Ion-Exchange Chromatography: Study with Video Lessons, Practice Problems & Examples

Ion exchange chromatography is a column chromatography technique used to purify proteins based on their net charge. Cation exchange chromatography targets positively charged proteins using a negatively charged stationary phase, typically composed of carboxymethyl (CM) functional groups. During the process, loosely bound cations are exchanged with the target proteins, allowing for effective separation. Negatively charged proteins elute first, followed by neutral proteins, while positively charged proteins remain bound, facilitating their purification. Salt can be added to elute these proteins by weakening ionic interactions, enhancing the efficiency of the purification process.

Ion-Exchange Chromatography

In this video, we're going to begin talking about ion exchange chromatography. So ion exchange chromatography is a type of column chromatography that purifies a protein based on the magnitude or the size of its net charge. And, in general, there are 2 main types of ion exchange chromatography. The first is cation exchange chromatography and the second is anion exchange chromatography. And just like we know cations are positively charged, cation exchange chromatography is generally used to purify positively charged proteins. And just like we know anions are negatively charged, anion exchange chromatography is generally used to purify negatively charged proteins. So, it makes it easy to remember in that respect. And so, if we know the overall net charge of our target protein, then ion exchange chromatography can be incredibly useful in our protein purification strategy. And so, first, moving forward, we're going to talk about cation exchange chromatography, and then afterwards, we'll talk about anion exchange chromatography. So, I'll see you guys in our next video.

Ion-Exchange Chromatography

So in general, ion exchange chromatography can sometimes be a little bit counterintuitive with all the different charges that we have going on. So in this video, we're going to break down cation exchange chromatography to make sure we understand how it works. And so, in our last lesson video, we said that just like cations are positively charged, cation exchange chromatography is used to collect and purify positively charged proteins. And so, in order to collect and purify positively charged proteins, cation exchange chromatography needs to use a negatively charged stationary phase or a negatively charged stationary resin, and resins are just the little beads that make up the stationary phase. An example of a negatively charged stationary resin are resins that have carboxymethyl functional groups attached, or just CM groups for short, for carboxymethyl. And so, all I want you guys to know is that CM groups are a type of negatively charged stationary resin used in cation exchange chromatography.

If we take a look at our example down below, what we'll see is that we've got our columns because we know ion exchange chromatography is a type of column chromatography. And so, inside our columns, we know that we have our stationary phase, so we can see our blue stationary phase inside all of our columns here. Notice that our stationary phase is made up of a bunch of beads or a bunch of resin, these circular things. And if we zoom in on one of them, what we'll see is that it's really like carboxymethyl functional groups that are attached, and they are negatively charged. Notice that we have all of these negative charges on our stationary phase. And then, what you'll also notice is that, before the cation exchange process even begins, what happens is we actually have these cations such as sodium ions that are loosely bound to the negatively charged resin. And so we can see that down below, we have these positively charged sodium cations that are very weakly bound to the negatively charged stationary resin. And so, these cations are there before we even begin the process. And so the reason that we call it cation exchange chromatography is because these loosely bound cations, during the process, they're actually exchanged with our target protein. The cations are exchanged with our target protein, and our target protein is going to be the one that we're trying to purify, which we know is going to be positively charged for cation exchange chromatography. And so that's exactly why it's called cation exchange chromatography, because cations are exchanged with the target protein.

To be able to figure out how cation exchange chromatography works, what we need to know is that positively charged proteins that we're interested in purifying, they actually bind to the negatively charged stationary resin. And if they bind to the stationary phase, which we know the stationary phase does not move, that means that the positively charged proteins are going to be less likely to move, so they are essentially not going to move as much as all of the other molecules in the column. So, you can think of them as being stuck inside the column. And so that means that the neutral or the negatively charged proteins, on the other hand, they are not going to interact with the stationary resin negatively charged, and they're going to pass right through. They literally just flow straight through the column and go through very quickly. And so, the greater the net negative charge on the protein, the faster and the earlier the protein will come out of the column. And remember, these are the unwanted proteins, because the ones that we're trying to collect and purify, which are the positively charged ones, are going to remain stuck inside the column, and it's only the ones that we don't want, the unwanted ones, that end up coming out.

