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 degrees 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 degrees C) separations.
Causon
Tim J
TJ
Australian Centre for Research on Separation Science, School of Chemistry, University of Tasmania, Private Bag 75, Tasmania, Australia.
Nordborg
Anna
A
Shellie
Robert A
RA
Hilder
Emily F
EF
D019840:2-Propanol / Q000737:chemistry*; D000097:Acetonitriles / Q000737:chemistry; D000818:Animals; D002417:Cattle; D002645:Chickens; D002853:Chromatography, Liquid / Q000379:methods*; D019544:Equipment Failure Analysis; D006736:Horses; D006358:Hot Temperature; D000432:Methanol / Q000737:chemistry*; D051379:Mice; D011137:Polystyrenes / Q000737:chemistry*; D055550:Protein Stability; D011506:Proteins; D012997:Solvents / Q000737:chemistry; D014783:Viscosity
D000097:Acetonitriles; D011137:Polystyrenes; D011506:Proteins; D012997:Solvents; C003771:Amberlite XAD-2 resin; D019840:2-Propanol; D000432:Methanol; C032159:acetonitrile