Handbook of Size Exclusion Chromatography and Related ...

Figure 10 Diol bonding reactions.
In a related paper, the same authors demonstrated the fractionation of milligram
quantities of polypeptides and proteins up to 50,000 dalton molecular weight, with
excellent recovery of biological activity on amide columns prepared from
LiChrosorb Si-100 silica (78). Miller et al. (79) synthesized an ether bonded phase
of the general formula ;Si O Si(CH 2) 3 O (CH 2 CH 2 O)n R,
where n ¼ 1, 2, or 3 and R ¼ methyl, ethyl, or n-butyl. Resulting phases allowed
the separation of proteins under hydrophobic interaction or SEC conditions.
Functional groups were bonded to the silica as trialkoxysilane reagents. The
reaction was performed in the presence of water to control the formation of a
bonded phase network that is more stable in aqueous solutions than those
produced from di- or monofunctional silanes (80). When operated in the SEC
mode, an ether bonded phase column showed stable elution volumes for basic
proteins in high ionic strength (0.5 M ammonium acetate, pH 6.0) mobile phase
after flushing the column for 40,000 column volumes. At low ionic strength
(0.05 M ammonium acetate, pH 6.0), the retention of lysozyme increased 2-fold
during the same experiment. Recently, Poppe and colleagues discussed the
inertness and stability of a maltose stationary phase (81). Effective shielding of the
silica surface was obtained by reacting maltose to aminopropyl bonded silica.
Stability against hydrolysis greatly improved by using acid-washed silica, by
adding a small amount of water to the silica before bonding with
aminopropylsilane, and by polymerizing the glucose units in the maltose groups
© 2004 by Marcel Dekker, Inc.