Optimization of the peak capacity per unit time

The peak capacity per unit time or chromatographic productivity, Pct, was calculated and optimized for four different sample mixtures (small molecules, peptides from protein digests, and mixtures of small and large intact proteins) analyzed on 4.6 mm I.D. columns packed with three different brands of core–shell particles. The calculations were based on the column permeabilities and on their van Deemter curves measured under isocratic conditions. The 3D plots of the peak capacity are provided as a function of the gradient steepness and the column length at three values of the inlet pressure and two temperatures. The maximum peak capacities obtained per unit time were 103 min−1 for small molecules, 111 min−1 for peptides, 63 min−1 for small proteins, and 22 min−1 for large proteins. They are all achieved for the shortest column lengths run at the maximum achievable flow rate (5 mL/min) and for an optimum gradient steepness, which had to be calculated. Remarkably, the best separation productivity of these columns cannot be achieved with modern UPLC instruments that deliver flow rates no larger than 5 mL/min. With instruments able to deliver a 15 mL/min flow rate and acquire data at 320 Hz, 4.6 mm × 50 mm columns packed with shell particles operated at 600 bar and a temperature of 60 °C, would give peak capacities per unit time of 256 min−1 for small molecules, 237 min−1 for peptides, 186 min−1 for small proteins, and 59 min−1 for large proteins.

► We derived the peak capacity per unit time in gradient elution of columns filled of shell particles.
► We considered four different mixtures of analytes covering a wide range of molecular weights.
► The calculations were based on the column permeabilities and their isocratic van Deemter curves.
► The results are particularly useful for the second dimension columns in 2D-LC.
► The best results are provided by short columns eluted at high velocity by steep gradients.