A spectrophotometer (2) comprising a source of radiation (6), preferably optical radiation, disposed to emit radiation at a plurality of wavelengths towards a sample in a sample holder (4) and a detection arrangement 8 for detecting the radiation after its interaction with the sample. The sample holder (4) is adapted to present a plurality of different path lengths for the emitted radiation through the sample. An arithmetic unit (10;10b) is operably connected to receive an intensity dependent output from the detection arrangement (8) and is adapted to store an intensity value of the detected emitted radiation indexed to its wavelength at two or more path lengths of the plurality of different path lengths and to calculate a value dependent on the ratio of the indexed intensity values at each of two path lengths by which an indication of the presence of a substance of interest withiA spectrophotometer (2) comprise a source of radiation (6), preferably optical radiation, disposed to emit radiation at a plurality of wavelengths towards a sample in a sample holder (4) and a detection arrangement 8 for detecting the radiation after its interaction with the sample. The sample holder (4) is adapted to present a plurality of different path lengths for the emitted radiation through the sample. An arithmetic unit (10;10b) is operably connected to receive an intensity dependent output from the detection arrangement (8) and is adapted to store an intensity value of the detected emitted radiation indexed to its wavelength at two or more path lengths of the plurality of different path lengths and to calculate a value dependent on the ratio of the indexed intensity values at each of two path lengths by which an indication of the presence of a substance of interest within the retained sample can be obtained.