in this video, we're gonna begin talking about H PLC, so H P. L. C is actually an acronym for high performance liquid chromatography. And it's a type of column chromatography that separates molecules in a column using an immensely high amount of pressure and resolution. And it uses automated computerized instrumentation for extremely effective separation of molecules. And the way that it gets this effective separation of the molecules is by using a high resolution column. And the high resolution column creates mawr interaction sites with the stationary phase. And so the mawr interaction sites there are, the greater the resolving in the separation power is going to be. And so because the molecules encounter mawr interactions with the stationary phase that actually slows the molecules down inside of the column. But the high amount of pressure that's applied to the column will actually increase the speed of the separation through the high resolution matrix in the column. And so what that means is that we get incredibly effective separation of the molecules at incredibly high speeds, and that makes H PLC the go to and the gold standard for separating most types of molecules. But because it uses automated computerized instrumentation that also makes H PLC an expensive technique to use and that limited to use for some research labs. And so it turns out that there are actually two main types of H p L C. There is normal Phase H PLC. And then there's also reverse phase H p l C. And in our next video, we're gonna talk about normal Phase H PLC, so I'll see you guys in that video.

So in our last lesson video, we said that there are two main types of h p l c that we're gonna talk about. And those are normal Phase H PLC and reverse phase of H PLC. And in this video, we're gonna focus on normal phase H P L C. Now normal Phase H PLC is specifically used to purify polar molecules, and the reason for that is because the stationary phase that's packed inside of the column is polar, whereas the liquid mobile phase that's used is non polar. And so it's the polar molecules that are gonna interact with the polar stationary phase. And if the polar molecules are interacting with the stationary phase, which remember does not move, then the polar molecules are gonna move mawr slowly through the column, and they're going to stay in the column longer, whereas the non polar molecules, on the other hand, are going to move through the column faster and they're going to dilute earlier from the column and so down below. In our example of normal Phase, H PLC, which will notice is that our column here is actually going horizontally. It's going side to side, which is different than our other column chromatography is that we talked about. And the reason for that is because it's really the high amount of pressure that moves the mobile phase through the column, and it doesn't really rely on gravity. It relies on the high amount of pressure that's applied to the column, and so notice that what we have over here on the far left is our mixed protein sample. So we have a mixture of proteins that we want to separate, and over here on the left, what we have is the input to the column. So this is where the mixture of samples originally begins is over here on the left. And when we start H PLC, the proteins are going to begin to separate through this high resolution matrix in the column, and they're going to make their way through the column till they get to the output on the right, where the proteins can be collected as they're separated. And so specifically for normal Phase H PLC. It's the stationary phase that is polar, whereas the liquid mobile phase is the one that is non polar. And so what this means again is that it's the polar proteins here that are going to interact with the polar stationary phase. And that means that these polar molecules are going to move more slowly through the column and they're going to stay in the column longer, whereas it's the non polar proteins that are going to move the fastest through the column and dilute the earliest. And so you can see here that the yellow proteins are non polar proteins, whereas the blue proteins are polar proteins and the ones in red here would be like the intermediate proteins. And so what you'll see here is that it's the non polar proteins that are going to allude first from our column. And so remember that we want to be using normal phase H p l C to separate out polar molecules. And the reason for that is because because the polar molecules stay in the column longer, they're gonna have mawr interactions with the stationary phase and mawr interactions with the mobile phase. And so the more interactions you have, the better the separation is going to be. And so, uh, that concludes our lesson on normal Phase H PLC. And in our next video, we're gonna be able to get a little bit of practice before we talk about reverse Phase H PLC. So I'll see you guys in that practice video.

