In this paper, a detailed continuum-based theoretical model is proposed to investigate the effects of near-wall potentials and hydrodynamic confinement on separation of charged macromolecules in channels of nanoscale dimensions. These wall effects are primarily confined within a few nanometers from the channel wall, and hence have negligible influences in the conventional electrokinetic separation methods that are routinely performed in micro-channels. However, in nanochannels, their zone of influence becomes significant in comparison to the channel height, thereby inducing certain nontrivial effects on the resultant separation characteristics. By executing a regular perturbation analysis, it is established that depending on the macromolecular size relative to the channel height and the extent of electrical double layer (EDL) interactions, the wall forces decide the speed of traverse and the extent of spreading (dispersion) of the macromolecular bands. These factors combine together to finally decide the separation characteristics (quantified by the resolution of separation) of the charged macromolecules in nanochannels. It is demonstrated that because of the near-wall effects, macromolecular pairs with less disparities in sizes give rise to higher values of resolution. Moreover, the wall-induced influences are shown to magnify the resolution for any given pair of macromolecules in the nanofluidic systems, thereby signifying greater separation efficiency.