When
preparing the HPLC mobile phase, there may be a need to adjust the
pH. In most cases, aren't we using a pH meter and adding acid or
base until the desired pH is achieved?
Relying on a pH meter
to prepare a "buffer" is fundamentally wrong. Because of the
buffering action, the pH does not change easily even if the added
amount is changed (that's why it's called a "buffering effect").
What's even more crucial for HPLC is the "ionic strength." With
ion-exchange columns and separation systems that involve ionic
interactions, insufficient ionic strength can affect peak shape and
reproducibility of retention and separation.
The method of
preparing the mobile phase based on the dependency of the pH meter,
assuming "it's fine as long as the pH matches," is incorrect.
In
biochemistry, when preparing a buffer, the correct method is to mix
aqueous solutions of acid and base of the same concentration (50mM
in this case) in a "volume ratio". With this, both ionic strength
and pH can achieve reproducibility. Once you monitor the mixing
ratio and pH for the first time, you can prepare quickly without
needing a pH meter from the second time onward.
For "ammonium
acetate" and "ammonium formate" and their buffers, which are
essential for LC-MS, they excel in volatility, so even if they seem
to be at a high concentration like 100mM, they mostly evaporate
during ionization. The ionic strength is also one third of that of
phosphoric acid. Because these organic salts are gentle on the
column, the use of acetic or formic mobile phases as pH adjusters
(buffers) is highly desirable.
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