So notice over here in our beaker, what we have is a protein mixture. And inside of our protein mixture, notice that we have these red, positively charged proteins, negatively charged proteins, and then we also have these black or gray, neutral proteins. And so we have a mixture of proteins with a bunch of different charges. And so if we take our protein mixture here and we pour it into our cation exchange chromatography column, essentially, what we will have is our mixture of proteins at the very top. And then we have our mobile phase inside of this container, at the top, and we know that we're going to be continuously adding the mobile phase throughout the entire process. And so, as we start to add more and more mobile phase, eventually what's going to happen is we're going to get separation of this protein mixture based on the charges of the proteins. With a cation exchange chromatography column, the negatively charged proteins do not interact with the negatively charged resin, and they come out of the column the fastest, and that's why we see them at the bottom. The neutral proteins, they come out next, so they come out the next fastest.

Down here at the bottom, what we can say is that it's the negatively charged proteins that are actually eluted from the column first, or come out of the column first before any of the other proteins. And so notice that throughout this entire process, the positively charged proteins are actually moving in the column, but they move much, much slower, and that's because the positively charged proteins are interacting with the negatively charged stationary resin. Even though they move through the column, they move incredibly slowly through the column, and that's what we're seeing. Positively charged proteins move incredibly slowly through the column. And so the proteins that move the slowest are going to be the ones that have the greatest positive charge because they interact with the negatively charged stationary phase the strongest. And the ones that, the positively charged proteins that move the fastest, they have a positive charge, but they just have a small positive charge. And so, they move through a little bit faster because they don't interact as strong with the negatively charged stationary phase. And so, the reason that we want to use cation exchange chromatography to collect and purify positively charged proteins and not negatively charged proteins is that, when we have our proteins that are stuck inside the column like this, they have more interactions with the stationary phase and with the mobile phase because we're continuously adding mobile phase the whole time. And so the more interactions you have with the stationary phase and the mobile phase, the better the separation is going to be. And so, if we're trying to collect and purify our target protein, we want to have incredibly good separation. And so we'll get better separation of positively charged proteins using a cation exchange chromatography column. And so, that explains why we want to use cation exchange chromatography for positively charged proteins.

And so, you might be wondering, how do we get these positively charged proteins out of the column? Now that we've gotten rid of all of these other proteins here, how do we collect these positively charged proteins? Now, we could continuously add more and more mobile phase, but that might take a long time because of how strong these interactions are with the negatively charged stationary phase. And so another way to be able to quickly get out our positively charged proteins is to elute our proteins later from our column, with the addition of salt. And so, we know from salting out, our salting-out topic in our previous lessons, that salts are able to reduce and decrease the strength of the ionic interactions. And so if we add salt, it's going to decrease the strength of the ionic interactions that hold the positively charged protein to the negatively charged stationary resin. And then that means that all of the positively charged proteins can be eluted quickly.

Down here in our example, what you'll see is that it's asking us which proteins are going to elute first during cation exchange chromatography. And so, we have protein A, which has a net charge of negative 4, and then we have protein B, which has a net charge of positive 2. And so we know that the proteins that elute first from the column are going to be negatively charged proteins. And so, that means that protein A is going to be the one to elute first. So, we can go ahead and highlight A here as our correct answer, give it a check to indicate A here is correct, and then we can cross off option B, which is not correct. So, this concludes our lesson on cation exchange chromatography, and we'll be able to get a lot more practice utilizing all of these concepts in our next practice videos. So, I'll see you guys there.

What is the order of elution of the following proteins from a cation-exchange chromatography column? 

Net charges of Proteins: Protein A = +1              Protein B = -2                Protein C = -5                Protein D = +3.

• A. A → B → C → D.
• B. D → A → B → C.
• C. C → B → A → D.
• D. B → C → D → A.

In a cation-exchange column at neutral pH, which peptide would elute last?

• A. A peptide that contains mostly Asp and Glu residues.
• B. A peptide that contains mostly Tyr and Trp residues.
• C. A peptide that contains mostly Ala and Gly residues.
• D. A peptide that contains mostly Lys and Arg residues.

Mixtures of amino acids can be analyzed by first separating the mixture into its components through ion exchange chromatography. Certain amino acids placed on a cation-exchange resin containing sulfonate groups (—SO₃^-) flow down the column slowly because of two factors that influence their movement: (1) ionic attraction between the sulfonate residues on the column and positively charged functional groups on the amino acids, and (2) hydrophobic interactions between amino acid R-groups and the strongly hydrophobic backbone of the polystyrene resin. For each pair of amino acids listed below, circle the amino acid that is eluted first from the cation-exchange column by a buffer at pH 7.

