Optimal mixing rate in reverse phase liquid chromatography. Experimental evaluations

The topic of this report is experimental verification of previously published theoretical predictions. The mixing rate (R_ϕ) is the temporal rate of increasing a fraction of stronger solvent in the mobile phase of gradient LC. The optimal R_ϕ (R_ϕ,Opt) is the one at which a required peak capacity (n) of gradient LC analysis is obtained in the shortest time. The key factors affecting R_ϕ,Opt are the sample molecular weight (M), the void time (t_M), and the column pressure condition - R_ϕ,Opt in a column operating below the instrumental pressure limit is 2-3 times higher than R_ϕ,Opt in a column operating at the instrumental pressure limit. Using previously proposed speed optimization criteria, t/sⁱ, where t, s and i are the analysis time, the separation capacity (a metric proportions to n), and the order (i=2 or i=4 for analyses operating below or at the instrumental pressure limit, respectively), parameter R_ϕ,Opt for analyses of small-molecule samples has been experimentally found. For both orders, the agreement with the theoretical prediction can be considered as very good. Base on the experimental results, we continue to recommend the earlier proposed theoretically based mixing rate of 5%/t_M as a default for all analyses of small-molecule samples (100M for larger molecules. Experimental evidence is also provided to explain why earlier published less accurate equations for peak capacity and analysis time are not suitable for theoretical prediction of R_ϕ,Opt.