Anion-Exchange Chromatography - Online Tutor, Practice Problems & Exam Prep

Anion exchange chromatography is a technique used to purify negatively charged proteins by utilizing a positively charged stationary phase, such as diethylaminoethyl (DEAE) resin. In this process, negatively charged proteins bind to the resin while positively and neutral proteins elute quickly. The separation occurs based on charge interactions, with the addition of salt later facilitating the elution of the target proteins by weakening ionic interactions. This method is essential for achieving better separation and purification of negatively charged proteins in biochemical applications.

Anion-Exchange Chromatography

In this video, we're going to talk about our second type of ion exchange chromatography, anion exchange chromatography. So, an ion exchange chromatography is essentially the opposite of cation exchange chromatography in terms of the charges. And so, if we were to go back to our cation exchange chromatography lesson, and literally swap all of the charges, so swap all of the positive charges with negative charges, and swap all of the negative charges with positive charges, we would have our anion exchange chromatography lesson. And so, that's great for us because all we need to do is focus on knowing just one type of ion exchange chromatography. And so if we know just one type of ion exchange chromatography, then we automatically know the other one because all we need to do is swap all of the charges. And so let's go through this lesson so you guys can see what I mean. And so, in our previous lesson videos, we said that just like anions are negatively charged, anion exchange chromatography is used to collect and purify negatively charged proteins. And so, in order to collect and purify negatively charged proteins, anion exchange chromatography needs to use positively charged stationary phase or positively charged stationary resin.

An example of positively charged stationary resin is resin that has diethylaminoethyl functional groups, or DEAE functional groups for short, for DEAE. Functional groups for short, for DEAE. And so all I want you guys to know is that DEAE is a type of positively charged stationary resin used in anion exchange chromatography. And so, if we go down and take a look at our example, notice that we have all of these columns again because, remember, ion exchange chromatography is a type of column chromatography. And packed inside of the columns, what we have is our stationary phase, which is this pink material that we see inside all of the columns. And so notice that the pink material here, the stationary phase, is made up of a bunch of beads or resin. And so if we were to zoom in on one of these resin, what you'll notice is that we'll have we have these DEAE functional groups on there, which are positively charged. And so that makes our resin, our stationary phase positively charged. So notice all of these positive charges on our resin.

Before our chromatography process even begins, what we have are negatively charged anions that are loosely bound and loosely attached to the positively charged stationary resin. And so, the reason this is called anion exchange chromatography is because it's these anions here that are going to be exchanged with our target protein. And our target protein is the one that we're trying to purify, which, remember, is going to be our negatively charged protein. And so our negatively charged proteins are going to remain essentially stuck inside the column, because they're going to be interacting with all of these positively charged stationary phase resins. And so they are going to move essentially not move and just move very, very slowly. Whereas, all of the neutral and the positively charged proteins, they do not interact with the positively charged resin. So they do not bind to that resin and that means that they're just going to pass through the column or flow through the column very, very quickly and they are going to be the first to elute. And so, the greater the net positive charges on the protein, the faster and the earlier those proteins are going to elute from the column. And remember, these are the unwanted proteins because we want to collect and purify the negatively charged proteins when we're using anion exchange chromatography.

And so, let's go down to our anion exchange chromatography example to clear all of that up. And so, again, what we have over here on the left in our beaker is our protein mixture. And notice, again, our protein mixture has positively charged proteins in red, negatively charged proteins in blue, and then the neutral proteins in gray. And so if we take our protein mixture and we pour it into our anion exchange chromatography column, we'll have our protein mixture at the top, and we have our mobile phase over here inside of the containers at the top. We know that we're going to be continuously adding mobile phase throughout our entire process. And so, as we start to add mobile phase to our column, what we're going to get is separation of our protein mixture based on the charges of the proteins. It turns out that the positively charged proteins are going to move through the column the fastest, and so that's why you see them at the bottom, and that's because the positively charged proteins do not interact with the positively charged stationary resin. And so, it's the neutral proteins that are actually going to elute second, so they're going to come out of the column the second fastest. We can see that it's the positively charged proteins that come out of the column first, and then after the positively charged proteins come out, it's the neutral proteins that come out later.

During this whole process, what you'll see is that the negatively charged proteins are moving through the column, but they're moving incredibly slowly. So the negatively charged proteins move through the column slowly and that's because they're interacting with the positively charged stationary resin. So, the negative proteins that move the slowest are going to be the ones that have the greatest net negative charge. And the ones that move the fastest, the negative proteins that move the fastest are going to be ones that have a negative charge, but they just have a tiny net negative charge. And so, they interact with the stationary resin, but they don't interact as strongly as the ones that have a greater net negative charge. And so the reason that we want to use an anion exchange chromatography column to collect and purify negatively charged proteins is because the negatively charged proteins, essentially, they remain stuck inside of our column. And when they're stuck inside the column, that means that they're going to have more interactions with the stationary phase and more interactions with the mobile phase, which is continuously aiding in the separation when we're using an anion exchange chromatography on negatively charged proteins. And so, if we want to purify our protein, we're going to want better separation. And so, that's why we want to use an anion exchange chromatography to collect and purify negatively charged proteins and not positively charged proteins. And so, down here at the bottom, what we can say is that it's the positively charged proteins that are actually going to elute first from the column, and those are the ones that we see coming out of the column first.

