Yarra GFC/SEC Columns

The void volume corresponds to the amount of mobile phase in an LC system. In most separation modes the void volume would be equivalent to the amount of mobile phase needed to elute a compound that doesn't interact with the stationary phase and is hence unretained. In size exclusion, analytes are not separated based on chemical interactions with the stationary phase but rather their ability, based on size, to enter pores in the stationary phase. Therefore, in size exclusion the terms total permeation and total exclusion volume have greater significance than void volume. The total permeation volume corresponds to the amount of mobile phase in the system, including that in the pores of the stationary phase, whereas the total exclusion volume does not include the mobile phase within the pores of the stationary phase. Consequently, the total permeation volume corresponds to the amount of mobile phase needed to elute a compound that is small enough to completely enter into the stationary phase pores and the total exclusion volume is the amount of mobile phase needed to elute a compound that is entirely excluded from the stationary phase pores.

Plot of KD vs. Log MW KD = (analyte elution time - time to elute the exclusion volume) / (time to elute total permeation volume- time to elute the exclusion volume). Log MW is simply the logarithm of the analyte's molecular weight (base 10)

Occasionally to facilitate a true entropic SEC separation a small amount of an organic modified such as ACN is required however more than 15% may result in de-naturing or unfolding of the protein to occur and should be avoided.

Although sodium azide can be added to a solution to prevent microbial growth it is not effective at removing growth that has already occurred. In a system is contaminated with microbial growth it is more effective to flush with 50:50 acetonitrile:water to remove the growth from the system. Note that the column should be flushed with water to remove any buffer salts (e.g. phosphate) before washing with high organic. It may also be necessary to clean or replace guard columns and in-line filters as these are likely to be blocked.

For ionic/polar compounds, adding a salt (typically 50-100mM) to the mobile phase is a good way to disrupt secondary interactions between analytes and the stationary phase that contribute to poor peak shapes. Commonly, for GFC, NaCl in used. For GPC, lithium bromide (LiBr) is used.

The most common application for SEC is aggregate analysis. This requires “native” conditions; i.e. physiological conditions. This requires a buffered solution at around pH 7 with a moderate amount of salt. A good starting point for mobile phase is a 100 mM Phosphate Buffer, pH 6.8. However, there are some instances where it is appropriate to optimize the mobile phase to improve chromatography. If it is suspected the protein is hydrophobic, a small amount of organic solvent (e.g. methanol, acetonitrile, isopropanol) can be added. This can be anywhere between 5-10%. If it is suspected that the protein may have strong ionic character (e.g. basic protein or protein with a high isoelectric point), the ionic strength of the eluent can be increased to improve chromatography. A 2X Phosphate Buffered Saline (i.e. 50 mM Phosphate, 300 mM Sodium Chloride, pH 6.8) is recommended to prevent secondary interactions. Note that increasing the salt will also increase hydrophobic interactions as well. Furthermore, varying the salt may improve the separation of one critical pair, but result in a loss of resolution of another.

Optimal salt concentration can vary depending on the nature of the compound in question. Typically, 150-250 mM works best, however you must balance the ionic and hydrophobic nature of the compound. The lower the salt concentration, the more ion-exchange will be observed. At higher salt concentration, more hydrophobic adsorption is observed due to salting of the protein. We typically recommend the customer uses 50 mM potassium phosphate and 250 mM KCl at pH 6.2 as a starting point to achieve the best results. Potassium is the preferred counter ion as it generates better peak shape and reduces interactions with any silanols at the surface of the particle.