Elsevier

Analytica Chimica Acta

Volume 630, Issue 1, 7 December 2008, Pages 47-56
Analytica Chimica Acta

Understanding the concept of concentration-dependent red-shift in synchronous fluorescence spectra: Prediction of λSFSmax and optimization of Δλ for synchronous fluorescence scan

https://doi.org/10.1016/j.aca.2008.09.056Get rights and content

Abstract

The phenomenon of concentration-dependent red-shift of fluorescence, observed in multifluorophoric systems at high concentrations, has been successfully used in the analytical fluorimetry of systems like petroleum derivatives, humic substances, biological fluids, etc. From a detailed investigation of the phenomenon, it is inferred that the primary cause contributing to this phenomenon is the inner-filter effect especially under right angle sample geometry condition. In this work, a method based on inner-filter effect has been proposed to obtain an optimal Δλ which give the most intense synchronous fluorescence spectral maximum for solutions containing fluorophores at higher concentrations. The applicability of the method has been evaluated for some single fluorophoric samples (coumarin-152, quinine sulphate, rhodamine-6G and fluorescein) at high concentrations. The proposed formula and methodology were applied to certain multifluorophoric systems like diesel, transformer oil and humic acid.

Introduction

Analytical fluorimetry with right angle sample geometry works well when the sample absorbance at the excitation wavelength is low (e.g. at low concentrations of the analyte) because of the linear dependence of fluorescence intensity with concentration. But when the absorbance is high (as for samples at high concentrations), the regular variation of intensity with concentration is lost mainly due to inner-filter effects [1], [2] and right angle geometry becomes unusable for analytical applications. Thus, there have been many attempts at correcting inner-filter effects [3], [4]. For highly concentrated samples, synchronous fluorescence spectroscopy (SFS) with right angle geometry was found to be highly advantageous and has been used successfully for many analytical applications [5], [6], [7], [8].

The phenomenon of concentration-dependent shift of synchronous fluorescence spectra has been profitably used to advantage the analysis of certain multifluorophoric systems. Analytical techniques have been developed using this context for the concentrated multifluorophoric samples. Petroleum products, one of the common multifluorophoric systems, have been widely investigated. John and Soutar used synchronous excitation spectrofluorimetry for the identification of crude oils and they observed that, the λmax of the normal and synchronous excitation spectra showed a marked shift to longer wavelengths as the concentration of the solutions increased [5]. At high concentrations, the excitation energy continue to cascade to larger fluorophores, producing greater red-shifts in the emission spectra due to extensive energy transfer [9], [10]. Ralston et al. studied the quantum yields of crude oils, where they have found that dilute solutions of light crude oils exhibit higher quantum yields than those of heavy crude oils [11]. Although they did not carry out any detailed investigation in to the cause of the phenomenon, they postulated that energy transfer processes becomes more probable at higher concentrations and emissions occurs predominantly from highly conjugated molecules.

The first report on red-shift cascade effect in three-dimensional fluorescence spectra was by Smith and Sinski when they investigated the concentration-dependent wavelength shifts in three-dimensional fluorescence spectra of petroleum samples [12]. As the solution strength is decreased, three-dimensional fluorescence maxima systematically shifted to shorter wavelengths. They describe the effect as ‘cascade’ effect, thereby implying excited state energy transfer to be the primary cause of the red-shift. Sinski et al. utilized the three-dimensional fluorescence red-shift cascade effect to monitor mycobacterium PRY-1 degradation of aged petroleum [13].

Patra and Mishra independently observed a similar behavior when they studied the synchronous fluorescence scan parameters of certain petroleum products [7], [8]. They documented that the excitation energy transfer results in shifting of synchronous fluorescence maxima with increasing concentration of the petroleum product. The correlation of this shift with concentration showed the possibility of using it as an analytical method to quantify the petroleum products in the environment. Kao et al. reported a comparison of fluorescence inner-filter effects for different cell configurations for anthracene solutions [14]. They observed that the right angle geometry exhibited the widest linear dynamic range and lowest detectable anthracene concentrations. The effect of sample geometry (front surface illumination, 45° and 90°) on synchronous fluorimetric analysis of petroleum products at a higher concentration was studied [7]. The 90°-angle sample geometry was found to give better analytical utility because it provided certain distinct characteristics to SFS spectra due to extensive inner-filter effect and resonance energy transfer. The observed higher sensitivity with right angle geometry immediately suggests that inner-filter effects play a significant role in the phenomenon of concentration-dependent red-shifts. Other energy degrading interactions like excited state energy transfer and quenching may also be contributing factors.

The concentration-dependent investigation of motor oils like diesel, petrol, kerosene, 2T oil and mobil showed a red-shift in λSFSmax[8], [15]. With dilution, the total synchronous fluorescence spectral (TSFS) contour maps measured at right-angle geometry of neat diesel samples shifted towards blue (shorter wavelengths) [16]. The total fluorescence spectra (TFS) of certain petroleum products like petrol and diesel produced a blue shift in the excitation and/or emission maximum on dilution with cyclohexane or kerosene [15], [17], [18], [19]. It has been concluded that the shift is a combined effect of various photo-physical processes such as resonance energy transfer, inner-filter effects, collisional fluorescence quenching, excimer or exciplex formation, etc.

The relative importance of inner-filter effect vis-à-vis other energy degrading mechanisms in causing concentration-dependent red-shift of concentrated multifluorophoric solutions have not been investigated in detail so far. An attempt to investigate this would necessarily involve understanding the inner-filter effects of selected single fluorophores at high concentrations where the effects of other energy degrading processes are negligible. In the first part of the work, fluorescence of single molecules (fluorophores) at high concentration has been studied. Subsequently, the developed method has been applied to certain multifluorophoric systems like diesel, transformer oil and humic acid.

Section snippets

Selection of fluorophores

For the initial studies, four fluorophores (quinine sulphate, rhodamine 6G, coumarin 152 and fluorescein) were selected which have different absorption and emission profiles. It is known that these molecules do not form excimers at high concentrations. Characteristics of the fluorescent molecules selected for the study are the following:

  • (a)

    Molecules showing a narrow absorption spectrum (e.g. rhodamine 6G) and a broad absorption spectrum (e.g. quinine sulphate and coumarin 152).

  • (b)

    Molecules with large

An expression for fluorescence intensity at the centre of the cuvette

In conventional right angle geometry, the fluorescence is usually observed from a volume at the centre of the cuvette. Although the fluorescence intensity is expected to vary along the light path in this observation volume, it can be assumed that for a cuvette of pathlength , the intensity of incident light at a pathlength of /2 (at the centre of the cuvette) will determine the fluorescence intensity. For a highly concentrated single fluorophoric sample, the longest wavelength edge of the

Acknowledgements

The authors thank the Council of Scientific and Industrial Research (CSIR) New Delhi for financial assistance. Divya thanks CSIR New Delhi for a fellowship.

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