PDA gives lot of Noise for 190 to 200nm when mobile phase used has Triethylamine as one component. Trying to Overlay spectra of standard & sample for Water’s PDA of 2998 or 2996.
Hello.
Triethylamine has a UV cutoff of about 235 nm, meaning when you use wavelengths below that number, you will inevitably get interference from it (increased baseline response).
For that range of 190-200 nm, you must try to use only water and acetonitrile mixture with reversed phase mode. Even better would be to stick to ranges starting from 205 nm and above if you have a choice.
You may not have a choice if that's previously approved method, the analyte's UV spectrum demands for that range, if you are doing identification run, or if you are doing peak purity detection (where you usually need 200-400 nm range).
For further assistance, try to send us more information about your run such as mobile phase composition, type of test (assay, impurities, identification), chromatograms, and tell us if you have room to modify your existing method or not.
What you observed is normal. TEA absorbs in the wavelength region you have selected (in fact, just about everything does, including your mobile phase! This is a basic fundamental of HPLC). BTW: Old TEA (not fresh) will absorb at much higher wavelengths too. No such valid comparisons or analysis are possible at the settings you have selected.
William Letter Thanks for your response, you have any idea what will happen to TEA by the time that it will cause more noise if we use the old one?
Do not use it in any form for low UV analysis. Not appropriate.
Yes, TEA degrades. Look for the brownish color. If you must use it for suitable applications, then purchase it in small volumes, after opening blanket with nitrogen, properly dispose of after a suitable time period.
The composition is
350ml of Buffer : 650ml Acetonitrile (Buffer contains Sodium Acetate, Triethylamine and water)
but it is the SOP of the method, the thing is that sometimes we see the noise and some times no.
Perhaps the SOP should be re-written? Anyone that develops a method with TEA for use at wavelengths between 190-200 nm should be questioned.
As mentioned earlier, when TEA ages, it absorb even more UV signal often resulting in increased noise. There may also be other concerns with the method that can only be identified on-site, not via a web forum. Please have someone experienced in liquid chromatography review your method on-site for more info.
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