Have you ever
scribbled something
down with a black pen on
a piece of paper, then
accidentally smudged
the paper with water?
What do you remember seeing?
Well, what happens is that
you might see the black ink
start to smudge and start
to see some colors that
aren't black at all, maybe
some dark blues and some darker
purples.
But why is this?
This is because black
dye is actually made out
of a bunch of different other
dyes and different components.
And what you've just
witnessed is a basic example
of paper chromatography.
Chromatography involves
taking some kind of mixture
and using either solid or
liquid to separate it out
into its different parts.
There are many different
kinds of chromatography,
but they all rely on
having a mobile phase
and a stationary phase.
Let's go over how
paper chromatography
works, since this is
the simplest kind.
In paper chromatography, the
stationary phase is the paper.
So here's your piece of paper.
You can draw a little
line at the bottom
and draw a spot for
where you're putting
on your dot of your sample.
Next, you'll want
to prep a beaker,
or actually, any kind
of container will do.
But in this, you'll be
putting in your mobile phase.
And your mobile phase can
be some kind of solvent.
It could be water,
or it could be
any organic solvent you want.
Now that you have this, you
pour a small amount of solvent.
Again, not too much, because
you don't want this to already
be above the level of your spot,
or else it will just all bleed.
And now when you
put in your paper
into this container,
what you'll get
is something that
looks like this,
with the spot being around here.
And what you'll
observe over time
is that through
capillary action,
this pink solvent will
travel up the piece of paper.
And as it does that, it'll
actually take some of the dyes
from that green spot with it.
And let's see what happens at
a few different time points.
At the first time
point, you might
see that it separated
into two spots.
This implies that it's composed
of two different components.
However, if this were
a very complex mixture,
you could even see five,
six, or a lot of other spots.
If you wait even longer,
this is what you'll see next.
You'll see that the spots will
continue traveling even farther
up the plate, and the
separation between them,
that distance will
increase even more.
So ultimately what
you've shown here
is that whatever was in
the green spot originally
wasn't just one compound.
It was two compounds.
And why do they
separate in this manner?
Well, the blue spot
traveled farther.
That means that it was pretty
attracted to that pink solvent
that was traveling along.
Whereas the yellow spot
didn't move quite as much,
which means it
was more attracted
to the paper for the
stationary phase.
This competition between
the stationary phase
and the mobile phase
pulling at the components
is what drives the
separation that
occurs in all different
kinds of chromatography.
So let's try to lay this
information out in a table.
We've talked about how
for paper chromatography,
the stationary phase is
a solid; the mobile phase
is some kind of
solvent, so a liquid;
and they're separating
it based on polarity,
meaning how attracted it is to
the paper versus the solvent,
depending on its
chemical properties.
The next kind of
chromatography that's
almost identical to
paper chromatography
is known as thin-layer
chromatography,
or TLC for short.
We'll see that all the
spots on this table
are pretty much the same.
The main difference
is that instead
of having a piece of paper,
you have a glass slide that
is coated with a
layer of silica gel.
This is a great preparative
tool that is commonly
used in the organic
chemistry lab.
To remind you that these two are
related, what I've drawn here
is a little beaker with a
small either plate or piece
of paper inside.
And the arrow shows that
the solvent is traveling up
the plate through
capillary action.
The next kinds of chromatography
we'll be going over
are column chromatography.
And in this case,
you have a column
that I've drawn right
here on the left.
And you fill it with some
kind of packing material
and dump in some
solvent as well.
You can lower your sample
that you want to separate out,
and what you'll
find is that as you
keep dumping in
more solvent, this
can separate into bands that
represent different compounds,
and they will travel
down the column.
So in basic column
chromatography,
you're usually using
something like silica gel
as your stationary phase.
Your mobile phase is
typically an organic solvent,
and again, you're separating
based on polarity.
In size-exchange chromatography,
your stationary phase
is composed of beads.
However, these
little beads actually
have some holes in the middle.
And because of that, with
size-exchange chromatography
these beads completely
fill the column,
and tiny compounds can
get through that center
of the hole, like so.
But really big
compounds kind of have
to go around and go
between the beads.
So what happens here is
that really small compounds
travel pretty far, pretty fast,
whereas large compounds take
a longer time to
come out the bottom.
In ion-exchange
chromatography, the beads
that are filling this column
have some kind of group on them
that is charged.
Compounds that have
the same charge
will be repelled by
the column, meaning
they'll travel pretty quickly.
But compounds that
have an opposite charge
will bind tightly to
the column and will
be more reluctant to come
out since they are so
attracted to the
stationary phase.
Affinity chromatography
is also pretty similar,
but this usually relies upon
very specific interactions,
such as between an
enzyme and a substrate,
and really relies on
this binding affinity.
Things that will bind
tightly to the enzyme
will probably just be
primarily the substrate,
and everything else will
just get washed right
off the column.
Later on, what you
can do is wash out
the compound of interest
that was previously bound
to the column, using something
that that molecule is
even more attracted to.
With HPLC, HPLC stands for
high-performance liquid
chromatography,
formerly known as
high-pressure liquid
chromatography.
This is essentially the same as
the basic column chromatography
that you see there in yellow.
However, with HPLC, it's
a more advanced technique
in that you're working with
very, very small quantities,
and the detector in the
machine is much more sensitive.
The last kind of chromatography
is gas chromatography.
Now, this looks pretty different
compared to the others.
And in this case,
your stationary phase
is a liquid, while
your mobile phase
is some kind of carrier gas
that's passing over the liquid.
So what happens is,
you inject your sample,
and it travels in a
coil tube into that box
known as the gas chromatograph.
That's a fancy name
for the equipment
used to run gas chromatography.
Inside the chromatograph
is a heated chamber
through which an
inert gas flows.
Here, the sample vaporizes
and enters the gas flow
onto the column.
The things that are
the most volatile,
meaning have a
lower boiling point,
are able to travel faster,
whereas things with a higher
boiling point take
longer to come out.
And so this is a
separation method
that's great when you have
differences in boiling point.
So we've gone over all
these different kinds
of chromatography, but
you could certainly
go into each of these
in much more detail.
