Chromatography Forum

Hi Ken

Your point 2: sadly, new does not necessarily mean clean. Try vials and caps from a different manufacturer. I agree that the autosampler is probably contiminated, but then the question becomes where did that contamination come from - unless you eliminate the source it will just come back again. Is there a reason that you are nut flushing the whole system with pure methanol, which you have already demostrated by injections of solvent blanks has the ability to mobilise the contaminant.

Peter

Was "forbidden" yesterday so here is another trial:
OK so Peter and I seem to agree that judging from your information it is most likely that you are injecting a contaminated blanc, rather than observing carry-over. Is it possible to run this without injecting anything?
Can you characterize the peak? Maybe it is not analyte at all. Strange coincidence? What does an injection of air look like?

Hi Peter and Hans, thanks for the reply.

1) I have used another vial and cap from different manufacturer; the contamination peak is still there.

2) I did not flush the system with methanol but I have flushed it with water and then ACN.

3) I have use other blanks ie newly opened, new vials etc but there is still the contamination peak.

4) I was pretty sure of the contamination peak due to:

i) Same RT.
ii) Same MRM transition ( using 3 MRM transition for my analyte)
iii) Same MRM ratio to the standard being injected.

I am currently flushing the system overnight, and baking my ion source in the hope that this will eliminate any possibilities. Is there any other suggestion?

Thanks again !!!

HI Ken

If you had provided the information in 4 from the outset we would have probably got to this point a lot quicker !

Agreed that you have carryover (in the strict sense) but with some strange features (particularly that the ghost peak stays the same with multiple blank injections), and that it is most likely coming from the autoinjector. You know that methanol dissolves the analyte, so purge everything with methanol. Depending on which instrument you have you might get a clean system quicker by changing some frits etc. Clearly whatever injector purge / needle wash protocol you have in your method is not doing its job so you need to revisit that. Since carryover peaks that do not diminish with repeated blanks point to very severe contamination you need to consider whether the amount of analyte that was abstracted from and bled back into the samples is likely to have compromised the results.

Peter

Try changing the sample loop, needle, etc. to be absolutely sure that you know the problem is in the autosampler. If you don't have another one, you could try removing the loop/needle and flushing with 1% nitric acid, then water, 1% ammonia, then water, DCM, then methanol. You could also try adding some acid (0.1% formic) or base (0.1% ammonia) to your autosampler rinsing solution.

To the unanswered questions (How does no inj. look, how does air inj. look) there is one more to be asked: How large is this peak compared standard or average size in samples? Unless you quantitation is miserable you would need an enormous deposit for a non-varient carry-over, especially if the carry-over peaks are relatively large.

try running the gradient twice, initiate with injection, then repeat without iinjection. the second gradient should elute the compound. then change the gradient-loading phase, slopes, and the max hold until the peak is not seen on the second gradient. then go back to the single gradient with the conditions you found eliminate the second peak.

Hi all, thanks for the reply.

After flushing and baking the source, I managed to bring down the contamination peak.

However, now, even after chaning the injection seat and using new fresh of buffers, we are still experiencing the carry over peak.

I am really unsure why this happens; even the system engineer and application specialist is not able to clear this issue as of now.

What I am trying to do is to really get another source of solvents for the buffers as I am suspecting maybe the bottles, solvents are contaminated one way or another - not sure how this will happens but I want to eliminate once and for all the contamination does not come from the buffer.

Any other advise, guys? Thanks so much.

Infusion of freshly prepared MP, then flow injection of it, see what happens.