A comprehensive investigation of the peak capacity for the reversed-phase gradient liquid-chromatographic analysis of intact proteins using a polymer-monolithic capillary column

•The maximum peak capacity is achieved at a flow rate 20 x higher than the optimal van Deemter flow rate.•The gradient volume has an important impact on overall peak capacity.•The use of TFA as ion-pairing agent leads to better mass loadability compared to FA. The present study reports on the analysis of different factors affecting the magnitude of the peak capacity for intact protein separations conducted in gradient reversed-phase liquid chromatography. Experiments were conducted using a 200 µm i.d. capillary styrene-co-divinylbenzene monolithic column that was developed in-house and was characterized by a mode globule cluster size of 1.2 µm and a mode macropore size of 1.0 µm (based on scanning electron microscopy). The monolith yielded a minimum plate-height value of 13.3 µm for uracil. The use of trifluoroacetic acid instead of formic acid as ion-pairing agent generally led to better peak symmetry, narrower peak widths which effect is protein-dependent, and improved loadability characteristics. The peak capacity has been systematically assessed at different flow rates and gradient duration. The highest peak capacity of 247 was obtained at a flow rate of 1 µL min−1 and a gradient time of 120 min, which corresponds to an optimal tG/t0 ratio of ∼60. While the optimum van Deemter velocity for intact proteins was approximated to be 0.065 µL min−1, the highest peak capacity was achieved at approximately 20-fold higher flow rate, depending on the gradient duration applied and the molecular weight of the proteins. The optimum velocity increased with decreasing gradient time and is a compromise between the magnitude of the mass-transfer contribution (decreasing the peak capacity with velocity) affected by molecular diffusion, and the increase in peak capacity induced by the more favorable gradient-volume ratio.