High temperature liquid chromatography of intact proteins using organic polymer monoliths and alternative solvent systems

Alternative approaches to conventional acetonitrile gradient methods for reversed-phase liquid chromatographic analysis of intact proteins have been investigated using commercial poly(styrene-co-divinylbenzene) monolithic columns (Dionex ProSwift™ RP-2H and RP-4H). Alternative solvents to acetonitrile (2-propanol and methanol) coupled with elevated temperatures demonstrated complementary approaches to adjusting separation selectivity and reducing organic solvent consumption. Measurements of peak area at increasing isothermal temperature intervals indicated that only minor (<5%) decreases in detectable protein recovery occurred between 40 and 100 °C on the timescale of separation (2–5 min). The reduced viscosity of a 2-propanol/water eluent at elevated temperatures permitted coupling of three columns to increase peak production (peaks/min) by 16.5%. Finally, narrow-bore (1 mm i.d.) columns were found to provide a more suitable avenue to fast, high temperature (up to 140 °C) separations.