Arsenic speciation in edible alga samples by microwave-assisted extraction and high performance liquid chromatography coupled to atomic fluorescence spectrometry

Twelve commercially available edible marine algae from France, Japan and Spain and the certified reference material (CRM) NIES No. 9 Sargassum fulvellum were analyzed for total arsenic and arsenic species. Total arsenic concentrations were determined by inductively coupled plasma atomic emission spectrometry (ICP-AES) after microwave digestion and ranged from 23 to 126 μg g−1. Arsenic species in alga samples were extracted with deionized water by microwave-assisted extraction and showed extraction efficiencies from 49 to 98%, in terms of total arsenic. The presence of eleven arsenic species was studied by high performance liquid chromatography–ultraviolet photo-oxidation–hydride generation atomic–fluorescence spectrometry (HPLC–(UV)–HG–AFS) developed methods, using both anion and cation exchange chromatography. Glycerol and phosphate sugars were found in all alga samples analyzed, at concentrations between 0.11 and 22 μg g−1, whereas sulfonate and sulfate sugars were only detected in three of them (0.6-7.2 μg g−1). Regarding arsenic toxic species, low concentration levels of dimethylarsinic acid (DMA) (<0.9 μg g−1) and generally high arsenate (As(V)) concentrations (up to 77 μg g−1) were found in most of the algae studied. The results obtained are of interest to highlight the need to perform speciation analysis and to introduce appropriate legislation to limit toxic arsenic species content in these food products.

