Chromatography Forum

Quick background:

I'm running a 2.1x100mm C18 column, 2.1x10mm guard column, flow 0.2 ml/min, 60-40 A:B to 0:100 A:B in ten minutes, flush 5 minutes at 100 B, back to starting conditions long enough to equilibrate, A:HPLC grade (bottled) water, B:ACN. Currently have about 700 injections on column, detector is Qtof, ESI-, MS-MS w/fragmentation to quantify. This is the setup we have been running for the last month, samples are groundwater cleaned up via SPE and in 50:50 H2O:ACN.

Last week (and for several weeks prior) back pressure has been ~1200, areas of standards and IS have been fairly consistent (~20 for IS). The last few days, the back pressure has climbed to as high as 1500 and yesterday it was only 1400. Delta PSI is about 15. The areas of our standards and IS have increased 3x in area from yesterday to today. My question is: as a result of "stuff" getting on the column, is the compound binding better, and more is getting held and then released? It seems so drastic for it increase 3x better sensitivity (IS = 60 area units). Retention time is the same for both days and hasn't changed when PSI was 1200 vs the now 1500. I've cross-posted in LC forum as well, since it seems like an LC issue more so than an MS issue.

Thanks,
Grant H.

How about your peak width? If retention and peak width are the same, I'd suspect the MS before the LC. I've had check standards fail on the high-side, even when the ion source should be dirty from running samples. There are other explanations not related to either, such as prep error, or evaporation from your vials.

One more question. Do you have any acid or buffer in your mobile phases? Without that, it would only take a small amount of mobile phase contamination to change your pH, and possibly your ionization efficiency.

To answer some of your other questions from the LC forum (I'll keep posting here since I started here): If you've eliminated sample-prep or autosamper error, and think this is an issue of increased ionization efficiency, *and* your analysis still meets the method criteria, then I would keep going until it no longer meets the criteria. On guard columns, I find that guard column failure is accompanied by increased backpressure, or poor peak-shape (broader peaks and tailing), or both. When to replace the guard is a judgement call based on the severity of these problems. The pressures you noted are not extreme, so if pressure increase alone is your problem, I would keep going.

>How about your peak width? If retention and peak width are the same, I'd suspect the MS before the LC. I've had check standards fail on the high-side, even when the ion source should be dirty from running samples. There are other explanations not related to either, such as prep error, or evaporation from your vials.

The RT and width are in fact the same. You're saying your check standards have given better response than the equivalent standard from the curve reported? I've seen that occasionally as well. I just don't think 50:50 ACN:H2O stored at 4C overnight would evaporate that much to show such an increase in response. The values of 3x increase I reported were from stds run to check MS calibration (drift) since we are so specific on the Q1 as to what it getting to the TOF, and calibration was fine. I ran a sample set and curve overnight and values are about 2x higher, not the 3x as reported in my original post. Maybe it is as simple as a getting better ionization due to the cone needing to be cleaned? Which seems backwards to me, but who knows? Thanks for the input.

Thanks,
Grant H.