Hi, everyone, my name is Dr. Meredith Herb and I'm with Miami University's Chemistry and Biochemistry Department. And today we're going to talk about thin layer chromatography or TLC. The purpose of our experiment today is going to be to learn about TLC. And then to use TLC to understand the polarity of different solutes. And how solvents can impact our results. For our procedure, we're going to squat two TLC plates with two different compounds, and then run those plates in two different solvent systems, one, which is going to be nonpolar and the other, which is a little bit more polar. And we're going to look at what types of results we get from those different solvent systems. And then we're going to analyze our results using UV light. So what exactly is thin layer chromatography, thin layer chromatography utilizes a small plate, in which there's a stationary phase kind of molded onto the plate, the stationary phase is usually polar compounds like silica, and then we use a mobile phase as well, the mobile phase is different types of solvent systems. So our stationary plate is put into a developing chamber, and the mobile phase is allowed to move up the stationary phase. And we look at how the different compounds that we are interested in interact with both the stationary phase and the mobile phase. So the key idea behind thin layer chromatography is that the different polarities of molecules and their intermolecular forces with one another have a big impact in the results that we are going to observe. polarity is the idea that different atoms have different electronegativities. And so a more electronegative atom will pull electron density towards itself, creating a dipole moment, whenever you have dipole moments, whether they're temporary or permanent, you can get intermolecular forces and intermolecular forces arise when you have two different molecules with electrostatic interactions, in which they're kind of being attracted to one another. three key types of intermolecular forces, dispersion forces, dipole dipole moments, sorry, dipole dipole interactions, and hydrogen bonding, which is the strongest intermolecular force. We're going to analyze our results like I mentioned using UV light. And then we are going to calculate what's called an RF and RF gives us an idea of how far a spot or a solar has traveled up the TLC plate. So it helps us to understand the interactions of the different compounds are interested in and how they interact with both the stationary phase and the mobile phase of a TLC. So our experiment is going to be looking at two compounds, asset analyst and employee acid. And both of these compounds are capable of hydrogen bonding. We're going to compare both of these molecules in two different solvent systems, one and at 20 hexane ethyl acetate mixture, this is going to be our more nonpolar solvent system because hexane only have dispersion forces, whereas ethyl acetate has those dipole dipole interactions. And the second solvent system is a 5050 mixture of hexane and ethyl acetate. And so this second system is more polar because we have more ethyl acetate present. We're going to look at the different RFI use and use those to kind of standardize our results and compare the two molecules to one another. So our key question is, does a high RF value equate to a polar or nonpolar molecule, so take a minute to think about it, and then I'll show you guys how to do the TLC experiment.
One of the key components of thin layer chromatography is the TLC or thin layer chromatography plate. The TLC plate has two sides. The front side is made up of silica gel, which is a pretty polar stationary phase. The backside is plastic, the plastic acts as a backing, and it kind of holds the silica gel into place. So when we do our experiments, we're going to be focusing on the silica gel side.
To prepare your TLC plate, you're going to always use pencil. We don't want to use pin on the TLC plate, because pin ink is made up of organic compounds that can dissolve in the organic solvents that we're going to be using to develop our plates. So at least use pencil. One of the first things that you're going to do to prepare your plate is to draw a line across the bottom of it. It's important that the line is not right on the very bottom of the plate, we want it to be a little bit of the ways up so that we are not going to put our plate into the developing chamber and have the solvent be above the line that we draw. I'm going to go ahead and draw a line Here's my line, make sure that you draw it very likely, you don't want to dig the pencil into the silica gel because it'll break off. However many spots or lanes, you're going to run in your TLC plate, usually make a little mark on the line to indicate where you're going to start your compounds at. I'll go ahead and do that. So you can see I've made two marks in my TLC plate, we're going a little closer. And those two marks are where I'm going to spot compound before putting my TLC plate into the developing chain.
So I'm going to show you guys how to go ahead and spot your TLC plates, you're gonna want to spot two TLC plates. One is for the first TLC that you're going to run in an ad hexane 20, ethyl acetate, mobile phase, and then the second TLC plate is going to be for a 50, hexane 50 ethyl acetate mixture for your mobile phase. So it's good to just go ahead and spot them right away at the beginning. The spotters that you're going to use for TLC are little pieces of glass too, that have been elongated, so that they have a very fine point. When we spot a TLC, we don't want to have a huge spot, we want to try and faintly spot our TLC. Okay, so I'm going to just dab the spotter into the standard. And I'm going to place the acid analysts on the left hand side.
