Isopropylammonium Formate as a Mobile Phase Modifier for Liquid Chromatography

Isopropylammonium formate (IPAF), a new alkylammonium formate (AAF) room temperature ionic liquid, has been synthesized from isopropylamine and formic acid and characterized as an organic solvent mobile phase replacement for reversed-phase liquid chromatography (LC). Characterization of IPAF solvent properties in water such as pH, conductivity, and viscosity, as well as its synthesis, is described. The LC polarity (P') and the solvent strength (S) parameters are determined to be 6.0 and 2.4, respectively, similar to those same parameters for methanol and acetonitrile. Application of this RTIL is demonstrated as an organic solvent replacement for reversed-phase LC to separate a test mixture of niacinamide, acetophenone and p-nitroaniline. The van Deemter plot profile for several columns of different dimensions, particle size, pore size and stationary phase are compared using an IPAF-water mobile phase. At flow rates above 2 mL/min, on-line mixing of the viscous IPAF with water appears not to be uniform. A flattening of the van Deemter profile is noted for particularly short (50 mm) wide bore (4.6 mm) columns packed with larger particles (10 µm). Small particle longer columns likely facilitated mixing at the beginning of the column generating typical linearly increasing van Deemeter curves. IPAF has been further shown as a function of temperature to be a non-denaturing modifier solvent for the separation of the protein cytochrome c from tryptophan compared to methanol. This is important to show, because the semi-preparative separation of native proteins using AAF mobile phases is the long-term goal of this research program.