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).

Prototyping of thermoplastic microfluidic chips and their application in high-performance liquid chromatography separations of small molecules.

The present paper discusses practical aspects of prototyping of microfluidic chips using cyclic olefin copolymer as substrate and the application in high-performance liquid chromatography. The developed chips feature a 60mm long straight separation channel with circular cross section (500µm i.d.) that was created using a micromilling robot. To irreversibly seal the top and bottom chip substrates, a solvent-vapor-assisted bonding approach was optimized, allowing to approximate the ideal circular channel geometry. Four different approaches to establish the micro-to-macro interface were pursued. The average burst pressure of the microfluidic chips in combination with an encasing holder was established at 38MPa and the maximum burst pressure was 47MPa, which is believed to be the highest ever report for these polymer-based microfluidic chips. Porous polymer monolithic frits were synthesized in-situ via UV-initiated polymerization and their locations were spatially controlled by the application of a photomask. Next, high-pressure slurry packing was performed to introduce 3µm silica reversed-phase particles as the stationary phase in the separation channel. Finally, the application of the chip technology is demonstrated for the separation of alkyl phenones in gradient mode yielding baseline peak widths of 6s by applying a steep gradient of 1.8min at a flow rate of 10µL/min.