And all you have to do just kind of spot it lightly. And you can kind of see it on the TLC plate, I'm going to do the same thing on the left hand side of the second plate. And you want to make sure that you don't cross contaminate, so only use one stopper for one compound. Next, I'm going to spot the employee with acid and that's going to be on the right side of each plate. So I'm going to use now a second spotter. Dip it in, and then gently spot. The second side spotter doesn't have a very good opening, so I'm gonna try a second one here. There we go. We spot you should be able to see the compound briefly when you spot it on the plate. Right. So now we have spotted both of our TLC plates as an analogue on the left employee look acid on the right. And so now the TLC plates themselves are ready to be run in the developing chamber. If you want to check and make sure that you have indeed spotted your compound heavily enough on your TLC plate, all you have to do is come over here to this UV lamp, we're going to use the shortwave UV light, and we can put our plate underneath and you can see our compounds are both on the plate they're showing up as purple. Okay, so that indicates that we definitely do have our compounds on here, and that we can then ensure that we're ready for the TLC. UV light works best for visualizing compounds that are conjugated, which means they have alternating pi bonds. So the alternating pi bonds allows them to absorb UV light, and thus we can see them using the UV lamp.
In the lab manual for part one, it talks about you running two TLC plates, you don't necessarily have to run them separately, you can run them both at the same time. If you want to save time, just have to be careful not to get things mixed up. You can use different size beakers and things like that, so that you can have two developing chamber setup. For the first TLC plate. We're using a mixture of hexane and ethyl acetate which are both available in your hood, you're going to use 80%, hexane and 20% ethyl acetate, so you can make the solution yourself. I made eight mils of hexane and combine that with two mils of ethyl acetate to get my 8020 mixture. The second TLC plate is going to be run in a 5050 mixture of the same two solvents. So to make my developing solvent here the mobile phase, I used five mils of hexane and five mils of ethyl acetate and mix them together. For our developing chamber, you can use any beaker and you're going to add about five mils or so into the bottom of the developing chamber. So I'm going to go ahead and do that. So here's the developing chamber for the 8020 hexane ethyl acetate Adding a little bit more than five mils, it's fine. And then we're also going to use a wick in our developing chamber. So it's just a piece of filter paper folded in half. And we're going to set that inside of our TLC developing chamber, you're going to try and adhere it to the sides, kind of like this. And the wick is going to help with the movement of the solvent up the TLC plate. So once we have the wick ready, we can go ahead and add our TLC plate, I'm going to use tweezers to do this. When you add your TLC plate to the developing chamber, you want to make sure that you try that add it in as evenly as possible so that the solvent front is even across the bottom. And you also want to make sure that the sides of the beaker are not touching the sides of the plate. Okay, so I'm going to go ahead and gently add this in a little rest up against the side of the plate. And then I'm going to gently put a piece of tin foil over the top of it. Right, so I'm going to leave that developing chamber right there. Next, I'm going to prep my 5050 mixture developing chamber, I'm going to pour some of the solvent in going to add my wick, we're going to use the tweezers again to try to as gently as possible put the TLC plate in. We're also wanting again to be even. Right, so that's in as well. Once we have our two plates developing, we're going to watch them and you'll be able to see the solid front moving up the TLC plate, we want to take the plate out of the developing chamber when the solvent front gets to about you know a little bit at the end of the TLC plate. So I'll try to bring the camera in closer to show you that when we get there, it usually takes several minutes for the solvent side to work its way up the TLC plate, and you can usually see it pretty well. So I'll bring the camera closer to give you guys a little bit better idea of what it looks like inside the developing chamber. But a lot of times this is just kind of a spot where you have to wait or work on prepping the next step of your experiment. While you're developing your TLC plate in the developing chamber, here is our 80, hexane 20, ethyl acetate plate and then chamber. Again, note that the back is up against the glass side of the beaker. None of the sides are touching the width or the sides of the beaker either. You can see the developing chamber right here. And you can see the solid front, working his way up the plate. For the 5050 hexane ethyl acetate, we can also see our plate in there, this one's a little bit harder to see just because of the angle. And again, we're looking for the solvent front to get just to the top of the plate, not all the way over the plate, but with a little bit of room left behind.
