Handbook of Size Exclusion Chromatography and Related ...

sample components can be varied by adjusting the amount of water in the mobile
phase. (Because sometimes a variation of as little as 10 ppm water can make the
difference between a good separation and no separation at all, alcohol is frequently
used to modify retention, which requires a larger volume and is thus easier to
control.)
Early successful attempts at reducing the activity of silanol groups on porous
glass supports included the use of mobile-phase modifiers (65) and coating the
surface with 20,000 dalton polyethylene oxide (66). A more permanent way to
deactivate silanol groups is to convert them through chemical reaction. Regnier
and Noel (5) first demonstrated that by reacting controlled porosity glass beads
with glycidoxypropyltrimethoxysilane, followed by opening the epoxide ring
under acid conditions, resulted in a hydrophilic surface suitable for analysis of
proteins, nucleic acids, and polysaccharides by a size exclusion mechanism. Other
examples of modifications are discussed here.
2.9 Chemical Modification
As mentioned in the introduction, the explosive growth of HPLC would not have
taken place without the recognition that instead of coating the stationary phase to
the silica surface, a permanent bonded phase would do away with some important
limitations of physically held phases (67–69). Among these limitations were slow
equilibration, decreasing retention as a function of time, and the inability to inject
samples dissolved in solvents that were miscible with the stationary phase. Early
investigations in bonded phase synthesis (68,69) employed esterification of
surface silanols to form a ;Si O C bond, which, however, was found to
hydrolyse in aqueous solutions (70). It was replaced by the silylation reaction,
leading to the formation of the more stable ;Si O Si C bond (71). Initial
bonded phase columns did not have the required physical stability and
reproducibility of retention and selectivity. Development of improved packing
and bonding procedures (72–74) corrected these weakenesses, resulting in the
design of reliable, automated HPLC-based analysers (75).
It is interesting to note that the first prepared HPLC bonded phase, named
C18 after the octadecylsilane bonding reagent, soon became the most popular
column type. According to a 1991 survey, this continues to be the case today with
almost half of all HPLC analyses being performed on this column type (76).
Chemical modification of the silica surface with long-chain alkyl groups creates a
nonpolar, hydrophobic surface that interacts with sample molecules through weak
dispersion (van der Waals) forces. Retention is in direct proportion to the
hydrophobic surface area of the molecule, and elution is accomplished with a
mobile phase consisting of a mixture of water and an organic solvent, such as
methanol or acetonitrile. The use of an aqueous mobile phase has greatly
simplified the injection of samples studied in the life and food sciences and related
© 2004 by Marcel Dekker, Inc.