Size Exclusion Chromatography - Online Tutor, Practice Problems & Exam Prep

Size exclusion chromatography, also known as gel filtration chromatography, purifies proteins based on size using porous beads in a column. Larger proteins elute first because they cannot enter the beads' cavities, taking a shorter path. In contrast, smaller proteins enter the pores, slowing their movement. This technique allows for the separation of proteins and can also determine the size of unknown proteins. Understanding this method is crucial for applications in proteomics and protein analysis.

Size-Exclusion Chromatography

In this video, we're going to talk about size exclusion chromatography. Size exclusion chromatography is another type of column chromatography used to purify a protein based on its size, of course. It is also known as gel filtration chromatography, and the reason for that is because the material, the stationary phase inside of the column, is made up of a gel, and it's used to filter proteins based on their size. We haven't yet talked about the technique gel electrophoresis. We will a little later in our course. But most of you have covered gel electrophoresis in your previous courses, so you might have a preconceived idea of how molecules are supposed to move through a gel. However, I'm here to tell you that the way that molecules move through a gel in gel electrophoresis is a lot different than the way that molecules move through a gel in gel filtration chromatography. Contrary to gel electrophoresis, it's actually the larger proteins that elute faster and earlier from the column than the smaller proteins. The smaller proteins take longer to move through the column and are eluted last.

The reason for this is actually pretty simple. The stationary phase inside of the gel filtration chromatography column consists of very porous beads. These porous beads have cavities in them that are engineered to be a very specific size, and different beads have different sized cavities. Let's take a look at our example below to clear this up. Notice that gel filtration chromatography is another type of column chromatography. We have our column here, and inside of our column, it is packed with a stationary phase, which is filled with these porous beads that have cavities engineered to be a very specific size. If we were to zoom in on these beads, what we would see is that the beads have passageways inside of them, and some of the pores are bigger than others. Some pores allow medium-sized proteins to go in, and other pores only allow small proteins to go in.

Notice in our protein mixture at the top here, what we have are large proteins in red, intermediate-sized proteins in green, and small proteins in light blue. The light blue proteins are able to enter all of the porous beads, whereas the intermediate proteins can only enter some of them. These porous beads create a longer path. The red proteins, which are very large, physically cannot fit into the pores of these beads. So they have to take another route around the beads. That's exactly the reason for the illusion of the proteins. Large proteins, since they cannot enter the cavities of the beads because they are literally just too large to fit, are going to take a shorter and faster route through the column. The small proteins that do enter the cavities are slowed down because they have to take a longer route through the column.

When we look at our column chromatography here, notice that our red proteins are eluting fairly quickly, and they end up coming out of the column fastest. The intermediate-sized proteins come out second fastest, and then the slowest proteins that come out are the smallest ones. So, the largest proteins are going to be the ones that elute from the column first. If we were to measure the absorbance of the molecules as they come out of the bottom of the column, note the chromatogram showing light absorbance on the y-axis and the elution time on the x-axis. The red protein has a short elution time, meaning that they come out of the column first. The green proteins, the intermediate-sized proteins, come out of the column next, and it's the smallest proteins that come out of the column last; they take the longest to come out.

It's essential to understand that size exclusion chromatography can separate proteins based on their size. Also, if we have proteins that we know exactly what their size is, we can use size exclusion chromatography to determine the size of an unknown protein. We'll be able to get a lot of practice with size exclusion chromatography in our next couple of videos. So I'll see you guys in those videos.

Which of the following statements is false?

• A. In ion exchange chromatography, the bound proteins are eluted using a salt solution.
• B. Gel filtration chromatography can be used to determine an unknown protein's relative molecular size/mass.
• C. In gel filtration chromatography, the smallest proteins are eluted from the column last.
• D. Separation of proteins in gel filtration chromatography is based on size & net charge of the proteins.
• E. None of them. All above statements are true.

A new protein of unknown structure has been purified & gel filtration chromatography reveals that the native protein has a molecular weight of 240 kDa. Chromatography in the presence of 6 M guanidine hydrochloride (GuHCl), a chaotropic agent that has a similar effect on proteins as urea, yields a single absorbance peak corresponding to a protein of M_r 60 kDa. Chromatography in the presence both of 6 M guanidine hydrochloride and 10 mM β-mercaptoethanol (β-ME) yields peaks for proteins of M_r 34 kDa and 26 kDa. Using this data, which option best describes the structure of this protein? Hint: sketch a visual of the protein after each chemical treatment.

• A. A homotetramer (4 identical 60 kDa subunits); each subunit is a heterodimer of 2 disulfide-linked chains (34 & 26 kDa).
• B. A heterooctomer (8 different subunits); four subunits each of 34-kDa & 26 kDa, all held together via disulfide bonds.
• C. A homodimer (2 identical 120 kDa subunits); each subunit is a a homodimer of 2 disulfide-linked chains (60 kDa each).
• D. A heterotetramer (4 different subunits); each subunit is a homodimer of 2 disulfide-linked chains (60 kDa each).

To answer the questions A, B & C below, use the provided chart with the properties of the four proteins.

A) What is the order of elution of the proteins from a size-exclusion chromatography column?

     a. A → B → C → D.

     b. D → B → A → C.

     c. B → D → A → C.

     d. C → A → D → B.

B) Which pH is best for separating the proteins using anion-exchange chromatography?

     a. pH = 6.                        b. pH = 7.                  c. pH = 8.

