Professor Dave here, let’s get some TLC.
We just learned about extraction, which is a technique we can use to separate compounds
on the basis of differing solubilities.
Now let’s learn about another separation technique called thin layer chromatography,
or TLC for short.
This separates compounds on the basis of differing polarity, so let’s go ahead and see how
this works.
In order to do TLC, we will need some TLC plates.
These have a thin layer of silica gel spread out on a glass surface.
It will be important to understand the structure of this silicate material, as this is what
will cause the components of the mixture to separate.
It is a network solid of silicon dioxide, with alternating silicon and oxygen atoms
in every direction, and at the surface of this material there will sit many hydroxyl groups.
These of course are polar, and can participate in dipole-dipole interactions with other polar
substances.
Now, in order to perform the separation, we will draw a line in pencil fairly near the
bottom of this plate, right on the silica.
Then, we take the solution containing the components to be separated, and insert a capillary tube.
Some of the solution will rise up into the tube by capillary action, and we will then
spot the plate with the contents of the tube.
To do this, press the bottom of the tube to the plate, right on the line we drew, firmly
enough that the solution gets deposited onto the plate, but not so firmly so as to disrupt
the silica.
Spot two or three times to load most of it on there.
Then, we will use some kind of solvent or solvent system to develop the plate.
We will talk about solvent selection in a moment, for now let’s just refer to it as
the solvent.
If we place a little bit of the solvent at the bottom of a beaker or developing chamber,
we can then place our spotted TLC plate in the beaker, leaning against the side.
The line with the spot must sit above the solvent, and not be submerged in the solvent,
or our sample will wash away, so make sure the pencil line is high enough and the solvent
is low enough that the spot sits slightly above.
Now cover the beaker with something so that solvent will not evaporate, and we can watch
the plate develop.
The solvent, which we call the mobile phase, will begin to rise up the plate, which we
call the stationary phase.
It does this by capillary action, just like we saw with the capillary tubes, and once
it hits the sample, it will drag those components up with it.
However, as the components move across the plate, they will interact with the hydroxyl
groups in the silica to differing degrees.
A substance that has polar functional groups, or a strong net dipole, will interact significantly
with the stationary phase.
We can say that this substance is strongly adsorbed to the stationary phase.
This will cause it to move more slowly along the plate, almost as though the interactions
make it sticky.
A substance that is largely nonpolar will not interact very well with the stationary
phase, and will instead move along the plate very close to the solvent front.
We can say that this substance is weakly adsorbed to the stationary phase.
Once the solvent is rather close to the top of the plate, but making sure not to let the
solvent reach the edge, we remove the plate and quickly use a pencil to mark the solvent
line, as this will not be visible in a few moments.
Then we let the plate dry.
Now it’s time to see how far each component traveled.
It is possible that the components are visible, but this is typically not the case, so we
usually have to use a special technique to see where they are on the developed plate.
Often we use a UV light, as certain functional groups in organic molecules will interact
with these wavelengths and glow under this light.
If using these, be extremely careful not to look directly at the light source, as this
can seriously damage your eyes.
Because of this, visualization is typically done either in a special box, or by using
handheld lamps to point the light downward, while using protective glasses as a precaution.
With the plate under the light, use a pencil to trace anything you see for later analysis.
Ideally there are circular dots, but there may also be lines, streaks, or splotches.
Just trace precisely what’s there.
Instead of UV light, sometimes we will have to use some kind of staining agent, like iodine,
anisaldehyde, or others.
These each react with a particular type of functional group and will cause the spots
to become visible, so we just use some tweezers to dip the plate in there, wash it off, and
put it on a hot plate until the spots show up, and then we have visual data to work with.
Now we can assign a retention factor, or Rf value, to any dot on the plate.
This represents the distance traveled by a particular substance as a fraction of the
distance traveled by the solvent front.
To get this, we just measure the distance from the line where spotting took place to
the center of the dot, and divide that by the distance from that same line to the line
at the top where the solvent front ended up.
This is why we do not let the solvent reach the end of the plate, otherwise we could not
get this second value, and we could not calculate Rf values.
We will get some value between zero and one, and we will do this for any spot on the plate.
If we actually intended to physically separate the mixture to collect its components, we
can remove the region of the gel each compound sits on and then perform an appropriate extraction.
But typically, TLC is used as an analytical technique.
We can use TLC to monitor a reaction.
Say we are performing a reaction where the reactant and product have significantly differing
polarities.
If we spot a plate with the solution in the reaction flask before adding any reagents,
we can get the Rf for the reactant.
Then, once the reaction has proceeded for some time, we can spot another plate and develop it.
If we get that same spot again, along with a new spot that has a totally different Rf,
we will know that some product has formed, but some reactant still remains.
If we wait a bit longer and do it again, and this time only the new spot shows up, and
not the first one, we know that the reactant is gone, and the reaction is complete.
Another reason we would use TLC is to select a solvent system for column chromatography,
which is a large-scale separation technique.
We will want to find a solvent system that maximizes the distance between two components
on a developed plate, because that will best enable us to separate them in a column, which
we will learn about in the next tutorial.
This solvent system is typically a mixture of two or more solvents like hexanes, ether,
ethyl acetate, or dichloromethane.
We may have to mix hexanes and ethyl acetate in a one to one ratio, or two to one, or five to one.
It’s usually difficult to predict what will work best, so just try something and see what happens.
If the two components end up very close together on the developed plate, this was a bad choice,
as they didn’t separate well.
If they separate a bit but end up towards the bottom of the plate, this is also bad,
as it means they travel very slowly across the stationary phase, and it will be a pain
to collect them.
The ideal solvent system will result in something like this, where the two components are separated
very well, one of them towards the top of the plate, and the other more near the middle.
This solvent system will be ideal for column chromatography, which is the technique that
we will use to actually physically separate all of the contents of our reaction flask,
once the reaction is complete and the organic products have been extracted.
This is an extremely important technique for any organic chemist, so let’s move forward
and learn about that next.