研究目的
To identify the absorbance value (Aλex^max_fl) that corresponds to the maximum observed fluorescence intensity for a specific spectrofluorometer, understand its correlation with fluorescence observation field (FOF) dimensions, and establish a methodology for determining FOF dimensions and Aλex^max_fl to correct for inner filter effects in fluorescence spectroscopy.
研究成果
The study provides a method to correct for inner filter effects in fluorescence spectroscopy by determining instrument-specific FOF dimensions and Aλex^max_fl. The Parker model accurately predicts observed fluorescence intensity and corrects for IFE, while the Lakowicz model overestimates fluorescence loss at higher absorbances. This approach enhances analytical accuracy in fluorescence measurements.
研究不足
The methodology assumes analytes obey Beer-Lambert's law; deviations (e.g., light scattering, aggregation, secondary photophysical interactions) can cause errors. IFE is more prominent at shorter wavelengths, requiring careful wavelength matching between instruments. The Lakowicz model is only valid for absorbance values up to approximately 0.7.
1:Experimental Design and Method Selection:
The study uses mathematical modeling based on Parker and Lakowicz inner filter effect (IFE) correction models to derive equations for observed fluorescence intensity as a function of analyte absorbance and FOF dimensions. Empirical verification is conducted using standard fluorophores.
2:Sample Selection and Data Sources:
Analytes include Quinine Sulphate (QS), Humic Acid (HA), Tryptophan (Trp), 3-Hydroxyflavone (3HF), and a urine sample spiked with Doxorubicin (DOX). Samples are prepared in triple distilled water.
3:List of Experimental Equipment and Materials:
Spectrofluorometers (instruments A and B, models not specified), Shimadzu V-2500 A1 UV-Visible Spectrophotometer, cuvettes with 1 cm path length, and chemicals purchased from Aldrich.
4:Experimental Procedures and Operational Workflow:
Absorbance spectra are measured using the spectrophotometer. Fluorescence emission is measured at specific excitation and emission wavelengths for each analyte. FOF dimensions are optimized empirically by comparing observed fluorescence with derived absorbance (Dabs) data.
5:Data Analysis Methods:
Data are analyzed using MS-Excel for plotting and comparison. Dabs is calculated using derived equations, and correlation with experimental data is assessed.
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