Chromatography Forum

Please, help me with some theory in HPLC!
How the concentration of buffer influence on the retention time of the analyte? In my case when I change the concentration of KH2PO4 buffer from 0.05M to 0.01M (pH=7.4), the retention time of the analysed substance decreases from 15 min to 18 min.
I need to explain this fact
I think, that increasing of buffer's concentration leads to growing of the ionic power of the solution,
but what is the result from this?

Thank you!

What is your analyte, the column, the mobile phase?
It´s a bit unusual to show that much change at such low ionic strengths. My suspicion is that you may have a pH problem.
Theoretically, the ionic strength effects are extremely complex. Among them are salting out or in, conformational changes, interferiing with ion exchange (SiO-...). Others might think of more. With the info you gave it is not possible to get more specific.

The analyte is azithromycin - 15-membered-ring lactone (macrolide) with basic nitrogen in the structure
the column is Luna C-18 Phenomenex 150*4.6, 5m
Mobile Phase: AcN-Me-buffer KH2PO4 (pH 7.4) =37.5%-37.5%-25%

all measurements with changing of buffer's concentration repeated for three times.

Thank you for attention!

Me is MeOH?
KH2PO4 has a pH near 4, so how did you adjust the pH? How did you determine the pH of the differently concentrated solutions?

If I understand you correctly, your retention increased, when you reduced the buffer concentration from 50 mM to 10 mM. Your buffer is a phosphate buffer at pH 7.4, your analyte contains two amino functions.

Your analyte is positively charged at pH 7. The silanols on the surface of the packing are negatively charged. You are getting some ionic interaction of your positively charged analyte with the negatively charged silanols. This ionic interaction increases, as you decrease the ionic strength.