Chromatography Forum

Hi All,

we are having a small but persistent and annoying problem in our lab: The UV and MS detectors are 0.2 min out of sync. The mass spec starts too early.

We have a Thermo PAL autosampler that is connected via the contact closure cable to an Accela pump, UV detector (off one cable) and to a Q Exactive mass selective detector (other end of cable).

Does anyone know how to correct this? Full size image: http://i.imgur.com/7mAFnT6.png

Also, we are seeing much broader peaks in MS compared to UV. Has anyone seen this phenomenon when using both detectors? We do not have a lot of capillary tubing between UV and MS (~3 ft of .005" i.d.). I suspect the ESI source with its metal needle causes the broadening. Any thoughts?

Thank you so much!

Arne

A bit of geometry allows calculation that your 90cm of tubing has a volume of about 11ul. The piece of information that I am missing is your flow-rate.
0.005" tubing is most suited to flow-rates less than 200uL/min (here's a link that contains a rate versus diameter table: http://www.mtc-usa.com/hplc/surefit.asp )
If you are running at 200uL/min, the 0.16min delay that you are seeing between PDA and MS corresponds to 32uL, which might mean that you have about 20uL of dead-volume somewhere (which would also give an enormous peak-broadening effect, I suspect much worse than you are seeing).

If you are running at 70uL/min, then the delay is just what you'd expect from that length of tubing, and the peak-broadening is probably just the diffusion and mixing that happened along the way.

My guess is that you're running at 100uL/min and the remaining volume is accounted for by the spray needle, but that's just a guess!

Good luck!

Hello,

thank you so much for the reply! It is very helpful.

I was running these samples at 350 uL / min. I can't believe I didn't specify that!

I got the same 11 uL volume from the PEEK tubing. I will check for dead volume after the UV detector again. That should of course give a much smaller time shift - on the order of 0.05 min. I wonder about the spray needle as well.

Do you know if there is a way to delay start time by x seconds to artificially align chromatograms? That will make matching of the traces and data analysis much easier. This is the first time ever that we have a system with two detectors in our lab. How is this usually handled? Does anyone have experience?

Thanks again,

Arne

I don't know about XCalibur, but some data analysis software have an option to shift a chromatogram by a given offset (Bruker DataAnalysis can do that, for example).
I often use that option to align UV an MS chromatograms.

For those interested: there is a setting in Thermo Xcalibur Qual Browser under Chromatogram -> Ranges to specify the offset of a trace.

Have you checked what is the concentration effect?Based on the image you have
high counts level in MS side and have anyway decent intensity in UV side.I have lots
of experience with UV and MS serial connections.Usually peaks were broader in MS than UV.Shorten capillaries and using low ID doesn't necessary resolve that problem.

VEBA

Are you running a split post UV? These are frequently the source of a mis-allignment, often as a result of the probe blocking over time. The MS does look to be rather broad it should be better as you are near the optimum flow rate in your system. It could be overloaded if it is not split; it is also worth playing with nebuliser / temperature settings to see if this can be improved.

Thanks for your input. The offset is caused by the way the system is wired. The real delay is <3 seconds. The picture I attached was from a sample that had a few very concentrated components (so we could also see components of lower concentration) - It is overloaded. There is no split post-UV. We think the tailing comes from the stainless steel needle. Do you inactivate the needles from time to time?