All analytical techniques are governed by the principles of physical chemistry, and chromatography is certainly no exception. By examining the underlying concepts of size exclusion chromatography (SEC), students can gain greater appreciation of and insight into the science and utility of thermodynamics, as well as solution properties of macromolecules. In this paper we will show how the separation mechanism that governs SEC, an unusual form of chromatography, can be explained using the tenets of thermodynamics. This paper gives educators access to an HPLC technique usually not included in undergraduate chemistry curriculum.