Optimización de los parámetros experimentales de la técnica de cromatografía líquida de alta eficiencia para la determinación de tartrazina, amarillo crepúsculo y rojo alura en muestras de alimentos
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Fecha
2013
Autores
Profesor/a Guía
Facultad/escuela
Idioma
es
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Editor
Universidad Andrés Bello
Nombre de Curso
Licencia CC
Licencia CC
Resumen
En este trabajo se optimizó los parámetros experimentales de la técnica de
cromatografía de alta resolución (HPLC) para la determinación de colorantes sintéticos
en muestras de alimentos (bebidas de fantasía, cereal, sopas, etc) utilizando una
columna con fase estacionaria C-18e y un detector espectrofotométrico UV-VIS-DAD
(arreglo de diodos). Se utilizó elución isocrática para la determinación de los
colorantes. Los colorantes sintéticos analizados fueron: tartrazina (E102), amarillo
crepúsculo (E110), carmoisina (E122), amaranto (E123), rojo ponceau (E124) y rojo
alura (E129).
Debido a la naturaleza hidrofílica de estos colorantes azoicos, su retención en la fase
estacionaria C-18 es baja eluyendo en tiempos relativamente cortos y obteniendo una
baja resolución. Por este motivo en esta unidad de investigación se propuso estudiar el
efecto de surfactantes (cromatografía micelar) y sales formadoras de pares iónicos
(cromatografía de pares iónicos) con el objetivo de lograr mayor interacción de los
analitos con la fase estacionaria. Para esto se utilizó un surfactante neutro (tritón X-
100), un surfactante catiónico (bromuro de acetipiridinio, CPB) y tetrafluorborato de
tetrabutilamonio (TFBTBA) el cual solvata a los colorantes.
Sin embargo, al agregar TFBTBA o tritón en la fase móvil, se obtuvo los resultados
esperados, pero luego de unas horas de realización de medidas, en las cuales se
mantenía constante la concentración de TFBTBA o tritón, las señales comenzaban a
tener áreas muy pequeñas y se desplazaban a tiempos muy largos. Luego de lavar la
columna durante varias horas con agua se restablecían las condiciones anteriores.
Esto mismo ocurrió con columnas que contenían fase estacionaria C-8, C-18 y C-18
encapada o encapsulada obteniendo similares resultados. Debido a que es muy
engorroso interrumpir las medidas para proceder a lavar o cambiar la columna varias
veces al día, se eliminaron estos compuestos de la fase móvil y se disminuyó el
número de colorantes a analizar, eligiendo para ello tartrazina, amarillo crepúsculo y
rojo alura que son los que con mayor frecuencia se encuentran en las muestras reales
de alimentos.
Se construyó curvas de calibrado y se determinó los límites de detección de la técnica.
Posteriormente se realizó la validación utilizando para ello agua mineral sin gas
dopada con 4,0 mg/L de tartrazina, con 8,0 mg/L de amarillo crepúsculo y 12,0 mg/L
de rojo alura obteniendo los siguientes valores: tartrazina 4,2 0,1 mg/L (ER: 5%),
amarillo crepúsculo 8,9 0,9 mg/L (ER: 11%) y rojo alura 12,5 0,4 mg/L (ER:2%).
Finalmente se aplicó la metodología en la determinación de estos analítos en bebida
de fantasía (Orange crush y Pap), cereales estrellitas de color naranja y una crema de
tomates Naturezza. La bebida Orange crush analizada contenía 4,5 0,1 mg/L de rojo
alura y la Pap 32,1 0,1 mg/L de tartrazina. La norma europea establece un nivel
permitido de 100 mg/L para las bebidas y jugos artificiales. Las estrellitas de color
naranja contenían 52,4 1,1 mg/kg de amarillo crepúsculo. De acuerdo a la
comunidad europea se acepta un máximo de 200 mg/kg de colorantes totales en
cereales. La crema de tomates contenía 104,6 mg/kg de tartrazina y 22,2 mg/kg de
amarillo crepúsculo, esto es preocupante debido a que el máximo total de colorantes
artificiales permitidos en sopas es de 50 mg/kg. Está en estudio bajar el nivel permitido
de amarillo crepúsculo, debido a que en la síntesis de este compuesto se obtiene
como impureza el Sudan I, el cual es cancerígeno.
