Experimental Techniques Explained: Thin Layer Chromatography (TLC)

Thin layer chromatography, or TLC, is a technique used for the separation and analysis of molecules in a sample (Note- NOT DNA!). It can be used on amino acids1, although in my lab it has been used to analyze the degradation (or lack of!) of large polymeric sugars by whole metabolically inactivated cells or by simple enzymes. Because of this, I’ll be focusing on the analysis of saccharides (sugars).

So far, good stuff! But! How does it work, and what the HELL do these results mean!?!

Background
First off, you can’t run a TLC without some samples, right? So your sample consists of your desired substrate (sugar) and bacteria/enzyme of interest mixed together and incubated at biological temperature (37° C). A small sample of this solution is taken at various time points and these form the samples to be run on the TLC. However if you are using a whole cell assay, with whole metabolically inactive cells, it is necessary to centrifuge the sample down so that the cells are spun down into a pellet, leaving the sugar-rich supernatant behind to be sampled.

How Does It Work?
If you’ve ever lived in the UK during your childhood, you’ve probably at some point watched a dot of ink travel up or across a piece of filter paper by the diffusion of water through said paper. Well- BAM! That’s pretty much TLC. It works on the same basic principles that this, SDS-PAGE and agarose gels run on; except we replace the electric current with a solvent that carries the sample up the TLC Plate by a distance relevant to their size.
First, samples are pipetted (around 2 μl) slightly above a penciled line drawn roughly a centimeter above the bottom of the TLC plate. It’s necessary to dry the samples onto the plate and repeat the pipetting to ensure there is a good concentration of the sample. The solvent used to run TLC plates is made from a mixture of water, propan-1-ol and acetic acid, nasty stuff! This is poured into a running tank, just enough to almost touch the penciled line. A lid must be placed on top of the tank while running to ensure that the solvent doesn’t simply evaporate into the air instead of diffusing up the TLC Plate. This setup is left to run until the solvent has ran all the way up the TLC Plate, where it is taken out of the tank, dried, and ran again (just like pipetting the samples). A resolving solution is used to resolve the plate via a 30 second soak after this second run and then the plate is placed in a heated oven for around 5 minutes until these lovely orange/brown spots are revealed.
Ta-da!

Reading your TLC Plate
“I see…I see…Wait, what do I see? And come to think of it, what is a TLC plate? You said it was like filter paper!” Sort of. A TLC plate is so-called because it is not simply a flimsy piece of paper. This could bend under the weight of the running solvent, ruining the run, so a metallic backing is added to the back of the silicon sampling layer to provide mechanical support. For the rest of this section I will be talking about reading TLC plates in the context of using whole cell assay samples, as this is where my experience is. Here are two of my own TLC plates.
Firstly, the standard ladder is made from various known sugars/polysaccharides so that the identity (or at least size) of any potential sugar products can be deduced, although this depends on the substrate used and the sugars included in the standard. The bands produced can be interpreted in a similar way to those on an SDS-PAGE. However, unlike in an SDS-PAGE gel if a band or blob is still visible at the pencilled line then this means that the substrate has not been degraded; the bacterium/enzyme does not break down the sample saccharide. Or, this may mean that the enzyme/cell takes a while to degrade it, and thus comes the need for samples at various time points of the previous whole cell assay.

 There are two general trends seen with whole cell assay TLCs. The first is an endo-acting pattern of activity, as can be seen here. The enzymes on the surface of the cell cut at/around the middle of the long sugar chain, thus a range of various products are made. Generally no monomers are produced as this is reflective of exo-acting behaviour.
TLC, Endo-acting Behaviour.jpg

Exo-acting enzymatic behaviour can be distinguished by the production of small monosaccharides, which are normally the last sugar in the standard. Larger products are not seen as the enzyme clips small sugar units off of the ends of the polymer, or from branches from the main chain. Both of these trends can be occurring together, however it is harder to tease apart the data for this situation, and more than a TLC is needed.

 TLC, Exo-acting Behaviour.jpg

And that’s it! I hope you’ve enjoyed this quick ‘run’ of TLC.

References;

Bhawani SA, Albishri HM, Khan ZA, Mohamad Ibrahim MN, & Mohammad A (2013). Surfactant Modified/Mediated Thin-Layer Chromatographic Systems for the Analysis of Amino Acids. Journal of analytical methods in chemistry, 2013 PMID: 24455427

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