Using C18 column on an Agilent 1100 series, what is the best fluorescence detection wavelengths am i to use to detect pregnanolone, allopregnanolone (progesterone metabolites) using a nongradient HPLC
Hi Innocent, do you have an UV/VIS spectrometer ? Make a solution of your compound and make a measurement. Instead of a UV/VIS spectrometer you can use your DAD. In both cases you will get a UV/VIS spectrum. - Markus -
The max wavelenght of pregnanolone in UV spectral is around 285nm. fluorencense spectral or the max emision signal has higher wavelenght than absorbance signal.
If the absorbance signal is gained at 285nm, the emision signal will be around 300-320nm. You can determine the max wavelengt by scanning emision spectral and absorbance spectral.
Tq. Wulan
its best to obtain the UV/Vis scan of your sample in the media you have dissolved it in and determine the wavelength at maximum absorbance.
Be sure you don't use Methanol in your mobile phase since it can quench the fluorescence reaction.
Steroid type molecules do not have intrinsic fluorescence and will need to be derivatized to do so.
Commonly used UV wavelengths are 210 nm (very non-selective but works for the steroids not having aromatic rings like your pregnanolone and allopregnanolone isomers ) and 260 nm with a wide band-pass (more selective for the estrogens but not very sensitive).
Smaller HPLC column particle size 3 um can help to achieve critical pair resolution.
Using non-selective detection and closely related chemical structures will probably require an LC method that uses gradient elution.
Wulan, Amir, Bruce and Duncan,
Thank you so much for your help on this issue.
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