Chromatography Forum

I recently evaluated analyses on an HPLC with an HPLC photodiode array detector that showed a systematic increase in the peak area ratio of 664/450 nm when the ratio was plotted as a function of the 450 peak area. These peak areas were obtained during analysis of multi-level chlorophyll a calibration standards and for which there were no co-elution problems, ghost peaks, and SNRs were adequate. This phenomenon was observed on a ThermoQuest detector using a flow cell with a 5 cm pathlength. I observe a constant ratio on my Agilent 1100 photodiode array detector as does another colleague of mine who has a Shimadzu (both of us have 1 cm flowcells) when these standards are analyzed. I am especially interested in this because the analyst with the ThermoQuest HPLC experiences non-linear calibration curves, where as I do not. I would really like to know if this changing ratio could be symptomatic of an inability to obtain a linear calibration. Any thoughts? (P.S. Chlorophyll a absorbs strongly at 450 and 664 and the standards used covered a wide concentration range).

Best guess...

Everyone is using the same set of standards.
As stated, not everyone is using the same flow cell.

The 5CM flow cell results will go "nonlilnear" first when approaching the high end of the standard curve. If the concentrations go low enough, it will also be the last one to go "nonlinear" at the low end of the curve. The "changing absorbance ratio" for one peak is probably symptomatic of nonlinearity associated with just one of the two wavelengths.

If you run a 1/5 dilution of the same set of standards on the Thermo, you should get pretty comparable results vs. Agilent, Shimadzu using the original standards.