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 - Online Tutor, Practice Problems & Exam Prep
Ion-Exchange Chromatography
Video transcript
Ion-Exchange Chromatography
Video transcript
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.
In a cation-exchange column at neutral pH, which peptide would elute last?
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.
Problem Transcript
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)