Gradient LC separation of macromolecules: theory and mechanism.

The theory and mechanism for the reversed-phase liquid chromatographic separation of macromolecules was somewhat vague and controversial until recently when the Flory-Huggins theory of dilute polymer solutions was used to provide the technique with a firm theoretical foundation. As a result of this treatment, the concept of a critical mobile phase composition (Xc) has been introduced to liquid chromatography. Macromolecular solutes below Xc are completely retained, while above Xc they are rapidly eluted. Because the capacity factor of a solute is related to Xc, traditional chromatographic parameters such as theoretical plate number have little meaning. A brief summary of the history of chromatographic polymer separations that foreshadows the current theory is given. A review and an evaluation of the current mechanistic status of macromolecular chromatography are presented. Also considered is the use of adsorption theory, "small molecule" theory, and solvophobic chromatographic theory for macromolecular separations. The theory and separation mechanism are relatively straightforward for synthetic polymers but tend to be more complex and less clearcut for biological polymers.