Laser Fluorescence Spectroscopy as a Method for Studying Humic Substance

Abstract

This paper summarizes the results obtained by fluorescence spectroscopic techniques with conventional and laser light sources for the characterization of the spectral luminescent properties of aquatic humic substances (AHS). The band profiles of corrected absorption, fluorescence emission, and fluorescence excitation spectra are compared in graphical form with unique functions used for the axes. In order to calculate the quantum efficiency of fluorescence, we used the Raman signal from water molecules as an internal reference. The temperature dependence of the fluorescence spectra of humic matter has been recorded in solid (−160°C to 0°C) and in liquid (0°C to +300°C) aqueous solutions. The behavior of the fluorescence band shape is discussed. A new method of nonlinear fluorimetry is proposed to enhance the capabilities of fluorescence spectroscopy. This method is based on the ORDER REPRINTS 88FILIPPOVA ET AL.fluorescence saturation effect manifesting itself under powerful laser pumping (excitation) conditions. The use of the technique allows one to determine certain photo-physical properties of molecules, as a complement to the normal spectral data. Using three different samples of aquatic humic substances with very similar fluorescence band shapes, it is demonstrated that these compounds have distinct molecular luminescent parameters and therefore, can be identified. The similarity of the fluorescence band shapes of humic substances in natural water of different types; the absence of any noticeable effect of temperature across a wide range and the fluorescence saturation on the fluorescence band contour can be explained. This assumes that in all of the samples of aquatic humic substances studied, with different molecular weight fractions, that there is a single dominant fluorescent functionality present.

Laser Fluorescence Spectroscopy: Application in Determining the Individual Photophysical Parameters of Proteins

“…The fluorescence signal from fluorophores of complex organic compound (COC) under powerful laser excitation is represented as the nonlinear function of the number of detected fluorescence photons N Fl (or fluorescence intensity I Fl ) on the photon fluxes F of pumping radiation (Filipova et al, 2001). The dependence N Fl (F) is called fluorescence saturation curve, its typical view is represented in the Fig.…”

“…where D , A and D , A are the lifetime and absorption cross section of the donor (denoted by D) and acceptor (denoted by A) as it defined above; K DA is the rate of the energy transfer from the excited donor to the unexcited acceptor; K SS is the rate of energy transfer from the excited donor to the excited acceptor (singlet-singlet annihilation (Fadeev et al, 1999) By solving systems (1a) and (1b) numerically, one can find the concentration of the fluorescent molecules in the excited state and calculate the number of fluorescence photons N Fl , emitted from the volume V after the action of the laser pulse (Filipova et al, 2001;Fadeev et al, 1999). The theoretical saturation curve for the model (1a) can be calculated from following equation:…”

“…For the same reason the induced processes from the excited states (two-photon absorption or photoizomerization, etc) have been excluded. , which can represent a part of exciting radiation directed to the reference channel of the detection system by a beamsplitter or a Raman scattering signal from water molecules (Fadeev et al, 1999 as a reference signal is used, then it is possible to find the fluorescence quantum yield of a complex organic compound (Filipova et al, 2001). …”

“…(2)), the photophysical parameters derived from the saturation curve relate only to a fluorescent molecule of COC; (ii) information on the concentration of fluorescent molecules is not used in deriving the photophysical parameters from the saturation curve (Banishev et al, 2009;Filipova et al, 2001). Thus, the method allows one to determine individual photophysical parameters of a molecule in the case when the following complex situation takes place: (i) the sample under study is a multicomponent ensemble of molecules, the absorption bands of its subensembles overlapping (i.e.…”

“…Indeed, given above-mentioned assumptions, one can write the following system of equations: RS (532) are the optical density of the protein solution and Raman scattering cross section of water (Filipova et al, 2001) at 570 and 532 nm, respectively.…”