What happend on RT of substane when PH of mobile phase increase & decrease in HPLC?
The retention time (rt) will be shifted little. n reversed phase HPLC, the retention of analytes is related to their hydrophobicity. The more hydrophobic the analyte, the longer it is retained. When an analyte is ionized, it becomes less hydrophobic and, therefore, its retention decreases. Acids lose a proton and become ionized when pH increases and bases gain a proton and become ionized when pH decreases. Therefore, when separating mixtures containing acids and/or bases by reversed phase HPLC, it is necessary to control the pH of the mobile phase using an appropriate buffer in order to achieve reproducible results. For the most robust methods, it is recommended that separations be developed at a mobile phase pH where the retention of analytes are little affected by changes in pH. When separating bases, for example, acidic mobile phases usually show better reproducibility than neutral mobile phases. Methylamphetamine is fully protonated at a pH less than 3 and its retention is not affe
