OPTIMUM HPLC PERFORMANCE continued
Klein’s equation estimates that about 80% of the stationary phase would be accessible.
Figure 3 shows the SEC calibration plot obtained by injecting individual peptide and protein standards on a small-pore 150-Å column. SEC plots like this supply specific size information relative to the pore size of the
column and can help users select HPLC columns that should provide adequate pore access and allow high-performance separations. Uracil (MW 112, point 12) elution volume estimates total column volume, and a monoclonal antibody (mAb) (MW 150 kDa, point 2) estimates excluded column volume. Since thyroglobulin (MW 667 kDa, point 1) lies outside the linear range, its elution volume is not an accurate indicator of size. The linear range of the semi-log plot estimates column pore volume. If the SEC calibration curve covers about two orders of magnitude, the 80% phase access point (a/r = 0.1) should be located near the midpoint of the linear plot, which intersects the calibration line at about MW 10,000. This is in agreement with the largest MW recommended earlier for HPLC on a 160-Å column. For the standards and column shown in Figure 3, larger molecules to the left of the midpoint would see less than 20% of the available stationary phase, while the smaller molecules to the right would see more than 80% of the available stationary phase on a 150-Å pore column. A larger-pore RP-HPLC column should be used for any sample component that lies to the left of the midpoint in Figure 3.
Giddings6 theorized that peak broadening occurs for larger molecules
Figure 2 – How the ratio of solute radius (a) to pore radius (r) impacts pore occupancy, KOC
, in pure SEC.5
because of pore crowding (radius-ratio of 0.1–1.0). He employed thermo- dynamic relationships to describe phase distribution in a bed of porous (or superficially porous) particles, where large molecules encounter wall constraints, gradually lose freedom of movement (decreasing entropy) and finally become excluded entirely from the pores. According to Gid- dings, this gradual exclusion process interferes with retention equilibria, slows mass transfer rate, and creates peak broadening. Wagner et al.3 reported the effect of 1000-Å pores for separations of mAbs and other large proteins and provide supporting evidence that diffusion rate (efficiency) of large molecules drops rapidly within the radius-ratio region of 0.1–1.0.
Figure 3 – SEC calibration curve for peptides and proteins. Conditions— column: Zenix SEC-150 (Sepax Technologies, Newark, DE), 4.6 × 300 mm; 3 µm; mobile phase: 0.2 M potassium phosphate, pH 7.0; flow rate: 0.25 mL/min; temperature: 25 °C; injection volume: 0.5 µL; instrument: Nexera (Shimadzu Scientific Instruments, Columbia, MD); detection: pho- todiode array (PDA), 215 nm and 280 nm. Peak identities: 1) thyroglobu- lin, 2) Sigma MAb, 3) IgG, 4) bovine serum albumin (BSA), 5) ovalbumin, 6) myoglobin, 7) ribonuclease A, 8) bovine insulin, 9) neurotensin, 10) Vitamin B12, 11) angiotensin II, 12) uracil.
AMERICAN LABORATORY 24 JUNE/JULY 2017
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