Comparison of common mobile-phase volume markers with polar-group-containing reversed-phase stationary phases

A systematic study of the behavior of several common mobile-phase volume markers using traditional and polar-group-containing reversed-phase stationary phases is presented. Examined mobile-phase volume markers include two neutral molecules, uracil and thiourea, concentrated (0.10 M) and dilute (0.0001 M) KNO3, and D2O. Mobile-phase volumes are examined over the entire reversed-phase mobile-phase range of 100% water to 100% methanol or acetonitrile. The behavior of these mobile-phase volume markers is compared with a maximum theoretical value (i.e. the void volume), as determined by pycnometry. The data suggest that: (i) uracil begins to fail as a mobile-phase volume marker in mobile phases below about 40% strong solvent for polar group containing phases; (ii) in nearly all cases, the mobile-phase volume measured dynamically is smaller than the pycnometric void volume; (iii) a significant dependence of measured mobile-phase volume on salt concentration is seen on the polar endcapped phase, which is not observed on the traditional and embedded polar group phase; and (iv) D2O does not work well as a mobile-phase volume marker with polar-group-containing phases, possibly due to interaction with the stationary phase polar group.