Automated Programmable Generation of Broad pH Range Volatile Ionic Eluents for Liquid Chromatography

Many
of the universal detectors in liquid chromatography, including
mass spectrometry, must completely volatilize the chromatographic
eluent first before further processing and detection of the analytes.
A basic requirement is that the eluent does not contain a nonvolatile
dissolved component. However, separation of biomolecules must be conducted
in mostly aqueous media of compatible pH and ionic strength if their
biological activity must survive the separation process. Combinations
of ammonia with acetic and formic acids are commonly used as eluent
for this purpose but generally maximum concentrations that can be
tolerated are relatively low. Further, buffering is good only over
a limited pH range. We describe a system where the eluent is generated
in an automated pressure-programmed manner from high-purity gaseous
NH3 and CO2 through gas-permeable membrane devices.
This can be aided by the prior presence of formic/acetic acids in
the mobile phase to extend the attainable low pH limit. We outline
the fundamental pH, ionic strength, and buffer intensity considerations
and demonstrate the application of such eluents in the separation
of amino acids, proteins, and monoclonal antibodies. We also demonstrate
the use of dissolved CO2 as an ion-pairing agent in the
separation of chiral amines.