A new methodology, suitable for spectral measurements on polar, biologically important molecules, has been developed. The capability of the thallium bromide iodide ATR crystal to adsorb water has been examined and the hydrated crystal surface was used as a substrate for the adsorbed molecules. Diagnostic bands for adsorbed water are reported. A freeze-drying procedure was used to adsorb adenosine-5'-phosphate molecules on the hydrated ATR crystal, and FT-IR spectra of the system were recorded. Different hydrated species of adenosine-5'-monophosphate adsorbed on a water-screened ATR crystal have been observed. The broad bands at 935, 950, and 970 cm<sup>−1</sup> are assigned to phosphate groups adsorbed on the thallium bromide-iodide crystal screened by low, medium, and high water coverage, respectively. The high sensitivity of the method is emphasized. The proposed ATR method may be useful for the study of membrane-associated phenomena and models of real-life biological systems with a low solute concentration.

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