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Some may suggest that
"all compounds can be
analyzed using only acetonitrile and water as the HPLC mobile
phase," but this is incorrect because it does not allow for
the control of ionization in ionic compounds.
So, is it acceptable to use only acetonitrile and water for
non-ionizable compounds?
For many
common compounds, this may be possible. However, in the case of
silica-based columns, even if the solute itself is non-ionic,
retention and peak shape reproducibility may not always be achieved
with just water and acetonitrile. This is often due to the presence
of residual silanol groups on the silica surface, which can
interfere with separation.
Silanol groups (SiOH) are
acidic and can
undergo dissociation as follows:
SiOH <->
SiO⁻ + H⁺
On the silica surface, residual
silanol groups always exist and become negatively charged at neutral
pH. If a compound is basic,
it can interact ionically with the dissociated silanol groups,
leading to increased retention and potential peak tailing due to
surface heterogeneity in the stationary phase.
Additionally, dissociated silanol groups can also exert
ion-dipole interactions
on non-ionic compounds, which can result in poor reproducibility of
retention and peak shape.
Residual silanol groups are
minimized through the
end-capping process, but they are never completely
eliminated. Additionally, over time, the silica surface undergoes
hydrolysis due to
exposure to water/acetonitrile, leading to the regeneration of
silanol groups.
Even if the analyte is
non-ionic, the presence of
ionized silanol groups
on the silica surface necessitates the use of
pH modifiers in the
mobile phase to control dissociation, along with an appropriate
ionic strength to
ensure stable retention and peak shape reproducibility.
When analyzing non-ionic compounds,
adding 0.1% acetic acid to the mobile phase generally enables highly
reproducible analysis. Acetic acid is volatile and serves as a
suitable acid additive for silica columns, as it maintains the pH
above 3, preventing excessive silica hydrolysis. Some applications
even report that adding up to 10% acetic acid to the mobile phase
does not cause column damage.
For ionic compounds, active
control of both mobile phase pH and ionic strength is essential.
Failing to do so can lead to poor reproducibility in retention and
peak shape.
It is strongly recommended to avoid using a pH meter when preparing
mobile phase buffers, as slight variations in the acid-to-base ratio
can impact ionic strength and retention. To prolong column lifespan,
it is preferable to prepare buffers using volatile salts, such as
ammonium acetate or ammonium formate.
(Reference)
Do Not Use a pH Meter for Mobile
Phase pH Adjustment
<How to Prepare Volatile Buffers>
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