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

•Isopropylammonium formate (IPAF) can replace organic solvent mobile phase modifiers for liquid chromatography of proteins.•Proteins can more effectively be maintained in their native form using IPAF than using acetonitrile (MeCN).•Enzyme activity of lactate dehydrogenase is evident using IPAF as the solvent as compared to MeCN.

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 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.