I have a problem in hplc analysis of L-cystine analysis that the peak of both sample and standard have negative absorbance.
This the first trial .
-UV detector at 500nm.
-Mobile phase phosphate buffer:methanol 920:80.
I try to change polarity, try to dissolve sample and standard in mobile phase and try to change wavelength and there is no change
Detection of cysteine at 500nm is not very common as it has no chromophore able to be monitored at the wavelength. Do you use derivatized sample?
Negative peaks (if your interpretation of the chromatogram is correct) can be a result of lower abs coefficient than the mobile phase at selected wavelength. The assay of the substance is however possible after inverting (rescaling with factor -1) the chromatogram to get positive peak. You should also consider that negative peak could occur as a result of solvent present in the sample which undergoes chromatography together with the analyte - run blank sample (sample solvent) to verify it.
For L-cysteine (as well as other AAs), when underivatized, I would suggest short wavelength 200-220nm and the mobile phase containing ACN rather than MeOH (lower cut-off). Derivatization (FMOC, fluorescamine, AQC etc) gives you a chance to detect AA at longer, more specific wavelength and offers higher sensitivity of the method.
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