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This paper provides a contribution to FTIR analysis of calcium sulphate-based grounds of paintings when a mixture of compounds with different degrees of hydration is present. The study is based on the analysis with that technique, electron microscopy coupled with energy-dispersive X-ray spectrometry and X-ray diffraction of both the grounds of 16th-century Portuguese paintings and reference samples prepared with anhydrite and calcium sulphate dihydrate bound in an animal glue solution. It is shown that the interpretation of the degree of hydration of calcium sulphate ground samples using FTIR cannot be based solely on the study of the hydroxyl bands, as is usual in routine work, but that small deviations of the S–O vibration bands are an important indicator of the presence of anhydrite in the grounds.