On the extra-column band-broadening contributions of modern, very high pressure liquid chromatographs using 2.1 mm I.D. columns packed with sub-2 μm particles.

Abstract

The efficiencies of two narrow bore columns (100 mm and 50 mm × 2.1 mm) packed with 1.7 μm totally porous BEH-C(18) particles were measured on two very high pressure liquid chromatographs (Acquity from Waters and 1290 Infinity HPLC System from Agilent) operating at maximum pressures of 1034 and 1200 bar, respectively. The probe compounds were a mixture of uracil, acetophenone, toluene, and naphthalene eluted in a 50/50 (v/v) solution of acetonitrile and water at 303 K with a flow rate of 0.40 mL/min. The apparent efficiencies of columns, which lumps the consequences of band broadening due to the column and the system contributions, may depend much on the extra-column volumes of the instruments used. Actually, it is known for a long time that the apparent column performance is strongly affected by the instrument characteristics, including the diameter of the connecting tubes, the injection technique (with or without needle seat capillary), and the detection cell volume. When the 1290 Infinity HPLC System is equipped with a needle seat, an inlet and an outlet connecting capillary tube with inner diameters around 115 μm, its extra-column variance for a 0.1 μL injection volume is 9.2 μL(2) while that of the Acquity instrument is 6.9 μL(2). Minor modifications suggested by their respective manufacturers allowed significant reductions of these variances, to 6.2 and 3.9 μL(2), respectively. Yet, in their optimized configurations and for weakly retained compounds (k≃1), these modern, sophisticated instruments cannot provide more than 75% (1290 Infinity) and 85% (Acquity) of the maximum efficiency of a 2.1 mm × 50 mm BEH column. For more strongly retained compounds (k>4), in contrast, they are both able to provide more than 95% of the maximum expected efficiency.