Professor Dave again, let’s run a column.
When we do chemistry in the lab, it’s never as neat and tidy as it is on paper.
Any reaction will have unwanted side products, unreacted starting material, and other impurities
in the product mixture.
We need a way to separate everything so that we can isolate the desired product.
And often times, that way is column chromatography.
In the previous tutorial we learned about thin layer chromatography, and a column will
operate by precisely the same principles, so if you haven’t seen that tutorial yet,
absolutely make sure to watch that one first.
If you’re up to speed on the general principles, let’s go ahead and see what a column is
all about.
As we said, in the laboratory, things are complicated.
We might run a reaction, quench it via aqueous acidic workup to neutralize any ions in solution,
and perform an extraction to isolate the organic layer.
In that organic layer is your target molecule, but also other stuff, mdepending on the nature
of the reaction.
But we don’t want that other stuff, we want just the target molecule, so we can get a
nice clean NMR spectrum and make sure we’ve got what we think we do, and possibly go on
to the next step in a synthesis.
The way we will do this is column chromatography.
As we said, this works just like TLC, in that we will use a silica gel to act as a stationary
phase, and then the components will separate as they move through the column based on how
they interact with the hydroxyl groups in the gel.
The only difference is that instead of moving up a plate, they move down the column, and
we don’t just separate a little bit, we put all of our material in there and do it
all at once.
Let’s go over the basics for setting these up.
First, at the bottom of the column, we place a little cotton plug using a wire.
Not too much or the column will drain too slowly, just enough to make sure silica will
not fall out.
Then we clamp the column in place, and add a layer of sand to fill the curved bottom section.
Next we fill up the column about a third of the way with our solvent system, which is
sometimes called eluent.
This is the solvent system we selected by TLC, the one that gives us the optimum separation
of the components in the mixture.
Ideally we can identify our target molecule by assessing its polarity and comparing this
to the Rf values on the plate, this will help us know what’s happening to our desired
compound as we run the column.
Now we take a beaker of our solvent, and pour in the dry silica gel, which is a powder,
making sure to do this in a hood, so as not to inhale the silica.
Stir things up until we get a slurry, and this will act as the stationary phase in our column.
It’s a little tricky, but use a spatula to get it in the column, adding more solvent
to the beaker as you go if necessary.
Use something to tap gently on the side of the column until all the silica settles at
the bottom, and use a pipette to rinse the sides with eluent to make sure it all gets
down there.
Start letting the column drip into a flask to help the silica settle, but don’t let
the solvent level run below the top of the silica.
Pushing compressed air through a tube fitted with a stopper at the top of the column can
help things go faster during this stage, or any other stage during the separation if it
seems necessary.
While packing the column, use more solvent if you need to, we just need the stationary
phase packed down tightly.
It is very important that the stationary phase is completely smooth, there can be no cracks
or uneven sections.
Once that’s all set, we place another layer of sand on top of the column, and then allow
the solvent to drip until it sits just between the sand and the stationary phase, without
going below.
Now we are ready to load the column.
We take our isolated mixture, and we dissolve it in the absolute smallest amount of solvent
possible that will enable us to pipette it onto the column, just a few drops if possible.
While getting it in there, try not to get any of it on the sides of the column, we really
want it to drip down directly onto the sand.
We can then drain just a little bit of solvent, so that the mixture will move through the
sand and just enter the stationary phase, so it sits just below the sand, in a nice
even band.
Make sure the solvent level lowers just enough that none of the mixture is left behind in
the sand, or this part will not separate properly, as anything that is not loaded onto the stationary
phase will become very dilute once solvent is added and therefore impossible to separate.
We need our sample to be very concentrated in a thin band at the top of the stationary
phase for separation to work.
Now we add the solvent.
At first, we have to carefully pipette the solvent into the column, squirting it onto
the sides at the top so that it gently flows down.
We do not want to disturb the sand or the gel whatsoever.
Do this until the solvent level is several inches above the sand.
At this point, you can carefully pour in more solvent from a beaker, just continue to ensure
that the sand is not disturbed, and in general once we begin to run the column, never, ever
let the solvent level go below the sand, or the stationary phase will dry out and crack.
The solvent level should always be well above the sand, so monitor and add more as necessary.
Now the sample is loaded and we are ready to start.
Let the column start to drip, and we will collect in a series of small flasks, each
of which is referred to as a fraction.
We don’t have to be too careful with the first fraction, because we know that our sample
is still way up at the top of the column, and none of the components will travel faster
than the solvent, but depending on how polar the solvent system is, we will soon want to
pay a bit more attention.
As we push eluent through the column, the components will begin to move with it, and
separate as they go, just like what happens on a TLC plate.
Try to find an optimal flow rate, we can’t go too fast or too slow, or it will not separate properly.
Just a few drops per second should be fine.
Often we can clearly see bands of different colors moving down the stationary phase, and
that makes things nice and easy, as we will know when to be more careful with our fractions.
When it looks like we are collecting organic material, we want to get fractions of a smaller
volume, maybe ten milliliters or even less, to maximize our chances of a clean separation.
We can gauge the concentration by color, but don’t be afraid to collect lots of fractions
if you need to, even up to 20 or 30 is no problem provided you have enough flasks to do so.
Keep adding more eluent and pushing it through until you’re confident you’ve collected
everything you need, and then you can push the rest out and let it go dry.
Sometimes the nature of the eluent can be modified as you go.
It’s not uncommon to utilize a gradient of polarity, starting with a less polar solvent
system to gently kick things off, and gradually increase the polarity throughout the course
of the column, especially if you think you’ve collected the relevant fractions, and you
want to get what’s left out of there more rapidly.
But one way or another, now we’ve got our fractions, and it’s time to analyze them.
We will do this by TLC, so make that pencil mark, add five to ten vertical notches to
each plate and number them so that you know which fractions they correspond to.
Each line gets a spot from the corresponding fraction, and then develop the plates.
As we said, hopefully we know which spot represents our desired product, based on its polarity
compared with anything else that might be in there, unreacted starting material, predicted
side products, or what have you.
If you know which one it is, then all you have to do is take all of the fractions that
gave TLC data showing only that spot and no others, and combine them.
That’s why we want to use very small fractions right when we think our product is exiting
the column, because there could be fractions on either side that contain our product along
with something else, and we want to minimize the amount of our product that we have to
throw away due to impurity, because we don’t want anything else to be collected, only our target.
Combine all the winning fractions in a flask suitable for a rotavap, evaporate the solvent
away, and there you have your product, purified and isolated.
Now you’re ready to get an NMR spectrum and know for sure if your reaction went as planned.
So that’s a basic introduction to column chromatography.
It may seem complicated, and to be honest, it definitely can be, and your first column
will probably go very poorly.
But don’t get discouraged, learn by watching more experienced labmates, practice, and soon
it will seem like a trivial and mundane task.
Now with extraction and chromatography under your belt, you’ve got a few of the quintessential
organic chemistry techniques down, so let’s go ahead and learn one more.