Protein Protocols Protein Protocols

HPSEC 577
Fig. 2. Plot of K d vs log molecular weight for those proteins listed in Table 1 with K d values
between 0.1 and 0.9 (i.e., all except glucagon and thyroglobulin). Chromatography was carried
out as described in the caption to Fig. 1. V 0 was determined to be 6.76 mL from the elution
peak of blue dextran. Vt was determined from the elution peak of glycine and from the negative
peak given by injecting water, both of which gave a value of 11.99 mL. The regression line
y = –0.4234x + 2.4378, r 2 = 0.9657 was computed using all the points shown.
exclusion chromatography is carried out at a low pH, the opposite behavior is found, with
highly cationic proteins being eluted early and anionic ones being retarded. To explain
this behavior, it has been suggested that at pH 2.0 the column may have a net positive
charge (11). To reduce ionic interactions it is necessary to use a mobile phase of high ionic
strength. On the other hand, as ionic strength increases, this promotes the formation of
hydrophobic interactions. To minimize both ionic and hydrophobic interactions, the
mobile phase should have an ionic strength between 0.2 and 0.5 M (12).
2. The support used in size-exclusion chromatography consists of particles containing pores.
The molecular size of a solute molecule determines the degree to which it can penetrate
these pores. Molecules that are wholly excluded from the packing emerge from the column
first, at the void volume, V 0. This represents the volume in the interstitial space (outside
the support particles) and is determined by chromatography of very large molecules,
such as blue dextran or DNA. Molecules that can enter the pores freely have full access to
an additional space, the internal pore volume, V i. Such molecules emerge at V t, the total
volume available to the mobile phase, which can be determined from the elution volume
of small molecules. Hence V t = V 0 + V i. A solute molecule that is partially restricted from
the pores will emerge with elution volume, Ve, between the two extremes, V 0 and V t. The
distribution coefficient, K d, for such a molecule represents the fraction of V i available to it
for diffusion. Hence
V e = V 0 + K dV i
and K d = (V e – V 0)/V i = (V e – V 0)/(V t – V 0)
3. These models give rise to various plots that should result in a linear relationship between
a function of solute radius, usually R S, and some size-exclusion chromatography parameter,
usually K d (13). However, none has proven totally satisfactory. Recently it has been
suggested that size-exclusion chromatography does not require pores at all, but rather that