In this work experimental parameters of chromatography technique (HPLC ) for the determination of synthetic dyes in food samples (juices, jellies , soups , etc.) using a column with stationary phase C -18 and spectrophotometric detector UV–VIS diode array were optimized. The synthetic dyes analized were tartrazine (E102), sunset yellow (E110), Carmoisine (E122), Amaranth (E123), Ponceau red (E124) and Allura red (E129). Due to the hydrophilic nature of these azo dyes, the retention times are very small and the resolution is not good, so in this study was carry out the determination in the presence of surfactants (micellar chromatography) and compounds which form ion pairs (ion pair chromatography) with the objective of obtain higher interaction of the analytes with de stationary phase. For this a neutral surfactant (triton X-100), a cationic surfactant (cetylpiridinium bromide, CPB) and tetrabutylammonium tetrafluoroborate (TFBTBA), which solvate the dyes were added to mobile phase. However, when Triton or TFBTBA was added into the mobile phase, we obtained the expected results, but after a few hours of realization of measures, which remained constant TFBTBA or Triton concentration, the signs began to have very small areas and displaced to very long time. After washing the column with water for several hours the above conditions were restored. We performed measurements with stationary phase columns containing C-8, C-18 and C-18 endcapped obtaining similar results. Because it is very cumbersome to interrupt the steps and proceed to wash or change the column several times by day, these compound were removed from the mobile phase and decreased the number of colorants to analyze choosing tartrazine, sunset yellow and Allura red which are most often found dyes in food samples. Calibration curves were constructed and determined detection limits of the technique. Later, the validation using spiked mineral water was carried out. It was doped with 4,0 mg/L of tartrazine, 8,0 mg/L of sunset yellow and 12,0 mg/L of allura red. The following values were obtained: tartrazine 4,2 ± 0,1 mg/L (RE:5%), sunset yellow 8,9 ± 0,9 mg/L (RE: 11%) and allura red 12,5 ± 0,4 mg / L (RE:4%). Finally, the methodology was applied in the determination of these analytes in fancy drink (Orange crush and Pap), orange star cereals and tomatoes soap. The orange crush drink analyzed contained allura red 4,5 ± 0,1 mg/L, Pap drink tartrazine 32,1 ± 0,1 mg/L and orange cereals contained sunset yellow 52,4 ± 1,1 mg/kg. European Community accepts a maximum of 100 mg/L for drinks and artificial juices and 200 mg/kg in cereals. For the other hand, the tomato cream contained 104, 6 ± 0,4 mg/kg of tartrazine and 55,3 ± 0,5 mg/kg of sunset yellow. These values are troubling because the upper level accepted for soaps is 50 mg/kg. Principally because in the last years the level of sunset yellow will be lowered due in the synthesis of compounds is probable obtain Sudan I which is cancerigen.
In this work experimental parameters of chromatography technique (HPLC ) for the determination of synthetic dyes in food samples (juices, jellies , soups , etc.) using a column with stationary phase C -18 and spectrophotometric detector UV–VIS diode array were optimized. The synthetic dyes analized were tartrazine (E102), sunset yellow (E110), Carmoisine (E122), Amaranth (E123), Ponceau red (E124) and Allura red (E129). Due to the hydrophilic nature of these azo dyes, the retention times are very small and the resolution is not good, so in this study was carry out the determination in the presence of surfactants (micellar chromatography) and compounds which form ion pairs (ion pair chromatography) with the objective of obtain higher interaction of the analytes with de stationary phase. For this a neutral surfactant (triton X-100), a cationic surfactant (cetylpiridinium bromide, CPB) and tetrabutylammonium tetrafluoroborate (TFBTBA), which solvate the dyes were added to mobile phase. However, when Triton or TFBTBA was added into the mobile phase, we obtained the expected results, but after a few hours of realization of measures, which remained constant TFBTBA or Triton concentration, the signs began to have very small areas and displaced to very long time. After washing the column with water for several hours the above conditions were restored. We performed measurements with stationary phase columns containing C-8, C-18 and C-18 endcapped obtaining similar results. Because it is very cumbersome to interrupt the steps and proceed to wash or change the column several times by day, these compound were removed from the mobile phase and decreased the number of colorants to analyze choosing tartrazine, sunset yellow and Allura red which are most often found dyes in food samples. Calibration curves were constructed and determined detection limits of the technique. Later, the validation using spiked mineral water was carried out. It was doped with 4,0 mg/L of tartrazine, 8,0 mg/L of sunset yellow and 12,0 mg/L of allura red. The following values were obtained: tartrazine 4,2 ± 0,1 mg/L (RE:5%), sunset yellow 8,9 ± 0,9 mg/L (RE: 11%) and allura red 12,5 ± 0,4 mg / L (RE:4%). Finally, the methodology was applied in the determination of these analytes in fancy drink (Orange crush and Pap), orange star cereals and tomatoes soap. The orange crush drink analyzed contained allura red 4,5 ± 0,1 mg/L, Pap drink tartrazine 32,1 ± 0,1 mg/L and orange cereals contained sunset yellow 52,4 ± 1,1 mg/kg. European Community accepts a maximum of 100 mg/L for drinks and artificial juices and 200 mg/kg in cereals. For the other hand, the tomato cream contained 104, 6 ± 0,4 mg/kg of tartrazine and 55,3 ± 0,5 mg/kg of sunset yellow. These values are troubling because the upper level accepted for soaps is 50 mg/kg. Principally because in the last years the level of sunset yellow will be lowered due in the synthesis of compounds is probable obtain Sudan I which is cancerigen.
Notas
Tesis (Licenciado en Química)
Palabras clave
Cromatografía Líquida, Colorantes en los Alimentos