I injected BPA and estrogens, and used a 100mm column to separate them. Detected by MS in sim mode.
Then I found out, for example: for BPA, there are two peaks: normal peak as expected with small retention time and another peak later (long RT).
Does anyone know why?
Thanks.
thanks for your insteresting.
but it is pure standard, we did not have this problem on UV detector.
The second peak could origin from a fraction of the standard binding not to the column material but strongly to some other hydrophobic surface within your HPLC setup. The reason why you observe it merely with MS but not with UV is the increase in sensitivity. For making sure, it is indeed your standard and not some impurity, you could try to run in MS/MS mode instead of SIM to identify the substance.
To suppress the second peak you could try adding a second substance with different retention characteristics to saturate the potential hydrophobic surface inn your HPLC setup.
That's interesting I am not sure why this could be but I wonder if it might be the enolate of BPA. I think the only way to check this might be to collect the peaks and do IR or NMR. Good luck. Looking forward to hearing what these peaks are.
thanks guys.
I just use Milli Q water and Acetonitrile or MeOH as my mobile phase.
And the second peak come out when the ACN or MeOH is reaching 100% at the end of my chromatograme.
I will try MS/MS then. thanks a lot.
Hi Chang-Er,
I have a simple idea that the second peak could be due to remaining analyte. Did you changed the injection volume/analyte amount per run? Does the second peak still occur if do an empty run w/o any injection?
The second peak occurs when you do flushing/cleaning with high amount of polar solvent (which is recommended, we do it as well, e.g. 60% B after 30 min, 100% B after 35 min, 0% B after 40 min). The BPA could stick to the column too much and gets off only with a strong force of the solvent. You could try other column types like C18, C8, C5, phenyl-hexyl, CN, NH2... I attached a link for you.
If you would have access to HPLC-MS, this would be perfect.
http://www.phenomenex.com/Products/HPLCDetail/luna
Dear Chang-Er,
Another possibility is that SIM mode in MS is detecting BPA once (eluting early) and an equivalent fragment from an estrogen (eluting later). Which m/z channel are you monitoring for BPA? Are the peaks you see Gaussian in shape or do they have significant tailing? What is your ionization source? + or - mode MS?
Neutral BPA has m/z 228. I think loss of methyl in EI-MS leaves the +ion at m/z 213.
http://webbook.nist.gov/cgi/cbook.cgi?ID=C80057&Mask=200
Estrogen (ethinyl estradiol) also has a high-intensity peak at m/z 213.
http://webbook.nist.gov/cgi/cbook.cgi?ID=C57636&Units=SI&Mask=200#Mass-Spec
You might be detecting BPA and an estrogen in the same mass channel for SIM. Using multiple reaction monitoring is like the MS/MS version of SIM and it may resolve this (by monitoring another fragment for the estrogens).
NOTE: BPA will only form a negative ion (enolate) in water at approx. pH > 9 which doesn't sound like your mobile phase. Even if it did, you would not see the ion in positive mode MS. I think that only one of your peaks is BPA.
Take care,
Matthew
http://webbook.nist.gov/cgi/cbook.cgi?ID=C57636&Units=SI&Mask=200#Mass-Spec
http://webbook.nist.gov/cgi/cbook.cgi?ID=C80057&Mask=200
There might be two isomers of the same ion or molecule which have been separated in your column as shown in two peaks.
thanks a lot Matt. the second peak is wide and tailing, I use ESI-.
many thanks guys.
I tried MRM, and the second peak is not observed, so it might some impurity from either standard or mobile phase.
Awesome!
A wide and tailing peak would normally translate into poor quantification of that analyte in chromatography, but because you were using SIM mode MS, it can also suggest a kind of "cross-talk" between monitored mass channels (i.e. two or more analytes producing the same MS peak and thus ), which is what I think you had. You didn't want the second peak anyway, so the Gaussian shape is more important for the first peak!
MRM significantly reduces this mass channel cross-talk because every mass channel that is monitored now has an associated fragment mass channel (so two channels for each analyte). The probability that two or more analytes will have the same entry channel AND the same fragment channel is exceedingly low, so your fragment channel must have filtered out the second peak and only allowed BPA through.
Whatever the molecule was, it was producing an ion at the same m/z as BPA, but the fragments (or other peaks) it produces in MRM are not the same as BPA's fragments. That is why MRM worked. The question now is of the origins of the second peak!
All the best!
Matthew
i would agree with Laura Ferrando, as i had faced similar problem too... my target peak appeared to be two peaks .i.e. in ionised form, certain molecules at a pH dessocite into eqilibration state thereby resulting two peaks...i then later changed the buffer and pH resulting in better separation and peak purity
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