Alright. So at first glance, this practice problem looks really, really challenging because it gives us so much information. But really, all it's asking us to do is for each pair of amino acids that's listed down below, it wants us to circle the amino acid that elutes first from the chromatography column, and it tells us that we're specifically using cation exchange chromatography columns. And so all we need to do is look at each of these pairs of amino acids and circle the amino acid that elutes first or comes out of the column first. So that's pretty easy to do. Recall that with cation exchange chromatography, which I've drawn over here, it uses negatively charged stationary resin, which are these, highlighted, negatively charged blue balls that I'm showing here. We know that positively charged proteins and amino acids, which are in red, are going to interact the strongest with the negatively charged stationary resin. So we can see them interacting really strongly and they're not going to move as much as all of the other amino acids. So because they are going to not move as much, they're going to stay in the column longer and they're going to elute last from the column. So with the positively charged amino acids, recall from our previous lessons that these are going to be the ones to elute last. As a reminder, we'll put "elute last" over here for the positively charged amino acids. Now, the negatively charged amino acids on the other hand are essentially going to repel that stationary phase. And because they repel the stationary phase, they're not going to interact with it at all and they're going to flow right out as fast as possible with the mobile phase. So these negatively charged proteins and amino acids are going to be the ones to elute first from the column, so they will elute first. As a reminder from our previous lessons, we'll put "elute first". Then, we know that the neutral amino acids, which are in between, they are going to essentially elute in between the two. We can put that these will "elute second". So really, that's all the information that we need to recall from our previous lessons to answer this problem. All we need to do is compare each amino acid and figure out which one of these groups it falls into and which one's going to elute first. Looking at our first pair here, we have aspartic acid and lysine that we're comparing, and aspartic acid's one letter code is D and lysine's one letter code is K. And from our mnemonic, dragons eat knights riding horses, we know that aspartic acid is a negatively charged amino acid, and lysine is a positively charged amino acid. Because the positively charged amino acid of lysine elutes last from the column, and the negatively charged amino acids of aspartic acid elutes first from the column, that tells us that we can circle aspartic acid since it's going to elute first here. So that is going to be the one that elutes first in this pair, and that's it for part A. So pretty easy, right? All we did was apply those same rules that we already knew from our previous lessons. And now we can do the same for part B. Looking at this pair, we have arginine, whose one-letter code is R, and methionine's one letter code is M. Methionine is a nonpolar, hydrophobic amino acid with a neutral charge, and neutral amino acids are going to elute second, whereas arginine is a positively charged amino acid, and positively charged amino acids elute last. So that means that methionine is going to elute first, and we can go ahead and circle methionine as being the correct answer since arginine is positive and it elutes last. Now moving on to part C. So pretty easy so far, and we're just going to keep moving through. We're comparing glutamic acid and valine. Glutamic acid's one letter code is E, and valine's one letter code is V. Valine is again a nonpolar, hydrophobic amino acid and it's neutral, so it's going to elute second. Whereas glutamic acid is a negatively charged amino acid, and so, because it's negatively charged, it's going to elute first from the column. So all we need to do is circle glutamic acid, for this problem, for this part, because it's going to elute first from the column since it's negatively charged. And now we're moving on to part D, we're comparing glycine and leucine. Glycine's one letter code is G, and leucine's one letter code is L. Both glycine and leucine fall into the nonpolar hydrophobic amino acids, and they are both neutral amino acids. So now all we need to do is use another level of comparison between glycine and leucine. Since they fall into the same group, we need to figure out what it is about their R groups that allows one to elute first before the other. Glycine's R group is literally just a hydrogen. It glides right on through. And leucine's R group is a loose extension of valine, so it's CH2 and then you have the valine at the end with two methyl groups. Essentially, gon;\

Give the order of elution of the following peptides when using cation-exchange chromatography at pH 7.2.

Peptide #1: A-D-G-H-E.           Peptide #2: K-L-M-R-A.           Peptide #3: M-D-L-I-V.             Peptide #4: I-L-R-P-M.

