On recording the true absorption spectrum and the scattering spectrum of a turbid sample: application to cell suspensions of the cyanobacterium Anabaena variabilis

An integrating sphere is often used for recording the absorption spectrum of a turbid sample. If the sample is placed inside the sphere, scattering losses are eliminated, but the recorded spectrum suffers from other distortions. These distortions can be avoided by positioning the sample outside the sphere; but, since some of the scattered light escapes the detector, the recorded spectrum suffers from residual scattering losses. A method proposed by Latimer and Eubanks more than 30 years ago (Arch. Biochem. Biophys. 98 (1962) 274), is put to a quantitative examination, which has shown that one can obtain, by recording two spectra at different distances from the sphere, not only the true absorption spectrum but also the scattering spectra of the sample. Conditions for the validity of the basic assumption underlying the method are investigated by examining suspensions containing various concentrations of cells of the cyanobacterium Anabaena variabilis, and it is shown that the calculated absorbance is proportional to the number density of the cells. The application of the method for quantitative spectrophotometric analysis of pigments in cell suspensions is discussed.