The value of k was calculated from tr and the column dead time (t0) using the following equation: k =(tr-t0)/t0 (1) To determine t0, three candidate tracers were measured at each φMeOH: uracil, DL-glyceric acid (GA), and D-(-)-quinic acid (QA). The previous studies determined t0 using uracil, which is mostly neutral (pKa, 9.4). The measured retention time of uracil at low φMeOH was slightly longer than that of GA and QA (Figure ), suggesting the possibility of weak retention for uracil. In addition, anionic compounds may undergo an anion exclusion effect with silica-based stationary phases, which are negatively charged near neutral pH, and thus the use of an anionic tracer may be more appropriate. Since GA showed a slightly shorter retention time than QA, we decided to use tr of GA for t0.