Experimental studies on transitional separated boundary layers

Separated transitional boundary layers appear on key aeronautical processes such as the flow around wings or turbomachinery blades. The aim of this thesis is the study of these flows in representative scenarios of technological applications, gaining knowledge about phenomenology and physical processes that occur there and, developing a simple model for scaling them. To achieve this goal, experimental measurements have been carried out in a low speed facility, ensuring the flow homogeneity and a low disturbances level such that unwanted transitional mechanisms are avoided. The studied boundary layers have been developed on a flat plate, by imposing a pressure gradient by means of contoured walls. They generate an initial acceleration region followed by a deceleration zone. The initial region is designed to obtain at the beginning of the deceleration the Blasius profile, characterized by its momentum thickness, and an edge boundary layer velocity, defining the problem characteristic velocity. The deceleration region is designed to obtain a linear evolution of the edge velocity, thereby defining the characteristic length of the problem. Several experimental techniques, both intrusive (hot wire anemometry, total pressure probes) as nonintrusive (PIV and LDV anemometry, high-speed filming), have been used in order to take advantage of each of them and allow cross-validation of the results. Once the boundary layer at the deceleration beginning has been characterized, ensuring the desired integral parameters and level of disturbance, the evolution of the laminar boundary layer up to the point of separation is studied. It has been compared with integral methods, and numerical simulations. In view of the results a new model for this evolution is proposed. Downstream from the separation, the flow near to the wall is configured as a shear layer that encloses low momentum recirculating fluid. The region where the shear layer remains laminar tends to be positioned to compensate the adverse pressure gradient associated with the imposed deceleration. Under these conditions, the momentum thickness remains almost constant. This laminar shear layer region extends up to where transitional phenomena appear, extension that scales with the momentum thickness at separation. These transitional phenomena are of inviscid type, similar to those found in free shear layers. The transitional region analysis begins with a study of the disturbances evolution in the linear growth region and the comparison of experimental results with a numerical model based on Linear Stability Theory for parallel flows and with data from other authors. The results’ coalescence for both the disturbances growth and the excited frequencies is stated. For the transition final stages the vorticity concentration into vortex blobs is found, analogously to what happens in free shear layers. Unlike these, the presence of the wall and the pressure gradient make the large scale structures to move towards the wall and quickly disappear under certain circumstances. In these cases, the recirculating flow is confined into a closed region saying the bubble is closed or the boundary layer reattaches. From the reattachment point, the fluid shows a configuration in the vicinity of the wall traditionally considered as turbulent. It has been observed that existing integral methods for turbulent boundary layers do not fit well to the experimental results, due to these methods being valid only for fully developed turbulent flow. Nevertheless, it has been found that downstream from the reattachment point the velocity profiles are self-similar, and a model has been proposed for the evolution of the integral parameters of the boundary layer in this region. Finally, the phenomenon known as bubble burst is analyzed. It has been checked the validity of existing models in literature and a new one is proposed. This phenomenon is blamed to the inability of the large scale structures formed after the transition to overcome with the adverse pressure gradient, move towards the wall and close the bubble. El estudio de capas límites transicionales con separación es de gran relevancia en distintas aplicaciones tecnológicas. Particularmente, en tecnología aeronáutica, aparecen en procesos claves, tales como el flujo alrededor de alas o álabes de turbomaquinaria. El objetivo de esta tesis es el estudio de estos flujos en situaciones representativas de las aplicaciones tecnológicas, ganando por un lado conocimiento sobre la fenomenología y los procesos físicos que aparecen y, por otra parte, desarrollando un modelo sencillo para el escalado de los mismos. Para conseguir este objetivo se han realizado ensayos en una instalación experimental de baja velocidad específicamente diseñada para asegurar un flujo homogéneo y con bajo nivel de perturbaciones, de modo que se evita el disparo de mecanismos transicionales no deseados. La capa límite bajo estudio se ha desarrollado sobre una placa plana, imponiendo un gradiente de presión a la misma por medio de paredes de geometría especificada. éstas generan una región inicial de aceleración seguida de una zona de deceleración. La región inicial se diseña para tener en al inicio de la deceleración un perfil de capa límite de Blasius, caracterizado por su espesor de cantidad de movimiento, y una cierta velocidad externa a la capa límite que se considera la velocidad característica del problema. La región de deceleración está concebida para que la variación de la velocidad externa a la capa límite sea lineal, definiendo de esta forma una longitud característica del problema. Los ensayos se han realizado explotando varias técnicas experimentales, tanto intrusivas (anemometría de hilo caliente, sondas de presión total) como no intrusivas (anemometrías láser y PIV, filmación de alta velocidad), de cara a aprovechar las ventajas de cada una de ellas y permitir validación cruzada de resultados entre las mismas. Caracterizada la capa límite al comienzo de la deceleración, y garantizados los parámetros integrales y niveles de perturbación deseados se procede al estudio de la zona de deceleración. Se presenta en la tesis un análisis de la evolución de la capa límite laminar desde el inicio de la misma hasta el punto de separación, comparando con métodos integrales, simulaciones numéricas, y proponiendo un nuevo modelo para esta evolución. Aguas abajo de la separación, el flujo en las proximidades de la pared se configura como una capa de cortadura que encierra una región de fluido recirculatorio de baja cantidad de movimiento. Se ha caracterizado la región en que dicha capa de cortadura permanece laminar, encontrando que se posiciona de modo que compensa el gradiente adverso de presión asociado a la deceleración de la corriente. En estas condiciones, el espesor de cantidad de movimiento permanece prácticamente constante y esta capa de cortadura laminar se extiende hasta que los fenómenos transicionales aparecen. Estos fenómenos son de tipo no viscoso, similares a los que aparecen en una capa de cortadura libre. El análisis de la región transicional comienza con un estudio de la evolución de las vii viii RESUMEN perturbaciones en la zona de crecimiento lineal de las mismas y la comparación de los resultados experimentales con un modelo numérico y con datos de otros autores. La coalescencia de los resultados tanto para el crecimiento de las perturbaciones como para las frecuencias excitadas queda demostrada. Para los estadios finales de la transición se observa la concentración de la vorticidad en torbellinos, de modo análogo a lo que ocurre en capas de cortadura libres. A diferencia de estas, la presencia de la pared y del gradiente de presión hace que, bajo ciertas condiciones, la gran escala se desplace hacia la pared y desaparezca rápidamente. En este caso el flujo recirculatorio queda confinado en una región cerrada y se habla de cierre de la burbuja o readherencia de la capa límite. A partir del punto de readherencia se tiene una configuración fluida en las proximidades de la pared que tradicionalmente se ha considerado turbulenta. Se ha observado que los métodos integrales existentes para capas límites turbulentas no ajustan bien a las medidas experimentales realizadas, hecho imputable a que no se obtiene en dicha región un flujo turbulento plenamente desarrollado. Se ha encontrado, sin embargo, que pasado el punto de readherencia los perfiles de velocidad próximos a la pared son autosemejantes entre sí y se ha propuesto un modelo para la evolución de los parámetros integrales de la capa límite en esta región. Finalmente, el fenómeno conocido como “estallido” de la burbuja se ha analizado. Se ha comprobado la validez de los modelos existentes en la literatura y se propone uno nuevo. Este fenómeno se achaca a la incapacidad de la gran estructura formada tras la transición para vencer el gradiente adverso de presión, desplazarse hacia la pared y cerrar la burbuja.

