Size Exclusion HPLC
Size-Exclusion HPLC (SE-HPLC or SEC) separates analytes on the basis of a combination of their hydrodynamic size, diffusion coefficient, and surface properties. In SEC, the stationary phase is composed of inert particles packed into a dense three-dimensional matrix within a glass or steel column. The mobile phase can be pure water, an aqueous buffer, an organic solvent, or a mixture of these. The stationary-phase particles have small pores and/or channels which will only allow species below a certain size to enter. Large particles are therefore excluded from these pores and channels, and their limited interaction with the stationary phase leads them to elute as a ‘totally-excluded’ peak at the beginning of the experiment. But the smaller analyte particles, which can fit into the pores, are removed from the flowing mobile phase, and the time they spend immobilized in the stationary-phase pores depends, in part, on how far into the pores they can penetrate. Their removal from the mobile phase flow causes them to take longer to elute from the column and results in a separation between the particles based on differences in their size. Thus, only particles in a size range comparable to that of the stationary-phase pores will be fractionated. Figure 1 illustrates the process involved in an SEC experiment.
Figure 1. A mixture of small (red) and large (blue) analyte particles are injected onto a size-exclusion HPLC column. Stationary phase beads are shown in black. The far left image depicts a sample being injected onto a size exclusion column. Progressive images represent snapshots of the elution process progressing in time from left to right. Both types of sample particles enter the column at the same time. As the particles flow through the column, the small red particles enter the pores on the stationary phase beads while the blue particles are too large to enter the pores of the beads. The large blue particles therefore spend little time within the beads, causing them to traverse the column more quickly. The result is that the largest particles elute first, while the smaller particles elute later. Ideally, the elution order is from largest to smallest.
A wide variety of size exclusion columns are commercially available, and the choice of the best one for any particular application depends on a number of factors, such as the chemical and physical properties of the analyte mixture, the fractionation range of the stationary phase, the desired degree of resolution, and the amount of analyte to be injected onto the column. At Analytical Ventura we have experience with a wide variety of size-exclusion columns and conditions, and we will be happy to assist you in determining which column will provide the best results for your project.