Generic gradient elution RPLC analyses

Optimal flow rate (F_opt) in reversed phase LC (RPLC) analysis using the same column with the same solvent at the same pressure substantially depend on the molecular weight (M) of the solutes in the sample. The same is true for the optimal mixing rate (R_ϕ,opt) – the temporal rate of changing the solvent composition at the column inlet – in gradient elution RPLC. General equations describing the dependence of F_opt and R_ϕ,opt on M in columns of an arbitrary internal support structure (packed, open, pillar array, etc.) are developed and treated herein as equations for constructing generic RPLC analyses. As an example, parameters of generic analyses of the samples with molecular weights 300, 3000 and 30,000 in packed columns with 1.25 µm, 2.5 µm and 5 µm particles at 400 bar, 800 bar and 1500 bar pressures at optimal and suboptimal flow of water/ACN and water/MeOH solvents are tabulated. Kinetic performance advantages of ACN vs. MeOH as well as the advantages of using F_opt are quantified.