do not over spot the plate. After you have run the TLC, if there is a large streak this means your spot was too concentrated. Your spots should appear as below:

  7. Time to run the TLC. This is reasonably straightforward. Place your solvent system in a small glass jar or beaker. It is a good idea to only have 0.5-1cm of solvent in the bottom of your jar so that the level sits just below the baseline of your TLC plate.
    If you like [MSG EDIT: you should!] you can line the beaker with some absorbent paper (this will saturate the atmosphere of your beaker with solvent fumes allowing the plate to run quickly and smoothly. It is a good idea not to lean the TLC plate against the paper as it may leech solvent onto the plate). Place a watchglass or lid over the jar. Take your TLC plate by the top with a pair of tweezers and, after removing the lid of your jar, gently place the plate into the beaker try to make sure the plate is inserted levelly so that the solvent runs up the plate evenly.
  8. Once the solvent level reaches 0.5cm from the top of the plate you can remove the plate from the jar. Gently mark the plate at the solvent front (i.e. the level the solvent reached on the plate) with a pencil.
  9. Now you can develop your plate. This can be done with either UV light (most plates are fluorescent at 254nm) or using a dip. [MSG EDIT: UV light is especially useful as a visualisation tool if you know your product/starting material is aromatic or contains conjugated bonds]. To dip, simply take the chosen dip (selection of a dip depends on your compound) dip your plate quickly into the dip up to the solvent front and remove. Ensure that all the dip drips back into the dip jar to save on making up more dip and gently heat the plate with either a heat gun or on a heat plate. [MSG EDIT: Make sure your plate is free of solvent before dipping. I usually use a heat gun to blow dry the plate before dipping, and when I use a higher boiling solvent (such as ethyl acetate or methanol), I blow dry my plate for longer before dipping. A common mistake made by students is not heating the plate sufficiently. The colour of the spots depends on the dip you're using. When using a PMA (or Goofy's) dip, the spots should come up as dark blue spots on a green/blue background. Heat the plate until you see this. When the plate starts to turn dark blue (or just black), that's too much heating: stop. When using a permanganate dip, the spots should come up bright yellow against a pink/purple background. If the background goes yellow, then that's more than sufficient heating.]
  10. Now you can see your spots on the TLC plate. HOORAY! The next step is simply perfecting the method for your particular reaction.
  11. Find a solvent system that works for your particular reaction - This can often take some time. Basically, you want a system that is polar enough to move your spots up off the baseline but not take any spots off the plate. A good system to start with is 15% Ethyl Acetate (EtOAc) in Petroleum Spirits (PetSp) or Hexane. In this case the EtOAc is the polar solvent. If the spots are seen to move too far up the plate then lowering the amount of this polar solvent should lower the distance the spots travel. (See the OrganicOverdose explanation of this in the following post). Ultimately, you should have a TLC plate that looks something like this:

    Here you can see that some SM remains and that the reaction mixture contains a new product. The co-spot shows both spots which indicates that the final R spot is not a false positive. It is usually a good idea to try to get the topmost spot to fall below halfway up the plate. (Reasons for this will come up later). [MSG EDIT: As a rule of thumb, the spot you're trying to isolate should be just less than 1/3 up the plate, ie Rf ~0.3)

    To measure the Retention Factor (Rf) of the spot is as follows:
    Rf = distance of spot/distance of solvent front
    the solvent front is the distance you let the solvent move up the plate. This means that no matter how far up you let the solvent front move the Rf of a particular spot should remain the same (to a degree). This means that it is very important to mark the solvent front when you have finished running the TLC and before you begin to develop the TLC.
  12. Once you have all these points down pat you can run a TLC every reaction to make sure that the reaction is complete.
Important points to remember:
  • Mark your plates gently with a pencil (don't use pen: it will be very embarrassing)
  • Spread your spots out evenly and leave a good distance from the side of the plate.
  • Don't overspot the plate. If you have a large streak on the plate it is a good indication of overconcentration. [MSG EDIT: Remember, TLC is a visualisation tool for your reaction. As long as you can see the spots, it's fine, so there's no need to make spots extra intense. Less intense and smaller spots means better resolution, which is always good, especially if you have spots which are very close running]
  • Don't overfill your TLC jar above the baseline of the TLC plate.
  • Don't lean your TLC plate against the absorbent paper in the TLC jar.
  • Make sure your solvent system is good you want decent separation between spots (this will be covered at a later date)
  • Be sure to mark your solvent front before developing your TLC.
  • Don't leave your plate in the dip. It is a quick dip and then removal.
  • When developing your plate under UV use a pencil to mark in the spots that appear under UV light.
To sum all of these points up we have created the following video:

Please Note that the video has no sound at present but we will add a commentary to the video soon.

[MSG EDIT: If you're an undergrad who wants to ask questions about anything on this post or laboratory techniques in general, feel free to post them on the comments section. OO and I (and I'm sure grad students around the world who visit here) are more than happy to answer your questions and give you a helping hand]


  1. why must we not overfill our TLC jar above the baseline of the TLC plate.

  2. if the solvent level is higher than the baseline, then whatever chemicals you spotted on the silica will diffuse out into the running solvent rather than being carried up the silica plate

  3. Can u kindly send me the video .. i cant see it here

    I have been doing TLC for sugars, but i get a smear/large spot in the centre of plate. in addition to diluting the sample what suggestion would u give for better separation?

  4. What is a co-spot lane? Why is it useful if we are looking at the TLC of a reaction mixture?