Study on the dependence of fluorescence intensity on optical density of solutions: the use of fluorescence observation field for inner filter effect corrections

摘要： This work reports the identification of absorbance value of an analyte, at the excitation wavelength, that corresponds to maximum of the observed fluorescence intensity obtainable for a certain instrument operating with right-angle fluorescence measurement (Aλex^max_fl). The Aλex^max_fl value depends on the fluorescence observation field (FOF) dimensions of the concerned spectrofluorometer. As the FOF varies from instrument to instrument, this work presents a simple method for obtaining FOF dimensions. With the knowledge of FOF and absorbance of analyte at the excitation wavelength (Aλex) and emission wavelength (Aλem), we deduced a derived absorbance spectral parameter (Dabs). The observed fluorescence intensity of an analyte is proportional to the Dabs. Upon differentiating Dabs w.r.t. Aλex, the value of Aλex^max_fl for the concentred spectrofluorometer is obtained and subsequently can be used for maximizing fluorescence sensitivity. It is observed that when the FOF is a point at the centre of a 1 cm path-length cuvette, the Aλex^max_fl value is 0.87; with progressive widening of FOF, the Aλex^max_fl value increases gradually till ~1. The proposed methodology is established using two well-known inner filter effect (IFE) correction models (Parker and Lakowicz model). The Dabs obtained from Parker model corresponds well with the observed fluorescence data, while the Dabs obtained using Lakowicz model overestimates the loss of fluorescence due to IFE. Using equations derived from Parker model, the correction of observed fluorescence intensity for IFE can be achieved. It is further demonstrated that the commonly used Lakowicz model loses its correction-efficiency at absorbance values ≥ 0.7.