Silver (Ag) films of varying thickness were simultaneously deposited using physical vapor deposition (PVD) onto six infrared (IR) substrates (BaF<sub>2</sub>, CaF<sub>2</sub>, Ge, AMTIR, KRS-5, and ZnSe) in order to correlate the morphology of the deposited film with optimal SEIRA response and spectral band symmetry and quality. Significant differences were observed in the surface morphology of the deposited silver films, the degree of enhancement provided, and the spectral appearance of <i>para</i>-nitrobenzoic acid (PNBA) cast films for each silver-coated substrate. These differences were attributed to each substrate's chemical properties, which dictate the morphology of the Ag film and ultimately determine the spectral appearance of the adsorbed analyte and the magnitude of SEIRA enhancement. Routine SEIRA enhancement factors (EFs) for all substrates were between 5 and 150. For single-step Ag depositions, the following ranking identifies the greatest SEIRA enhancement factor and the maximum absorption of the 1345 cm<sup>−1</sup> spectral marker of PNBA at the optimal silver thickness for each substrate: BaF<sub>2</sub> (EF = 85 ± 19, 0.059 A, 10 nm Ag) > CaF<sub>2</sub> (EF = 75 ± 30, 0.052 A, 10 nm Ag) > Ge (EF = 45 ± 8, 0.019 A, 5 nm Ag) > AMTIR (EF = 38 ± 8, 0.024 A, 15 nm Ag) > KRS-5 (EF = 24 ± 1, 0.015 A, 12 nm Ag) > ZnSe (EF = 9 ± 5, 0.008 A, 8 nm Ag). A two-step deposition provides 59% larger EFs than single-step depositions of Ag on CaF<sub>2</sub>. A maximum EF of 147 was calculated for a cast film of PNBA (surface coverage = 341 ng/cm<sup>2</sup>) on a 10 nm two-step Ag film on CaF<sub>2</sub> (0.102 A, 1345 cm<sup>−1</sup> symmetric NO<sub>2</sub> stretching band). The morphology of the two-step Ag film has smaller particles and greater particle density than the single-step Ag film.
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