Delay Volume Calculation Methods - Results Disparity

Hi all,

I used below method to estimate dwell volume of our system and used initial signal liftoff time (@2 mins), half gradient time and step elution response time (@14 mins) to calculate dwell volume and ended up with three different values.

1 - Liftoff point following gradient start (2.2 mins ~400 µL - this one is closer to the volume stated in the pump spec sheet)

2- Midpoint of the gradient (@60%B ~550 µL)

3- Final step signal profile starting at 14 mins (both yielding approx. 330 µL delay volume)

Which one is the correct measurement / generally accepted method for dwell volume calculations? Is this disparity normal? I have seen all the points above utilized in papers and application notes.

Thanks in advance.

Flow Rate: 2 mL/min

Column Comp: 40C joined w. ZDV union

Signal: 265 nm, 4nm (Ref: 480 nm, 50 nm) at 10 Hz

Inj. Vol: 0 µL

Backpressure ~120 bar

A: Ultrapure water

B: 0.1% acetone in ultrapure water


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