Optimal mixing rate in linear solvent strength gradient liquid chromatography. Balanced mixing program

The mixing rate (R-phi) is the temporal rate of increase in the solvent strength in gradient LC. The optimal R-phi (R-phi,R-opt) is the one at which a required peak capacity of gradient LC analysis is obtained in the shortest time. The balanced mixing program is a one where, for better separation of early eluting solutes, the mixing ramp is preceded by a balanced isocratic hold of the duration depending on R-phi. The improvement in the separation of the earlier eluites due to the balanced programming has been evaluated. The value of R-phi,R-opt depends on the solvent composition range covered by the mixing ramp and on the column pressure conditions. The R-phi,R-opt for a column operating at maximum instrumental pressure is different from R-phi,R-opt for a column operating below the instrumental pressure limit. On the other hand, it has been shown that the difference in the R-phi,R-opt values under different conditions is not very large so that a single default R-phi previously recommended for gradient analyses without the isocratic hold also yields a good approximation to the shortest analysis time for all conditions in the balanced analyses. With or without the initial balance isocratic hold, the recommended default R-phi is about 5%/t(0) (5% increase in the solvent strength per each t(0)-long increment in time) for small-molecule samples, and about an order of magnitude slower (0.5%/t(0)) for protein samples. A discussion illustrating the use of the optimization criteria employed here for the techniques other than LSS gradient LC is included. (C) 2016 Elsevier B.V. All rights reserved.