Methanol Pressure and Mobile Phase recovery

[Zoraku]

Hi all.

So I have been consistently running a method with the following method parameters:
Inj vol: 10uL
Flow: 1ml/min
UV: 240nm
Isocratic 78/22 MeOH/H2O
Column: Thermo Hypersil Gold C18 100x4.6mm 3u particle size

Initially I have had no problems at all with sample analysis (analysis of Pyrethrin I and Pyrethrin II, to be specific). Now, over time, I have seen the Pyrethrin I peak start to front and my pressure has continually increased. I was originally running around 250bar. Now the system will go as high as 310bar.

If I run the system with ACN/H2O instead of MeOH, I don't see this type of pressure build up - this isn't too problematic as I know the solvents have different viscosities. I just have trouble understanding why the same mobile phase is causing such a large pressure built up all of a sudden.

I have run 100% acn through the column, I have run 50/50 acn/water, and other variations in attempt to clean the column if methanol wasn't eluting everything from the sample, but that didn't help.

My second questions:

When I run the above method with ACN/H2O instead of MeOH/H2O, I see significant sample recovery differences.

For examples: ACN/H2O - 3.87% (4% nominal)
MeOH/H2O - 3.97% (4% nominal)

Same sample, same standards, but I have significant recovery problems when running ACN/Water.

The thing is, in all the research I've done, most people seem to be running some variation of ACN/Water gradient or isocratic when analyzing pyrethrin on HPLC.

Anyway, I know I have quite a bit here, a second perspective or opinion would be great to hear. I'm the only one that does any analytical chemistry in my lab so I don't really have anyone to bounce ideas or theories off of except myself.

[Perreman]

Regarding your first question:
How does the chromatography look? Is there no fronting at all on Pyrethrin II? I was just considering if what you see is the beginning of a clogged column inlet, but then you would see "shoulder-fronting" on both peaks.

Anyway, if you could add a chromatogram it might be helpful.

[Gerhard Kratz]

Please check if your pump and valves get the 1ml/min flow out!
I think at 70/30 MeOH/Water this MP has a maximum back pressure.
Your fronting can come from not correct solvent composition.

[Zoraku]

So I did experiment with a few things. I think the fronting may be because my mobile phase is not adequately flowing through the column. I ran 70/30 MeOH/Water with NO column connected, and was getting about 250bar of pressure just through the pump.

I tried both running MeOH/Water separately at 70/30 and I also premixed and degassed and I get significant back pressure doing it both ways.

I think it might just be the mobile phase composition and if I'm not getting adequate flow through the column, that could have an effect on my recovery and not the mobile phase composition per se.

Edit: What I forgot to mention, is that initially my pressure started off as being acceptable, around 240 - 260 bar and as I continue the duration of running the mobile phase, the pressure continues to build up, until it gets to over 340bar and my system keeps shutting down from high pressure.

[tom jupille]

If you are getting 250 bar with no column, there is a problem! Possibly a plugged frit ir screen inside the pumping system or a defective/mis-calibrated transducer.

[hossam kamal]

https://www.youtube.com/watch?v=zr7Lvah8l4A
please check for this link , it is agilent troubleshooting demo for back pressure change and flow problems
i hope it can be helpful in your situation

[tkubowicz]

Hello

If you have 250bar without column there could be blockage:

- in pump (frits, valves)
-in sampler (needle/needle seat/rotor seal). Just check it by manually switching from mainpass to bypass and you will see if it is needle/needle seat.
- column compartment if you have valve installed

Regards

Tomasz Kubowicz

[Zoraku]

Yea, I realized there was most likely a problem within the system at that pressure with no column.

It turns out it is something specifically to line "D" of my quarternary pumping system. Lines A, B, C all pump and flow just fine. So, that problem is solved.

As for the recovery, I have fixed that problem as well. Let's just say my math was a little off haha.

I ended up changing my mobile phase completely anyway to ACN/Water at an equal eluotropic strength. Everything has worked out in the end. Thanks for any assistance you all have provided.

[BostonFSE]

You did the right thing by replacing the column with a union to troubleshoot.
If the solenoid on "D" has inconsistent drip rate out of purge valve when purging (compare to A,B,C) then you may need a new MCGV. However, most people only use line D for seal wash, so I think you are fine.

[Zoraku]

We have another specific line for seal wash on this instrument. To be honest I don't plan on having a method that will require more than 3 buffer lines for an analysis so for the time being with my work-load, troubleshooting the actual problem for line D will have to wait.

I will definitely check on the solenoid though, as well as all other parts of the pump that I can single out related to line D only.

Thanks for your suggestion.