Study on the dependence of fluorescence intensity on optical density of solutions: the use of fluorescence observation field for inner filter effect corrections†
In this study, we report the identification of absorbance value of an analyte at the excitation wavelength that corresponds to the maximum of the observed fluorescence intensity obtainable for a certain instrument operating with right-angle fluorescence measurement (). The value depends on the fluorescence observation field (FOF) dimensions of the concerned spectrofluorometer. As the FOF varies from instrument to instrument, this study presents a simple method for obtaining FOF dimensions. Using the knowledge of FOF, 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. While differentiating Dabs w.r.t. Aλex, the value of for the concentred spectrofluorometer was obtained and subsequently could be used for maximizing fluorescence sensitivity. It was observed that when the FOF was a point at the centre of a 1 cm path-length cuvette, the value was 0.87 with a progressive widening of FOF, the value increased gradually till ∼1.0. The proposed methodology was established using two well-known inner filter effect (IFE) correction models (Parker and Lakowicz model). The Dabs obtained from the Parker model corresponded well with the observed fluorescence data; however, the Dabs obtained using the Lakowicz model overestimated the loss of fluorescence because of IFE. Using equations derived from the Parker model, the correction of observed fluorescence intensity for IFE could be achieved. Furthermore, it is demonstrated that the commonly used the Lakowicz model loses its correction efficiency at absorbance values of ≥0.7.