Chromatography Forum

Hi again guys!

I am running a new method with a 3um eclipse c18 plus column. Mobile phase is 95% (2%tea) 5% water.

when i inject the standard diluent (ALONE), which is 20% Methanol (to dissolve the API) and then made to volume with Mobile phase i get three negative peaks. Very clean negative peaks at 4/6 and 8 minutes.

When i inject a stardard obviously including this diluent, the peaks turn the other way and become positive. As you can imagine this is very annoying and making a problem as they can be mistaken for degradents etc..

Any ideas? Heellpppp!

Thanks again.

What you see is mainly an refractive index effect in the flow cell of your UV detector. I guess the wavelength you messure is below 210nm.
Use a wavelength above 210nm and try to get a similar solvent composition as you use in your mobile phase. Good luck.

What you see is mainly an refractive index effect in the flow cell of your UV detector. I guess the wavelength you messure is below 210nm.
Use a wavelength above 210nm and try to get a similar solvent composition as you use in your mobile phase. Good luck.

Thankyou for your reply. Do you have any idea as to why these could 'flip' with the addition of an API to the solution. I.e. why do they seem negative in a diluent injection then positive with my analyte solutions?

Thanks again

Hi:

Maybe you're using hplc with the option "reference correction" turned on, you must deactive it.

To do this go to the method parameters options> multiple wavelenghts (or something similar) and unmark the option reference correction

its work for me in a shimadzu device