Functionalized nanoparticles are widely used for various medical applications, defined as nanomedicine. Notwithstanding, surface functionalization remains poorly understood. Distributions of surface ligands per particle are rarely considered or measured for the appropriate methods are lacking. This research aims to establish a procedure for qualitatively analyzing said distributions. The nanostructures of interest are peptide, and polyethylene glycol (PEG) functionalized fluorescent silica nanoparticles, Cornell Prime dots (C’ dots). Average surface ligands per particle were measured as shown in previous work.Then these C’ dots were fractionated by size with Gel Permeation Chromatography (GPC) and separated by surface chemistry with High Performance Liquid Chromatography (HPLC). Peptide distributions per particle were indiscernible from PEG; this finding indicates that PEG functionalization is not as homogenous as previously considered, and contributes significantly to surface heterogeneity. Additionally, despite uncoupling the contributions of functionalized particle sizes, results indicated size had little impact on surface ligands per particle distributions.