Ion-Exchange Chromatography - Video Tutorials & Practice Problems

Ion-Exchange Chromatography

in this video, we're gonna 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 two main types of ion exchange chromatography. The first is cat ion exchange chromatography, and the second is an ion exchange chromatography. And just like we know, cat ions are positively charged. Cat ion exchange chromatography is generally used to purify positively charged proteins. And just like we know, an ions are negatively charged. An ion 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, uh, in our protein purification strategy. And so, first moving forward, we're gonna talk about cat ion exchange chromatography, and then afterwards, we'll talk about an ion 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 of the different charges that we have going on. So in this video, we're gonna break down cat ion exchange chromatography to make sure we understand how it works. And so, in our last lesson video, we said that just like cat ions are positively charged. Cat ion exchange chromatography is used to collect and purify positively charged proteins. And so, in order to collect and purify positively charged proteins, cat ion exchange chromatography needs to use a negatively charged stationary phase or a negatively charged stationary resin and resin, or just a little beads that make up the stationary phase. And so, an example of a negatively charged stationary resin, our resins that have car boxy metal functional groups attached, or just see em groups for short for car, boxy method. And so all I want you guys to know is that CME Group's are a type of negatively charged stationary resin used in cat ion exchange chromatography. And so 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 of our columns. We know that we have our stationary phase so we can see our blue stationary phase inside of all of our columns here and notice that are stationary phase is made up of a bunch of beads or a bunch of resin. These circles circular things. And if we zoom in on one of them, what we'll see is that it's really like car boxy metal or C M functional groups that are attached and they're negatively charged. So notice that we have all of these negative charges on our stationary phase and then which will also notice is that before the cat ion exchange process even begins, What happens is we actually have these, uh, cat ions such as sodium ions that are loosely bound to the negatively charged present. And so we can see that down below that we have these positively charged sodium cat ions that are very weakly bound to the negatively charged stationary resin. And so this is thes Catalans air there before we even begin the process. And so the reason that we call it Cat ion exchange chromatography is because these loosely bound cat ions during the process there actually exchanged with our target protein. So the cat ions are exchanged with our target protein, and our target protein is gonna be the one that we're trying to purify, which we know is going to be positively charged for cat ion exchange chromatography. And so that's exactly why it's called cat ion exchange chromatography because cat ions air exchange with the target protein. And so to be able to figure out how does cat ion exchange chromatography works? What we need to know is that the positively charged proteins that we're interested in purifying they actually bind to the negatively charged stationary resin. And if they bind toe the stationary phase, which we know the stationary phase does not move. That means that the positively charged proteins are gonna be less likely to move. So they're essentially not going to move as much as all of the other molecules in the column. So they're gonna you can think of them as being stuck inside of 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 state the negatively charged stationary resin. So they do not bind to that negatively charged stationary resin, and they're gonna pass right through there, literally just gonna flow straight through the column and go through very, 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 air the unwanted proteins because the ones that were 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. And so let's take a look at our example down below to be able to clear up all of this stuff that we said about cat ion exchange chromatography. So notice over here and our beaker. What we have is a protein mixture and inside of our protein mixture, notice that we have these red, positively charged proteins. Here we have blue negatively charged proteins, and then we also have these black or gray, uh, 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 cat and exchange chromatography column, essentially, what we'll have is our mixture of proteins at the very top. And then we have our mobile phase inside of this container, um, at the top, and we know that we're gonna be continuously adding the mobile phase throughout the entire process. And so as we start to ADM or, um, or mobile phase, eventually what's gonna happen is we're gonna get separation off these of this protein mixture based on the charges of the proteins. And so with a CATA and exchange chromatography column, the negatively charged proteins do not interact with negatively charged resin, and they come out of the column the fastest. And that's why we see them at the bottom and the the neutral proteins. They come out next so they come out the next fastest. And so what we'll see is if we continue to ADM or more mobile phase as we go. The negatively charged proteins come out of the column first, so they are eluded from the column first. And then, after the negatively charged proteins come out, it's the neutral proteins, the black ones here that come out next after the negatively charged proteins and so down here at the bottom. What we can say is that it's the negatively charged proteins that are actually eluded 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. And so 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 gonna 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, ah, positive charge, but they just have a small, positive charge. And so they moved 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 a cat ion exchange chromatography to collect and purified, positively charged proteins and not negatively charged proteins is because when we have our proteins that are stuck inside the column like this, they have mawr interactions with the stationary phase and with the mobile phase, because we're continuously adding mobile phase the whole time. And so the mawr interactions you have with stationary phase in 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 toe have incredibly good separation. And so we'll get better separation of positively charged proteins using a cat ion exchange chromatography column. And so that explains why we want to use a positively charged Um, uh, that explains why we want to use cat ion exchange chromatography for positively charged proteins. And so you might be wondering, How do we get these positively charged proteins out of the column? So 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 ADM or um, or mobile phase, but that might take a long time because of how how strong these interactions are with the negatively charged stationary phase and so another way to be able to quickly get out are positively charged. Proteins is to dilute 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 assaults 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 positive, positively charged proteins can be eluded quickly. And so, uh, down here, in our example, what which will see is that it's asking us which proteins are going to allude first during a cat ion exchange chromatography. And so we have protein A, which has a net a net charge of negative four, and then we have Protein B, which has a net charge of positive, too. And so we know that the proteins that pollute first from the column are going to be negatively charged proteins. And so that means that protein A it's gonna be the one to allude first. And so we can go ahead and highlight a here, as are correct. Answer. Give it a check to indicate a here is correct. And then we can cross off option B, which is not correct. And so this concludes our lesson on cat ion exchange chromatography, and we'll be able to get a lot more practice utilizing all of these concepts and 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.