Column Chromatography

in this video, we're gonna talk about our fifth and final step in our protein purification strategy, and that's column chromatography so literally. We've saved the best for last. And that's because chromatography is the most effective separation technique that we've talked about so far in this course, and we've saved it for last intentionally, when the sample size is relatively small and so recall that when we first did protein extraction we obtained are crude extract from ourselves, which was a big mixture of all of the cell components. And then each purification technique that we talked about removed some unwanted cell components and unwanted proteins. So we were able to reduce our sample size. And the reason we want a small sample size during chromatography is because some of the materials required for some types of chromatography, especially the more sophisticated types of chromatography, can be relatively expensive, especially when you use them in a large scale on large sample sizes. And so the larger the sample sizes, the mawr expensive. The overall process and technique is going to be. And so the tendency is toe always reduce the cost by using the cheaper techniques first, such assaulting out to reduce the sample size before we use arm or sophisticated and more expensive chromatography techniques. And so what you'll see is that column Chromatography is literally just a separation technique that uses a column, and it literally uses a vertical column, and we'll be able to see that in our example below. And it does that to purify very specific target molecules from a mixture of molecules and so column Chroma chromatography uses two different types of materials. The first type of material that it uses is called a stationary phase. And so the stationary phase literally is a solid material that is immobile, and that just means that it does not move throughout the entire chromatography process. It is stuck inside the column, and it is stuck in its same exact position throughout the entire process. Now, the second type of material used in column chromatography is the mobile phase. And just like it sounds, the mobile phase is a liquid material that is mobile, and that means that it does move during chromatography, and we'll be able to see that down below in our example. And so the idea for column chromatography is that the mobile phase actually flows over top of the stationary phase and interacts with different components in the sample mixture to separate those components. And so, in our example below of column chromatography notice that what we have are a bunch of vertical columns, and each of these columns is packed with a stationary phase that does not move. And so examples of common types of stationary phase includes silica, and so inside of the state are column is the stationary phase, and what you'll notice is that the first step is to add our protein mixture at the top of our column. So this little gray blob up here is our protein mixture. And so the second step is going to be to continuously add mobile phase throughout the entire process. And so the mobile phase is inside of these little bottles up here and notice that we are continuously adding the mobile phase throughout the entire column chromatography process. And remember the mobile phases mobile and it flows. It actually moves, so the stationary phases stuck. But the mobile phase is liquid, and it was going to trickle all the way down and drip out through the bottom of our column. And so, essentially, what happens is the mobile phase will start to interact with the proteins in our mixture to separate out the proteins and eventually, over time, as we, ADM or and Mawr Mobile phase. What happens is we get better and better separation of our proteins. And eventually what will happen is the mobile phase is going to pull and, uh, essentially allow for all of the materials to be alluded at the bottom of the column and by this word alluded here. What we really mean is that it is collected and removed from the column and so allude. Here is a very common word or a piece of terminology that is used with chromatography. And so again, all it means is that it's just coming out of the bottom and being collected. And so you may be wondering, exactly how is it that we get this protein separation in the column? And the way that it works is that the different components inside of our protein mixture will have different affinities for the mobile phase and for the stationary phase. And that's what allows for separation and by this word, affinities here what we really mean our attractions. And so if you have a strong affinity for something, that means that you have a strong attraction for it. And so each of these components in the mixture have different attractions. Uh, strengths, um, for the mobile phase and stationary phase. And that's really what allows for the separation. And so, essentially, what you'll see is that if we continuously run mawr and more mobile phase were able to dilute each of the components and, um, at the bottom of our column and collect them and separate tubes so that we have are purified purple proteins are purified orange protein and are purified, uh, teal protein here. And so protein purification is ideally complete after our column chromatography. And it turns out that there are actually several different types of chromatography that separate proteins based off of different properties, some separate based off of charge, some separate based off of size. Others polarity. And so we're gonna talk about different types of column chromatography, uh, moving forward in our course. But first, before we get there, let's get a little bit of practice, so I'll see you guys in those practice videos