Jeffrey Perkel has been a scientific writer and editor since 2000. He holds a PhD in Cell and Molecular Biology from the University of Pennsylvania, and did postdoctoral work at the University of Pennsylvania and at Harvard Medical School.
When researchers are searching through cellular components for specific enzyme or protein activities, they have no choice but to start with relatively crude mixtures. That’s the proverbial haystack, biologically speaking. Their job: Find the needle.
Unfortunately, crude protein mixtures are, shall we say, complicated. There’s the protein you want, plus a whole bunch of stuff you don’t. The best way to separate the two is liquid chromatography. Several options have been developed, including affinity chromatography, gel filtration and reversed-phase chromatography. (See GE Healthcare’s free handbook, Strategies for Protein Purification, #28-9833-31, for a nice summary of different chromatography approaches.)
One popular option for protein separation is ion exchange chromatography (IEX), which separates molecules by charge. In the case of proteins, charge is a function of the molecule’s amino acid composition and the pH of the buffer relative to the molecule’s isoelectric point (pI). Under the right conditions, the technique can separate molecules differing by just one charged group.
IEX resins do exactly what the technique’s name implies: They grab molecules based on their charge and release them when buffer conditions change such that the column resin “exchanges” one molecule (the protein) for another (a charged ion in the buffer). (GE Healthcare also publishes a handbook specific to IEX, Ion Exchange Chromatography & Chromatofocusing: Principles and Methods, #11-0004-21.)
Here are some variables to consider when selecting an IEX resin.
Cation or anion?
The most obvious variable in selecting an IEX resin is charge: Under the buffer conditions used, is your protein positively charged (a cation) or negatively charged (an anion)? Cation exchange resins are negatively charged, to capture positively charged molecules. Anion exchange resins are positively charged to capture negatively charged molecules.
IEX chromatography resins can also be classified as “strong” or “weak.” These classifications refer to how easily the resin exchanges ions as buffer conditions (e.g., salt concentration or pH) change. According to Strategies for Protein Purification, “A strong ion exchange medium has the same charge density on its surface over a broad pH range, whereas the charge density of a weak ion exchanger changes with pH.” In other words, a strong ion exchanger tends to hold onto bound molecules as buffer conditions change, but weak ion exchangers do not.
Porous or non-porous?
IEX, like all chromatography, relies on the relative affinity of a molecule for the stationary phase of separation, or resin, vs. the mobile phase (the buffer). The “resin” is essentially a functionalized bead of some sort, and those beads can have different properties.
One such property is porosity, which influences resolution and binding capacity. According to Ion Exchange Chromatography & Chromatofocusing, “High porosity offers a large surface area covered by charged groups and so ensures a high binding capacity. High-porosity is also an advantage when separating large biomolecules. Non-porous matrices are preferable for extremely high resolution separations when diffusion effects must be avoided.”
For instance, GE’s Sepharose resins are comprised of porous agarose, whereas its MiniBeads™ are made of a non-porous polystyrene/divinyl benzene mix.
Bead size?
Although many variables contribute to the quality of separation achieved in any given experiment, in general there exists an inverse relationship between bead size and resolution. In other words, all else being equal, a well-packed column of 3-um beads will produce sharper chromatographic peaks than a column made of 30-um beads.
The key phrase in that correlation is “well-packed.” Both pre-packed columns and loose beads are available, and you can pack your own column if you so choose. But be advised: According to Ion Exchange Chromatography & Chromatofocusing, “Columns that are packed unevenly, too tightly, too loosely or that contain air bubbles will lead to channeling (uneven passage of buffer through the column), zone broadening and hence loss of resolution.”
Loading capacity?
Another key variable to consider with a column, especially an off-the-shelf, pre-packed column, is loading capacity. There’s a limit to how much protein can be loaded on the column while maintaining good separation.
Obviously, how important this limitation is depends on the stage of your separation. If you are doing a first-pass cleanup of a crude extract, you’ll probably need to load more protein than at later stages of a purification protocol.
In any event, Ion Exchange Chromatography & Chromatofocusing recommends loading just a fraction of the capacity: “In general, loading 20-30% of the total binding capacity of the column gives optimal resolution with gradient elution. Sample loads can often be increased if resolution is satisfactory or when using a step elution.”
The image at the top of the page is from Bio-Rad Laboratories: "Purification of whey proteins with similar isoelectric points (pIs) on Nuvia Q media."