C) In what order would the proteins elute from the anion-exchange chromatography column?

     a. A → C → D → B.

     b. D → A → B → C.

     c. B → D → C → A.

     d. C → B → D → A.

Alright. So here's a three-part practice problem broken up into parts a, b, and c. Notice above, it says to answer questions a, b, and c below, use the provided chart with the properties of the four proteins. In this chart below, we have proteins a, b, c, and d in the first column. The second column has the molecular weight (MW) for each of the four proteins. The third column has the isoelectric point (pI) for each of the four proteins. We're given all of the charges for these proteins. There's no need for us to estimate the net charge, compare the pH to the \( pK_{a} \)s of all the ionizable groups, or even use the Henderson-Hasselbalch equation. At first glance, this problem might look challenging, but it's a lot easier than it appears, simply because we're given all the information we need. Another point to notice is that some of the proteins at these pHs have double-digit charges, which is something we haven't seen yet in our lessons. The reason they have double-digit charges is that these proteins have large molecular weights. The larger the molecular weight, the more amino acids. The more amino acids, the more potentially ionizable groups. With large proteins, all the ionizable groups collectively could give the proteins a double-digit charge, which is typical for large proteins at specific pHs. Notice that part a here is asking us the order of elution of the proteins from a size exclusion chromatography column. The answer options have the first protein to elute from the column on the far left and the last protein to elute from the column on the far right. Recall that size exclusion chromatography, the elution order, is a bit counterintuitive, where it's actually the larger proteins that elute from the column first, and the smaller proteins take a longer route through the column and elute last. Because size exclusion chromatography separates proteins based on their molecular weight, we're only going to focus on the column here with the molecular weight of each protein for part a. Protein b has the largest molecular weight, so it's going to be the first to elute from our column. Then we need to order the rest of the proteins based on their molecular weight. First, it will be protein b followed by protein d, a, and then protein c, which has the smallest molecular weight, will elute last. So all we need to do is look for the answer option that has this order of elution for the proteins; that, of course, is option c. So we can indicate that option c here is correct and all the others are incorrect. Part b is asking which pH is best for separating the proteins using an ion exchange chromatography. Recall that anion exchange chromatography is used to collect and purify negatively charged proteins. In terms of which pH is best, at pH 6, we only have one negatively charged protein. At pH 7, we have two negatively charged proteins. But at pH 8, notice that we have three negatively charged proteins. Since most of our proteins are negatively charged at pH 8, that means that pH 8 will be the best pH for separating the proteins using anion exchange chromatography. So we can indicate that option c here is correct, and all of the others are incorrect. Part c is asking in what order the proteins would elute from the anion exchange chromatography column. With anion exchange chromatography, negatively charged proteins are going to interact with the positively charged stationary phase, so they're going to remain stuck inside of the column and move very slowly through the column, whereas all other proteins that are not negatively charged are going to flow through the column really fast and be the first to elute. Protein A here is the only one that does not have a negative charge, so it's going to be the first protein to elute. All the rest of our proteins are negatively charged, so they're all going to remain stuck inside the column. What's going to determine their order of elution is which one interacts the strongest with the stationary phase and which one interacts the weakest. The proteins that have the smallest magnitude in their net charge are going to be the ones that interact the weakest with the stationary phase. Since protein C has the smallest net charge, it's going to be the next to elute, followed by protein D, which has the next smallest net charge. And then protein B, which has the greatest magnitude in its net negative charge, is going to be the last to elute. So all we need to do is look for this order of elution and notice that matches answer option a. We can indicate here that a here is correct, and all the others are incorrect. This concludes this practice problem. I'll see you guys in our next video.

Here’s what students ask on this topic:

What is size exclusion chromatography and how does it work?

Size exclusion chromatography (SEC), also known as gel filtration chromatography, is a technique used to separate molecules based on their size. It involves a column filled with porous beads. Larger molecules cannot enter the pores and thus travel through the column faster, eluting first. Smaller molecules enter the pores, taking a longer path and eluting later. This method is useful for purifying proteins and determining the size of unknown proteins.

Why do larger proteins elute faster in size exclusion chromatography?

In size exclusion chromatography, larger proteins elute faster because they cannot enter the pores of the beads in the column. Instead, they travel around the beads, taking a shorter and more direct path through the column. Smaller proteins, on the other hand, enter the pores and are slowed down, resulting in a longer elution time.

What are the applications of size exclusion chromatography?

Size exclusion chromatography is widely used in proteomics and protein analysis. It is employed to purify proteins, separate protein complexes, and determine the molecular weight of unknown proteins. Additionally, it can be used to analyze the size distribution of polymers and to study protein-protein interactions.

How can size exclusion chromatography be used to determine the size of an unknown protein?

To determine the size of an unknown protein using size exclusion chromatography, a calibration curve is created using proteins of known sizes. The elution times of these known proteins are plotted against their molecular weights. The elution time of the unknown protein is then measured and compared to the calibration curve to estimate its size.

What is the difference between size exclusion chromatography and gel electrophoresis?

Size exclusion chromatography and gel electrophoresis both separate molecules based on size, but they operate differently. In size exclusion chromatography, larger molecules elute faster because they cannot enter the pores of the beads. In gel electrophoresis, smaller molecules move faster through the gel matrix. Additionally, size exclusion chromatography is used for purification and size determination, while gel electrophoresis is primarily used for analyzing and separating nucleic acids and proteins.