Recent experimental studies show that electrokinetic phenomena such as electroosmosis and electrophoresis can be used to separate nanoparticles on the basis of their size and charge using nanopore-based devices. However, the efficient separation through a nanopore depends on a number of factors such as externally applied voltage, size and charge density of particle, size and charge density of membrane pore, and the concentration of bulk electrolyte. To design an efficient nanopore-based separation platform, a continuum-based mathematical model is used for fluid. The model is based on Poisson–Nernst–Planck equations along with Navier–Stokes equations for fluid flow and on the Langevin equation for particle translocation. Our numerical study reveals that membrane pore surface charge density is a vital parameter in the separation through a nanopore. In this study, we have simulated high-density lipoprotein (HDL) and low-density lipoprotein (LDL) as the sample nanoparticles to demonstrate the capability of such a platform. Numerical results suggest that efficient separation of HDL from LDL in a 0.2 M KCL solution (resembling blood buffer) through a 150 nm pore is possible if the pore surface charge density is ∼ −4.0 mC/m2. Moreover, we observe that pore length and diameter are relatively less important in the nanoparticle separation process considered here.