Retention of Ionizable Compounds on HPLC. 12. The Properties of Liquid Chromatography Buffers in Acetonitrile−Water Mobile Phases That Influence HPLC Retention

Abstract

The addition of acetonitrile to aqueous buffers to prepare RP HPLC mobile phases changes the buffer properties (pH and buffer capacity). This variation is studied for ace tate, phosphate, phthalate, citrate, and ammonia buffers in acetonitrile-water mixtures up to 60% in acetonitrile (v/v). Equations are proposed to relate pH and buffer capacity change of these buffers to the initial aqueous pH value and to the volume fraction of acetonitrile added. It is demonstrated that the pH change of the buffer depends not only on the initial aqueous pH of the buffer and on the percentage of acetonitrile added but also on the particular buffer used. The proposed equations allow an accurate prediction of this ionization for the studied buffers. Since the retention of acid/base compounds shows a strong dependence of their degree of ionization, the equations are used to predict the change in this ionization with addition of acetonitrile when the RP HPLC mobile phase is prepared. This prediction allows estimation of the retention of an acid/base compound in a particular acetonitrile-water buffered mobile phase.

Chromatographic behavior of epirubicin and its analogues on high-purity silica in hydrophilic interaction chromatography

“…The stronger retentions of the solutes with acetonitrile mobile phase reveals that, besides adsorption, ion exchange interactions between solutes and stationary phase exist as well, i.e. the effects of organic modifier on the dissociation of buffer compound and the pK a of the silica silanols also contribute to retention properties in HILIC [24,25]. The variation in elution order of the solutes in the two solvents is a reflection of complexity of the interferences between these effects.…”

“…Moreover, the influences of organic modifier on the dissociation of buffer compound and the pK a of both analytes and silanol groups of silica have played a role in the resolution of solutes [24,25]. The combination of these effects makes it rather difficult to unravel our experimental results with common theoretical models in HPLC.…”

Development of Zwitterionic Hydrophilic Liquid Chromatography (ZICⓇHILIC-MS) Metabolomics Method for Shotgun Analysis of Human Urine

“…This may have reduced retention on RPLC, but increased retention on HILIC as evident from the results. The composition of aqueous mobile phase can affect the buffering capacity [21]. The pKa of weak bases decreases with an increase in the organic component in mobile phase whereas the pKa of weak acids increase.…”

Reversed‐Phase HPLC

“…There have been many studies about the determination of pK values of acids [61][62][63][64][65] in acetonitrile/water mixtures. Several models have been used to relate the pK with the solvent composition [66,67].…”

“…Typically, the pH of acidic buffers upon the addition of acetonitrile or methanol increases to higher values, and the pH of basic buffers upon the addition of acetonitrile or methanol decreases to lower values. On the basis of IUPAC rules and recommendations [68,69], Bosch and co-workers have studied the different pH scales that are employed in pH measurement of hydro-organic mixtures [64]. The three different pH scales are usually considered the w w pH, s w pH, and s s pHs.…”