Chemometric methods are very useful in analyzing complex mixtures. In this thesis two methods are presented that allow resolution of overlapping spectra from mixtures without knowledge of the pure component spectra. It also includes applications of these methods, in which the spectroscopic properties of the fluorescein system are characterized.<p /> The first method is applicable to samples that contain interacting components and is used to study thermodynamic equilibria. It uses the equilibrium expression relating the components and a prerequisite for this method is that the equilibrium expression is known. If the exact expression is not known, it might be used to distinguish between different equilibrium models. The method is easy to use and gives unique solutions.<p /> The other method is based on the Procrustes rotation of matrices. It requires that two kinds of spectra, composed of the same spectral profiles but different intensities of the components, can be measured on each sample. An advantage of this method is that it is not restricted to samples that contain interacting components, and requires no knowledge of equilibrium expression. The solution is also always found in a single step by diagonalizing one matrix.<p /> Using these two techniques, we have characterized the spectroscopic properties of fluorescein. The protolytic equilibrium constants that relate the four protolytic forms of fluorescein and the absorption spectra of the four protolytic species are determined by the equilibrium constraint method. The Procrustes rotation method is used to determine the fluorescence excitation and emission spectra of the fluorescein mono- and dianion. The methods are also used to study the changes of the spectroscopic properties of fluorescein upon attachment to nucleic acids
