Effects of sample dimension and dye distribution characteristics in absorption microspectroscopy

Models and the relevant equations giving optical absorbance of an analyte (i.e., dye) confined in a small volume with different shapes and sizes (sample dimension, D) are given, and factors governing absorbance are discussed. Dye absorbance is shown to be dependent on the three-dimensional sample structure: films (1-D), tubes (2-D), and spheres (3-D). The magnitude of the dimension effect on absorbance is determined by the size of both the sample and the probe beam. Furthermore, dye distribution characteristics in 2-D and 3-D systems also affect absorbance; a dye distributed exclusively to the surface layer of a 2-D or 3-D sample gave an absorbance 0.50- or 0.33-fold of that homogeneously distributed, respectively. If a dye is distributed in the inner volume of the sample alone, absorbance increases over that for homogeneous distribution. Effects of sample dimension, dye distribution characteristics, and the size of the probe beam in absorption microspectroscopy are discussed on the basis of the proposed models, and an experimental check of the models is presented.