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 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 mu m 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 mu m and a mode macropore size of 1.0 mu m (based on scanning electron microscopy). The monolith yielded a minimum plate-height value of 13.3 mu m 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 mu L min(-1) and a gradient time of 120 min, which corresponds to an optimal t(G)/t(0) ratio of similar to 60. While the optimum van Deemter velocity for intact proteins was approximated to be 0.065 mu L 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. (C) 2019 Elsevier B.V. All rights reserved.