In order to get these negatively charged proteins out of the column, what we'll what we can do is, we can either continuously add more and more mobile phase, but that might take a long time, and we have to use resources, mobile phase, which could be expensive. And so, a fast way to be able to get out the negatively charged proteins is to later elute these proteins from the column just by the addition of salt. And so we know from our salting out lesson in our previous videos, that salt has the ability to decrease the strength of ionic interactions. And so, if we add salt, these negatively charged proteins are going to decrease the strength of their interactions with the positively charged resin. And so that's going to allow them to be quickly eluded from the column and to come out of the column so that we can collect them.

Anion-Exchange Chromatography Example

And so in our example down below, it asks us to circle the peptide below that elutes, that elutes last during anion exchange chromatography if the pH is about 7 or neutral or physiological pH. And so we know that with anion exchange chromatography, we're collecting and purifying negatively charged proteins, and it's those negatively charged proteins that are going to elute last from the column. And so, really what we're looking for in our peptides down here are which ones are going to have the greatest negative charge. And so, notice that the charges are not provided for either of these 2 peptides, which means that we're going to need to estimate the net charge of these peptides. And recall that the way that we estimate the net charge of a peptide is by considering all of its ionizable groups. And so that means that we're going to need to recall our 7 amino acids with ionizable r groups, and that mnemonic for memorizing the seven 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, and we don't really care about it because it's not going to contribute to the net charge, and that's really all we care about when it comes to ion exchange chromatography. And so, again, we don't really care about the structure, we care about the net charges. And we know that each peptide is going to have an ionizable amino group, and it's also going to have an ionizable carboxyl group. And we know that at a pH of 7, which is physiological pH, that the backbone of a peptide is going to be a zwitterion, which means that it has 2 opposite charges. And we know that the amino group is going to be positively charged, so we'll put a positive charge over here to just consider that positive charge on the amino group. And we can do the same for this peptide over here, positive charge for the amino group. And then the carboxyl group, we know, is going to be negatively charged, so we can put a negative charge for our carboxyl group. So now, all we need to do is consider the ionizations of the r groups. And so, again, if it's not one of these amino acids here, then we're going to, completely ignore it because it's not going to contribute to the charge. And so, glycine's one letter code here is g, and g is not part of our mnemonic over here. So that means that we can ignore its r group. And the same goes for alanine, whose one letter code is a, and that's not up here, so we can ignore that. And then aspartic acid's one letter code is d, and d is up here and aspartic acid is a negatively charged, r group, so we can go ahead and put a negative charge for that. Now lysine's r group, one letter code is k. And k is found up here as a positively charged protein. And so, we can put its r group down here with a positive charge. Glutamic acid or glu, its one letter code is an e. And e is a negatively charged protein, so we can go ahead and put its r group down here as a negative charge. And then serine is s, and s is not up here, so we can ignore it. And so now that we have all of the ionizable groups represented, all we need to do is sum all of the net charges. So we have 2 positive charges and we have 3 negative charges, which means that the total net charge on our peptide here is going to be negative one for this peptide on the left. And so we can do the same thing for the peptide on the right here, and notice that leucine's one letter code is l, which is not part of the 7 ionizable amino acids. Same goes for threonine and isoleucine, whose one letter codes are t and I. They are not found up here in our mnemonic. And then histidine is a positively charged protein. Its one letter code is h, and it's found here. And so histidine has a positive charge, so we can go ahead and put a positive charge there. Glycine's one letter code is g, which is not found up here. And arginine is r, and so it's a positively charged protein. And so we can put its r group down here as a positive charge. So now we have all of the ionizable groups represented, so all we need to do is total the net charges. So we have 3 positive charges and one negative charge, which means that the total net charge over here is positive 2. And so now that we have the net charges, we can determine which one is going to elute last from the anion exchange chromatography. And remember, it's the one that has the greatest negative charge that's going to elute last. So that means that peptide number 1, which has a negative one charge, is going to be the one to elute last, and so we can go ahead and circle peptide number 1 over here as our correct answer. And so that concludes this practice here, and we can, move on to our practice problems where we'll be able to get a lot of practice on these concepts. So I'll see you guys there.

Which amino acid elutes last from an anion-exchange column at physiological pH?

• A. Lysine.
• B. Alanine.
• C. Glutamate.
• D. Asparagine.
• E. Glycine.

Use the chart to determine which tripeptide would elute last from an anion-exchange column at pH = 9.3.

• A. Tyr-Lys-Met.
• B. Gly-Pro-Arg.
• C. Asp-Trp-Tyr.
• D. Asp-His-Glu.

Which type of ion exchange chromatography would be best to separate a mixture of histidine and arginine? 

