Two classes of micron-sized stationary phases have been encountered in this sec- tion: silica particles and cross-linked polymer resin beads. Both materials are porous, with pore sizes ranging from approximately 50 to 4000 Å for silica particles and from 50 to 1,000,000 Å for divinylbenzene cross-linked polystyrene resins. In size-exclusion chromatography, also called molecular-exclusion or gel-permeation chromatography, separation is based on the solute’s ability to enter into the pores of the column packing. Smaller solutes spend proportionally more time within the pores and, consequently, take longer to elute from the column.
The size selectivity of a particular packing is not infinite, but is limited to a moderate range. All solutes significantly smaller than the pores move through the column’s entire volume and elute simultaneously, with a retention volume, Vr, of
Vr = Vi + Vo ……………..12.32
where Vi is the volume of mobile phase occupying the packing material’s pore space, and Vo is volume of mobile phase in the remainder of the column. The maxi- mum size for which equation 12.32 holds is the packing material’s inclusion limit or permeation limit. All solutes too large to enter the pores elute simultaneously with a retention volume of
Vr = Vo ……………..12.33
Equation 12.33 defines the packing material’s exclusion limit.
In between the inclusion limit and the exclusion limit, each solute spends an amount of time in the pore space proportional to its size. The retention volume for a solute is
Vr = Vo + DVi ……………..12.34
where D is the solute’s distribution ratio, which ranges from 0 at the exclusion limit to 1 at the inclusion limit. The validity of equation 12.34 requires that size exclusion be the only interaction between the solute and the stationary phase responsible for the separation. To this end, silica particles used for size exclusion are deactivated as described earlier, and polymer resins are synthesized without exchange sites.
Size-exclusion chromatography provides a rapid means for separating larger mol- ecules, including polymers and biomolecules. Figure 12.35 shows the application of size-exclusion chromatography for the analysis of protein mixtures. In Figure 12.35a, a column packing with 300 Å pores, with an inclusion limit of 7500 g/mol and an exclu- sion limit of 1.2 x 106 g/mol, is used to separate a mixture of three proteins. Mixtures containing a wider range of formula weights can be separated by joining together sev- eral columns in series. Figure 12.35b shows an example spanning an inclusion limit of 4000 g/mol and an exclusion limit of 7.5 x 106 g/mol.
Another important application is for the determination of formula weights. Calibration curves of log(formula weight) versus Vr are prepared between the exclu- sion limit and inclusion limit (Figure 12.36). Since the retention volume is, to some degree, a function of a solute’s size and shape, reasonably accurate determinations of formula weight are possible only if the standards are carefully chosen to mini- mize the effect of shape.
Size-exclusion chromatography can be 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.
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