Chromatography Forum

we have two old pumps for our HPLC. I just noticed recently that the flow rate was not stable. I set the total flow rate to 1 ml/min, but as pressure goes up with higher concentration of aqueous phase, the flow rate decreases. Without column attached, both pumps can deliver the flow rate as set. Does it mean our pumps are near the end or their lives? Or, would cleaning solve the problem? Any thoughts are highly appreciated.

How do you know your flowrate is changing? Do you have a flow meter?

If you have a meter and you get less flow at higher pressure I would suspect a system leak somewhere. Try capping the outlet of your column, apply some pressure with pump and when it gets to near operating pressure turn the pump off. If the pressure drops rapidly you have a system leak (injector seal, pump seals, tubing)

Thank so much for replying. no, I don't have a flow meter. I used a graduated cylinder and a stopwatch to measure the liquid volume at the outlet. MeOH and H2O are gradient mobile phase. I get 1 ml/min flow with 100% MeOH at pressure ~100 bar. As H2O concentration ramps up to MeOH-H2O (50:50), the pressure stops at ~150 bar and the flow rate is about 0.5 ml/min. Both pumps deliver 1 ml/min without column attached. We have another exactly same HPLC system, and the pressure on that one can reach 230 bar with 1 ml/min MeOH-H2O (50:50). Weird thing is I didn't see any leaks from either pumps.

I'll try what you suggested, but just a little worried about cracking the old bones in the pumps.

In your sysytem a 1 ml/min flow with 100% MeOH 100 bar (1450 psi) is high if there is no column in place.

If there is a column in place what is the phase, particle size and column dimensions.

300 Bar (4300 psi) would be a very high pressure for a 1 mL/ min flow of 50:50 MeOH:water. Even if you had a 25 cm 5 µm C18 column I would expect only about half that pressure.

Check your tubing sequentially for blockages.

The pressure at 100 bar was with column attached. Without column, the pressure would be about 4 -5 bar (we have a short guard column). The column we have is a Keystone Betasil C18, dimension 2.0 X 100 mm, particle size 5 um, pore size 100 angstrom. The pumps are Shimadzu LC-10AS.

I just received two new ODS columns this afternoon, they are 4.6 mm in ID. I'll put them on next Monday and see if I'll have pressure lowered and flow rate stabled.

Thanks a lot for the suggestions.