So now that we've covered normal Phase H PLC in this video, we're gonna focus on reverse Phase H PLC. And reverse Phase H PLC is really just the reverse of normal phase H PLC in terms of the polarities of the stationary phase and the mobile phase. And so with reverse Phase H PLC, it's actually the stationary phase that is non polar this time, and we know that the stationary phase does not move. And so the stationary phase is immobile, and the non polar stationary phase that's in mobile actually immobilizes the non polar molecules inside of the column. And that means that the non polar molecules do not move through the column as fast. And so the way that the non polar stationary phase interacts with the non polar molecules is via the hydrophobic effect, which remember from our previous lesson. Videos allows non polar molecules to clump and interact with each other. And so, in reverse Phase H PLC, it's actually the liquid mobile phase that is polar, and so the liquid mobile phase is polar, and we know that the mobile phase flows through the column quickly and it flows over the stationary phase And so the result of reverse Phase H PLC is that non polar molecules remained in the column longer, whereas the polar molecules that arm or solid soluble, they interact with the mobile phase that moves quickly through the column. And so they get eluded faster and earlier from the column. And so, in our example of reverse Phase H PLC below, you'll notice again we have a horizontal column instead of a vertical column. And that's because with H P L. C. There's a high amount of pressure that's applied to the mobile phase that pushes the mobile phase through the column so it doesn't. The mobile phase movement doesn't rely on gravity. It relies on high pressure that's being applied to the count. And so notice that on the far left over here, what we have is our mixed protein sample, and the mixed protein sample enters our input side over here, which is on the left. And so we have our mixed sample on the left, and as the sample moves through this high resolution column, the proteins begin to separate until they get to the output side. Over here on the right, where the proteins the separated proteins can be collected. And so again, with reverse Phase H PLC. It's actually the stationary phase this time that is non polar, and it's actually the mobile phase that is polar this time. And so what this means is that polar molecules that air in blue here, polar molecules or polar proteins are going to interact with the polar mobile phase and flow out of the column the fastest. And so that's exactly what we see here. It's the polar proteins that are going to allude from the column first, and so what you'll see is that it's the non polar molecules, on the other hand, that move through the column the slowest this time. And that's because the stationary phase that does not move is non polar. And so that means that the non polar proteins are gonna interact with the non polar stationary phase via the hydrophobic effect. And that's going to slow them down in the column and allow the non polar molecules to elude the last from the column. They'll be the last to come out, and so you can see here how this is literally the reverse of normal Phase H PLC and So if you know normal phase H p l C, then you automatically know reverse Phase H PLC because it's literally the reverse. And so this concludes our lesson on reverse Phase H PLC and in our next video will be able to get some practice, so I'll see you guys there.

So now that we've covered both normal phase and reverse Phase H PLC and this video, we're going to focus on an H PLC chromatograph AM. And so when proteins air separated via H PLC, the results of the protein separation can be plotted onto a data plot called a crime Mata Graham. And so a chromatic Graham plots the illusion time on the X axis or the amount of time it takes for the separated molecule to dilute from the column versus the light, absorb INTs of each separated molecule on the Y axis. And so the light absorb INTs is an indicator of the amount of the separated protein that is present. And so the greater the light absorbent is the mawr of that separated molecule that's present. And so if we take a look at our example down below on the right side over here, notice What we have is a chromatic graham, where we have the illusion time on the X axis and we have the light absorb INTs on the Y axis, and so, for the illusion time it increases from left to right and so shorter illusion times means that these molecules alluded earlier from the column and longer illusion times mean that these molecules alluded later from the column and with the light absorbent that increases from bottom to top. And so greater light absorbent means that their arm or of the molecule present and lower light absorbent means that there's less of the molecule that's present. And so, in this entire example, over here, what we have is just the entire process for H p l C. Just to help you guys, uh, understand h p l c just a little bit better. And so over here on the far left, what we have is a flask that contains the mobile phase and eso What we have is the mobile phase reservoir, and, uh, notice that over here what we have is a pump. And so this is a pump delivery system for the mobile phase. And so really, it's this pump here that is the most expensive portion off the H p l C. Because what it does is it takes the mobile phase, and it pumps the mobile phase into our chromatograph, um, column. And so it pumps the mobile phase at an incredibly high pressure. And so that is why we're able to get the high pressures due to this pump. And so notice down Over here, what we have is our mixed protein sample. So this is the protein sample that we want to separate. And we have a sample injector which is able to take our mixed protein sample and inject it into our column. And so notice up here. What we have are these two columns. The first column represents our column at time. Zero. So this is initially at the start of our H PLC and noticed that at the start of HP LCR protein mixture is over here by the inputs are protein mixtures is black blob here and over time. So after about 10 minutes of pumping the mobile phase through our column at an incredibly high pressure, what will happen is our proteins will begin to separate, so we can see that are proteins are separating as they move through this high resolution column. And so they moved towards the output. And as the proteins moved towards the output, they can be detected by a detector. And so, uh, the detector here can translate the information that's being detected to a computer and the computer can translate the information from the detector into a peak on a chromatic graham. And so notice that it's the proteins that dilute first from the column, like this yellow protein here that is plotted onto the chromatic graham first. And then the protein that come out next, like the red protein will be plotted onto the chromatograph next. And then the proteins that dilute last from the column will be plotted onto the chromatograph on the far right because they have. They take the longest amount of time to allude from the column. And so, uh, that is how we get our chromatograph, Um, and so notice that the chromatic Graham has a bunch of these different peaks. And at the top of the peaks we have the amino acid, one letter code that is being identified. And so this is also able to identify some types of modified amino acids. So notice here we have C M. C, which is a modified amino acid. It's a modified Sistine, so it's a car boxy metal Sistine. Not that you guys need to know that, but to just know that it's not, um, just regular amino acids It's also modified amino acids that could be detected. And over here we have another modified amino acid that is a meth I inning sull, Fox Side. And so all of these other one letter codes are just the regular one letter amino acids that we are familiar with already. And so it's these amino acids that eluded first from the column on the far left, and the ones on the far right are the ones that elude last from the column. And so in our next video will be able to get a little bit of practice with H P L C chromatograph, Um, so I'll see you guys in that practice video.