We’ve already examined chromatography as an efficient process for separating components of a
mixture on the basis of differing polarity. This occurs due to different degrees of interaction
with a stationary phase as components are pushed along with a mobile phase. But for TLC, we are
only able to separate the tiny bit of the mixture that is spotted onto the plate. We will often
want to perform chromatography on large samples. For this we will need column chromatography, a
commonly used technique in chemistry, biology, and industry, which we have covered from a conceptual
standpoint over in the lecture-based series, so be sure to review that video for additional context.
To perform column chromatography, we will need a column. That’s this. This will be loaded with the
stationary phase. The mobile phase is a solvent solution that moves down through the column along
with your sample, just as solvent moved up the plate in TLC. You can then collect fractions as
they come out of the column, using a series of small flasks, such that the components of the
mixture will arrive in different fractions due to their differing rates of travel down the column,
according to how well they interact with the stationary phase. The most common type of column
chromatography in a chemistry lab is silica gel column chromatography. Solvent is added to the gel
to form a slurry, and this slurry of silica gel acts as the stationary phase, while the solvent
system that passes through the column acts as the mobile phase. The stationary phase can separate
compounds based on their differences in polarity, precisely the same way as the TLC plate, only now
moving through a 3D matrix rather than along a 2D surface. The key concept is still adsorption. The
materials being separated are attracted to the silica beads to differing degrees depending on
their polarity, because of all the hydroxyl groups protruding from their surfaces, which can make
hydrogen bonds or other types of dipole-dipole interactions with polar molecules, which will
cause them to move more slowly through the column. Less polar molecules will move more quickly since
they won’t interact as well with the stationary phase, and are thus pushed along with the solvent.
Now it’s time to perform the technique. We’ll start by packing our glass column. As you can see,
the column has a stopcock at the bottom, so make sure it’s closed. We’ll then add a
cotton ball to the bottom. This is so that our gel doesn’t go through, as it is a fine powder
and can easily slip through when we open the stopcock. Using a glass rod like this one,
we’ll push the cotton down like this. Let's then secure our column to a ring stand and clamps.
Next we will add a fine layer of sand so that it fills the curved bottom section and forms a
level surface on top of the cotton ball. Add some solvent to clean the sand off the sides
by using a Pasteur pipette. We want all the sand to go at the bottom and not mix with our silica.
Make sure the sand level is as horizontal as possible. If it’s a little slanted,
you can just gently tap the side of the column like this.
Now we're going to prepare the gel by adding 25 milliliters of our solvent to the silica
powder. The solvent we’re using is also known as the eluent for column chromatography.
An appropriate solvent for column chromatography can be chosen by performing TLC on your mixture in
various solvents and combinations, such as hexanes and ethyl acetate. You can then choose one that
gives the best separation. Since the TLC plate also utilizes silica, just like in our column,
the separation pattern will therefore be the same. In today’s experiment, we’re using a 1:1
acetone/ethanol mixture as our solvent. Let’s add it to the silica powder. The size of your
column and volume of your mixture will determine how much silica and solvent you use. You need a
level tall enough to allow complete separation of your samples, and you should aim for this filling
up about two thirds of the column. Let’s stir the mixture with our rod until we get a nice slurry.
After we’ve made a slurry of our silica, we’ll add it to our column by using a glass funnel.
You want to keep mixing with a stir rod right up until you pour it in quickly like this.
Use more solvent to remove the rest of the silica from the beaker, and also to wash any gel off the
sides of the column. For reference, this is called wet loading. An alternative is dry loading which
consists of adding the dry powder to the column and then the solvent after. This technique can
cause a lot of bubbles that interfere with the separation, and tends to be less effective,
so wet loading is suggested. As you add the gel to your column, be sure to tap on the
column gently to make sure it stays flat on top, and to get rid of any bubbles or uneven packing.
After we've packed our column, we’ll open the stopcock to remove some of the solvent.
