Lesson Video: Chromatography Chemistry

Video Transcript

In this video, we will learn about chromatography, a technique that’s used to separate substances in a mixture. We’ll learn the theory behind chromatography, how to set up an experiment, and how to interpret the results of a chromatography experiment.

Perhaps you’ve performed an experiment like this or seen it performed. You take a pen or a marker, place a spot of ink on a piece of paper or a coffee filter, then you place that piece of paper in a cup of water and sit back and watch. You’ll see the water slowly climb up the piece of paper, and the dot of ink might move as the water moves up the piece of paper. But as the water continue to move up the paper, the ink will begin to separate. Eventually, the water will travel all the way up the paper, and all of the components that made up the ink will be separated.

From this experiment, we can see that the ink in the marker wasn’t made of just one color. Rather, it’s made of a mixture of different pigments, and this experiment was able to separate them out. This experimental technique is called chromatography. It’s quite simple in its setup, but it’s very effective at separating out these different components in the ink of a marker. So how does it do this? Let’s break this experiment down. There’s two main components to this experiment — well, three, if you count your analyte, that is, the substance that you’re trying to separate.

The first is called the stationary phase which, in this case, is the paper. The second is the mobile phase which, in this case, was the water. You can remember which one is which because the stationary phase, the paper, doesn’t move during the experiment. And the mobile phase, the water, moved up the stationary phase during the experiment, so it was mobile. So how does the paper and water separate the ink in the marker? Well, when we first write on the paper with the marker, the pigment particles in the ink become absorbed on the paper. This is an attraction between the pigment particles that make up the ink and the paper. Now this attraction isn’t a permanent attraction, so that means when we put the paper in water and the water starts to travel up the paper, some of the pigment particles will start to go into the solution.

Now, because the molecules that make up these pigments are different, they’re going to be attracted to the paper differently. Some will be more attracted to the paper than others because of these differences in attraction between the pigment molecules and the paper. As the water moves up the paper and some of the pigment molecules become dissolved in the water, they will move at different rates. Pigment molecules that are less attracted to the paper will dissolve in the water more readily. This means that they will travel with the water quickly, so they’ll move further up the paper. Those that are more attracted to the paper will move more slowly because they’re stuck to the paper, so they won’t travel as far.

How easily the molecule dissolves in water also has an effect. If it dissolves in water easily, then it can travel with the water quickly and it will move up the paper further. But if it doesn’t dissolve in water easily, it will take time for it to dissolve, meaning that it won’t travel as quickly with the water, and so it won’t move as far off the paper. The combination of these two effects, the attraction of the molecules to the paper or the stationary phase and the attraction of the molecules to the water or the mobile phase, is what causes chromatography to be able to separate out the components of a mixture.

If the component in the mixture is either more attracted to the stationary phase or less attracted to the mobile phase, it will move very slowly up the stationary phase, so it won’t travel very far. It will stay at the bottom. But if the component is either less attracted to the stationary phase or it dissolves more easily in the mobile phase, that will cause it to move quickly up the stationary phase, so it will travel farther and end up near the top of the stationary phase at the end of the experiment.

Now, the paper-and-water chromatography experiment was able to separate out ink in a marker really well. But there’s other chromatography techniques that are used in a chemistry lab. One of the most commonly used techniques is called thin layer chromatography, or TLC. It’s quick, easy to use, and very similar in setup to the paper chromatography that we just looked at. Because TLC gives such great visual results, it’s often used for things like seeing if a product in your experiment has formed yet and determining what amino acids might have been in a protein sample that was broken apart.

Now, there are other chromatography techniques that use the same basic theory, like gas chromatography, column chromatography, and HPLC that can be used to physically separate the components in a mixture. But they aren’t the subject of this video, so we won’t be going over them. We’ll just focus on TLC. Thin layer chromatography is performed very similarly to paper chromatography. This time, instead of paper, our stationary phase will be what’s called a TLC plate. The TLC plate is just a plastic or glass plate that’s coated in a thin layer of silica gel.

To perform the experiment, we spot a small amount of our sample onto the plate, and then we’ll draw a line on the plate to indicate where the samples started off. We’ll want to make sure that this line is in pencil since, as we just learned, any pen that we use will be separated by the chromatography experiment, so it won’t ultimately be useful for marking where we started off. Then we’ll want to place the TLC plate into a solvent, which will be acting as the mobile phase. Sometimes the solvent will be water, but frequently it’s a mixture of organic solvents. We’ll also want to make sure that the solvent is below the line that we’ve drawn so that the sample doesn’t immediately dissolve into the solvent when we first put the plate in.

We’ll also want to cover the beaker so that the solvent doesn’t evaporate, since most of the solvents used in TLC experiments evaporate quite easily. Then we’ll let the plate develop so that the sample can separate. We’ll want to stop the experiment and pull the plate out of the solvent before the mobile phase reaches the end of the plate. Otherwise, the separation will not be as good and the different components might start to run into each other. Once we remove the TLC plate from the solvent, we’ll want to mark where the solvent ended up. We don’t want to delay in doing this too long because eventually the solvent will evaporate, and we won’t be able to see where it ended up.

