A good nanoflow LC system starts with a HPLC pump capable to operate at an flow rate (F) that is in balance with the hold-up volume of the pump (Vm). Too low flow rates result in a poor, non-linear and delayed gradient. The appropriate flow rate for a particular pump rate can determined from the delay in time in which the composition in the outgoing flow (C2) reaches the input composition following a step-change from C1 to C2 as described by:
t = (Vm /F) . ln [(C1-C0)/(C1-C2)] ~ 6.4 (Vm /F)
or Vm= (F x t)/6.4
The delay, t, in which the back pressure stabilizes at a new level can be determined from pressure time course following a step-change in the solvent composition. A delay of 3 min corresponds to a flow rate that is about 2 times the hold-up volume. The flow rate should be > 3 times the hold-up volume of the pump.