            His: pK₁ = 1.8, pK₂ = 9.3, pK_R = 6.0                     Arg: pK₁ = 1.8, pK₂ = 9.0, pK_R = 12.5

• A. Anion-exchange chromatography at pH = 2.
• B. Anion-exchange chromatography at pH = 4.
• C. Cation-exchange chromatography at pH = 2.
• D. Cation-exchange chromatography at pH = 4.
• E. Cation-exchange chromatography at pH = 9.

Stationary resin compounds with carboxymethyl (CM) and diethylaminoethyl (DEAE) groups are shown below. Indicate which one is likely used in a cation exchange column and which one is likely used in an anion exchange column. Considering the following peptide at pH 7, should DEAE or CM groups be used as the stationary resin to purify the peptide? 

Peptide: G-R-W-K-R-H

So this practice problem says, stationary resin compounds with carboxymethyl or Centimeters groups and diethylaminoethyl or DEAE groups are shown below. And it says to indicate which one is likely used in a cation exchange column and which one is likely to be used in an anion exchange column. Now, looking down below on the left over here, we can see that we have a carboxymethyl resin or Centimeters resin here. And recall from our previous lessons on ion exchange chromatography that carboxymethyl resin is associated with a negative charge. And so a negatively charged stationary resin will attract positively charged proteins. And we know that we use a cation exchange column in order to collect and purify positively charged proteins. Now on the right over here, we have the DEAE resin, and recall from our previous lessons on anion exchange chromatography that DEAE resin has a positive charge associated with it. And so a positively charged stationary resin is used to attract negatively charged proteins. And so an anion exchange chromatography column is used to collect and purify negatively charged proteins. And that's why we have cation and anion here. Now, in the second part of our problem up above, it says, considering the following peptide at pH 7, should diethylaminoethyl or carboxymethyl groups be used as the stationary resin to purify the peptide? And then it gives us the peptide with this following sequence here. And what we'll need to recognize is in this sequence, that there are multiple amino acids that have a positive charge. So you can see that arginine, lysine, arginine, and histidine are all amino acids that have positive charges on them. And the rest of the amino acids, glycine, and tryptophan, are neutral amino acids. Glycine is a nonpolar amino acid, and tryptophan is an aromatic amino acid. But because most of the amino acids, 3 out of 5, have a positive charge associated with them, that means that our peptide is going to have a positive charge associated with it at pH 7. And so, because we have a positively charged peptide, we're going to want to use a cation exchange column, because cation exchange chromatography columns are used to collect and purify positively charged proteins and peptides. And so what that means is we're going to want to use a carboxymethyl stationary resin because that's what's found in cation exchange columns. And so the correct answer for this problem is actually carboxymethyl, or group number 1 over here. So we can go ahead and give it a check to indicate that this is the correct answer. And that concludes this practice, so I'll see you guys in our next video.

Here’s what students ask on this topic:

What is anion exchange chromatography and how does it work?

Anion exchange chromatography is a technique used to purify negatively charged proteins. It utilizes a positively charged stationary phase, such as diethylaminoethyl (DEAE) resin. In this process, negatively charged proteins bind to the positively charged resin, while positively charged and neutral proteins elute quickly. The separation occurs based on charge interactions. To elute the target negatively charged proteins, salt is added to weaken the ionic interactions, allowing the proteins to be collected. This method is essential for achieving better separation and purification of negatively charged proteins in biochemical applications.

What is the role of DEAE resin in anion exchange chromatography?

DEAE (diethylaminoethyl) resin plays a crucial role in anion exchange chromatography as the positively charged stationary phase. The DEAE functional groups on the resin beads provide positive charges that attract and bind negatively charged proteins. This interaction allows for the separation of proteins based on their charge. Positively charged and neutral proteins do not bind to the DEAE resin and elute quickly, while negatively charged proteins remain bound until eluted by adding salt, which weakens the ionic interactions. This selective binding and elution process is key to purifying negatively charged proteins.

How are proteins eluted in anion exchange chromatography?

In anion exchange chromatography, proteins are eluted by adding salt to the mobile phase. The salt ions compete with the negatively charged proteins for binding sites on the positively charged DEAE resin. This competition weakens the ionic interactions between the proteins and the resin, allowing the negatively charged proteins to be released and eluted from the column. The elution process can be controlled by gradually increasing the salt concentration, which helps in achieving better separation and purification of the target proteins.

What types of proteins are purified using anion exchange chromatography?

Anion exchange chromatography is specifically used to purify negatively charged proteins. These proteins have an overall negative charge at the pH of the mobile phase used in the chromatography process. The negatively charged proteins bind to the positively charged DEAE resin, allowing for their separation from positively charged and neutral proteins, which elute quickly. This technique is essential for isolating and purifying proteins with a net negative charge, which are often of interest in various biochemical and research applications.

Why is salt used in the elution process of anion exchange chromatography?

Salt is used in the elution process of anion exchange chromatography to weaken the ionic interactions between the negatively charged proteins and the positively charged DEAE resin. The salt ions compete with the proteins for binding sites on the resin, reducing the strength of the protein-resin interactions. This allows the negatively charged proteins to be released and eluted from the column. Using salt for elution is a quick and efficient method to collect the target proteins, ensuring better separation and purification.