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In this study we report the effect of fuel type (biodiesel vs. methane), flame structure and flame height (inner-cone vs. outer-cone), and the percent of oxygen content in the oxidizer stream for the formation of hydrophobic carbon layers using co-flow diffusion flames. It was found that a flame formed using a gaseous fuel (methane) over a vaporized liquid fuel, Canola Methyl Ester (CME), has significant structural differences that enable vastly different deposition behavior of soot layers on the surface of solid substrates. Due to its larger pyrolysis zone (taller inner-cone), the CH4/air flame has a smaller region that supports uniform soot deposition of hydrophobic carbon layers (C-layers) compared to the CME/air flame. When a solid substrate is placed within the pyrolysis zone (inner-cone) of a flame the resulting layer is non-uniform, hydrophilic, and consists of undeveloped soot. However, when outside the pyrolysis zone, the deposited soot tends to be uniform and mature, ultimately creating a hydrophobic C-layer consisting of the typical microscale interconnected weblike structures formed of spherical soot nanoparticles. The effect of oxygen content (35% and 50% O2) in the oxidizer stream for the formation of hydrophobic C-layers was also studied in this work. It was found that oxygen enrichment within the CME flame alters the structure of the flame, hence affecting the morphology of the formed C-layer. Under oxygen enrichment the central region of the deposited C-layer is composed of a weblike structure similar to those seen in the air flames; however, this central region is bordered by a region of densely compacted soot that shows signs of significant thermal stress. At 35% O2 the thermal stress is expressed as multiple microscale cracks while at 50% O2 this border region shows much larger cracks and macroscale layer peeling. The formed C-layers under the different flame conditions were tested for hydrophobicity by measuring the contact angle of a water droplet. The morphology of the C-layers was analyzed using scanning electron microscopy.