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.

Pressure profile during a gradient analysis on a 25cm long, 25 umID column packed with 3 um particles. At the end of the gradient of 60%B at t=30 min, the eluens composition is switched back to 100% A (A=water ; B=acetonitrile both with +0.1 M acetic acid). The backpressure corresponding to 100% A is reached after 2.5 min. The hold-up volume at 200 bar is calculated as (2.5 x 0.4)/6.4 or 0.15 mL.  HPLC pump: Agilent 1100 equipped with Nanoseparations īDeansī switching system.