Handbook of Size Exclusion Chromatography and Related ...

the decline in permeability is permanent. Similar results (not shown) were found
for YMC-GEL 120A silica, which started to deviate from linearity at 5000 psi. It
was shown earlier that the pore volumes of LiChrospher Si-100 and YMC-GEL
SIL120A S5wereconsiderablyhigher thanthat for SupelcosilLC-Si.Astandard
proceduretostrengthen silicaistosinter theparticles athightemperature (23,63).
Asaresult,thedistributionoftheporesshiftstowardlargersizes,andifperformed
in the presence of ahigh-melting salt, pore volume can be maintained.
2.7 Silanol Groups
The strong affinity of silica toward polar solutes, which makes it an excellent
choice as an adsorbent in adsorption chromatography,is responsiblefor it being a
less than ideal column packing material for size exclusion chromatography.The
amorphous nature of silica is reflected in the random distribution of various
chemical structures on the surface, as shown in Fig. 9(23). Free silanols are
isolated from other hydroxyl groups by an O O bond distance larger than
0.30 nm, that is, the average bond distance between two hydrogen-bonded silanol
groups. Vicinal and geminal silanols are not commonly discriminated and are
referred to as bound silanols. Because silica is hydroscopic at room temperature, it
contains physically adsorbed water. Heating under vacuum at 473 K for several
hours drives off most of this water. At higher temperatures, however, condensation
of bound silanols results in the formation of siloxane bonds. The total
concentration of silanol groups (free and bound) on silica is about 8 mmol/m 2 .Of
these groups, the free silanol groups constitute the premier adsorption and reaction
sites. The bound silanol groups play a secondary role in the adsorption process.
It is well known in HPLC that silica-based packings have two important
shortcomings: the silica matrix is not stable at alkaline pH, and most silane-bonded
phases can be cleaved at a pH below 2. After chemical modification, approximately
4 mmol/m 2 of silanol groups remains unbonded. These residual silanol groups are
negatively charged above pH ’ 3 and, when accessible, may interact with positive
charges on a polymer surface. Because of the use of organic solvents in GPC,
chemical stability of the silica is not a concern. The limited stability at high pH,
however, is a potential problem in GFC. In general, proteins are most stable at
pH 7–8, which is the upper limit of the accepted pH range for silica-based packing
materials. By removing metal impurities in the starting material, several
manufacturers have been able to produce highly purified silicas, although it is not
yet clear whether ultrapure silica particles have the same chemical stability as
standard silica particles. Taking the opposite approach, silica particles when
covered with 1 mmol/m 2 of zirconium oxide before performing the diol bonding
reaction, allowed extended operation at pH 8 or greater without degrading the
column performance (64). An alternative approach involves the preparation of
polymerized bonded phases. The bonded layer makes the ;Si O Si bond less
© 2004 by Marcel Dekker, Inc.