Chromatography Forum

Hello,
I have quite highly variable pressure with my waters 2695 instrument. There are no visible leaks. I use mobile phase consisting of 70% Methanol and 30% Phosphate buffer (pH-3.0). The flow rate is 1ml/min. The pressure reaches to 1300 psi drops to arnd 800psi sometimes arnd 60-100 psi and then comes back to 1300. I am at my wits end. I use fresh mobile phase and have a inline degasser as well. I do dry prime and wet prime and have also replaced the head plunger seals. Would appreciate any suggestion.

Thanks !!

guessing one check valve's not closing properly...

have you watched the flow stream? is it constant?

Another possibility is particles in the solvent keeping the check valve from closing completely. Are you filtering the solvent? I found fresh solvent from newly opened bottles have a surprising amount of particulates.

Another possibility ...keeping the check valve from closing completely.

...one check valve's not closing properly...

Where's the difference?

Nevermind, now two votes for the same cause...

Check Valves
Inline Filter
Solvent Frits

Did you change the check valves? What about the inline-filter? It tends to clog with buffers. Are you cleaning the system regularly with for example 70%Methanol 30%Water to remove the build up of buffer? Also you may want to change the solvent frits, especially for the buffer solvent line. If it is an isocratic method, you may want to try pre-mixing the mobile phase.

@ Hollow... Thank u...I havn't changed the check valves, wud definitely try dat. The flow stream is not constant. I did try collecting the Mobile phase after it passed thru detector and the volume was not 1 ml / min. It was more like 500-600ul.

@ Jack Silver.. Thank u . I dint realize that newly opened bottles hv particulates too.. I don't filter it.. guess I hv 2 start doing dat from now...

Check Valves
Inline Filter
Solvent Frits

Did you change the check valves? What about the inline-filter? It tends to clog with buffers. Are you cleaning the system regularly with for example 70%Methanol 30%Water to remove the build up of buffer? Also you may want to change the solvent frits, especially for the buffer solvent line. If it is an isocratic method, you may want to try pre-mixing the mobile phase.

Thanks 4 d suggestions.. I havn't changed d check valves yet. I did try to declog the inlet filter in with 100% methanol and clean the system regularly with 50 : 50 MeOH : Water. I follow an isocratic method and I am using pre - mixed mobile phase. I will try changing the solvent frit for the mobile phase line and change the check valves.. Hope all ur suggestions work for me ..fingers crossed..

Thanks ..It worked.. There was a problem with the outlet check valve , I replaced it and the pressure is stable now...However, nw I vl hv 2 figure out the injection problems . Whenever I try to inject sample , a stop message appear on the HPLC screen showing Absorbance Detector fault. We hv a Waters 486 tunable detector and the wavelength I am working with is 227nm.
I did not find any reference for this error message. Please lemme know if u hv any ideas hw to go abt this..Thanks

Thanks ..It worked.. There was a problem with the outlet check valve , I replaced it and the pressure is stable now...However, nw I vl hv 2 figure out the injection problems . Whenever I try to inject sample , a stop message appear on the HPLC screen showing Absorbance Detector fault. We hv a Waters 486 tunable detector and the wavelength I am working with is 227nm.
I did not find any reference for this error message. Please lemme know if u hv any ideas hw to go abt this..Thanks

Go to display on the HPLC. In the method editor. Remove the detector from the method.