Solubility of Buffers in Aqueous–Organic Eluents for Reversed-Phase Liquid Chromatography

Address correspondence to P.W. Carr. hen preparing binary eluents containing both an organic solvent and a buffer for a buffer reversed-phase liquid chromatography (LC), one must know the solubility of the buffer in the organic cosolvent to avoid precipitation of the buffer salt. For example, one must know the highest percent organic cosolvent (that is, %B, v/v) in which a buffer salt remains soluble. Obviously, one can test the solubility of a buffer at a desired %B by preparing a small volume of the desired eluent and waiting a sufficient time to see if a precipitate forms. Although buffer solubility is determined readily, this method is tedious and time-consuming. One always can avoid precipitation of the buffer salt by using a very low concentration of buffer salt ( 10 mM) in the aqueous portion of the eluent. However, the resulting low buffer capacity can lead to slow equilibration, irreproducible retention, and poor peak shapes (1). Furthermore, both retention and selectivity are functions of the salt concentration for ionizable solutes (2–5). Thus, knowing the relationship between the buffer’s solubility limit and volume fraction of organic cosolvent is useful in reversedphase LC. There is a paucity of information in the literature on the solubility of common buffers in typical hydro-organic mixtures. We expect that buffer solubility should follow the same trend as the solubility of cations in water (NH4 K+ Na+). Therefore, we studied the three most common organic modifiers in combination with five of the more common buffer systems, including: ammonium acetate at pH 5.0, W ammonium phosphate at pH 3.0 and pH 7.0, and potassium phosphate at pH 3.0 and pH 7.0. We limited our studies to inorganic buffer salts, as they are less soluble in typical organic cosolvents compared with organic buffer salts. We also avoided sodium salts because they are expected to have lower solubility, which makes them less useful for reversed-phase LC.