When
ionizable molecules are analyzed with inverted phase chromatography, the
buffers usually need to separate them. The buffer resists the change in pH so
that compounds and silica (column) will be ionized consistently, resulting in
reproducible results of chromatography. A buffered mobile phase is selected to
maintain consistent retention time (RT) and selectivity to separating acids and
bases. Buffer salts effectively decrease peak tailing by masking silanols for
basic analytes. They also decrease the interactions of potential ion exchange
with unprotonated silanols. To separate the most basic compounds in reversed
phase-HPLC, it is recommended that the buffer concentration should be in the range
of 10 - 50 mM.
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