HPLC Analysis of Insulin (Species Variants) on Ascentis® Express Phenyl-Hexyl
CONDITIONS
column
Ascentis Express Phenyl-Hexyl, 15 cm x 2.1 mm, 2.7 μm particles (53338-U)
mobile phase
[A] 70:30, water with 0.1% TFA:acetonitrile with 0.09% TFA; [B] 55:45, water with 0.1% TFA:acetonitrile with 0.09% TFA
gradient
0 to 100% B in 7.5 min
flow rate
0.3 mL/min
pressure
3960 psi (273 bar)
column temp.
30 °C
detector
UV, 215 nm
injection
0.5 μL
sample
200 μg/mL in 0.1% formic acid
Description
Analysis Note
These three insulins have more than 76% identical positions in a primary structure alignment. The human and porcine proteins are most similar with 85% identity. The Phenyl-Hexyl phase provides an alternate retention and selectivity to a C18 phase, particularly the C18 phase on a larger pore silica, Ascentis Express ES-C18
Legal Information
Ascentis is a registered trademark of Merck KGaA, Darmstadt, Germany