LC-MS: Peaks in DAD signal, none in TIC.

[ZeroAir]

Hi,

I an injecting a prepared sample of ergosterol (UV=282nm) into my HPLC. It elutes and I see a peak from the strong peak on the diode array detector. I am using an Agilent Active Splitter and sending some of the flow from the DAD to an MS. ESI source, 0.1% acetic acid solution from the isocratic pump. Unfortunately, I see no peaks in the TIC, just background noise. There is also visible condensation on the windows looking into the spray chamber... not sure if this is good or bad.

I have done the following to attempt to pin-point the error:
-verified that there is spray from the nebulizer
-verified that the capillary voltage is correct
-tuned the MS (successful)
-verified vacuum pressures
-checked drying gas flow/temp
-verified fragmentor settings
-looked for electronics errors (none reported)
-concentrated the sample (it should DEFINITELY register on the MS)

I am considering: starting a run, and once the sample passes the DAD for the first time, stopping the pumps, and re-routing the post active splitter tubing (the one that goes A.S. ->MS) back into the DAD, then starting pumps again. This would verify that the splitter is working correctly. However, before I do this I thought I would ask for opinions about the condensation in the spray chamber... doesn't seem like that should happen under vacuum

Thanks in advance,

Luke

[sav]

Hi, Luke
See theory of API (ESI etc.).

You can use APCI (APPI) for ionization of ergosterol or use derivatization (or adduct formation with Ag+ etc.) of ergosterol (-OH > -OX) with ESI.

Best regards, Alex.

[ZeroAir]

Thanks Alex - will do.

About the condensation though - is that supposed to be there?

L