ABSTRACT

I. INTRODUCTION Gel permeation chromatography/size exclusion chromatography (GPC/SEC) is a liquid column chromatographic technique that segregates molecules based on their effective molecular sizes in solution, i.e., their hydrodynamic volumes. The stationary phase particles (often polymeric, but sometimes based on silica) contain pores of various sizes. Solute molecules that are smaller than the available pores are able to enter the stationary phase particles and, thus, reside within the particles for a finite time, then leave the pores and enter the mobile phase where they can travel along the column, carried by the mobile phase, until they enter pores further along the column. Molecules that are larger than the available pores cannot enter the pore structure. They travel through the column exclusively in the mobile phase and elute first. Very small molecules can enter virtually every pore they encounter and, therefore, elute last. Intermediate-size molecules travel through the column at a rate that is in proportion to the fraction of the time they spend in the mobile phase. The sizes, and sometimes the shapes, of the mid-size molecules regulate the extent to which they can leave the mobile phase and enter the pores. So the largest molecules in a sample will elute first, followed by smaller molecules, sequentially, according to size, and finally by the smallest molecules. GPC/SEC is particularly well suited to separate high molecular weight substances, including biological macromolecules and synthetic polymers. The resultant data are generally used to calculate the molecular weight averages (number-average, weight-average, viscosity-average, z-average, z + 1-average) as well as the molecular weight distribution of the molecules contained in a sample.