Terrible LC baseline

[ccahalane]

Hello all,

I am currently attempting the USP 36 monograph for the limit of impurity J in Atracurium Besylate.

Mobile Phase A is 20:5:75 ACN:MeOH:75mM KH2PO4 buffer @ pH 3.1
Mobile Phase B is 20:30:50 ACN:MeOH:75mM KH2PO4 buffer @ pH 3.1
All mobile phase components are HPLC grade and were filtered through 0.45 um membranes.
The gradient starts at 80% A, detecting at 217 nm. Using a new Hypersil BDS C18 column which had been rinsed with at least 20 volumes of mobile phase.

My baseline is atrocious. It just keeps fluctuating by +/- 25 mAU...grassy. Can anyone give me any ideas for troubleshooting? I've already tried remaking the mobile phases with the same results.

[DR]

air bubble in the flow cell?

how steady is your pressure (under constant %A)?

how many hours on your lamp?

[LCbob]

Has this method been running ok before on the system? You may have contamination, especially measuring at 217nm. Did you previously run a normal phase method with immiscible solvents.

Check baseline with pump flow off.

Hope this helps

[Meerkat]

Here are my suggestions for getting some traction on your chrom problem.
At this juncture we can conclude that the problems are NOT related to your column nor your m.p. preparation. Furthermore, it is not likely yoiur instrument nor sample matrix. Thus, the problem is related to most likely, the idiosyncrasies of the particular U S P method you are taksked with running. The U S P methods are notorious for being "out-dated" and "wishy-washy" on some analyses.

So, here is my ultimate suggestion to you my fellow chrom voyager: Please go back to the USP monograph and look at the end of it, there is a link and pho # of the "technical liason" for that particular analytical procedure. Email the technical liason and ask them questions---from my experience in doing this I have been able to get copies pdf's of the original test method sponsor's data, chrom run logs, and all related sample preparation documents in order to replicate the test method sponsor's results. Rem'ber the USP has a vested interest in helping you succeed because they need for you/your company to keep purchasing their expensive USP Reference Standards!*

*opps, thot you had problems with their sop, but on re-read your problem is related to lack of signal equilibration on your UV detector*

[ccahalane]

air bubble in the flow cell?

how steady is your pressure (under constant %A)?

how many hours on your lamp?

Hi DR - Thank you for the response

The pressure is steady under constant %A (@ 119 bar)
I just replaced my UV lamp on 7/1 and passed all tests in the Self Test diagnostics
As for an air bubble in the flow cell, how would I check for that and/or get rid of it?

[ccahalane]

Has this method been running ok before on the system? You may have contamination, especially measuring at 217nm. Did you previously run a normal phase method with immiscible solvents.

Check baseline with pump flow off.

Hope this helps

Hi LCbob - Thanks for the response. And thanks in advance to any more advice you can give

I've never run the method before and the system I'm using is dedicated to running reverse phase only.

I'm watching the baseline right now with the pump flow off and it looks identical to when the pump is on. What can I conclude from that?

[ccahalane]

Here are my suggestions for getting some traction on your chrom problem.
At this juncture we can conclude that the problems are NOT related to your column nor your m.p. preparation. Furthermore, it is not likely yoiur instrument nor sample matrix. Thus, the problem is related to most likely, the idiosyncrasies of the particular U S P method you are taksked with running. The U S P methods are notorious for being "out-dated" and "wishy-washy" on some analyses.

So, here is my ultimate suggestion to you my fellow chrom voyager: Please go back to the USP monograph and look at the end of it, there is a link and pho # of the "technical liason" for that particular analytical procedure. Email the technical liason and ask them questions---from my experience in doing this I have been able to get copies pdf's of the original test method sponsor's data, chrom run logs, and all related sample preparation documents in order to replicate the test method sponsor's results. Rem'ber the USP has a vested interest in helping you succeed because they need for you/your company to keep purchasing their expensive USP Reference Standards!*

*opps, thot you had problems with their sop, but on re-read your problem is related to lack of signal equilibration on your UV detector*

Hi Meerkat - Thank you for the response.

I'd been monitoring the baseline for over an hour when I wrote my original post. And I've been monitoring for the past half hour now as I'm writing this response. But I suppose it's possible that it will take more time to equilibrate the column/detector.
I will definitely try to contact the technical liaison. Hopefully I can get some helpful information from them as well.

[ccahalane]

After checking for leaks and loose fittings, I finally found the problem!

It was a dirty flow cell! Cleaned it with water, then MeOH, then dried with some canned air and now my baseline is beautiful.

Hope someone finds this useful in the future...