Today we are talking about column chromatography. Everything we are doing
should be done in a hood because we are using volatile organic solvents.
For video purposes we are doing it on the bench top but remember: always work in a hood.
You have already familiar with TLC which is a separation technique that
uses silica gel chromatography. TLC is an analytical technique, meaning that all
you get out of this technique is information. You're using way too little
compound for it to be practically useful. If you want to have a sizable amount of
compound you have to scale it up. So, to scale it up you don't use just a very
thin layer of silica on a plastic backing, you use a lot of silica and you
pack a column of it. Column chromatography is a very touchy technique.
There are three phases in column chromatography: first you pack the column (that is you prepare it
for your experiment) then you load it (which is the equivalent of spotting the
TLC plate - that is you put your compound on the silica gel), and then you run it
(which is the equivalent of putting your TLC plate in the TLC chamber and
running it up. The only difference between TLC and column
chromatography is that in TLC your solvent goes up by capillary action, in
column chromatography your solvent goes down by gravity, but that's the only
difference. The columns we have here are actually very handy because the stopcock
comes out. The very first thing you want to do is take a very small cotton plug and put
it at the bottom of the column, and then you carefully reinsert the stopcock. This will
prevent the sand from going down the stopcock. Then you want to clamp the column,
you put a powder funnel on top and you put in sand.
The silica gel column must be a perfect cylinder so you want to put in enough
sand to go past any curvature in the side of the column. The solvent that you
are going to use in order to pack the column is the solvent that you will use to run
the column so it's going to be a mixture of hexane ethyl acetate that you already
determined by TLC. You start by pouring a little bit of your mixture on
top of the sand and if you see there is some air coming up. Then you tap the side
of the column to make sure the sand is perfectly flat.
You will need approximately 14 grams of silica gel. Dry silica gel has a density
of about 0.5 so there's no need to really weigh it, it's going to be
approximately 28 milliliters. So if you use a 30 milliliters beaker and you go
somewhere in between 25 and 30 is plenty accurate, so that's what we are going to do.
That's approximately the right amount. We transfer it into a bigger beaker and you
want to make a slurry with your solvent so you add enough solvent... oops, sorry -
that's not a big deal - then you add enough solvent to make a very runny slurry,
fairly liquid, and you want to stir it very well. Then you need to pour it
into the column while you are stirring to keep the gel in suspension, so you do this.
Then you can add some more solvent to rinse the beaker, and it's not important
that not all the silica gel is going into the column; this is totally
inconsequential. Now you open the column and you let your solvent flow through.
You are going to tap on the column with a plastic object to allow the silica gel
to settle. It's important that the silica gel never goes dry.
Now that you have ran a column worth of solvent through the column, you want to
close the stopcock and you need to repeat the operation. The solvent that
ran through a column during packing is clean, so you can reuse it. It is extremely
important not to disturb the surface of the silica gel when you are pouring the
solvent. At the very beginning you can use a pipette and add solvent very slowly,
and then, when you have enough solvent to buffer the fall, you can add a little faster.
You reopen the stopcock and you let all the solvent go through again.
Make sure you stop before the surface of the silica gel, because it's very important
that the silica gel never goes dry. To be able to watch the silica gel settling,
it is actually helpful to mark the level of the silica gel with a sharpie.
While the solvent is going through you keep gently tapping the site to help settling.
In theory, you should keep running solvent through the column until the
silica gel stops settling. When you have about five millimeters of solvent on the
column head, you want to close the stopcock, and you need to add a little sand on the
top of the column in order to protect the silica gel - a couple of
millimeters are enough.
Like that. Then with a pipet you may want to rinse the side of the column.
Let the solvent drain until the sand goes dry...
...and you want to stop right there. So the sand is dry but the silica gel is not.
At this point your column is packed and is ready to be loaded. Now you are
ready to load your compound. In our case the compound is a liquid so it's very
easy: you want to use a long Pasteur pipette and drop it right in the middle
of the dry sand.
Then you open the stopcock and you let the compound enter the column -
which means you go with the sand dry - and you stop right there. Now you want to
rinse the walls of the container and the pasteur pipet that you use to load, so to
make sure that all the your compound is loaded. So you take a clean Pasteur
pipette, you take about one ml of solvent, you put it in and you wash the walls.
Then you put this solution again at the top of the sand. Be careful not to disturb
the surface of the silica gel when you do this.
Then you open the stopcock and you let the solution enter your column.
And you go again dry with the silica gel. At this point your column is loaded
and you can start running it. So you can add your eluting solvent very slowly at
the beginning - so you don't disturb the surface of the silica gel -
and then more briskly until the column is full.
Now you are ready to run, so you can open the stopcock and you will start
collecting small fractions in test tubes. And this concludes your column
chromatography video.