Carlo Tancon: Please NEVER use any software feature such as "Reference Wavelength" with your HPLC system. Two HPLC systems which appear to be similar or the same are in fact ALWAYS different. Different in many ways and you can not compare the results obtained from one to the other without a detailed evaluation (which requires someone with an advanced level of experience). Please get some help with your HPLC method as the problem you have observed requires that you obtain DAD training and have someone on-site to review your method(s). However, the most likely reason for your observation is that you have turned 'ON' and set (probably not set at all, just using some manufacturer filled in values, which have nothing to do with your method) the UV/VIS detector's Reference Wavelength feature. That software feature should ALWAYS be turned OFF. It was designed to destroy the original raw data and replace the data with a new set (but you will never know what the original data was) so it invalidates ALL data collected as well as the method itself as invalid. *I will post a free article taken from the HPLC training classes I teach on this topic here on RG which explains this in more detail .

• REFERENCE WAVELENGTHS (as used in HPLC UV/VIS): https://hplctips.blogspot.com/2011/03/reference-wavelengths-as-used-in-hplc.html

Please review this article and share with others to prevent invalid HPLC methods from being developed and used.

Check http://ccc.chem.pitt.edu/wipf/Web/LCMS%20trouble%20shooting.pdf