Chromatography Forum

Hello.
I am developing a method for analysis of 15 PAHs in water.
I am using HPLC-ELSD from Agilent, with fluorescence detection (FLD G7121A).
My mobile phase of choice is a gradient consisting of water and acetonitrile, and the column I am using is Zorbax eclipse PAH 3x 250 mm, 5um.
I managed to work through the programming of wavelengths and all the other parameters to get a pretty good chromatogram with great reproducibility. However, I am having a hard time separating Indeno[1,2,3-cd]pyrene and dibenz[a,h]anthracene, the two PAHs known for overlapping. At first, I only got a kind of "pregnant-looking" peak , but I played around a bit with my gradient at that point in runtime (lowering the ACN concentration with a gentle oven temperature rise, and setting the peak broadening to a higher frequency), which lead to a separation of kind. Now I have two very ugly looking peaks, with an area value four times lower that the original "pregnant" peak.
I am developing a method for the first time, and it is quite challenging, especially because of many parameters (15 of them) in four different types of water.
I am also aware that my column is not the best for the job, it would be better if it were less than 5um, but I have to work with what I have.

Can you please give me some advice how to get those two to separate nicely?
Thank you so much!

Did you try the separation conditions recommended by the column manufacturer? The usual peak elution order is D[a,h]A - B[ghi]P - Ind[1,2,3-cd]P for such columns.

It is easier first to adjust the separation conditions using a UV detector (e.g. at 210 or 252 nm; of course, the concentrations of the analytes must be chosen high enough for the UV detection) or an FLD with zero-order light (polychromatic) from the excitation and emission gratings of the detector. After the separation conditions have been chosen, the FLD Ex/Em wavelengths timetable can be created.

Try to change the gradient, but set the constant column temperature. Try to use different values of the column temperature. The selectivity ("shape selectivity") of the PAH-columns depends significantly on temperature. Lower temperatures (20, 15, or 10 С) can improve the selectivity.