In column chromatography, it is important that you don’t let your column run dry,
meaning all of the gel must always be submerged in solvent. In other words, the solvent line must
always remain above the stationary phase. As you progress with the technique, you will always keep
an eye on the solvent line and add more solvent when necessary to keep the column wet. If it is
not wet, it can cause cracking of the silica which will cause unreliable separation. For right now,
let’s allow the solvent line to go down until it’s above the silica gel line like this.
We're now ready to load the sample. Today, we're separating a mixture of methyl red and bromocresol
green. You may know them as indicators that are used in titration, but if you look at
their structures, you can see that they have different functional groups, which allows them
to be separated based on their polarities. You can see that bromocresol green has many polar
groups and that makes it more polar than methyl red. We are using these chemicals today because
they are brightly colored, and we can therefore visualize them as they move through the column,
which will help us understand what is happening. This will usually not be the case, typically we
can’t see compounds as they move through the column, which makes things more challenging.
When adding your sample to the column, if you have isolated the organic products from
a chemical reaction to form an oil, we want to dissolve that oil in as small an amount of our
solvent as possible. This will allow us to load the sample as a thin band, which will result in
good separation. If we use to much solvent, the compounds will be very spread out, and it will
be harder to separate them. Today our mixture is already ready to load. To load the sample,
we have to be careful. We usually take it into a glass pipette and then lower the pipette as close
to the top of the column as possible, slowly letting it out to make sure that our layer is
even. We have to be gentle because we don’t want to disturb the silica layer. In this case, we’re
adding a quarter of a milliliter of a mixture of our two samples, and we can allow it to run down
part of the side of the column to load gently. After loading the sample, and verifying that
all of it has been adsorbed onto the column, we can add another layer of sand as a protective
layer. Let’s add about a centimeter. We can now fill up the column with our eluent.
We’re now ready to run our column and collect the fractions. Let’s open the stopcock. We don’t
have to start collecting fractions immediately as there is no way for our compounds to immediately
be exiting the column, but soon after beginning, you will want to begin collecting fractions in
numbered vessels. Typically these will be small Erlenmeyer flasks, but today we will be using
test tubes. Start collecting and just move to the next one whenever the current one is full,
or sooner if you think that a new substance is about to exit the column. It is very important
that you monitor the solvent level as you collect your samples. When you open the stopcock,
the solvent level will start to go down. Remember that we can’t let our column dry, so we have to
add more solvent as the level goes down. We also want to be careful when adding more solvent,
as we do not want to disturb the column. Add more before the level gets all the way down to the gel,
and add carefully, letting it run down the sides. Continuing, we can see that our mixture is
starting to separate into individual components. Again, since we have colored chemicals,
it will be easier to observe the separation. The dark green mixture we started with is separating
into blue and yellow colors; this is why we have to collect fractions. If we just collect these
in one container, they will mix together again. This way we isolate the components
in separate fractions. At first you will be collecting only solvent, but as you can see,
the yellow band is getting closer to the bottom. Keep adding the eluent and collecting
fractions until you have collected all your compounds. When the compounds are not visible,
you will have to perform TLC to find out what’s in each fraction. You can test many fractions on one
plate, and all the fractions that show data for a particular compound can be combined, and the
solvent evaporated, to give you a pure, isolated product. If you know for sure which compound you
want, you can dispose of the rest, but sometimes you will want to isolate two or more products.
There are times where rather than TLC, you will want to analyze the fractions by absorbance
spectroscopy, UV, or a colorimetric assay to figure out the identities of any compounds.
But in this particular case, we were able to isolate these compounds using just our eyes.
Column chromatography can get much more complex than this. In several forms of chromatography,
we use more than one solvent system, either in succession, or applied as a gradient,
usually increasing in polarity over time to push out substances that travelled more slowly.
Furthermore, the material in the column can be agarose, acrylamide,
or some other compound depending on what we’re separating. In the lab,
we can use columns to separate things such as peptides or drugs based on aspects such as size,
charge, polarity, or other affinities. The packing, mixing, and collection of these can
also be automated in large scale academic and industrial processes. A sophisticated example
of column chromatography is FPLC, or fast protein liquid chromatography, a technique that is used to
separate proteins. But for now, that concludes an introduction to column chromatography.