In this video, we're gonna begin talking about HPLC. So HPLC 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 more interaction sites with the stationary phase. And so the more interaction sites there are, the greater the resolving and the separation power is going to be. And so, because the molecules encounter more 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 HPLC the go to and the gold standard for separating most types of molecules. But, because it uses automated computerized instrumentation, that also makes HPLC an expensive technique to use, and that limits it to use for some research labs. And so it turns out that there are actually two main types of HPLC. There is normal phase HPLC, and then there's also reverse phase HPLC. And in our next video, we're gonna talk about normal phase HPLC. So I'll see you guys in that video.
2
concept
HPLC
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So in our last lesson video, we said that there are 2 main types of HPLC that we're gonna talk about, and those are normal phase HPLC and reverse phase HPLC. And in this video, we're gonna focus on normal phase HPLC. Now, normal phase HPLC 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 nonpolar. And so it's the polar molecules that are going to 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 going to move more slowly through the column and they're going to stay in the column column longer, whereas the non polar molecules, on the other hand, are going to move through the column faster and they're going to elute earlier from the column. And so down below in our example of normal phase HPLC, what you'll notice is that our column here is actually going horizontally. It's going side to side, which is different than our other column, chromatographies 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 HPLC, 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 HPLC, it's the stationary phase that is polar, whereas the liquid mobile phase is the one that is nonpolar. 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 nonpolar proteins that are going to move the fastest through the column and elute the earliest. And so you can see here that the yellow proteins are our nonpolar proteins, whereas the blue proteins are our 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 nonpolar proteins that are going to elute first from our column. And so remember that we want to be using normal phase HPLC 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 more interactions with the stationary phase and more interactions with the mobile phase. And so the more interactions you have, the better the separation is going to be. And so, that concludes our lesson on normal phase HPLC. And in our next video, we're gonna be able to get a little bit of practice before we talk about reverse phase HPLC. So I'll see you guys in that practice video.
3
Problem
Problem
What is the order of elution (first → last) of the following amino acids in normal-phase HPLC: Phe, Gly, Glu.
A
Phe → Gly → Glu.
B
Phe → Glu → Gly.
C
Glu → Phe → Gly.
D
Glu → Gly → Phe.
E
Gly → Phe → Glu.
F
Gly → Glu → Phe.
4
concept
HPLC
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4m
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So now that we've covered normal phase HPLC, in this video, we're going to focus on reverse phase HPLC. And reverse phase HPLC is really just the reverse of normal phase HPLC in terms of the polarities of the stationary phase and the mobile phase. And so with reverse phase HPLC, it's actually the stationary phase that is nonpolar this time. And we know that the stationary phase does not move, and so the stationary phase is immobile and the nonpolar stationary phase that's immobile actually immobilizes the nonpolar 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 nonpolar stationary phase interacts with the nonpolar molecules is via the hydrophobic effect, which, remember from our previous lesson videos, allows nonpolar molecules to clump and interact with each other. And so, in reverse phase HPLC, 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 HPLC is that non polar molecules remain in the column longer, whereas the polar molecules that are more soluble, they interact with the mobile phase that moves quickly through the column and so they get eluted faster and earlier from the column. And so in our example of reverse phase HPLC below, you'll notice again, we have a horizontal column instead of a vertical column, and that's because with HPLC, 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 column. 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, those, 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 HPLC, it's actually the stationary phase this time that is nonpolar, and it's actually the mobile phase that is polar this time. And so what this means is that polar molecules that are in blue here, 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 elute from the column first. And so, what you'll see is that it's the nonpolar 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 nonpolar, And so that means that the nonpolar proteins are gonna interact with the nonpolar stationary phase via the hydrophobic see 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 HPLC. And so if you know normal phase HPLC, then you automatically know reverse phase HPLC, because it's literally the reverse. And so this concludes our lesson on reverse phase HPLC. And in our next video, we'll be able to get some practice. So I'll see you guys there.
5
Problem
Problem
What is the order of elution (first → last) of the following amino acids in reverse-phase HPLC: Ala, Arg, Leu.
A
Leu → Ala → Arg.
B
Leu → Arg → Ala.
C
Arg → Leu → Ala.
D
Arg → Ala → Leu.
E
Ala → Leu → Arg.
F
Ala → Arg → Leu.
6
Problem
Problem
What is the basis for the separation of proteins by the following techniques?
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7
concept
HPLC
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So now that we've covered both normal phase and reverse phase HPLC, in this video, we're gonna focus on an HPLC chromatogram. Chromatogram. And so when proteins are separated via HPLC, the results of the protein separation can be plotted onto a data plot called a chromatogram. And so a chromatogram plots the elution time on the x axis, or the amount of time it takes for the separated molecule to elute from the column versus the light absorbance of each separated molecule on the y axis. And so the light absorbance is an indicator of the amount of the separated protein that is present. And so the greater the light absorbance is, the more 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 chromatogram where we have the elution time on the x axis and we have the light absorbance on the y axis. And so for the elution time, it increases from left to right. And so, shorter elution times means that these molecules eluted earlier from the column, and longer elution times mean that these molecules eluted later from the column. And with the light absorbance, it increases from, bottom to top, and so greater light absorbance means that there are more of the molecule present, and lower light absorbance 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 HPLC, just to help you guys, understand, HPLC 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, so what we have is the mobile phase reservoir. And, 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 of the HPLC, because what it does is it takes the mobile phase, and it pumps the mobile phase into our chromatogram, 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 0, so this is initially at the start of our HPLC. And notice that at the start of HPLC, our protein mixture is over here by the input, so our protein mixture is this 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 our proteins are separating as they move through this high resolution column. And so they move towards the output, and as the proteins move towards the output, they can be detected by a detector. And so, 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 chromatogram. And so notice that it's the proteins that elute first from the column, like this yellow protein here, that is plotted onto the chromatogram first. And then the proteins that come out next, like the red protein, will be plotted onto the chromatogram next. And then the proteins that elute last from the column will be plotted onto the chromatogram on the far right because they have, they take the longest amount of time to elute from the column. And so, that is how we get our chromatogram. And so, notice that the chromatogram 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 CMC, which is a modified amino acid. It's a modified cysteine, so it's a carboxymethylcysteine. Not that you guys need to know that, but to just know that it's not, just regular amino acids, it's also modified amino acids that can be detected. And over here, we have another modified amino acid that is a methioninesulfoxide. 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 eluted first from the column on the far left, and the ones on the far right are the ones that elute last from the column. And so, in our next video, we'll be able to get a little bit of practice with HPLC chromatograms. So I'll see you guys in that practice video.
8
Problem
Problem
In the following HPLC chromatogram, which amino acid was the third substance eluted from the column?