Impact of frit dispersion on gradient performance in high-throughput liquid chromatography.

The impact of porous frits (2.1 mm i.d. × 1 mm thick, 0.2 μm porosity, 20% void, 0.69 μL) in short 0.5-5 cm long × 2.1 mm i.d. columns packed with sub-2 μm superficially porous particles on gradient performance was investigated using a low dispersive i-class ACQUITY UPLC system (25 cm × 75 μm outlet tube + 250 nL optical cell). In order to maximize data accuracy, the sample dispersion through a single frit was measured from four independent methods: (1) plate height subtraction, (2) peak capacity versus efficiency plot, (3) flow reversal, and (4) direct measurement. Frit dispersion increases non-linearly with increasing flow rate. The corresponding volume variances of small molecules were measured at 0.16 ± 0.04, 0.24 ± 0.05, and 0.31 ± 0.06 μL at flow rates of 0.1, 0.2, and 0.3 mL/min, respectively. These observed variances are lower than and consistent with the maximum volume variance of 0 . 69 ∼ 0.5 μL expected for a mixer-like behavior. The peak capacity of short columns were then calculated for mixtures of peptides using a general model of gradient elution by considering (or not) the actual analyte dispersion taking place in the outlet frit, in the post-column connecting tube, and in the detection cell. The results show that the sole presence of the outlet frit is responsible for about 50% loss in peak capacity (relative to the expected gradient performance in absence of frit and post-column tube) for a 1 cm long column operated under standard gradient steepness. The actual structure and volume of column frits needs adjustment if one wants to take full advantage of the true performance of short high-throughput columns packed with sub-2 μm particles.