Order of Elution: _______________, _______________, _______________, _______________

                                 (1   ^st to elute)                                                                             (Last to elute)

Alright. So this practice problem wants us to give the order of evolution of the following peptides when using cation exchange chromatography at pH 7.2. And we're given the sequences for each of these 4 peptides and these blank spaces below where we can provide the order of elution of these 4 peptides from the first to elute from the column to the last to elute from the column. And so we know that cation exchange chromatography separates proteins based on the magnitude of their net charges. But notice that we're not actually given any of the net charges for any of these peptides, which means that we can break this practice problem up into 2 general steps. And in the first step, step number 1, we're going to estimate the net charges for each of the predominant structures for each of these peptides. And in step number 2, we're going to use the estimated net charges from step number 1 to predict the order of elution of these peptides from the cation exchange chromatography column. And so the good thing here is that for step number 1, we already know how to estimate the net charges of peptides from our previous lesson videos. And in the past when we did this, we separately compared each of the ionizable group pKas to the pH of the solution. Now we have the pH of the solution as 7.2, but notice we're not given any of the pKas for any of the ionizable groups, which means that you either had to have memorized all of the pKas for all of the ionizable groups, which your professor is not likely going to expect you guys to do, or you have to use some kind of amino acid pKa chart or table. And you guys are more than welcome to use an amino acid pKa chart or table to estimate the net charges of each of these peptides like we did in our previous practice problems. But the reason that I didn't provide a pKa charter table for this practice problem is because we really don't need one. And that's because we're using cation exchange chromatography at pH 7.2, which is right around physiological pH, which means that the ionizable groups are going to have the ionizable states as we originally memorized them when we first introduced the amino acids. And so we know, for instance, at physiological pH, each of these peptides is going to have an ionized amino group on the n terminal end. And so we can put a positive charge to represent the amino group that is ionized at physiological pH for each of these peptides. And, in a similar fashion, we know that on the carboxyl end, there's going to be an ionized carboxylate group at physiological pH for each of these peptides as well, so we can put a negative charge here to represent that. And so now that we have the amino and carboxyl groups taken care of for each of these peptides, we now only need to focus on the R groups. And recall the mnemonic that helps us memorize the amino acids with ionizable R groups is just yucky crazy dragons eat knights riding horses. And so if it's not one of these 7 amino acids, then its r group is not ionizable. It's not going to contain a charge and it's not going to contribute to the net charge of the peptide. So that means that we can essentially not worry about it if it's not part of this group. And so looking at our first amino acid residue alanine, notice it's not part of this group, so we don't have to consider it in the net charge, and we can move on to the next one. And the next one here is D, which is for aspartic acid, and that is part of our mnemonic. And we know that at physiological pH of 7.2, aspartic acid's r group has a negative charge, so we can put a negative charge here. Now glycine is not part of our mnemonic, so we can skip it. Histidine is part of our mnemonic, and we know that at physiological pH, it's going to have a positive charge on it. And then glutamic acid here is going to be part of our mnemonic as well, and it's gonna have a negative charge at physiological pH. And so now all we need to do to get the net charge of this peptide is to sum up all of these charges. So we've got 3 negative charges and 2 positive charges, which comes out to a total net charge of negative one. Now moving on to peptide number 2, K is part of our mnemonic down below, and so we know it's gonna have a positive charge at physiological pH. Now leucine and methionine are not part of our mnemonics, so we can skip those. But arginine is, so it has a positive charge, so we can go ahead and include that. And then alanine is not part of our mnemonic, so essentially here we have 3 positive charges and only one negative charge. So the net charge on peptide number 2 is going to be positive 2. Now with peptide number 3, we have methionine, which is not part of our mnemonic down below, and then we have aspartic acid, who is part of our mnemonic and we already did it earlier, we know it has a negative charge. And then notice that leucine, isoleucine, and valine are not part of our mnemonic, so we can skip those r groups. So essentially, we have 2 negative charges and one positive charge, which means that we have a total net charge of negative one on peptide number 3. And then with peptide number 4, notice that isoleucine and leucine are not part of our mnemonic, so we can skip those. And arginine, we already did earlier, we know that it has a positive charge, so we can put a positive charge on arginine here. And then proline and methionine are not part of our mnemonic, so we can skip those. So essentially, we have 2 positive charges over here and one negative charge. So our total net charge for peptide number 4 is plus 1. Alright. So now we have finished step number 1. We've estimated the net charges. So we can go ahead and give step number 1 a check here. So now all we need to do is use the estimated net charges from step number 1 to predict the order of elution from the cation exchange chromatography column. And recall that cation exchange chromatography separates or is used to collect and purify proteins that are positively charged. And so the positively charged proteins are gonna be the ones that get stuck in the column and move really, really, really slowly through the column. And so the positively charged proteins are gonna be the ones to elute last, and the ones that have a greater positive charge are gonna be the ones to elute last from the column. And so notice that peptide number 2 has the greatest positive one Now, peptide number 4, notice, has the next greatest positive charge, so it's going to be the second to last here to elute. And then what you'll notice is that both peptide number 1 and peptide number 3 have identical charges. They are both negative one. And so the next step that we're going to need to realize is we're going to have to take into account this note here that we should always take into account, and that's the fact that histidine's pKr is pretty close to physiological pH. And because we're using pH of 7.2, which is close to physiological pH, we need to take that into consideration. And so histidine's pKr is 6.0. And so you're not expected to memorize all of the pKrs, but this is definitely a good pKr to know for histidine. And so what you'll know is that histidine here, because it has a physiological, because it has a pKr of 6 that is pretty close to the pH of 7.2, it's actually going to have a partial charge at, near physiological pH. It's going to be partially charged. And so yes, it is going to have a positive charge, but it's going to have a partial charge. So let's just put a plus 0.5 here, just so that we know that it's going to be a partial charge. And so what that means is when we total the net charge, it's really going to be more like a negative 1.5 charge instead of a negative one. And so, now when we do that and take that into consideration here, we can pretty clearly see that peptide number 1 is actually going to be the first to elute because it has the greatest negative charge. And remember, in a cation exchange chromatography column, the negatively charged proteins elute first. And the greater the negative charge, the greater the magnitude of the negative charge, the earlier it will come out. So that means that peptide number 1 will be the one to elute first, and then, of course, peptide number 3 will be the one to elute at this position here. And so what that means is that this here is the answer to our problem, And that concludes this challenging problem, and I'll see you guys in our next video.

