pH affects the electrophoretic mobility in capillary electrophoresis in several ways. The migration order of analytes is influenced by their charge and the strength of the EOF. At high pH levels (where EOF is significant) cations migrate first, followed by neutrals, and then anions. Increasing the pH above 9.75 leads to a greater presence of neutral NH3 molecules, which reduces the overall mobility of solutes in capillary electrophoresis. Neutral molecules, however, remain unseparated due to their lack of net charge. Anions still migrate towards the cathode due to the stronger EOF. At lower pH levels, EOF is reduced, allowing for the measurement of both cations and anions. Specifically, lowering the pH below 9.75 increases the movement of the solute in capillary electrophoresis by increasing the presence of positively charged NH4+ ions.
The pH of the buffer solution also affects the charge of analytes, particularly for complex compounds like peptides. Choosing the optimal pH involves considering the analyte's pKa, with a recommended deviation of at least two units for complete ionization. Highly alkaline pH levels can lead to rapid EOF, potentially causing incomplete separations. Protein separations can be feasible at acidic pH levels, where the capillary wall is uncharged, thus preventing electrostatic interactions. However, hydrophobic interactions may still occur, affecting separation efficiency.