Estudios de especiación de arsénico y acumulación de metales en muestras de interés medioambiental : Arsenic speciation and metal accumulation studies in environmental samples

Se ha estudiado la determinación de especies de arsénico y de contenidos totales de arsénico y metales pesados, específicamente cadmio, cromo, cobre, níquel, plomo y cinc, en muestras de interés medioambiental por su elevada capacidad acumuladora de metales, concretamente algas marinas comestibles y plantas terrestres procedentes de suelos contaminados por la actividad minera. La determinación de contenidos totales se ha llevado a cabo mediante espectrometría de emisión atómica con plasma de acoplamiento inductivo (ICP‐AES), así como por espectrometría de fluorescencia atómica con generación de hidruros (HG‐AFS), para bajos contenidos de arsénico. Las muestras fueron mineralizadas en medio ácido y calentamiento en horno de microondas. Los métodos fueron validados a través de su aplicación a materiales de referencia de matriz similar a la de las muestras, certificados en contenidos totales de los elementos seleccionados. Los resultados obtenidos mostraron su elevada capacidad de bioabsorción, especialmente en relación a los elevados contenidos de arsénico encontrados en algunas especies de algas pardas (Phaeophytas). En las plantas, se calcularon los factores de translocación, acumulación y biodisponibilidad de los elementos estudiados, permitiendo identificar a la especie Corrigiola telephiifolia como posible acumuladora de plomo e hiperacumuladora de arsénico. La determinación de especies de arsénico hidrosolubles en las muestras objeto de estudio, se llevó a cabo por cromatografía líquida de alta eficacia (HPLC) acoplado a ICP‐AES, HG‐ICP‐AES y HG‐AFS, incluyendo una etapa previa de foto‐oxidación. Los métodos desarrollados, mediante intercambio aniónico y catiónico, permitieron la diferenciación de hasta once especies de arsénico. Para el análisis de las muestras, fue necesaria la optimización de métodos de extracción, seleccionándose la extracción asistida por microondas (MAE) con agua desionizada. Asimismo, se realizaron estudios de estabilidad de arsénico total y de las especies hidrosolubles presentes en las algas, tanto sobre la muestra sólida como en sus extractos acuosos, evaluando las condiciones de almacenamiento adecuadas. En el caso de las plantas, la aplicación del diseño factorial de experimentos permitió optimizar el método de extracción y diferenciar entre las especies de arsénico presentes en forma de iones sencillos de mayor movilidad y el arsénico más fuertemente enlazado a componentes estructurales. Los resultados obtenidos permitieron identificar la presencia de arseniato (As(V)) y arsenito (As(III)) en las plantas, así como de ácido monometilarsónico (MMA) y óxido de trimetilarsina (TMAO) en algunas especies. En la mayoría de las algas se encontraron especies tóxicas, tanto mayoritarias (arseniato) como minoritarias (ácido dimetilarsínico (DMA)), así como hasta cuatro arsenoazúcares. Los resultados obtenidos y su estudio a través de la legislación vigente, mostraron la necesidad de desarrollar una reglamentación específica para el control de este tipo de alimentos. La determinación de especies de arsénico liposolubles en las muestras de algas se llevó a cabo mediante HPLC, en modo fase inversa, acoplado a espectrometría de masas con plasma de acoplamiento inductivo (ICP‐MS) y con ionización por electrospray (ESI‐MS), permitiendo la elucidación estructural de estos compuestos a través de la determinación de sus masas moleculares. Para ello, fue necesaria la puesta a punto de métodos extracción y purificación de los extractos. La metodología desarrollada permitió identificar hasta catorce especies de arsénico liposolubles en las algas, tres de ellas correspondientes a hidrocarburos que contienen arsénico, y once a arsenofosfolípidos, además de dos especies desconocidas. Las masas moleculares de las especies identificadas fueron confirmadas mediante cromatografía de gases acoplada a espectrometría de masas (GC‐MS) y espectrometría de masas de alta resolución (HR‐MS). ABSTRACT The determination of arsenic species and total arsenic and heavy metal contents (cadmium, chromium, cooper, nickel, lead and zinc) in environmental samples, with high metal accumulator capacity, has been studied. The samples studied were edible marine algae and terrestrial plants from soils polluted by mining activities. The determination of total element contents was performed by inductively coupled plasma atomic emission spectrometry (ICP‐AES), as well as by hydride generation atomic fluorescence spectrometry (HG‐AFS) for low arsenic contents. The samples studied were digested in an acidic medium by heating in a microwave oven. The digestion methods were validated against reference materials, with matrix similar to sample matrix and certified in total contents of the elements studied. The results showed the high biosorption capacity of the samples studied, especially regarding the high arsenic contents in some species of brown algae (Phaeophyta division). In terrestrial plants, the translocation, accumulation and bioavailability factors of the elements studied were calculated. Thus, the plant species Corrigiola telephiifolia was identified as possible lead accumulator and arsenic hyperaccumulator. The determination of water‐soluble arsenic species in the samples studied was carried out by high performance liquid chromatography (HPLC) coupled to ICP‐AES, HG‐ICP‐AES and HG‐AFS, including a prior photo‐oxidation step. The chromatographic methods developed, by anion and cation exchange, allowed us to differentiate up to eleven arsenic species. The sample analysis required the optimization of extraction methods, choosing the microwave assisted extraction (MAE) with deionized water. On the other hand, the stability of total arsenic and water‐soluble arsenic species in algae, both in the solid samples and in the water extracts, was studied, assessing the suitable storage conditions. In the case of plant samples, the application of a multivariate experimental design allowed us to optimize the extraction method and differentiate between the arsenic species present as simple ions of higher mobility and the arsenic more strongly bound to structural components. The presence of arsenite (As(III)) and arsenate (As(V)) was identified in plant samples, as well as monomethylarsonic acid (MMA) and trimethylarsine oxide (TMAO) in some cases. Regarding algae, toxic arsenic species were found in most of them, both As(V) and dimethylarsinic acid (DMA), as well as up to four arsenosugars. These results were discussed according to the current legislation, showing the need to develop specific regulations to control this kind of food products. The determination of lipid‐soluble arsenic species in alga samples was performed by reversed‐phase HPLC coupled to inductively coupled plasma and electrospray mass spectrometry (ICP‐MS and ESI‐MS), in order to establish the structure of these compounds by determining the corresponding molecular masses. For this purpose, it was necessary to develop an extraction method, as well as a clean‐up method of the extracts. The method developed permitted the identification of fourteen lipid‐soluble arsenic compounds in algae, corresponding to three arsenic‐hydrocarbons and eleven arsenosugarphospholipids, as well as two unknown compounds. Accurate mass measurements of the identified compounds were performed by gas chromatography coupled to mass spectrometry (GC‐MS) and high resolution mass spectrometry (HR‐MS).

