Relationship between the Concentration of a Fluorophore and its Fluorescence Intensity: Determination of Unknown Concentration of an Analyte
Detection and quantification are two common uses of fluorimetry. Fluorimetry is extremely useful for the determination of concentrations that are too small to be easily determined by spectrophotometry. Because of the low detection limits, fluorescence spectroscopy is routinely used for the quantification of aromatic, or highly unsaturated organic molecules present at trace concentrations, especially in biological and environmental samples. The technique can be extended to a wide variety of non-fluorescent organic and inorganic compounds via derivatization procedure or tagging the analyte with a fluorescent compound. Fluorescence spectrometry has high sensitivity. Sensitivity depends on the fluorescence quantum yield of analyte/fluorophore. For intensely fluorescent compounds, detection limits of 10-10 to 10-12 M can be achieved by using commercial spectroflurometers. Sensitivity can be improved by minimizing the blank signals (such as impurity fluorescence and Rayleigh and Raman scatter) and enhancing the intensity of the exciting radiation, the sensitivity of the detector, etc. It should be noted that a few problems like self-quenching, excimer formation, etc. should be avoided in fluorimetry. Now-a-days, the ultimate limit of detection i.e., detection of a single-molecule has been achieved by using some special fluorescence measurement techniques.