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In this research study, the interaction of particles with substrates of different roughness magnitude was investigated. Particle surface treatment, relative humidity (RH), and surface roughness levels were controlled in order to achieve separation of different particles by applying removal forces. Three different approaches to reproducibly roughen surfaces were used. Initially, glass disks were laser engraved to create a reproducible, controlled roughness substrate. However, the laser engraving method produced surface features that were much greater in scale than the particles. These scale differences were such that the substrates produced were not of value to this research. The second option investigated to induce reproducible substrate roughness was to scratch the glass disk using sandpapers of known grain size. A third approach to establish reproducible roughness was to use fine stainless-steel wire mesh substrates. In tests with sanded glass disks, the interfacial energy of plasma-cleaned (hydrophilic) glass beads had a high variation at 40% RH, showing non-uniformity of area of contact between particles and substrates. As the RH increased, it was expected that the interfacial energy of hydrophilic particles would increase, but this behavior was not observed. In addition, comparing the interfacial energy results of hydrophilic particles with hydrophobic particles, a region with significant interfacial energy difference was not identified. In the case of the stainless-steel mesh substrate, the mesh asperities and particle dimensions were comparable. Thus, the smaller particles had more area of contact with the substrate than the larger particles. For the plasma-cleaned (hydrophilic) beads, the recovery values had an average of 92.5% recovery when the RH was between 46 and 85%. For the hydrophobic beads, the average recovery was 19.0% when the RH was between 46 and 75%. Thus, the hydrophobic characteristic of the particle influenced its lower interaction with the mesh substrate. The difference in recovery can be exploited to achieve separation of particles based upon adhesive forces.