Chromatography Forum

Here are the mobile phase:

Bottle A: H2O
Bottle B: MeOH
Bottle C: ACN
Bottle D: MeOH

Yesterday I equilibrated the system with 70:30 MeOH:H2O and noticed that pressure ripple from 1 to 10 bar. Baseline looked like a constant wave form.

The first step I did is to release the purge value, purge all channels. Hence I find that there is a stable pressure/baseline for Channel B/C/D. Still have ripples in A (H2O).

I excluded the problems come from inlet/outlet check valves and pump seals because there was ok in Channel B/C/D. But ripple still appeared in channel A.

Then I guess there is a problem in proportion control (Agilent called it Multi-Channel Gradient Valve, MCGV).

I swap the bottle of A with D this moring, now A is MeOH; D is H2O. The ripples disappeared and pressure is stable as before.

Do you know the reason behind?

I think you are true. Problem is probably with MCGV valve channel A. Try to flush it directly with syringe (when valve is opened) or ask Agilent representative for repair/exchange.

If you have enough time and experience, you can perform gradient test with acetone. Procedure is described in manual and performed during OQ. It will tell you definitively if problem is there.

Edit:

I'm sorry, I overlooked this "...I swap the bottle of A with D this moring, now A is MeOH; D is H2O. The ripples disappeared and pressure is stable as before...."

In that case it seems that H2O is not properly degassed but as you told below....

Hi,
Alterantively, you can run a test aq-MeOH gradient directly and detect MeOH at 210 nm (instead of using aceton in aq).

So it pumps OK with MeOH on A?
Now switch water back on A, purge at least 30mL and then see if it is stable.
Check that the degasser is switched on and you can hear the vac pump operating at intervals.
Could the glass sinter on channel A be partially blocked? Check it by disconnecting the tubing from bottle A at the degasser and the water should freely flow out. If not it's blocked and needs cleaning or replacing.
Check that the degasser connections are tight so they cannot draw any air bubbles in. (but not overtight - the plastic torque tool is recommended for tightening the connections).

Remember also to ALWAYS purge ALL channels, even if you're only using one or two....

Hello Bob,
The degasser is always switch on and vac pump will have "2 stage" fluttering sound. (No LED light means operating in good condition)

Filter in bottle A are not blocked. I disconnected the junction between degasser outlet and MCGV inlet and it is free flow. And I tried to lift up the filter above the water level for a while and place back. Small gas bubble flow steadily into the degasser.

What is the actual pressure when the flow is through the column and you see this 10 bar pressure ripple?

Rob

The actual pressure is 55 bar.

Yesterday I tried running samples. Channel A: MeOH, Channel D: H2O, ratio: 70:30; pressure 58 bar. That's no problem for the 1st run.

For the 2nd run, the pressure was 58 bar but sudden dropped to 1 bar and ripples appeared.

I called the service engineer and he suggested to check the flow rate for every channels, make sure that MCGV is ok.

For MeOH, ACN (A/B/C) are ok but H2O (D) was not. When I switched H2O to Channel A, now the flow rate of A was not ok. And now the MeOH (D) is back to normal.

Why does the flow rate problem follows H2O?

The answer is partial Active Inlet Value cartridge blocked. It permits less viscous solvent (MeOH/ACN) to flow through. When H2O is added, maybe some thin film dirt hinder the movement of the ruby ball inside.

I sonicated the cartridge in MeOH for 10 min. Problem was solved.

What I learn is: Don't exclude the potential problem that was excluded previously.

How often do you change your water? Can it be algae?

About 3-4 days. I discard the remaining H2O, rinse the bottle and fill the new one.

Yes, it maybe algae. Sometimes I can see black spots when I replace PTFE frit in the purge valve. But I will flush the system with 100% MeOH every day-end.

About 3-4 days. ...

Wow, we change water daily after bad experiences. Use several crystalls of sodium azide iuf you want use water longer.

Thanks Jackus,

Will sodium azide alter the separation performance? It will come out with breakthrough peak. But, if the analyte is ionic or high polar compounds, will it affect the early peaks?

We have been through this many times: One does not need something like sodium azide in pure water. It is impossible for microbes to multiply in pure water, if you have them there it is a clear sign that you don´t keep your system clean.

Oh I see.
Jackus, just curious about how bad experiences you got. Could you share your experiences to us? Thanks!