Sometimes your sample won’t have colored components in it like if we’re performing a chromatography experiment with amino acids. In this case, we can use a chemical called ninhydrin to make the spots visible. The ninhydrin reacts with the different amino acids differently, so we’ll end up with visible spots on the TLC plate. We can also use iodine vapor, which can stain the spots on our TLC plate and make them visible for us.

So what can we do with the developed TLC plate? Well, we can use our results to identify what components were in the mixture that we separated. One quantitative way that we can do this is by calculating the R f value, which is the distance that each spot reached divided by the distance that the mobile phase traveled. So, for example, let’s say that from where we spotted our sample to where the mobile phase reached was 10 centimeters. The distance from where we spotted our sample to where the blue spot on the bottom reached was two centimeters. And the distance from where we spotted our sample to where the yellow spot on the top reached was eight centimeters.

For this spot on the bottom, the R f value would be the distance that spot traveled, which was two centimeters, divided by the total distance the mobile phase traveled, which was 10 centimeters. This would give us an R f value of 0.2. For this yellow spot on the top, the R f value would be the distance that that spot traveled, which was eight centimeters, again divided by the distance that the mobile phase traveled, which was 10 centimeters. So the R f value would be 0.8. We can look up R f values for various substances in tables, so we can use this to identify what components were in our mixture.

There’s another thing that we can do that’s more qualitative. Let’s say we know that the sample we’re starting off with contains one of four amino acids. What we could do is spot not only our sample but also each of the four amino acids that we know that it could be when we’re setting up our experiment. That way, after we’ve developed our plate, we can just visually examine it to identify which amino acid we had. Here, we can see that our sample ended up at the same exact level as amino acid number three. So our sample contained amino acid three. Of course, if we didn’t have amino acids for reference, we could still calculate the R f value for our sample and identify it that way.

So that’s everything we need to know about setting up a chromatography experiment, why chromatography experiments work, and what to do with the results of chromatography experiments. So let’s try some practice problems.

Chromatography usually involves two different phases. What names are given to these two phases?

Chromatography is an experimental technique that we can use to separate components of a mixture. To perform a chromatography experiment, we place our sample on the stationary phase, which could be something like a piece of paper or a TLC plate or something more complicated for other forms of chromatography. Then, we place our sample that’s on the stationary phase in a container filled with the mobile phase, which can be water or some other kind of solvent. As the mobile phase moves up the stationary phase, interactions between the sample in the stationary phase and the sample in the mobile phase will cause the sample to be separated into the components that make it up.

So the names of the two phases involved in chromatography experiments are mobile and stationary.

The given chromatogram shows that substance B has traveled further up the chromatography paper than substance A. What properties of a substance affect how far it will travel up the chromatography paper?

When we perform a chromatography experiment, the first thing that we do is place our samples on the chromatography paper. The particles in the sample get adsorbed to the chromatography paper, which means they get stuck to it. But this adsorption is not permanent. When we place the paper in the solvent, which could be water or something else, the solvent will begin to travel up the paper. As the solvent travels up the paper, some of the particles in the sample will dissolve in the solvent and travel up the paper with the solvent.

But what if the substance doesn’t dissolve very well? Well, that means that it won’t travel as far with the solvent as the solvent moves up the paper. So it seems that one factor that definitely affects how far a substance will travel up the paper is the solubility of the substance in the solvent. If it doesn’t dissolve in the solvent very well, it won’t be able to travel as far. And if it dissolves really easily, it will travel with the solvent as it moves up the paper very well.

Now, the other thing that’s going on besides how easily the substance dissolves in the solvent is how attracted the substance is to the paper. After all, adsorption is an attraction to the paper. The substance is stuck to it. So if the substance is really attracted to the paper, it won’t be able to move as far as the solvent travels up the chromatography paper. And if the substance is not very attracted to the paper, it will move more easily with the solvent. So the other property of a substance that affects how far it will move up the paper is how attracted it is to the paper. So knowing this, we can tell that substance B either dissolves more easily in the solvent or it’s less attracted to the paper than substance A.

Now it’s time to wrap this video up with the key points for this lesson. Chromatography is an experimental technique that we can use to separate substances in a mixture. Chromatography experiments consist of two phases: the stationary phase, which can be paper or a TLC plate, and the mobile phase, which is a solvent that travels up the stationary phase. The separation that we get of the different substances depends on the attraction between the substance and both the stationary phase and the mobile phase. One quantitative thing that we can do to help us identify the substances that we separate in chromatography experiments is calculate an R f value for each substance by taking the distance that the substance traveled divided by the distance that the mobile phase traveled.