in this video, we're gonna talk about our second type of ion exchange chromatography and ion exchange chromatography. So an ion exchange chromatography is essentially the opposite of cat ion exchange chromatography in terms of the charges. And so if we were to go back to our cat ion 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, then we would have our an ion 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 an ions are negatively charged. An ion exchange chromatography is used to collect and purify negatively charged proteins. And so, in order to collect and purified negatively charged proteins and ion exchange chromatography needs to use positively charged stationary phase or positively charged stationary resin and an example of positively charged stationary resin is resin that has di ethyl amino ethyl functional groups or D E e functional groups for short for E. And so all I want you guys to know is that D E. E is a type of positively charged stationary resin used an an ion 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, which will notice is that way, have these D e functional groups on there, which are positively charged and so that makes our resin are stationary phase positively charged to notice all of these positive charges on our resin. And so before our, uh, chromatography process even begins, what we have are negatively charged and ions that are loosely bound and loosely attached to the positively charged stationary resin. And so the reason this is called an I N exchange chromatography is because it's these and ions here that are gonna be exchanged with our target protein. And our target protein is the one that we're trying to purify, which, remember, the one that we're trying to purify is going to be are negatively charged protein, and so are negatively charged. Proteins are gonna remain essentially stuck inside the column because they're gonna be interacting with all of these positively charged stationary phase resonance. And so they're gonna 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're going to be the first to allude. And so the greater the net positive charges on the protein the faster, and the earlier those proteins are going to allude from the column. And remember, these are the unwanted proteins because we want toe collect and purify the negatively charged proteins when we're using and I m exchange chromatography. And so let's go down to our an ion exchange chromatography example to clear all that up. And so again, what we have over here on the left and our beaker is our protein mixture and notice again. Our protein mixture has positively charged proteins and red, negatively charged proteins of blue and then the neutral proteins and gray. And so if we take our protein mixture and we poured into our anti and 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, and we know that we're gonna 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. And 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 resident. And so it's the neutral proteins that are actually going to dilute second. So they're going to come out of the column the second fastest, and so we can see that down here at the bottom, we can see 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 and during this whole process. Which will see is that the negatively charged proteins are moving through the column, but they're moving incredibly, 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. And so it's 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 gonna be the 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 wanna use an an ion 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 gonna have mawr interactions with the stationary Phase and MAWR interactions with the mobile phase, which is continuously added throughout this whole process. And mawr interactions with stationary and mobile phase means that we're going toe have better separation when we're using, um, an an ion exchange chromatography on negatively charged proteins. And so if we want to purify our protein, we're gonna want better separation. And so that's why we wanna use an ion exchange chromatography to collect them, 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 dilute first from the column, and those are the ones that we see coming out of the column first and So in order to get these negatively charged proteins out of the column what what? What we can do is we can either continuously ADM or more mobile phase. But that might take a long time. And we have to use Resource is we have to use mobile face, which could be expensive. And so, ah, fast way to be able to get out. The, uh, negatively charged proteins is to later allude these proteins from the column just by the addition of salt. And so we know from our, um, salting out lesson, uh, in our previous videos, that salt has the ability to decrease the strength of biotic interactions. And so if we add salt, these negatively charged proteins are going thio decrease the strength of their interactions with the positively charged resin. And so that's going to allow them to be quickly alluded from the column and to come out of the column so that we can collect them