The effects of iron catalyst thickness on the fabrication and performance of microfabricated, binder-free, carbon nanotube (CNT)-templated, thin layer chromatography (TLC) plates are demonstrated. The iron catalyst was deposited at thicknesses ranging from 4 to 18 nm in increments of 2 nm. Its thickness plays a key role in governing the integrity and separation capabilities of microfabricated TLC plates, as determined using a test dye mixture. Atomic force microscopy and scanning electron microscopy show that smaller and more numerous catalyst nanoparticles are formed from thinner Fe layers, which in turn govern the diameters and densities of the CNTs. The average diameter of the Fe nanoparticles, Dp, is approximately six times the initial Fe film thickness, tFe: Dp ≈ 6tFe. After deposition of relatively thick silicon layers on CNTs made with different Fe thicknesses, followed by oxidation, all of the resulting CNT-templated SiO2 wires had nearly the same diameter. Consequently, their surface areas were very similar, although their areal densities on the TLC plates were not because thinner catalyst layers produce denser CNT forests. For tFe = 6 nm, nanotube growth appears to be base growth, not tip growth. Best TLC separations of a test dye mixture were obtained with plates prepared with 6 or 4 nm of catalyst. Calculations suggest a loss of surface area for TLC plates made with thicker Fe layers as a result of fewer, thicker CNTs, where the density of silica nanotubes (device surface area) goes approximately as 1/tFe2. While the focus of this paper is toward a greater understanding of the processing conditions that lead to the best TLC plates, a baseline separation of three analgesics (caffeine, phenacetine, and propyphenazone) is shown on a normal phase TLC plate grown with 6 nm of iron.