Chromatography Forum

Hello everyone,
I suspect that my system is not providing accurate flow rates.

What is the best test I can use to measure the real flow rate?

The pump is a low pressure mixing system.

Thank you in advance.

Best way would be to replace your column with a calibrated flow meter. Second best way (and good enough) would be to collect water from the waste tube with a stopwatch and weigh the water.

Hello

Disconnect the pump from everthing else. Prime it with the mobile phase. Run the pump at 1 ml/min for exactly 10 min, collect the output in a 10 ml specific gravity bottle, if available, or a calibrated measuring cylinder.

There should be an SOP for this in the pump manual.

Usually the vendor will do this for you, as part of his IQ/OQ/PQ routine.

Rgds,

S.K. Srinivas

The easiest way is to to pump your system with your mobile phase and column in place (just like you are going to use it) and collect from the waste line into a volumetric flask. Use a stopwatch to measure the time it takes to fill to the line and calculate the flow rate from that. For 1 mL/min I use a 5 mL volumetric.

If you establish your flow rate is OK, you can then investigate your solvent mixing system and your check valves. I suspect that you are getting varying retention times, so the first thing you wish to check is the flow. The last time we had variability in retention times on a gradient assay, it was a malfunctioning active inlet valve. Of course, before that we did use the oportunity to replace piston seals and one scored piston, but that was overdue anyway.

Thank you all for your help.

I actually need to check my flow rate because I am comparing between DryLab simulated chromatograms and experimental ones and I noticed that larger error occurs between the two when using different flow rate than the one used to generate the simulation.

Second best way (and good enough) would be to collect water from the waste tube with a stopwatch and weigh the water.

I applied this method in triplicates @ 3 different flow rates and I got accurate results for the theoritical Flow (less than 1% error).

The easiest way is to pump your system with your mobile phase and column in place (just like you are going to use it) and collect from the waste line into a volumetric flask. Use a stopwatch to measure the time it takes to fill to the line and calculate the flow rate from that. For 1 mL/min I use a 5 mL volumetric.

I also tried this in triplicates but the error here was much higher (about 33% less than the theoritical)


The large variation between the two methods makes me a bit worry

I am also wondering about the error in DryLab simulation when using different flow rate values

Any suggestions or comments are very appreciated.

It is interesting that you get 2 different results. I find that when doing any kind of system testing, it is important to do the test much like you run the system. In this case it means running mobile phase (which may have buffers and certainly organic with a different viscosity that 100% water) through the column (running the system at operational backpressures). An error of 33% would indicate to me that some kind of problem is evident (leak, check valve, seal, GPV).

based on these observations, you either have a leak towards the outside somewhere or a checkvalve that leaks back at elevated pressure