Infrared (IR) spectroscopy is widely used for studies of temperature-dependent properties of liquids and solutions, such as thermal denaturation of proteins and other molecules of biological interest. The variation of the spectroscopic signals with temperature can be affected by the changes in the optical path length due to the thermal expansion of the components of the sample cell. In this report we investigate the temperature dependence of the optical path length for a liquid IR sample cell of a design typical for aqueous solution experiments. The path lengths were measured from the interference fringes, both in dry cells and with cells partially filled with water. We found that the optical path length variations are significant, on the order of several percent within the temperature range used (0–87 °C). Several commercially available spacers (Teflon, mylar, and lead) and gaskets (Teflon, lead, silicone rubber, Viton, and neoprene) were tested to find materials with either the smallest or most reproducible effect. Teflon, due to its phase transition (known as the “knee point”) near room temperature, leads to abrupt changes in path length when used as either spacer or gasket component. On the other hand, Teflon is preferred for its inertness, while several of the other tested materials, most notably lead, are not practically usable due to adhesion to the cell windows upon heating and contact with the aqueous sample. The combination that yielded the most reproducible results, with minimal complications due to adhesion, was Teflon spacer with neoprene gaskets. The implications of the optical path length changes for the temperature-dependent IR experiments and their possible corrections are discussed.

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