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  • 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