In this article, we investigate the effects of pH, ionic strength, and channel height on the mobility and diffusivity of charged spherical particles within planar microfluidic channels. Specifically, we report results of a broad experimental study on the transport and separation behavior of 50 and 100 nm spherical carboxylated polystyrene nanoparticles, confined in 20 μm, 1 μm, and 250 nm deep fluidic channels. We find that pH, ionic strength, and channel height have coupled impacts on mobility changes. In particular, we show that, depending on pH, the dependence of particle mobility on channel size can have opposing behavior. In addition, we also show that at the nanoscale, at lower ionic strengths, there is a substantial increase in mobility, due to enhanced electric fields within the nanochannels. These effects are important to understand in order to avoid potential downfalls in terms of separation efficiency as well as design for better tuning of separation performance in micro- and nanochannels. Finally, we propose a method to estimate the effective zeta potential of spherical particles from measured electrophoretic mobility data. This could prove useful in characterizing a heterogeneous collection of particles having a distribution over a range of values of the zeta potential.