Operating Parameters (F, T, $\Delta\Phi$, $t_G$, Gradient Segment) #

HPLC and UHPLC For Practicing Scientists, 2nd Edition, P255

Flow rate (F) does not affect analyte retention (k) or selectivity (𝛼) in an isocratic analysis, but it can be an important factor affecting both average retention (k*) and selectivity (𝛼) within gradient elution (Section 2.6.2). Increasing column temperatures (T) reduces retention and solvent viscosity in RPC and can have significant effects on selectivity. The gradient range (ΔΦ) of the strong solvent (typically %B) is defined as “final %B – initial %B” and is usually determined from the initial work using a broad gradient scouting analysis.

A narrower gradient range is useful to increase the resolution of analytes around the main component. Gradient time ($t_G$) in conjunction with ΔΦ defines the gradient slope (ΔΦ/$t_G$). An increase in tG often increases the overall resolution of complex samples, as the peak capacity is roughly proportional to the square root of tG for broad linear gradients. Both T and $t_G$ can be continuously varied, and their combined change offers an exceptional opportunity for fine-tuning separations using retention modeling software programs (e.g. DryLab, LC Simulator, or ChromSword). For purity analysis of complex samples, a multiple-segment gradient approach is an effective strategy as shown in Section 10.4.3.