Laserchrom HPLC - pH Buffer Guide. The importance of pH in HPLC. For some separations the effect of changing pH is minimal. However for acids and bases, the effect of even a very small change in pH is very significant. Changing the pH changes the degree of ionisation of molecules in solution. It thus affects their polarity, and as a consequence it changes their retention times in an HPLC separation. Buffers. MES Buffer - Biology-Online. Buffers for pH control (c) Rob Beynon, University of Liverpool.