Online SEC/MS Analysis of Proteins on Unix(TM) SEC-300: Effect of Temperature

Online SEC/MS Analysis of Proteins on Unix™ SEC-300: Effect of Temperature

Conditions

column Unix SEC-300; 30 cm x 4.6 mm I.D., 1.8 μm particles (Z777302)
column temp. as indicated
mobile phase 100 mM ammonium acetate, pH 7.0 with ammonium hydroxide
flow rate 0.3 mL/min
pressure 2610 psi (25 °C); 2170 psi (35 °C); 1730 psi (50 °C) (180 bar (25 °C); 150 bar (35 °C); 119 bar (50 °C))
sample protein mix, 100 μg/mL, 100 mM ammonium acetate, pH 7.0 with ammonium hydroxide
injection 1 mL
detector MSD, ESI(+), 100 - 3000 m/z

Description

Analysis Note SEC has historically been used as an initial purification step prior to a secondary polishing step leading to final characterization. This limitation in SEC is due to the use of high concentrations of non-volatile salts in the mobile phase. Recently, mass spectrometry (MS)-friendly salts have been employed in SEC, enabling the analyst to perform online SEC/MS for the analysis of proteins and peptides. This application demonstrates the importance of temperature in such an assay. Increasing the temperature of the column yielded improved efficiency and resolution between the protein analytes.
Featured Industry Pharmaceutical (small molecule)
Legal Information Unix is a trademark of Sepax Technologies
suitability application for HPLC

Materials

     
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