Analysis and characterisation of fullerene nanoparticles

Abstract

Fullerenes or Buckyballs are a group of carbon nanoparticles, classified as engineered nanoparticles. The advent of fullerene nanoparticles in commercial, industrial and biomedical applications raises concern about their potential ecological and human health risks. Fullerenes have not been regulated, although the European Commission (European Commission, 2002), the European Parliament (European Parliament, 2008) and the US Environmental Protection Agency (EPA, 2015) have prioritised legislation on nanomaterials handling and disposal. Given the current lack of studies regarding their presence, fate and behaviour in consumer products and environmental matrices and their associated human and environmental risks it is becoming increasingly important to be able to characterise and quantitate fullerene nanoparticles, especially derivatives, in a wide range of matrix types.

To fill these knowledge gaps, one of the objectives of this thesis was the development of analytical methodologies for the determination of pristine and surface modified fullerenes by ultra-high performance liquid chromatography coupled to MS (UHPLC-MS) and by nonaqueous capillary electrophoresis (NACE) and micellar electrokinetic capillary chromatography (MECC) with UV-Vis detection in different environmental matrices (water and sediment samples) and cosmetic products, respectively. In addition, in this thesis the size and shape characterisation of surface modified fullerenes in aqueous solutions of different pH and ionic strength values was studied by combining several techniques (CE, AF4-MALS and TEM).

Regarding the analysis of fullerenes, the use of a sub-2 µm C18 column, toluene-methanol as a mobile phase and of APPI ionisation source allowed to develop a UHPLC method coupled to MS/(MS) for the analysis of five pristine (C60-C84) and three surface modified fullerenes (PCBM, PCBB and C60-pyrr) in less than 4.5 min showing high sensitivity and selectivity. Furthermore, the employment of H-SIM mode for pristine fullerenes (mass resolving power >12,500 FWHM), and SRM mode for fullerene derivatives, allowed achieving MLODs lower than most of those previously reported in the literature. For the extraction of the water samples, liquid-liquid extraction (LLE) with toluene and the addition of salt is proposed obtaining recoveries higher than 83 %. For sediments we propose the use of pressurised liquid extraction (PLE) performed at a high extraction temperature (150 °C) using one extraction cycle of 10 min achieving good recoveries (70-92 %). The developed methodology allowed us to report for the first time the presence of C60-pyrr, PCBM and PCBB in sediments (2.0 - 8.5 ng Kg-1 levels) and of PCBM and PCBB in pond water samples (0.1- 5.1 pg L-1 levels).

Two CE methods, a non-aqueous (NACE) and a micellar (MECC) method have been developed. LOQs at mg L-1 levels were obtained with both methods making possible their application for the analysis of cosmetic products. Both methods (NACE and MECC) were applied for the quantitation of C60 in cosmetic products and the results are comparable to those obtained by LC-MS (in an anti-aging serum).

With respect to the characterisation of fullerene aggregates, the use of AF4-MALS demonstrated that fullerene aqueous solutions contain particles of different aggregation degree in agreement with TEM micrographs. The size determination of the studied compounds by AF4 at different salt concentrations demonstrated that the enhanced aggregation of fullerenes with the increase in the ionic strength could explain the broad, multiple and distorted peaks obtained in MECC which were more obvious at high buffer concentration. The hydrodynamic radii of polyhydroxy-fullerenes increased more than 5 times and those of the carboxy-fullerene derivatives up to 180 nm (3rd peak) (for C60-pyrr tris acid) for 0.1M NaCl. The propensity of fullerenes to aggregate in both aqueous solutions and organic solvent mixtures justify the electrophoretic peak profiles observed in MECC (i.e., broad peaks at high electrolyte concentration) and their higher retention in C18 columns using toluene-acetonitrile compared to toluene-methanol mobile phases (due to the formation of bigger aggregates), respectively.

Los fulerenos son cada vez más utilizados en medicina (administración de fármacos), en procesos ambientales (remediación) y en la industria (células solares) debido a sus propiedades estructurales y electrónicas únicas. En este contexto, es importante aumentar el conocimiento actual con respecto a las características, el comportamiento, el destino y la toxicidad de los fulerenos, una nueva clase de contaminantes emergentes orgánicos. Por lo tanto, es cada vez es más importante ser capaz de caracterizar y cuantificar las nanopartículas de fulerenos en una amplia gama de tipos de matriz y para este fin, se necesita el desarrollo de métodos analíticos para su análisis cuantitativo y cualitativo. Además de la determinación de las concentraciones de fulerenos en matrices complejas, se requiere su caracterización en términos de grado de agregación, distribución del tamaño y morfología de la superficie en soluciones acuosas de diferentes características (fuerza iónica y pH) con el fin de proporcionar herramientas para establecer su riesgo.

En esta tesis se han establecido metodologías para el análisis de ocho fulerenos en muestras ambientales (agua y sedimentos) por cromatografía líquida acoplada a espectrometría de masas y se ha detectado por la primera vez la presencia de tres derivados de fulerenos. Además, se ha desarollado metodología para el análisis de fulerenos hidrofobicos y solubles en agua mediante la electroforesis capilar en medio no-acuoso (NACE) y la cromatografía capilar electrocinética micelar (MECC) y los metodos se han aplicado para la determinación de C60 en productos cosmeticos.

Por último, se ha estudiado el comportamiento de agregación de los fulerenos en soluciones acuosas de diferentes características (pHs, fuerzas iónicas) mediante diferentes técnicas (electroforesis capilar (CE), asymetrical flow-field flow fractionation (AF4), microscopia electrónica de transmisión (TEM)). En este contexto se han determinado los tamaños de los agregados formados en las condiciones evaluadas, y la morfología de las partículas.