Chromatography Forum

I'm trying to apply a method for quantification of glycoalkaloids (Tomatine and tomatidine).

I'm using a HPLC/PAD unit (Gilson) and a ACE 3 C18-AR.

Detection: 202 and 205 nm

Which reference value I should use for this wavelenght?

Thanks for your atention.

What is your mobile phase composition? Is it retained with RPLC? Have you considered HILIC?

I'm using Methanol and CH3COOH

Thanks for your atention.

Please provide your chromatographic conditions. Is your method isocratic or gradient? What are the column dimensions, temperature and flow rate?

What are the retention times of your 2 analytes? Please post a chromtogram if possible.

Instructions for embedding chromatograms in a post are shown here:

viewtopic.php?t=2617

"Which reference value I should use for this wavelength?"

Do you mean what detector reference wavelength should be used? If so, why do you think you need one? A reference wavelength is not always needed or helpful. Try it first without a reference. If the baseline drift is too high, 360nm +/- 50nm commonly used

You don't need hilic for tomatine and tomatidine. They are well-retained on C18. I don't envy you trying to detect them by UV, though. I use LC-MS for the job.

Below is an LC-MS application for a-solanine & a-chaconine from potato. This separation was done using Cadenza CD-C18:
http://www.imtaktusa.com/site_media/fil ... TI189E.pdf

As already stated, it will be difficult to do this analysis with UV detection.