Here’s what students ask on this topic:

What is ion exchange chromatography and how does it work?

Ion exchange chromatography is a technique used to purify proteins based on their net charge. It involves a column filled with a stationary phase composed of charged resin beads. There are two main types: cation exchange chromatography, which targets positively charged proteins using a negatively charged stationary phase, and anion exchange chromatography, which targets negatively charged proteins using a positively charged stationary phase. Proteins with the opposite charge to the stationary phase bind to it, while others pass through. By adding a mobile phase or salt, the bound proteins can be eluted and collected.

What is the difference between cation exchange chromatography and anion exchange chromatography?

Cation exchange chromatography targets positively charged proteins using a negatively charged stationary phase, typically composed of carboxymethyl (CM) functional groups. Anion exchange chromatography, on the other hand, targets negatively charged proteins using a positively charged stationary phase, often composed of diethylaminoethyl (DEAE) groups. The choice between the two depends on the net charge of the target protein. In both methods, proteins with the opposite charge to the stationary phase bind to it, allowing for their separation and purification.

How are proteins eluted in ion exchange chromatography?

Proteins are eluted in ion exchange chromatography by gradually changing the conditions within the column. One common method is to increase the concentration of salt in the mobile phase. The salt ions compete with the bound proteins for binding sites on the stationary phase, weakening the ionic interactions and allowing the proteins to be released. Another method is to change the pH of the mobile phase, which can alter the charge of the proteins and the stationary phase, leading to elution. These techniques help in efficiently collecting the target proteins.

What are the applications of ion exchange chromatography in biochemistry?

Ion exchange chromatography is widely used in biochemistry for protein purification, separation of nucleotides, and purification of enzymes. It is particularly useful in isolating proteins with specific charge properties, making it essential in research and pharmaceutical industries. The technique is also employed in water purification, where it removes ions and impurities. Additionally, it is used in the analysis of complex biological samples, helping to identify and quantify various biomolecules based on their charge characteristics.

Why is salt used in ion exchange chromatography?

Salt is used in ion exchange chromatography to elute bound proteins from the stationary phase. The salt ions compete with the proteins for binding sites on the charged resin beads, weakening the ionic interactions that hold the proteins in place. This process, known as salting out, allows the proteins to be released and collected more efficiently. By adjusting the salt concentration, researchers can control the elution process, ensuring that the target proteins are separated and purified effectively.