"Ghost" peaks during gradient

[Jackus]

Please can comeone help me to improve baseline behaviour during gradient? The first chromatogram is sample, second blank inject. Analytical column was previously carefully flushed with pure acetonitrile. In such chromatogram I'm not able to recognize what to integrate as impurity and what are ghost peaks. When I use methanol based phases (in diferrent method), the gradient profile looks smoothed. Is there any explanation for such behaviour?

Thank you for your time.

[philippem]

Hi Sadilek,

According to me it is caused by impurities in the water. You start your gradient with 66 % water , so impruties can adsorp on the column, assuming you are doing PR chromatography , and as you gradually change your mobule phase composition (higher % organic phase) you elute the concentrated impurites onto your column.

So pay attention to the quality of the water used in the gradient



regards

philippe

[Jackus]

I was in the conviction that our ultrapure water system is in good condition. We use Millipore Milli-Q system with UV lamp and TOC monitor. TOC in output water is 4-6 ppb. I will test water for injection too.

[DR]

Search the forum for the word "Empore" - you will find threads about the use of Empore Extraction disks. Use these to pretreat your water before making your A phase. Despite your water being down in the 4-6PPB range, an occasional styrene monomer or plasticizer trace coming off the final filter will frequently cause what you're seeing, especially at lower wave lengths.

All WFI will get you is ~"pyrogen free Milli-Q" grade - probably no better than what you're using.

[Bruce Hamilton]

If you suspect the water, why not try a few runs after long elution of high-water-content mobile phase, before running three gradients one after the other?.

I'd be wary of the IPA purity as well, and check that in a similar way. Ensure that any membrane degassers are extremely well flushed with high water content mobile phases, as water TOC values <10 should give minimal grief, even at 220nm. I'd also be looking to see if the impurity areas change if you inject some large volumes of high water content blanks - suggesting instrument/column contamination .

Main water issue is to throughly flush water from point-of use, I like to waste about 500-1000 mLs each time, if it hasn't been dispensed that day.

Please keep having fun,

Bruce Hamilton

[Jackus]

Thank you for ideas. I'll try to test for instrument contamination first. I have never heard about Empore disks, I'll test it too and let you know what solved my problem.
Regards

Edit: when I searched this forum I found lot of similar questions. So sorry for duplicates

[Kostas Petritis]

A good way to identify if it is your mobile phase A that causes problems is to try different equilibration times. If you get nearly increased baseline increases/perturpations with increased equilibration times then probably it is something in your mobile phase if not, probably something else...