Effect of parallel segmented flow chromatography on the height equivalent to a theoretical plate III : influence of the column length, particle diameter, and the molecular weight of the analyte on the efficiency gain

The effects of column length on performance in segmented flow chromatography were tested. Column efficiencies were measured for 4.6mm I.D. 3, 5, 7.5 and 10cm long columns packed with 3.0μm Hypurity-C18 fully porous particles and of 4.6mm I.D. 5, 10, 15 and 25cm long columns packed with 5μm Hypersil GOLD C18 particles. For each column length and particle type, two different configurations were tested: (1) both the inlet and outlet column endfittings were standard and (2) the inlet endfitting was standard but the outlet endfitting allowed parallel segmentation of the exiting flow into a central and a peripheral coaxial region. The segmentation flow ratio was set at 45% (for 3μm) and at 43% or 21% (for 5μm). Four samples were used, naphthalene, toluene, butylbenzene, and insulin, which has a ten times smaller diffusion coefficient than the small molecules.The column performance for the low molecular weight compound is significantly improved at velocities above the optimum value when the outlet flow rate is segmented because longitudinal diffusion and mass transfer resistance of this compound in the stationary phase are negligible sources of band broadening at reduced linear velocities between 5 and 25. At high flow rate (4mL/min), the long-range eddy dispersion terms are about 3.9, 3.2, 2.6, and 1.8h unit lower for the 3, 5, 7.5 and 10cm long columns, respectively. The longer the column, the lower the efficiency improvement because the border effects are smaller. This result was not systematically observed for the columns packed with 5μm particles because the transverse dispersion is larger. In contrast, the gain in column efficiency is marginal for insulin because the mass transfer mechanism of this compound is mostly controlled by the slow diffusivity of insulin across Hypurity-C18 particles.