Time-resolved fluorescence polarization anisotropy of multimodal samples: the asphaltene case

In this work we describe an application of the time-resolved fluorescence polarization anisotropy (TRFPA) technique to the analysis of asphaltenes, complex mixtures of high-molecular weight compounds, typically present in petroleum oils. Our asphaltene samples consist of nanometer-sized polydispersed particles, whose lighter fraction showed a relatively high fluorescence quantum yield. Most of the fluorescence intensity observed from the complex sample originated from a well defined sample fraction presenting a large fluorescence yield. Consequently, the TRFPA analysis only provided the average size of more fluorescing particles, that, in our case, were the smaller ones. Larger and less fluorescing aggregates did not significantly contribute to the TRFPA signal. Hence, to overcome intrinsic limitations of the TRFPA technique in characterizing multimodal samples, we preliminarily fractionated our complex samples by means of size exclusion chromatography (SEC), thus obtaining nearly monomodal fractions of the original samples. This procedure allowed to estimate also the size of less fluorescing and larger particles. A comparison of particle size estimate by means of TRFPA and SEC methods was also used to acquire information about occurrence of aggregation phenomena, and about the kind and strength of the chemical bonds linking chromophores to each other or to their parent particle.