I performed a H/D exchange by adding D2O to my sample, then analyzed it with RP-HPLC/MS (targeted SIM) using ACN/D2O as mobile phase, and discovered that the retention time of the isomers of my molecules had changed.
Hello Zahraa,
I have no experience in using D2O instead of H2O in RP-HPLC, but from my experience, deuterated molecules (used as internal standards) always show a slightly earlier retention time than the un-deuterated molecules, this can - in extreme cases- even lead to a baseline separation (e.g. Bisphenol A vs. d16-Bisphenol A). Consequently, from the chromatograms you have shown, this would fit perfectly: the retention times shift app. 0.2 - 0.5 min later... given the fact that the solvent you used (D2O) is slightly more polar than H2O... hope this helps as explanation!
Considering differences in properties like viscosity, density and pH for both the solvents, retention time should slightly change. Basically, a major change may be due to their pH difference. (7.0 for H2O & 7.44 for D2O)