Buffer Selection in HPLC

Buffers added to aqueous solvents can be used to control the ionization state of an acidic or basic analyte thereby changing its retention. Ion exchange techniques require that an analyte be ionized and buffers can be used to ensure this. In reverse-phase separations, ionized species show the least retention and neutral species the most. A buffers can only be used to reliably control retention if it is used within ? 1.5 pH units of its pKa, with some buffers having multiple buffer ranges such as phosphate. Only buffers which are volatile are suitable for mass spectrometric detection techniques. The table below lists different buffers and their useful ranges and their suitability for mass spectrometric detection.



Typically 10-50 mM buffer concentrations are used. Higher concentrations run the risk of salt precipitation at higher organic modifier concentrations which could block the column, affecting pressure, efficiency and peak shape.