Chromatography Forum

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

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.

Thanks Alex - will do.

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

L