**Analysis of Olive Oils by Fluorescence Spectroscopy: Methods and Applications**

Ewa Sikorska1, Igor Khmelinskii2 and Marek Sikorski3

*1Faculty of Commodity Science, Poznań University of Economics, 2Universidade do Algarve, FCT, DQF and CIQA, Faro, 3Faculty of Chemistry, A. Mickiewicz University, Poznań, 1,3Poland 2Portugal* 

## **1. Introduction**

62 Olive Oil – Constituents, Quality, Health Properties and Bioconversions

Sinelli, N., Cerretani, L., Di Egidio, V., Bendini, A. & Casiraghi, E. (2010). Application of near

Torrecilla, J.S., Rojo, E., Domínguez, J.C. & Rodríguez, F. (2010). A novel method to quantify

Tena, N., García-González, D.L. & Aparicio, R. (2009). Evaluation of virgin oil thermal

Vandeginste, B.G.M., Massart, D.L., Buydens, L.C.M., De Jong, S., Lewi, D.J. & Smeyers-

Wold, S., Sjöström, M. & Erikkson, L. (2001). PLS-regression: a basic tool for chemometrics.

Wrolstad, R.E., Acree, T.E., Decker, E.A., Penner, M.H., Reid, D.S., Schwartz, S.J.,

Zou, M.Q., Zhang, X.F., Qi, X.H., Ma, H.L., Dong, Y., Liu, C.W., Guo, X. & Wang, H. (2009).

*Chemometrics and Intelligent Laboratory Systems,* Vol. 58, pp. 109-130.

*Agricultural and Food Chemistry*, Vol. 57, pp. 6001-6006.

*Journal of Agricultural and Food Chemistry*, Vol. 58, pp. 1679-1684.

Vol. 42, pp. 369-375.

Amsterdam–chapter 33.

Hoboken.

*Chemistry*, Vol. 57, pp. 10505-10511.

(NIR) infrared and mid (MIR) infrared spectroscopy as a rapid tool to classify extra virgin olive oil on the basis of fruity attribute intensity. *Food Research International*,

the adulteration of extra virgin olive oil with low-grade olive oils by UV-Vis.

deterioration by fluorescence spectroscopy. *Journal of the Agricultural and Food* 

Verbeke, J. (1998). *Handbook of Chemometrics and Qualimetrics,* Elsevier Science BV,

Shoemaker, C.F. & Sporns, P. (2005). *Handbook of Food Analytical Chemistry, Pigments, Colorants, Flavours, Texture, and Bioactive Food Components,* J. Wiley & Sons,

Rapid authentication of olive oil adulteration by Raman spectrometry. *Journal of the* 

Fluorescence spectroscopy is a well established and extensively used research and analytical tool in many disciplines. In recent years, a remarkable growth in the use of fluorescence in food analysis has been observed (Christensen et al., 2006; Sadecka & Tothova, 2007; Karoui & Blecker, 2011). Vegetable oils including olive oil constitute an important group of food products for which fluorescence was successfully applied. Fluorescence is a type of photoluminescence, a process in which a molecule, promoted to an electronically excited state by absorption of UV, VIS or NIR radiation, decays back to its ground state by emission of a photon. Fluorescence is emission from an excited state, in which the electronic spin is equal to that in the ground state, and typically equal to zero. Such transitions are spin allowed, and occur at relatively high rates, typically 108 s-1 (Lakowicz, 2006).

A unique feature of fluorescence, distinguishing it from other spectroscopic techniques, is its inherently multidimensional character (Christensen et al., 2006). Excitation of molecules results from absorption of radiation at the energy corresponding to the energy difference between the ground and excited states of a given fluorophore. Subsequently, radiation at a lower energy characteristic for the specific molecule is emitted during its deactivation. Thus, fluorescence properties of every compound are characterized by two types of spectra: excitation and emission. This feature and the fact that not all of the absorbing molecules are fluorescent both contribute to higher selectivity of fluorescence as opposed to absorption spectra.

Another important advantage of fluorescence is its higher sensitivity. In contrast to absorption measurements, the emitted photons are detected against a low background, making fluorescence spectroscopy a very sensitive method. The sensitivity of fluorescence is 100-1000 times higher than that of the absorption techniques, enabling to measure concentrations down to parts per billion levels (Guilbault, 1999).

The fluorescent analysis of olive oils takes advantage of the presence of natural fluorescent components, including phenolic compounds, tocopherols and pheophytins, and their oxidation products. Oils are complex systems and therefore conventional fluorescent

Analysis of Olive Oils by Fluorescence Spectroscopy: Methods and Applications 65

scanning of both excitation and emission wavelengths, keeping a constant difference between them. Synchronous scanning fluorescence spectroscopy is very useful for the analysis of mixtures of fluorescent compounds, because both excitation and emission characteristics are included into a single spectrum. Although it provides less information than the excitation-emission matrix, it may still present a viable alternative to the total luminescence measurements due to its inherent simplicity and rapidity. A set of synchronous spectra recorded at different wavelength intervals may be concatenated into a total synchronous fluorescence spectrum. In such spectra fluorescence intensity is plotted as a function of the excitation wavelength and the wavelength interval. Both single wavelength interval and total synchronous fluorescence spectra were used for studies of olive oils (Sikorska et al. 2005a; Poulli et al. 2005). The relation between various kinds of fluorescence

300 400 500 600 700

em 300 nm 330 nm 670 nm

Excitation spectra

ex [nm]

 10 nm 30 nm 60 nm

Synchronous spectra

spectra of a virgin olive oil is presented in Fig. 1.

 ex 280 nm 300 nm 400 nm

Synchronous spectrum

Emission spectra

300 400 500 600 700 800

extra virgin olive oil (1%, v/v, in n-hexane) are shown as an example.

em [nm]

Emission spectrum

Excitation spectrum

Fig. 1. Different types of fluorescence spectra; fluorescence spectra of a diluted sample of

Numerous factors affect measured fluorescence intensity and spectral distribution. These factors are related to the nature and the concentration of fluorophores, their molecular environment, and scattering and absorption effects. They may be immeasurably important in complex natural systems, such as oils, and have to be taken into account when measuring and interpreting the fluorescence spectra. Fluorescence intensities are proportional to the concentration over only a limited range of optical densities (Lakowicz, 2006). To obtain proportionality between the fluorescence intensity and the fluorophore concentration, the absorbance at the excitation wavelength should be below 0.05 and close to zero in the emission spectral region. At higher concentrations, the inner filter effects have to be taken into account. These effects may decrease the observed fluorescence intensity by either

300

400

ex [nm]

500

techniques, relying on recording of single emission or excitation spectra, are often insufficient if directly applied. In such cases, total luminescence or synchronous scanning fluorescence techniques are used, improving the analytic potential of the fluorescence measurements. With contributions from numerous analytes, the autofluorescence of olive oil exhibits numerous overlapping bands. Such complex spectra should be analyzed using multivariate and multiway methods.

Analytical applications of fluorescence to olive oils include discrimination between the different quality grades, adulteration detection, authentication of virgin oils, quantification of fluorescent components, monitoring thermal and photo-oxidation and quality changes during storage.

In this chapter the application of fluorescence spectroscopy to qualitative and quantitative analysis of olive oils is reviewed. Methodological aspects of fluorescence measurements and analysis of fluorescence spectra are also discussed.
