As is expected with a group of organic acids, lower pH will reduce the degree of ionization and make the components more hydrophobic and thus more strongly retained. The simulated chromatograms were generated from the data of reference 5. When using pH to control an LC separation, it is especially important to obey the fundamental rules of buffer usage. Outside this range, buffering is marginal, meaning that methods using those conditions will be less robust. Another variable that can provide useful changes in selectivity is column temperature.
Most workers consider temperature only in terms of retention time changes.
Table I lists the conditions for this separation. Temperature control is important for reproducible retention times. Same as in Figure 2. The simulated chromatograms were generated from the data of reference 5. It is less appreciated that changes in temperature can cause changes in selectivity in LC separations.