HPLC: Study with Video Lessons, Practice Problems & Examples

High Performance Liquid Chromatography (HPLC) is a powerful technique for separating molecules using high pressure and resolution. It includes two main types: normal phase HPLC, which separates polar molecules using a polar stationary phase and nonpolar mobile phase, and reverse phase HPLC, which does the opposite. The results are visualized in a chromatogram, plotting elution time against light absorbance, indicating the presence of separated proteins. Understanding these principles is crucial for effective protein analysis and purification in research and industry.

HPLC

In this video, we're going to 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. It uses automated computerized instrumentation for extremely effective separation of molecules. The way it achieves this effective separation of the molecules is by using a high-resolution column, which creates more interaction sites with the stationary phase. The more interaction sites there are, the greater the resolving and the separation power is going to be. Because the molecules encounter more interactions with the stationary phase, that actually slows the molecules down inside of the column. However, 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. 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. However, because it uses automated computerized instrumentation, HPLC is also an expensive technique to use, and that limits its use to some research labs. It turns out that there are actually two main types of HPLC: there is normal-phase HPLC, and there is also reverse-phase HPLC. In our next video, we're going to talk about normal-phase HPLC. So, I'll see you guys in that video.

HPLC

So in our last lesson video, we said that there are 2 main types of HPLC that we're going to talk about, and those are normal phase HPLC and reverse phase HPLC. In this video, we're going to focus on normal phase HPLC. 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. 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 longer, whereas the nonpolar molecules, on the other hand, are going to move through the column faster and they're going to elute earlier from the column.

Down below in our example of normal phase HPLC, you'll notice 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. 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. 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. Over here on the left, what we have is the input to the column. This is where the mixture of samples originally begins, is over here on the left. 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.

Specifically, for normal phase HPLC, it's the stationary phase that is polar, whereas the liquid mobile phase is the one that is nonpolar. 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. You can see here that the yellow proteins are our nonpolar proteins, whereas the blue proteins are our polar proteins. The ones in red here would be, like, the intermediate proteins. What you'll see here is that it's the nonpolar proteins that are going to elute first from our column.

Remember that we want to be using normal phase HPLC to separate out polar molecules, and the reason for that is that because the polar molecules stay in the column longer, they're going to have more interactions with the stationary phase and more interactions with the mobile phase. The more interactions you have, the better the separation is going to be. That concludes our lesson on normal phase HPLC. In our next video, we're going to 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.

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.

HPLC

So now that we've covered normal phase HPLC, in this video, we're going to focus on reverse phase HPLC. 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. With reverse phase HPLC, it's actually the stationary phase that is nonpolar this time. 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, meaning that the nonpolar molecules do not move through the column as fast. 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. In reverse phase HPLC, it's actually the liquid mobile phase that is polar. The liquid mobile phase is polar and we know that the mobile phase flows through the column quickly, flowing over the stationary phase. The result of reverse phase HPLC is that nonpolar molecules remain in the column longer, whereas the polar molecules that are more soluble interact with the mobile phase that moves quickly through the column, and so they get eluted faster and earlier from the column.

In our example of reverse phase HPLC below, you'll notice again we have a horizontal column instead of a vertical column, because with HPLC, there's a high amount of pressure applied to the mobile phase that pushes the mobile phase through the column. The mobile phase movement does not rely on gravity, but instead on the high pressure being applied to the column. Notice that on the far left over here, we have our mixed protein sample, and the mixed protein sample enters our input side over here on the left. 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 separated proteins can be collected. 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. 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. That's exactly what we see here. It's the polar proteins that are going to elute from the column first.

The nonpolar molecules, on the other hand, move through the column the slowest this time, and that's because the stationary phase that does not move is nonpolar. That means that the nonpolar proteins are going to interact with the nonpolar stationary phase via the hydrophobic effect of the column. They'll be the last to come out. You can see here how this is literally the reverse of normal phase HPLC. If you know normal phase HPLC, then you automatically know reverse phase HPLC because it's literally the reverse. This concludes our lesson on reverse phase HPLC. In our next video, we'll be able to get some practice. So, I'll see you guys there.

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.

What is the basis for the separation of proteins by the following techniques?

So this practice problem asks, what is the basis for the separation of proteins by the following techniques? So really, it's just asking what property of the protein is being used to separate them. And the techniques are gel filtration chromatography, affinity chromatography, ion exchange chromatography, chromatography, and reverse phase HPLC or high performance liquid chromatography. Recall that gel filtration size exclusion chromatography separates proteins based on their size. And so it's the largest proteins that actually elute from the column first. So it's a little bit counterintuitive. Now, for affinity chromatography, the property that's being used is the ligand binding affinities. So the ligand binding affinity is the property that is being used to separate the protein. And so, what that means is that only the proteins that have an affinity for the ligand will stay in the column. All of the other proteins that don't bind to the ligand are washed out of the column quickly. Now, for ion exchange chromatography, this focuses on the net charge of the protein. And so, for cation exchange chromatography, it's used to collect and purify positively charged proteins. But anion exchange chromatography is used to collect and purify negatively charged proteins. And the ones that we're trying to collect and purify are going to be the ones that are stuck and remain in the column the longest, whereas all of the other proteins elute the fastest. And so with both reverse phase HPLC and regular phase HPLC, it's actually the polarity of the proteins that is being used to separate them. And so with reverse phase HPLC, it's the nonpolar molecules that remain in the column and take the longest to elute, but with regular phase HPLC, it's the polar molecules that remain in the column and take the longest to elute. Essentially, these are the basis of separation for the proteins in the following techniques. And this concludes this practice problem, so I'll see you guys in our next video.