Chemical speciation and mobilization of copper and zinc in naturally contaminated mine soils with citric and tartaric acids

A one-step extraction procedure and a leaching column experiment were performed to assess the effects of citric and tartaric acids on Cu and Zn mobilization in naturally contaminated mine soils to facilitate assisted phytoextraction. A speciation modeling of the soil solution and the metal fractionation of soils were performed to elucidate the chemical processes that affected metal desorption by organic acids. Different extracting solutions were prepared, all of which contained 0.01 M KNO3 and different concentrations of organic acids: control without organic acids, 0.5 mM citric, 0.5 mM tartaric, 10 mM citric, 10 mM tartaric, and 5 mM citric +5 mM tartaric. The results of the extraction procedure showed that higher concentrations of organic acids increased metal desorption, and citric acid was more effective at facilitating metal desorption than tartaric acid. Metal desorption was mainly influenced by the decreasing pH and the dissolution of Fe and Mn oxides, not by the formation of soluble metal–organic complexes as was predicted by the speciation modeling. The results of the column study reported that low concentrations of organic acids did not significantly increase metal mobilization and that higher doses were also not able to mobilize Zn. However, 5–10 mM citric acid significantly promoted Cu mobilization (from 1 mg kg−1 in the control to 42 mg kg−1 with 10 mM citric acid) and reduced the exchangeable (from 21 to 3 mg kg−1) and the Fe and Mn oxides (from 443 to 277 mg kg−1) fractions. Citric acid could efficiently facilitate assisted phytoextraction techniques.

A three-dimensional self-adaptive hp finite element method for the characterization of waveguide discontinuities

We propose the use of a highly-accurate three-dimensional (3D) fully automatic hp-adaptive finite element method (FEM) for the characterization of rectangular waveguide discontinuities. These discontinuities are either the unavoidable result of mechanical/electrical transitions or deliberately introduced in order to perform certain electrical functions in modern communication systems. The proposed numerical method combines the geometrical flexibility of finite elements with an accuracy that is often superior to that provided by semi-analytical methods. It supports anisotropic refinements on irregular meshes with hanging nodes, and isoparametric elements. It makes use of hexahedral elements compatible with high-order H(curl)H(curl) discretizations. The 3D hp-adaptive FEM is applied for the first time to solve a wide range of 3D waveguide discontinuity problems of microwave communication systems in which exponential convergence of the error is observed.