Everything You Need To Know About Chromatography for the USABO, Part 1: Column and Affinity Chromatography
USABO Opens is this Monday!!! AAAAAAAAAAA
Let’s begin with the basics. Column chromatography is the simplest form of chromatography, so it’s a good context to learn the basic terms. The goal of chromatography is separation of molecules by some property. For column chromatography, the property that differs is molecular polarity: in other words, if a molecule is hydrophilic or hydrophobic.
Column chromatography requires both a mobile phase (eluent) and stationary phase. The polarity of each phase depends on the goal of our chromatography experiment. If we want to purify the polar part of the sample, then we need a polar stationary phase and a nonpolar mobile phase. This allows us to slow the motion of the polar part of the sample, because the polar part of the sample will be interacting with the stationary phase instead of moving through the column. The opposite is true if we want to purify the nonpolar portion of a sample: the stationary phase will be nonpolar to slow the flow of nonpolar components in the sample.
Essentially, we are slowing the flow of the part of the sample we want to isolate. This means that the desired part of the sample will fall out near the end. So, when we take multiple fractions of the sample throughout the time period of the experiment, our desired portion will come out near the end. The act of our sample (accompanied by mobile phase/eluent) exiting the column is known as elution. The mix of the eluent and our sample that exits the column is called the eluate.
There are four other kinds of chromatography relevant to USABO: affinity chromatography, size-exclusion chromatography, ion exchange chromatography, and high-performance liquid chromatography. All of these have a mobile phase, stationary phase, and an elution step. Additionally, fractionation over time remains an important concept for all of these variations. In this article, I’ll go over affinity chromatography, and I’ll go over the rest next week.
Affinity chromatography is similar to column chromatography: the molecule of interest has an affinity for the stationary phase (which, recall, is stuck to the column), and the mobile phase just passes through. However, the molecule of interest usually doesn’t just fall out on its own: rather, it remains stuck to the stationary phase until we do something about it. For protein affinity chromatography, we can use an acid to denature the protein bond and release the molecule of interest. The molecule of interest will elute with the acid as its mobile phase, and this is what we will collect. There is no time-based fractionation approach for this method.
Another method for affinity chromatography elution can be demonstrated through examining affinity chromatography for recombinant proteins. Recombinant proteins are genetically engineered proteins with custom attached sequences. One sequence that can be attached is that of glutathione S transferase (GST). The substrate to this enzyme is glutathione (GSH), which is used for the stationary phase. When the sample is poured through the column, the recombinant proteins with a conjugated GST will have an affinity for the GSH stationary phase. To elute the GSH-bound proteins of interest, we can use free GSH; the proteins of interest will then detach from the stationary phase GSH and bind the free GSH. We can then collect the eluted fraction.
Another interesting, somewhat similar, elution method involves adding a histidine tag to the proteins of interest. This tag is simply a poly-histidine sequence. The stationary phase would be nickel ions (which histidine has an affinity for). Elution occurs using free histidine (or a similarly-shaped molecule, imidazole), which competes with the proteins of interest for the stationary phase nickel. Think carefully about the difference between this and the GST: don’t get them confused.
That’s all I got for this week, and stay tuned for part 2! Also GOOD LUCK EVERYONE ON USABO!!! Here’s a cool article about this stuff: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5206469/
