High pressure during equilibration before first injection

<p>I'm using an H-class UPLC. My method typically runs at about 10,000-11,000 psi. About 50% of the time we observe that when setting up the system for a new run, the pressure during equilibration is closer to 14,000 psi. It remains that high no matter how long we equilibrate (up to an hour) and even though the column temp has stabilized. However, immediately following the first injection, the pressure drops back down to about 10,000 psi and remains in the expected range throughout the run. Any ideas how we can avoid the high pressure during equilibration?</p><p></p><p>Here are some method details:</p><p></p><p>Injection volume: 1.5 uL</p><p>Diluent 50:50 ACN/water</p><p>Gradient start 300 uL before injection</p><p>Initial composition: 93% MPA (aqueous), 7% MPB (90:10 ACN/MeOH)</p><p>Pre- and Post-injection wash: 6 sec</p><p>Flow: 0.8 mL/min</p><p>Column: BEH C18, 1.7 um, 2.1x100 mm</p><p>Column Temp: 60 C</p>


  • RD

    I can't think of any reason the pressure would be 4000 psi higher during equilibration at initial conditions versus what you observe at those same conditions when making injections.

    Are you still having problems ?

    Are you using any additives in your mobile phase (salts ? acids ? bases ?).

  • This may not be the exact reason, but it does at least need to be ruled out because it is a possibility.

    93% aqueous is getting a little high for a column that is not designed to run under highly aqueous conditions. Is it possible that the column you are using is dewetting and you are experiencing partial phase collapse, which results in higher pressure. This will be maintained until you make your first injection. When that first injection is made, the gradient kicks in and you no longer have the dewetting problem you have during equilibration...

    I guess one way to rule this out without doing any additional work is by your chromatography. If the chromatography of that first injection different from all other injections. I would think that you would suffer from a loss of analyte retention time, plates, and/or peak shape if you are experiencing dewetting problems.

  • KWB

    Since this is a gradient method, it might be more efficient to run gradients before the first injection instead of long equilibrations. This might solve the high pressure problem, especially if it is caused by dewetting. In any case, it will leave the column in a more appropriate state for the first injection. With Empower use the 'Condition Column' function in Run Samples with an appropriate run time. With MassLynx, you can make blank injections or use the 'Inlet Prerun' column or both.