In this video, we're going to talk about our 5th and final step in our protein purification strategy, and that's column chromatography. So we've saved the best for last. 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. Recall that when we first did protein extraction, we obtained our crude extract from our cells, which was a big mixture of all of the cell components. Each purification technique that we talked about removed some unwanted cell components and unwanted proteins, so we were able to reduce our sample size. 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, can be relatively expensive, especially when used on a large scale with large sample sizes. The larger the sample size is, the more expensive the overall process and technique is going to be. There is always a tendency to reduce the cost by using cheaper techniques first, such as salting out, to reduce the sample size before we use more sophisticated and more expensive chromatography techniques.
Column chromatography is literally just a separation technique that uses a column. It uses a vertical column. Below in our example, we can see that in column chromatography, 2 different types of materials are used. The first type is a stationary phase, which is a solid material that is immobile, meaning it does not move throughout the entire chromatography process. It is stuck inside the column and remains in its same exact position. The second type of material is the mobile phase, which is a liquid that is mobile, meaning it does move during chromatography. In column chromatography, the mobile phase flows over the stationary phase and interacts with different components in the sample mixture to separate those components.
In our example below of column chromatography, notice that what we have are vertical columns, each packed with a stationary phase that does not move. Common types of stationary phase include silica. Inside the column, the first step is to add our protein mixture at the top. The second step is to continuously add mobile phase throughout the entire process. The mobile phase is inside these bottles, and we continuously add it throughout the entire column chromatography process. The mobile phase is mobile and it flows; it moves down the column and drips out through the bottom. What happens is the mobile phase interacts with the proteins in our mixture to separate the proteins, and over time, as we add more mobile phase, we get better separation of our proteins. Eventually, all of the materials will be eluted at the bottom of the column. The term "elute" here means that they are collected and removed from the column.
Protein separation in the column results from different components in the protein mixture having different affinities for the mobile and stationary phases. "Affinities" here means attractions. If a component has a strong affinity for something, it has a strong attraction to it. Each of these components has different attraction strengths for the mobile phase and the stationary phase, which allows for separation. If we continuously run more and more mobile phase, we are able to elute each of the components at the bottom of the column and collect them in separate tubes, leading to purified purple protein, orange protein, and teal protein. Protein purification is ideally complete after our column chromatography. There are actually several different types of chromatography that separate proteins based on different properties, such as charge, size, or polarity. We're going to talk about different types of column chromatography 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.
