negative and/or positive baseline

[kaurnavy]

I am using Beckman coulter gold HPLC and recently I changed a lot of parts of the instrument viz. piston, flow cell etc. The pump seems to be working perfectly as I get a good flow rate, however there has been some problem and I guess that is because of flow cell. I am getting either extremely negative or positive baseline reaching to about 500000 mAU. The baseline starts at zero and then suddenly it either shoots up or falls down. I tried to clean the flow cell using water as mentioned in the manual, which says pull water through the flow cell's outlet line. I tried doing that but I could not pull water, all I got was air. I am not sure if that is the air entrapped in flow cell or the loosely connecting union and flow cell outlet line, as I doubt that much air can be entrapped in the flow cell. Also when I tried pushing the water through the outlet line I could see water going from the other end.

Can anyone suggest me what should I do to solve that problem? Can it be some kind of contamination in the flow cell?

Thanks

[GregK]

Hmm. I've never seen that happen on any of ours. My suggestion: get rid of the Beckmans and buy Agilents or Waters.

Is it a quick spike up and then back down, or does it jump up (or down) and slowly come back? A quick spike suggests an electrical problem, IMO. I have no idea how to diagnose or fix that. Slow return is maybe some contamination that washes out, maybe some salt or bacteria? Try flushing everything with water, IPA.

Beckman techs are okay to talk to on the phone, try calling them and asking. I've done that before, and got info for free. (They told me a bunch of things to try, and when I said "Yeah, did that all already", they said "Try replacing them again." )

I'm sure someone will come along that is more experienced than me with these things, but I figured I'd get my $0.02 in...

-Greg

[kaurnavy]

Thanks Greg for your reply..

I called Beckman coulter service guys 'n' number of times and they have been very helpful too but still the problem is unsolved.

The beaseline starts at zero and stays there for 10 sec and then either shoots up or dips to 500000 mAU and then it stays there for the rest of the run time i.e. 10 min. I tried this w/ and w/o column. I donno what is happening. Although flow cell is new am still thinkin of cleaning it with nitric acid. Also, could it be a fault of lamp coz I donno how old it is?

Thanks

[GregK]

The older (few years maybe) lamps we had around here had a mercury "clock" on the cable attached to the lamp, with a little bubble that moved as the lamp got older. I think they got rid of it because it was their responsibility to properly dispose of the mercury, and everyone was throwing them in the trash...

Are you getting any peaks in you run? Maybe the detector is maxed out? In my experience a max out detector will give a real "spiky" baseline, going up and down a few mAu, but if you have a huge jump, you probably wouldn't see that.

Have you ran the wavelength calibration?

If you have the budget, try replacing the lamp. I don't know how much Beckman wants for them; we get ours from CTS (ctshplc.com) for about $400.

You mentioned you just replaced a bunch of parts, was it running okay before the maintenance?

As the Beckman guy I talked to once said, "That's a real head-scratcher you got."

Good luck.

-Greg

[kaurnavy]

It was a series of events:
HPLC working perfectly...then suddenly lot of noise and undulating baseline
I figured leaking flow cell- replaced it
then split peak-changed column
performed maintenance replacing piston, filters etc- Result: good flow rate
Alas, lost my baseline after maintenance....getting this weird problem now...thought problem with the alignment of flow cell...tried fixing that following instructions from manual...got counts b/w 10,000 to 50,000 counts (which is required)..but still the PROBLEM..

now the only thing I can think of replacing is lamp...and I hope that works...donno if I can get another one right now though...

Any more suggestions!

[HW Mueller]

What does the baseline do when there is no flow?

[kaurnavy]

No flow as in with pump switched 'off'? I thought detector should not be switched 'ON' w/o any flow...

Haven't tried it though...

[GregK]

If you turn off the flow and watch the response, you can tell if the noise is in the mobile phase flow or just noise from the lamp. I hadn't thought of that... If the baseline does the same thing without flow, then you'll know its the lamp; if it doesn't do the same thing, then you have contamination. At least that's what I'm thinking... If I'm wrong, someone please correct me.

Yeah, I've always been told not to leave the detector on without flow, but this is just for a short time (couple of minutes). Extended periods with the lamp on and flow off is bad (still not 100% sure why, overheating the flow cell maybe?), but short periods are okay.

[kaurnavy]

I tried running ACN through a union and then stopped the flow to detector. When I stopped the flow what I got was lot of noise between 1.5 to about 3 mins and then I again started flow through detector



and one interesting observation was when I ran ACN with the column, I started getting these synchronous peaks. Dont know what these are?



If anyone has any idea please do let me know.

Thanks




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[GregK]

Stupid web filter at work ; can't see the images you have posted. Hopefully, I'll remember to come back to the site when I'm at home (no filter there!). Sorry I can't be more helpful!

[bisnettrj2]

Flow cell bubble? Or the mysterious "bad board in the detector". I had an Agilent MWD give me fits a while back, and the problem repeated itself with no flow - ended up being a bad board according to Agilent. Since you're obviously not using this instrument due to this issue, you could program in a couple no-injection very long (360 minutes?) blank runs. Do one with your normal mobile phase (isocratic to simplify things) at the beginning of the day tomorrow, and one with zero flow and the flow cell removed from the detector (not sure if you can do this on your system and still acquire data, but you can do it on an 1100) at the end of the day for the same amount of time. Compare the runs on Monday. If it's the electronics or the optics or the flow cell, the problem should show up in both runs. If it's the pump or something else upstream of the detector, the second run should be relatively flat. If it's the lamp, it will still show up in both, but I think it's rare for a lamp to do what you're describing.

[GregK]

The second one looks like a flow cell bubble. This first one... I have no idea, sorry.

Is this the only LC you have? Maybe try and switch flow cell or lamp with another, working, LC? That's what I'd do, but we have 3 Beckmans and 7 Agilents.

[HW Mueller]

The repetitious peaks appear to be a somewhat strange sawtooth pattern, which I have seen only in the form of a rise and then sudden drop back to ~ the baseline. These are due to a bubble forming in the flow cell which is then swept out when big enough. Here there is a relative slow decrease after the apex, but then comes the sudden drop. Can not imagine that it is anything else but one bubble after another forming in the cell.
The first chromatogram could also be an air phenomenon, a bubble not forming in the cell, but being swept into it. The mess obtained when the flow is stopped looks like what I have seen in stationary solvent-air mixtures. One investigates this sort of thing by not only stopping the flow, but also variing it, using a different, strongly vacuum degassed solvent, maybe even pushing it through the cell with a syringe. The only real trouble I ever had in determining air interference was when I had a leak in a connection which "sucked" in air when under pressure with flow (Venturi).

[stev434]

Hi all,

Well for all to know this IS Beckman HPLC support and yes we try to be as helpful as possible. The second chromatogram you have is from either your back pressure regulator having an issue or the lamp. Since our lamps are pre-aligned and are made with a heat sink they need to be from us due to this option. I recently had a customer with this same problem and replacing the lamp with a lamp from us solved the problem. A rhythmic baseline like this is due to power issues possibly or the bubble problem mentioned above.

2 things:

1. Check your back pressure regulator to see if it's been corroded or not working. Replace if it is
2. Try buying a new lamp from us and use it as a control. That way you know if the lamp can be causing this issue or not.

Feel free to submit any questions to cesupport@beckman.com for additional help.

Sincerely,

Steve

P.S. The comment above wax possibly made by my colleague Dave. He lives in Virginia, USA and well they use these colloquialisms there