Assessment of the resolving power of hydrophobic interaction chromatography for intact protein analysis on non-porous butyl polymethacrylate phases

This study reports on the assessment of the separation performance of hydrophobic interaction chromatography for intact protein analysis using non-porous butyl polymethacrylate phases. The maximum peak capacity in inverse gradient mode was reached at a volumetric flow rate which was significantly (10–20 times) higher than the flow rate yielding the minimum plate height in isocratic mode, as the gradient volume dominates the peak-capacity generation. The flow rate yielding the maximum peak capacity increased with decreasing gradient volume, i.e., steeper gradients, and also depends on the magnitude of the mass-transfer contribution to peak dispersion (affected by particle size and molecular diffusion coefficient of proteins) at these high flow rates. The maximum peak capacity using a 100 mm long column packed with 4 µm particles for steep 7.5 min gradients was determined to be 60. Increasing the column length by coupling columns leads to better gradient performance than increasing the gradient duration for gradients of 60 min and longer. Using a coupled column system (2 × 100 mm long columns packed with 4 µm particles), the maximum peak capacity was determined to be 105, which was 33% higher compared to that of a single column while applying a similar gradient volume. Decreasing the particle size to 2.3 µm leads to higher peak capacities even though the column was operated at lower volumetric flow rate. The maximum peak capacity obtained with the 2.3 µm column was 128% higher than was obtained with the coupled column. Even at suboptimal conditions, the 2.3 µm column yields a higher peak capacity (14%) than when using two coupled columns packed with 4 µm at optimal conditions (gradient time of 120 min and a flow rate of 0.5 mL/min).