The ionic liquid isopropylammonium formate as a mobile phase modifier to improve protein stability during reversed phase liquid chromatography

Abstract

The room temperature ionic liquid isopropylammonium formate (IPAF) is studied as a reversed phase HPLC mobile phase modifier for separation of native proteins using a polymeric column and this protein stability is compared to that using acetonitrile (MeCN) as the standard organic mobile phase modifier. A variety of important proteins with different numbers of subunits is investigated, including non-subunit proteins: albumin, and amyloglucosidase (AMY); a two subunit protein: thyroglobulin (THY); and four subunit proteins: glutamate dehydrogenase (GDH) and lactate dehydrogenase (LDH). A significant enhancement in protein stability is observed in the chromatograms upon using IPAF as a mobile phase modifier. The first sharper peak at about 2 min represented protein in primarily the native form and a second broader peak more retained at about 5–6 min represented substantially denatured or possibly aggregated protein. The investigated proteins (except LDH) could maintain the native form within up to 50% IPAF, while a mobile phase, with as low as 10% MeCN, induced protein denaturation. The assay for pyruvate using LDH has further shown that enzymatic activity can be maintained up to 30% IPAF in water in contrast to no activity using 30% MeCN.

Ionic Liquids in Analytical Chemistry: Fundamentals, Advances, and Perspectives

“…Baseline separation of the enantiomers was achieved within 14 min, while the enantioseparation factor was observed to be 1.34. Zhou and co-workers applied isopropylammonium formate (IPAF) as a mobile-phase modifier for the separation of native proteins . A significant enhancement in protein stability was observed when using IPAF as a mobile-phase modifier compared to acetonitrile.…”

Simultaneous extraction and concentration of water pollution tracers using ionic-liquid-based systems

“…In all of these investigations no major interferences of the ILs as phase-forming components have been found. In addition to the use of ILs as extraction solvents, these have also been used as additives in mobile phases of liquid chromatography, showing to be able to improve the separation and resolution of this technique [32–34]. Although remarkable results have been reported with IL-based ABS [29–31], these works only address the extraction and concentration of individual compounds.…”

Capillary column coated with geminal ionic liquids as stationary phases for gas chromatographic separation

“…The aforementioned unique properties allow ionic liquids to be widely applied in various analytical applications including gas chromatography [2][3][4], high-performance liquid chromatography [5], solvent-based microextraction [6], sorption-based extractions [7,8], capillary electrophoresis [9,10], mass spectrometry [11], and electrochemical sensing systems [12].…”