Fluorescence of highly absorbing samples in transmission geometry with a commercial spectrofluorometer

NICOLÁS I. KRIMER; DARÍO RODRIGUES; HERNÁN B. RODRÍGUEZ; MARTÍN MIRENDA

Villa Carlos Paz

Encuentro; XIII Encuentro Latinoamericano de Fotoquímica y Fotobiología (XIII ELAFOT); 2017

Grupo Argentino de Fotobiología

The experimental adquisition of emission spectra and quantum yields under highly absorbing conditions generally represents a difficult task due to the presence of internal filter effects. In this sense, we have recently developed a simplified methodology to obtain: a) steady-state emission spectra and b) fluorescence quantum yields (FiF) of samples with high optical density at both excitation and emission wavelengths.[1] The experimental configuration consists of a commercial spectrofluorometer adapted to a transmission geometry, where the detection of emitted light is performed at 180° with respect to the excitation beam. The main goal of this approach is the use of short path-lengths ensuring small excitation and detection volumes, in order to guarantee overlaped excitation and detection regions for all optical densities. The procedure includes two different mathematical approaches to describe and reproduce distortions caused by reabsorption in both emission spectra and quantum yields.In this work we will discuss three different application cases for which our metodology is usefull: a) 9,10-diphenylanthracene (DPA) in toluene, b) quinine bisolphate (QBS) in sulphuric acid and c) the ionic liquid p-toluensulfonate of 1-butil-3-metilimidazolium (BMIMTOS). a) DPA in toluene was studied in a concentration range from 1 x 10-5 M to 1 x 10-2 M. This dye was selected to validate our methodology since it presents significant resorption and re-emission in concentrated solutions without self-quenching or aggregation phenomena. From the fluorescence intensity at 460 nm, where reabsorption is negligible, we obtained FiF ≈ 1 for the entire concentration range. On the other hand, FiF values, calculated from the integrated emission spectra, decrease as the concentration increases. We will show how this effect, caused by reabsorption, can be quantitatively described by the two mathematical approaches.[1]b) QBS in sulphuric acid was estudied in a concentration range from 1 x 10-5 M to 1 x 10-2 M.[1] This dye was selected to demonstrate that our methodology also constitutes a very simple and robust alternative to determine self-quenching constants, KSQ. QBS shows no aggregation and a large stokes-shift that prevents, in principle, reabsorption and re-emission events in concentrated solutions. However, the re-absorption of 6% observed in our experiments confirms that it is necessary to perform appropriated corrections on emission spectra for the accurate data processing. The value obtained after corrections, KSQ = 18.4 +/- 0.1 M−1, shows no significant differences with those obtained from steady-state and average lifetimes by another authors, pointing out the diffusional nature of the self-quenching phenomenon.[2]c) BMIMTOS constitutes a novel kind of fluorescent ionic room-temperature fluid medium showing a high density of fluorophores. For this reason, our metodology constitutes a valuable tool to evaluate the photophysics of this compound. We observed that the fluorescence of BMIMTOS enhances about 300% when temperature falls from 55 to 0 ºC. The increment is associated to excimer formation and strongly differs to those observed for toluene in the same temperature range. [1] N. I. Krimer, D. Rodrigues, H. B. Rodriguez, M. Mirenda, Anal. Chem. 2017, 89, 640.[2] N. I. Krimer, M. Mirenda, Methods Appl. Fluores. In press.