28.7: Size-Exclusion Chromatography

We have considered two classes of micron-sized stationary phases in this chapter: silica particles and cross-linked polymer resin beads. Both materials are porous, with pore sizes ranging from approximately 5–400 nm for silica particles, and from 5 nm to 100 μm for divinylbenzene cross-linked polystyrene resins. In size-exclusion chromatography—which also is known by the terms molecular-exclusion or gel permeation chromatography—the separation of solutes depends upon their ability to enter into the pores of the stationary phase. Smaller solutes spend proportionally more time within the pores and take longer to elute from the column.

A stationary phase’s size selectivity extends over a finite range. All solutes significantly smaller than the pores move through the column’s entire volume and elute simultaneously, with a retention volume, V_r, of

\[V_{r}=V_{i}+V_{o} \label{12.3} \]

where V_i is the volume of mobile phase occupying the stationary phase’s pore space and V_o is volume of mobile phase in the remainder of the column. The largest solute for which Equation \ref{12.3} holds is the column’s inclusion limit, or permeation limit. Those solutes too large to enter the pores elute simultaneously with an retention volume of

\[V_{r} = V_{o} \label{12.4} \]

Equation \ref{12.4} defines the column’s exclusion limit.

For a solute whose size is between the inclusion limit and the exclusion limit, the amount of time it spends in the stationary phase’s pores is proportional to its size. The retention volume for these solutes is

\[V_{r}=DV_{i}+V_{o} \label{12.5} \]

where D is the solute’s distribution ratio, which ranges from 0 at the exclusion limit to 1 at the inclusion limit. Equation \ref{12.5} assumes that size-exclusion is the only interaction between the solute and the stationary phase that affects the separation. For this reason, stationary phases using silica particles are deactivated as described earlier, and polymer resins are synthesized without exchange sites.

Size-exclusion chromatography provides a rapid means for separating larger molecules, including polymers and biomolecules. A stationary phase for proteins that consists of particles with 30 nm pores has an inclusion limit of 7500 g/mol and an exclusion limit of \(1.2 \times 10^6\) g/mol. Mixtures of proteins that span a wider range of molecular weights are separated by joining together in series several columns with different inclusion and exclusion limits.

Another important application of size-exclusion chromatography is the estimation of a solute’s molecular weight (MW). Calibration curves are prepared using a series of standards of known molecular weight and measuring each standard’s retention volume. As shown in Figure 28.7.1 , a plot of log(MW) versus V_r is roughly linear between the exclusion limit and the inclusion limit. Because a solute’s retention volume is influenced by both its size and its shape, a reasonably accurate estimation of molecular weight is possible only if the standards are chosen carefully to minimize the effect of shape.

Figure 28.7.1 . Calibration curve for the determination of molecular weight by size-exclusion chromatography. The data shown here are adapted from Rouessac, F.; Rouessac, A. Chemical Analysis: Modern Instrumentation Methods and Techniques, Wiley: Chichester, England, 2004, p 141.

Size-exclusion chromatography is carried out using conventional HPLC instrumentation, replacing the HPLC column with an appropriate size-exclusion column. A UV/Vis detector is the most common means for obtaining the chromatogram.