Handbook of Size Exclusion Chromatography and Related ...

silanol groups (52–54); even capped silica materials tend to exhibit some of this
property inasmuch as the “capping” process usually leaves some unmodified
silanols (51,52), and more silanols may be produced by erosion of capping groups
during use of the column (52). Some polymeric packing materials tend to be
negatively charged because of the presence of small numbers of carboxyl groups
(54). Proteins with net positive charges will therefore tend to be adsorbed on the
matrix, be retained longer on the column, and be assigned erroneously small
molecular sizes. Negatively charged proteins will (to a first approximation; see
below) tend to be repelled from the surface of the packing material, which
repulsion will result in their being denied access to some of the pore volume and
eluted earlier than would be expected on the basis of size alone.
For a given packing material, the most generally useful means of
suppressing electrostatic interactions with proteins is to vary the ionic strength of
the mobile phase until a region of ionic strength is encountered in which elution
volume is essentially independent of ionic strength (56–59). It should be kept in
mind that high ionic strengths tend to promote hydrophobic interactions; if a
simple minimum in elution volume is observed in the dependence of elution
volume on ionic strength, instead of a flat plateau of significant width, the results
may not mean that ideal SEC is taking place at the ionic strength producing the
minimum elution volume. Both electrostatic and hydrophobic binding to the
packing may be influencing the elution significantly, with the minimum elution
volume simply marking the ionic strength at which the sum of the two interactions
is at its minimum (60,61).
Another approach to suppressing electrostatic interactions is to adjust the
charges on the protein, the packing material, or both, by adjusting the pH of the
mobile phase (49,55–57). In (oversimplified) theory, if the positive and negative
charges on the protein can be equalized, so that the net result is an electrically
neutral molecule, there should be no electrostatic attraction or repulsion between
protein and packing material. In practice, however, one rather extensive evaluation
of this strategy found that the most nearly ideal SEC occurred when the mobile
phase pH was slightly above the isoelectric point of the protein (48).
Strategies based on protein pI values appear to work well in a number of
instances (55,56), although pH adjustment will not be an appropriate response to
non-SEC effects in the not-unlikely event that a given pH value is an integral part
of the experiment being conducted and not a variable that can be varied for purely
analytical reasons, or, as will be discussed below, in the event that protein stability
becomes a problem at the pH that would be chosen for chromatographic reasons.
Deviations from predictions based solely on net charges of proteins and packing
materials may also arise from chromatographic implications of the macromolecular
nature of proteins. In most cases, charged proteins cannot be represented
adequately as point charges equal to their net charges; the charged groups on the
exterior of proteins have definite distributions about quite appreciable diameters,
© 2004 by Marcel Dekker, Inc.