ask for help in contaimination in LC/MS

[zyfy]

I always has a peak at m/z 241-196.8 (MS/MS) even I bypass the autosampler. (directly hook the pump to the column).
I changed a new column, new guard column but no help. and since the peak is pretty good shape, I think it should come from either mobile phase or the pump tubing.
It is a binary pump, A is H2O with 5mM Formic acid, B is MeOH. I started at 20% MeOH and immediately increase to 100% MeOH after 10 seconds, and stay at there for 8 minutes, and then go back to 20% to stable for 5 minutes.
The peak came out at 7.5min with 2.0*150mm C18 5u column. If only guard column hooked in the system, the peak came out at 2 min. Thus, should be a pretty non-polar compounds. If I use isocratic mobile phase, what ever 50%,60%,70%, 80%,90% or 100% MeOH, I cannot get the peak. So, looks like the peak came from the Water source contaminations: with pretty high H2O content (80%), the contaminate(s) stuck on the beginning column, and washed out with high percent MeOH. However, I tried the commerical DI water with another clean container and still get the peak.
Also, the contaminates look like an acid: If I only use neutral water, I got nothing. it only showed up whenever I add 5mM acetic acid or formic acid.
I used pure MeOH to wash the whole tubing lines overnight but no use.
I used acetonitrile to replace the MeOH, but got same peak.
I have a tiple qual MS, but full scan didn't give me any clue eigher. only 241-196.8 has the peak.
any suggestion?

[Kostas Petritis]

Some random suggestions/thoughts:

Do you happen to use a polar endcapped column that leaks under acidic solution? Do you get these peaks with another type of column?

Did you eliminate the possibility of mobile phase container as the source of the contamination (by either using new glassware or washed by alternating hydrophilic to hydrophobic to hydrophilic washing steps)...

[zyfy]

Some random suggestions/thoughts:

Do you happen to use a polar endcapped column that leaks under acidic solution? Do you get these peaks with another type of column?

No, our lab don't have polar column. The only other kind of column is C8, I haven't tried it. I guess the same thing. why not?

Did you eliminate the possibility of mobile phase container as the source of the contamination (by either using new glassware or washed by alternating hydrophilic to hydrophobic to hydrophilic washing steps)...

I did wash the container with MeOH-Acetone-Hexanes-Acetone-MeOH-H2O.
and I did tried several other container with different source of water: distilled water, deionized water from other labs and same thing.

I used Acetonitrile washed the systems last night and run the same program today. In the begining the peak disappear, but after 1hour running with only 20%MeOH, the peak back again. the first injection has big peak, the next continue injections has kind of repeatable smaller peak. SO, looks like ACN did washed out something, but after accumulate a while, the peak came back again.

[Kostas Petritis]

How about the first part of my question? Do you use a polar encapped or a regular C18 column?

[zyfy]

How about the first part of my question? Do you use a polar encapped or a regular C18 column?

Actually it was answered in the last response, but it was in the quote.

No, we don't have any polar column actually.

[Kostas Petritis]

Do you have an online degasseur? If yes can you try to by-pass to see if this is the problem?

Have you switched the polarity to see what kind of ions you see there? For some reason 241 sounds familiar but I do not seem to remember what is it...

[zyfy]

Do you have an online degasseur? If yes can you try to by-pass to see if this is the problem?

Have you switched the polarity to see what kind of ions you see there? For some reason 241 sounds familiar but I do not seem to remember what is it...

It is a high pressure mixture binary pump system and so no degasser online since mobile phase mixed under the pressure after the pump. there are a dumper, pressure module and a mixture online.

negative mode was used. actually 241-196.8 is the picloram transition. while I am doing the picloram residue, I started to found even the blank also has the peak. the whole story started from there.

[Kostas Petritis]

This is a good piece of information which probably should have been given from the first post. Should I assume that the retention time of picloram residue and the "unknown 241-196.8 transition" is the same?

If yes, and you are confident that this is picloram and as started seeing this compound after analyzing it, the question is not what is this contamination and where did it came from but how it ended up contaminating the mobile phase and/or HPLC system.

As an amino acid (4-amino-3,5,6-trichloropicolinic acid) it would be best to use 90:10 methanol:water with 1% of formic acid to clean your HPLC system. I would clean check valves separately through sonication in acidic solution. If that does not work, try an aqueous solution of a volatile salt at high concentrations, followed by water washing....

[zyfy]

This is a good piece of information which probably should have been given from the first post. Should I assume that the retention time of picloram residue and the "unknown 241-196.8 transition" is the same?

If yes, and you are confident that this is picloram and as started seeing this compound after analyzing it, the question is not what is this contamination and where did it came from but how it ended up contaminating the mobile phase and/or HPLC system.

As an amino acid (4-amino-3,5,6-trichloropicolinic acid) it would be best to use 90:10 methanol:water with 1% of formic acid to clean your HPLC system. I would clean check valves separately through sonication in acidic solution. If that does not work, try an aqueous solution of a volatile salt at high concentrations, followed by water washing....

This peak shouldnot be picloram, though they are the same retention time in the first begining about couple months ago. At that time, I am kind of in the deadline, so I just choose another product ion and the blank become to blank. now I have several days nothing to do and go back and find the problem is still there. the gradient program in the original post was a concise one focus on this problem, not the original one. I will try the picloram with this concise program to see if they are the same Rt and report here again.

[lmh]

Loss of 44 is pretty common; CO2 from -COOH, and H2C=CHOH both have that mass. If you have more losses of 44, consider PEG.

[ColinB]

Hi, Without wanting to go into the deepest darkest places of my mind because it will make me ill. It is probably your water....

We have had a nightmare with water since last November, different manufacturers produce different grades of water but their testing procedures are completely laughable or non existent to the point where different batches from the same manufacturer can have different impurity profiles. Usually the impurities are phthalates (from plastic lids). The issue apparently is one of purity...the water is so pure that it absorbs impurities from everywhere that it can...depending on how long it has been exposed for.

Some things to think about:
How pure is your lab DI water.
Has it gone through a milliQ system? #
How pure is your Formic Acid?#
How pure is your methanol?
We have had so much trouble and done so much testing...degasser off and on, changed water to multiple batches from multiple manufacturers. We changed from Methanol to ACN due to the impurity profile of the Methanol.

Our current testing procedure is as follows:
Wash out all your lines with Methanol/IPA (50/50). We only used this at the very start when we first had issues. Degassers have a tendency to collect some of the impurities and then leach them into "clean" samples.

For the actual test!
Run 95/5 Water/ACN onto the top of your column for 45 minutes. This allows all water based impurities build up on the head of the column. Then run a standard blank. This will be the worst case scenario and then run 1-2 more blanks...when a stable blank profile is achieved then that is the best case scenario. You either accept it of find a different manufacturer.....we are still looking for a different manufacturer. BDH have a proper testing procedure but sometimes they appear to ignore it, although when they have a good batch, it is fantastic!! Waters are probably the best of the rest (at the moment) and we are currently having discussions with them to try and improve prcedures.
From my point of view there is nothing worse than having loads of chemists calling me to tell me that there are peaks in their supposedly clean sample! I hope this helps.