And when that happens, we're gonna pull our TLC plate out and immediately mark it with a pencil. It's really important to mark where your solvent front was at the end of the TLC so that you can then calculate the RF. And I'll show you guys how to do that when these are ready. Alright, so it looks like the 8020 hexane plate is ready to come out. The solvent front is just a little bit below the top of the plate. So I'm going to go ahead and pull it out. And right away, I'm going to mark that solid front where is the line across the top of the plate. Again, this is really important to do so that we can accurately calculate the RS. So you can kind of see here, I've got my solvent front marked, and then we're gonna wait for the TLC plate to dry and then we can go visualize it using the UV. Alright, I've taken the lid off of the 5050 hexane ethyl acetate mixture just to kind of show you the solvent front approaching the top of this TLC plate. I might let it go just a little bit longer before I take it out, but just an idea of the solvent dries what it looks like, and how you know when it's ready to take it out. So I might let it go just a tiny bit longer, but then I'm going to take it out and mark it and then we will go visualize this with the UV Alright, here is the 5050 plate, and I'm going to take it out and right away mark the solvent front pencil. And you can see the solvent front didn't quite get as high in this case, which is fine. That'll be corrected for when we calculate the RF of the spots. So let's go ahead and visualize our two TLC plates from part one. First, we're gonna look at the 8020 plate. And you can see that there are two spots on this plate. One is right here, it's tailing just a little bit, but I'm going to circle the densest part. And then the other is kind of down here close to the baseline. So when you look at this plate in the TLC, you always want to circle all of the spots that you see. For this case, I only see two spots. And that should make sense because we started with two pure compounds one spot for each compound night. So this is the 8020 TLC plate that you'll want to draw in your lab manual. I'll show it a little bit later to I'll draw it out as well and give you guys the RF values so I can calculate. And here is the 5050 mixture, the two spots are still present. Okay, well, there's one right here. And then there's one right here.
Notice that they've moved a lot farther up on the plate compared to the 8020 mixture. Okay, so the distance traveled has increased. And that's going to be significant when we go to analyze the results from this experiment. To calculate the RF of the spots from our two TLC plates, you're going to get out the ruler from your lab door. And we're going to use this guide in our lab manual. The first thing that you're going to do is you're going to measure the solvent front, which is the first line that you drew where we spotted our sample, all the way to the second line that we drew where the solvent was at when we pulled it out of the developing chamber. For our 8020 plate, the solvent front was six centimeters. The next thing you're going to do is you're going to measure where the spots are at. So my first thought the asset amyloid had a distance traveled of 0.3 centimeters, so it didn't move very much. A in the second spot, the employee with acid moved further and it finished at 1.0 centimeters. This second plate, which was the 5050, and a shorter solvent front, okay, so it was about five centimeters. So the five centimeters goes on the denominator, and then our acid amyloid traveled 1.5 centimeters, while our m totally like acid traveled 3.7 centimeters. So you can go ahead and use these numbers to calculate the RF values for our acid annalynne and our m tall you have acid.
Here the TLC results of our experiment, we have our two plates and the spots that we saw in the UV light, and the 8020 mixture of hexane and ethyl acetate, we can see that acid and lead had an RS of 0.05, which is quite low. And until you look acid had an RMS of 0.17. Next in the 5050 mixture, we see that acid and live had a 0.3 rF whereas acid had a 0.7 for Rs value. So what conclusions can we draw from our data. So our results clearly show that the acid anlin and ammend was an ns two h Bond had lower RF values compared to the inventory with acid which had an O h group in it. Generally, a molecule with a lower RF is more polar compared to a molecule with a higher RF value, especially when the stationary phase of the TLC plate is quite polar like ours is which is silica again, the lower RF value is indicating that the molecule interacted more with the polar stationary phase of the TLC plate. So acid in lead was interacting with the stationary phase instead of with the mobile phase, which was moving up the plate, whereas the inventory like acid dissolves more into the mobile phase, and therefore it moved up the plate more. As we increase the polarity of our mobile phase, going from 8020 to 6050. We saw that both molecules moved up the plate more indicating that they reacted and interacted more with the stationary phase as we increase the polarity. So overall, because as Alan had the lower RF value, we can conclude that it is more polar than that until you have acid was. I hope you guys enjoyed our experiment. If you have any questions, please feel free to look me up on the Chemistry and Biochemistry department's website.