title: 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 the 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 are 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 2min represented protein in primarily the native form and a second broader peak more retained at about 5-6min 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.
authors.affiliation: Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056, United States. Electronic address: zhoul3@miamioh.edu.
authors.forename: Ling
authors.initials: L
authors.lastname: Zhou
authors.forename: Neil D
authors.initials: ND
authors.lastname: Danielson
mesh_terms: D000097:Acetonitriles / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D056148:Chromatography, Reverse-Phase / Q000379:methods*; D005563:Formic Acid Esters / Q000737:chemistry*; D052578:Ionic Liquids / Q000737:chemistry*; D008956:Models, Chemical; D011437:Propylamines / Q000737:chemistry; D055550:Protein Stability; D021122:Protein Subunits / Q000737:chemistry / Q000302:isolation & purification; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification; D000644:Quaternary Ammonium Compounds / Q000737:chemistry
chemical_list: D000097:Acetonitriles; D005563:Formic Acid Esters; D052578:Ionic Liquids; D011437:Propylamines; D021122:Protein Subunits; D011506:Proteins; D000644:Quaternary Ammonium Compounds; C035263:2-propylamine; C032159:acetonitrile
keywords: High performance liquid chromatography; Ionic liquid; Mobile phase modifier; Protein stability