[ROSETTA]

Hi, I have run toluene on the HPLC and record the UV spectrum at 254nm, and get practically no adsorption.  Am I doing something wrong.
Thanks
Rosie

[agrobert]

What type of column are you running?  What is your mobile phase?  What is your loading solvent (if any)?  What is your flow rate?

[ROSETTA]

Hi there, 15cm x 4.6mm id.  C4 column.  mobile phase is acetonitrile-water (70:30v/v).  I am dissolving the sample in 70:30 methanol-water.  The concentration of the sample is 100µg/ml and the injection volume is 1 µl.
When I look at the sample at 212 nm I have a really sharp peak.
Any thoughts?
cheers
Rosie

[Arkcon]

Remember that an HPLC flow cell has a much shorter pathlength than the standard 1 cm cuvette, and analyte is often moving through quickly.  You'll have to be closer to lambda max, for starters.  Also, you'll have to consider the UV cutoff of your solvents -- 212nm is a little short, HPLC detector optics may not be efficient enough at that wavelength.  If I had to quantify toluene by HPLC instead of GC, I'd consider using a RI detector.

[fulkon]

Maybe this will help:

Look at these two specification sheets on toluene from sigma-aldrich (specifically under "UV Absorption").

http://www.sigmaaldrich.com/catalog/search/SpecificationSheetPage/SIAL/155004
http://www.sigmaaldrich.com/catalog/search/ProductDetail/FLUKA/89675

Toluene appears to have an absorption maxima at 285 or 286 nm.  Try that range.

Hope this helps!