A Little TLC

Now that we have collected our different fractions, it is time to begin to consider what may be in the different fractions and, in particular, whether there is potentially a new and exciting molecule in one of them.

The first step towards doing this involves a process called thin layer chromatography (TLC).  Before carrying out TLC each fraction is redissolved in a small volume of 'Magic Brew' (a mixture of methanol and dichloromethane).

In TLC a capillary tube is used to add small amounts of each sample onto a TLC plate.  A TLC plate is a thin piece of plastic covered in silica.  The plate has a horizontal line near the bottom and you place a labelled pencil mark where you want each sample to go on the line.  The aim is to get a nice small circle of sample on each location.  To achieve this you 'spot' a small amount of sample, let it dry, and then add some more sample.  (If you put too much sample on at the start it spreads out too far and you will not get good results.)

Once we have spotted all our samples onto the plate, the plate gets lowered into a glass container with a small amount of solvent in the bottom.  The solvent used was 5% methanol in dichloromethane.  A lid is placed on the container so that fumes from the solvent are kept in the container.

The solvent begins to rise up the plate and as it does so it carries molecules with it. The surface of the TLC plate is quite polar. This results in polar molecules sticking to the plate while molecules of lower polarity continue to travel up the plate with the solvent. You end up with the most polar molecules at the bottom of the plate and the least polar molecules at the top.

The plate to the right shows my three P. microcladioides fractions. The 100% fraction contains a number of coloured compounds which have traveled varying distances up the plate.  There appear to be no compounds in the 30% and 75% fractions but this this only because none of them are coloured.  To see more compounds the plate is put under 2 different wavelengths of UV light.

The image to the left shows the plate when placed under a 350 nm UV light source while the image on the right is of the plate under a 254 nm UV light source. As you can see, new spots have shown up on the plate.  These are for molecules that contain chromophores. A chromophore is a part of a molecule that absorbs certain wavelengths of UV or visible light and reflects other wavelengths. (Chromophores often contain alternating single and double bonds.)

You may notice that the spots for the 30% fraction are all fairly low.  This makes sense as you will recall that the molecules in the 30% fraction are the most polar ones and it is these polar molecules that stick to the relatively polar surface of the plate and therefore don't travel very far.