Experimental band compression factor of a neutral compound under high pressure gradient elution

To measure the gradient compression factor for a low molecular weight compound (caffeine, MW=194 g/L), 1 microL samples of solution were injected into a 2.1 mm x 100 mm column packed with bridged ethylsiloxane BEH-silica. These samples were successively run under isocratic and gradient elution modes. Both chromatograms were recorded with either a low (<250 bar) or a high pressure drop (>650 bar), corresponding to flow rates of 0.10 and 0.35 mL/min, respectively. Caffeine was eluted with a mixture of methanol and water at room temperature. The efficiency and the retention factors of caffeine on the BEH-C(18)column were measured as a function of the mobile phase composition under isocratic conditions. During the gradient elution, the methanol concentration was increased from 10 to 25% (v/v). The experimental compression factors measured are in excellent agreement with those predicted with an equation previously derived, which is valid at low flow rates and for smooth gradients. The negative relative difference observed between experimental and theoretical values at high flow rates originates from the compressibility of the eluent. Using a high gradient steepness to perform fast gradient elution limits the degree of band compression that can be achieved, even when the radial temperature gradient across the column is as small as 0.2K.