Handbook of Size Exclusion Chromatography and Related ...

Tris–HCl buffer, pH 8.0, whose ionic strength was adjusted to 0.5 by Li2SO4,was
used as the eluant. PVP was adsorbed by the column when water or buffer solution
was used as eluant; upon the addition of 10% (vol/vol) ethylene glycol to the
eluant, however, this interaction was eliminated.
Herman and Field synthesized monomeric diol onto E. Merck Lichrospher
SI-500 and reported in 1981 (10) the SEC of PVP with molecular weight 10,000.
Poor recovery (0–25%) of PVP was noticed using water as eluant. A 100%
recovery was obtained using 40% acetonitrile in 0.01 M KH2PO4, pH 2.1. A 100%
recovery of PVP was also reported using a TSK-PW-3000 column with 0.08 M
Tris buffer. Mori reported in 1983 (11) the SEC of PVP with molecular weights
from 11,000 to 1,310,000 AMU using two Shodex AD-80M/S columns with
dimethylformamide (DMF) and 0.01 M LiBr as eluant at 608C. Separation of PVP
based on hydrodynamic volume in this SEC system was demonstrated by the
applicability of universal calibration using PEO and polyethylene glycol as
calibration standards. Domard and Rinaudo grafted quaternized ammonium
groups onto silica gels with pore diameters 150, 300, 600, 1250, and 2000 A ˚ and
reported in 1984 (12) the SEC of PVP K-15, 25, 30, 60, and 90 using 0.2 M
ammonium acetate as the eluant. Some adsorption of PVP K-15, 25, 30, 60 and 90
was noticed by deviation from the universal calibration curve.
In 1984, Malawer et al. (13) conducted a thorough study on the SEC of PVP
K-15, 30, 60, and 90 using diol-derivatized silica gel column sets and aqueous
mobile phase modified with various polar organic solvents. A log-linear
calibration curve over three decades in molecular weights was obtained on a
specially constructed Electronucleonics gylceryl-CPG column set consisting of
two 75, 500, and 3000 A ˚ columns and was found to provide better recovery and
separation than the commercially available prepacked, 10 mm high-efficiency diolderivatized
silica gel columns. Methanol was found to be a better aqueous mobilephase
modifier to eliminate the adsorption effect than either dimethyl-formamide
or acetonitrile. The best recovery (.90%) and separation were obtained with a
mobile phase of 50:50 (vol/vol) methanol–water containing 0.1 M LiNO3.
In summary, when commercially available SEC columns are used,
successful SEC separation of PVP without polymer-column interactions has
been reported in either an aqueous environment (8) or DMF (11). However, as
indicated later, the aqueous environment has the advantage of providing better
separation at the low-molecular-weight end of the SEC peak, especially for the
lower molecular weight grades, PVP K-30 and K-15. Therefore, the remaining
discussion of PVP concentrates on the aqueous environment.
3.2 SEC/LALLS and SEC with Universal Calibration for PVP
In a continuation of an earlier work (13), Senak et al. reported in 1987 (4) the most
extensive SEC study on PVP to date with the determination of absolute molecular
© 2004 by Marcel Dekker, Inc.