Size Exclusion Chromatography
Compared to Reverse-phase, Size Exclusion Chromatography (SEC) or Gel Filtration is a bit more straightforward. As stated in the name it separates proteins based on size (or hydrodynamic volume). Imagine that you want to get the molecular weight of your peptide but there is a butt load of salt which will interfere with mass spectrometry since it relies on the mass: charge ratio.
Gel Filtration is a great for removing salt from your sample. Since you don't use harsh reagents you don't lose the biological activity of your protein. As usual there is a stationary phase and mobile phase. The stationary phase is made of particles or beads with pores of a specific size. The mobile phase is the buffer flowing through the particles and it carries the salts, proteins and everything else in the sample that you apply to the column. The smaller particles (or your tiny salt ions) will flow into the beads and get retained but the larger molecules (the proteins that you actually care about) will flow past the beads and get eluted. It's harder for the column to retain larger proteins so those proteins get eluted at a smaller buffer volume or retention time. The smaller proteins will get eluted with a larger volume of buffer or higher retention time. Since there is a linear relationship between the molecular weight of a protein and the elution volume, so you can use size exclusion to get a rough estimate of protein’s molecular weight.
Figure 1: Chromatogram with elution volumes for proteins of different sizes.
Figure 2: This is a bead of particle found in a typicla gel filtration column. The bead has tiny pores where small and adorabe solutes (like salt) can actually flow through the resin. Most of the proteins are too big to pass through these tiny pores so they actually flow through the column a lot faster, that’s why you need to less of your buffer to elute larger proteins.
Figure 3: Molecular weight vs Kav (or binding affinity of the protein for column)
A short list of boring but important jargon:
Void volume (V0): This the elution volume of stuff that doesn’t enter the pores or interact with the chromatography resin. These solutes just pass by the particles in the packed bed.
Internal volume (Vi): The volume of the pores within the gel.
Total volume (Vt): This is the total volume of your column. (It is usually the sum of internal and void volume)
Elution Volume (Ve): The volume of the buffer needed to elute a specific solute.
Stuff you need to keep in mind
Type of column and pore size: A longer column can give you a better separation of proteins, but it is the improvement in resolution is only proportional to the square root of the column length. Connecting columns with different pore sizes can help separate proteins with a wider range of molecular weights. You have to select your column based on the size of the target protein. Some columns can separate proteins with wide range of sizes (100-10 Kda) others are more suitable for separating smaller proteins (<10 Kda). The uniformity of the pore size is just important as pore size. Generally, if the pore size of the particles is larger you get poorer resolution.
Flow rate: Usually a slower flow rate leads to leads to better resolution or separation of proteins. However, one of the biggest drawbacks of size exclusion is the ridiculously long run time, since the flow rate tends to be slower than other forms like HPLC like Reverse-phase. The typical flow rate is usually between 0.5 ml/min and 1 ml/min.
Another time-consuming aspect of SEC is the equilibrating the column before use and washing it after use. Sometimes the more hydrophobic proteins end up aggregating and sticking to the particles in the column (like an annoying piece of gum) and you have to use really hydrophobic reagents to wash those proteins out (like Acetonitrile).
Reagents: The buffer you use doesn't affect resolution that much but you want to pick a buffer with low ionic strength and ideal for storing proteins (e.g. ammonium bicarbonate buffer). Usually the beads in the column are silica based so they are a bit hydrophobic and have a weak negative charge. So, what can happen is that proteins can end up getting retained by the anionic and hydrophobic beads, which gets in the way of eluting and collecting the proteins. Since you don't want any kind of ionic or hydrophobic interactions between the column and the solutes/proteins, adding a bit of salt to the solvent or mobile phase reduces these interactions and makes it easier to elute your proteins.
Another thing to keep in mind is that the shape of the protein affects how it moves through the column. A more rod like protein can move more easily than a more globular protein. BUT we want the column to separate the proteins based soley on size, so a tiny bit of a denaturing reagent (like SDS and guanidine hydrochloride) is added to the buffer. All of the proteins end up with a rod like shape and they are only separated based on size.
Primary sources
Barth, H. G., Jackson, C. & Boyes, B. E. Size Exclusion Chromatography. Anal. Chem. 66, 595–620 (1994).
Nagy, K. & Vékey, K. Separation methods. Med. Appl. Mass Spectrom. 61–92 (2008). doi:10.1016/B978-044451980-1.50007-0
Recommended readings
GE Healthcare. Size exclusion chromatography: Principles and Methods. GE Heal. Handbooks 139 (2012).
GE Healthcare. Size exclusion chromatography columns and media Selection guide. 1–10 (2016).