HPLC

So now that we've covered both normal phase and reverse phase HPLC, in this video, we're going to focus on an HPLC chromatogram. When proteins are separated via HPLC, the results of the protein separation can be plotted onto a data plot called a chromatogram. 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. The light absorbance is an indicator of the amount of the separated protein that is present. The greater the light absorbance, the more of that separated molecule is present. 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 the light absorbance on the y-axis. For the elution time, it increases from left to right. Shorter elution times mean that these molecules eluted earlier from the column, and longer elution times mean that these molecules eluted later from the column. With the light absorbance, it increases from the bottom to top, and greater light absorbance means that there are more of the molecule present, and lower light absorbance means that there's less of the molecule present. In this entire example over here, what we have is just the entire process for HPLC, just to help you guys understand HPLC a little bit better.

Over here on the far left, what we have is a flask that contains the mobile phase, the mobile phase reservoir. Notice that over here, what we have is a pump. This is a pump delivery system for the mobile phase. Really, it's this pump here that is the most expensive portion of the HPLC because it takes the mobile phase and pumps the mobile phase into our chromatogram column. It pumps the mobile phase at an incredibly high pressure. That is why we're able to achieve the high pressures due to this pump. Notice down over here, what we have is our mixed protein sample. This is the protein sample that we want to separate. We have a sample injector, which is able to take our mixed protein sample and inject it into our column. Notice up here what we have are these two columns. The first column represents our column at time 0, initially at the start of our HPLC. At the start of HPLC, our protein mixture is over here by the input, so our protein mixture is this black blob here. Over time, after about 10 minutes of pumping the mobile phase through our column at an incredibly high pressure, our proteins will begin to separate. They move towards the output, and as the proteins move towards the output, they can be detected by a detector. The detector here can translate the information being detected to a computer, and the computer can translate the information from the detector into a peak on a chromatogram.

Notice that it's the proteins that elute first from the column, like this yellow protein here, that are plotted onto the chromatogram first. Then the proteins that come out next, like the red protein, will be plotted onto the chromatogram next. The proteins that elute last from the column will be plotted onto the chromatogram on the far right because they take the longest amount of time to elute from the column. That is how we get our chromatogram. 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. This is also able to identify some types of modified amino acids. Notice here, we have CMC, which is a modified amino acid. It's a carboxymethylcysteine. Not that you guys need to know that, but just know that it's not just regular amino acids, it's also modified amino acids that can be detected. Over here, we have another modified amino acid that is methioninesulfoxide. All of these other one-letter codes are just the regular one-letter amino acids that we are familiar with already. 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.

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.

In the following HPLC chromatogram, which amino acid was the third substance eluted from the column?

• A. Aspartic acid.
• B. Leucine.
• C. Phenylalanine.
• D. Glutamic Acid.

Here’s what students ask on this topic:

What is High Performance Liquid Chromatography (HPLC) and how does it work?

High Performance Liquid Chromatography (HPLC) is a technique used to separate, identify, and quantify components in a mixture. It works by passing a liquid sample through a column packed with a stationary phase under high pressure. The stationary phase interacts with the sample components, causing them to separate based on their different affinities. The separated components are then detected and recorded as peaks on a chromatogram, which plots elution time against light absorbance. HPLC is highly effective due to its high resolution and the use of automated, computerized instrumentation.

What are the main differences between normal phase and reverse phase HPLC?

Normal phase HPLC uses a polar stationary phase and a nonpolar mobile phase, making it suitable for separating polar molecules. Polar molecules interact more with the stationary phase and elute slower, while nonpolar molecules elute faster. In contrast, reverse phase HPLC uses a nonpolar stationary phase and a polar mobile phase. Here, nonpolar molecules interact more with the stationary phase and elute slower, while polar molecules elute faster. Essentially, the polarities of the stationary and mobile phases are reversed between the two methods.

How is a chromatogram used in HPLC analysis?

A chromatogram is a data plot used in HPLC to visualize the separation of molecules. It plots elution time on the x-axis and light absorbance on the y-axis. Elution time indicates how long it takes for a molecule to pass through the column, while light absorbance reflects the amount of the molecule present. Peaks on the chromatogram represent different molecules, with their height indicating concentration. This allows researchers to identify and quantify the components in a sample.

Why is HPLC considered an expensive technique?

HPLC is considered expensive due to the sophisticated and automated computerized instrumentation it requires. The high-resolution columns, high-pressure pumps, and sensitive detectors contribute to the cost. Additionally, the need for high-purity solvents and regular maintenance further increases expenses. Despite the cost, HPLC's high efficiency, resolution, and speed make it the gold standard for many types of molecular separations in research and industry.

What types of molecules can be separated using HPLC?

HPLC can separate a wide range of molecules, including proteins, peptides, nucleic acids, small organic compounds, and pharmaceuticals. The choice between normal phase and reverse phase HPLC depends on the polarity of the molecules. Normal phase HPLC is ideal for polar molecules, while reverse phase HPLC is better suited for nonpolar molecules. This versatility makes HPLC a valuable tool in various fields such as biochemistry, pharmacology, and environmental science.