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Abstract(s)
No presente trabalho pretendeu-se estudar a aplicação das técnicas de espetrofotometria
UV-Vis e da cromatografia líquida em fase reversa acoplado ao detetor de UV para
deteção e quantificação de cisteína em amostras de cabelo, cedidas pelo CeNTI, e sujeitas
previamente a um processo de hidrólise ácida. Neste trabalho, ambos os métodos
desenvolvidos foram validados procedendo ao estudo de parâmetros como a gama de
linearidade, limites de deteção e quantificação, precisão e exatidão.
No método da espetrofotometria UV-Vis as amostras foram analisadas recorrendo a
um espetrofotómetro da PerkinElmer, Lambda 35 de duplo feixe. A resposta foi calculada
pela relação entre as absorvâncias dos vários padrões de cisteína e a concentração
dos mesmos. Efetuando a análise do coeficiente de correlação para a cisteína foi possível
verificar que a correlação entre as absorvâncias e a concentração dos padrões foi
linear, verificando-se um R2=0.9995. O limite de deteção e quantificação para a cisteína
foi de 0,002 mM e 0,005 mM, respetivamente.
No método de HPLC todos os padrões (cisteína e cistina) e amostras de cabelo foram
analisadas utilizando uma coluna C18, 5 μm, (15 cm e diâmetro de 4,6 mm), e uma fase
móvel composta por ácido tricloroacético (TCA) 0,01M e pH=2,2 com acetonitrilo na
proporção de 9,6:0,4. A resposta foi calculada pela relação entre área dos picos de cisteína
e cistina vs concentração de cisteína e cistina. Pela análise dos coeficientes de correlação
quadrática da curva de calibração para os padrões de cisteína e cistina constatou-
se que existe uma boa correlação entre a área e a concentração dos padrões, tendose
verificado um R2=0.9981 para a cisteína e R2=0.9978 para a cistina. O limite de deteção
registado para a cisteína apresentou o valor de 0,040mM, enquanto para a cistina o
valor foi de 0,021 mM. O limite de quantificação verificado par a cisteína foi de 0,120
mM e para a cistina foi de 0,064 mM.
Os resultados de validação aplicados para a linearidade, limites de deteção e quantificação
e exatidão das amostras analisadas demonstraram a aplicabilidade de ambos os
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métodos na deteção e quantificação de cisteína e cistina nas amostras de cabelo. No
entanto efetuando a comparação dos dois métodos, verifica-se que o método da espetrofotometria
foi aquele que permitiu detetar um maior número de resíduos de cisteína presente
nas amostras de cabelo.
In the present work we intended to study the application of UV-Vis spectrophotometry and liquid chromatography coupled to an UV detector for detection and quantification of cysteine in several hair samples, provided by CeNTI, and previously subject to the process of acid hydrolysis. In this work, both the developed methods were validated by the study of parameters such as the range of linearity, limits of detection and quantification, precision and accuracy. In the UV-Vis spectrophotometry method the samples were analyzed using a spectrophotometer PerkinElmer Lambda 35 with double beam. The response was calculated as the ratio between the absorbances of various patterns and cysteine concentrations. Performing the analysis of the correlation coefficient for cysteine it was also observed that the correlation between the absorbance and the concentration of the standards was linear, providing a correlation coefficient of R2 = 0.9995. The limit of detection and quantification of cysteine was 0.002 mM and 0.005 mM, respectively. The HPLC method analyzed all standards (cysteine and cystine) and hair samples by using a C18 column, with 5 microns (diameter 15 cm, 4.6 mm) and a mobile phase consisting of trichloroacetic acid (TCA) 0,01M and pH = 2.2 with acetonitrile in the ratio of 9,6:0,4. The response was calculated as the ratio of the peak area versus cysteine and cystine concentration of cysteine and cystine. For the analysis of correlation coefficients of quadratic calibration curve for patterns of cysteine and cystine was found that there is a good correlation between the area and the concentration of the standards, having been an R2 = 0.9981 for cysteine and R2 = 0.9978 to cystine. The detection limit for registered cysteine showed the value of 0.040 mM, while for cystine value was 0.021 mM. The limit of quantification was verified pair cysteine and 0.120 mM cystine was 0.064 mM. xi The results of validation applied to the linearity, limits of detection and quantification and accuracy of the samples demonstrated the applicability of both methods in the detection and quantification of cysteine and cystine in hair samples. However making the comparison of the two methods, the results presented by the spectrophotometry method allowed to detect a larger number of residues present in hair samples cysteine.
In the present work we intended to study the application of UV-Vis spectrophotometry and liquid chromatography coupled to an UV detector for detection and quantification of cysteine in several hair samples, provided by CeNTI, and previously subject to the process of acid hydrolysis. In this work, both the developed methods were validated by the study of parameters such as the range of linearity, limits of detection and quantification, precision and accuracy. In the UV-Vis spectrophotometry method the samples were analyzed using a spectrophotometer PerkinElmer Lambda 35 with double beam. The response was calculated as the ratio between the absorbances of various patterns and cysteine concentrations. Performing the analysis of the correlation coefficient for cysteine it was also observed that the correlation between the absorbance and the concentration of the standards was linear, providing a correlation coefficient of R2 = 0.9995. The limit of detection and quantification of cysteine was 0.002 mM and 0.005 mM, respectively. The HPLC method analyzed all standards (cysteine and cystine) and hair samples by using a C18 column, with 5 microns (diameter 15 cm, 4.6 mm) and a mobile phase consisting of trichloroacetic acid (TCA) 0,01M and pH = 2.2 with acetonitrile in the ratio of 9,6:0,4. The response was calculated as the ratio of the peak area versus cysteine and cystine concentration of cysteine and cystine. For the analysis of correlation coefficients of quadratic calibration curve for patterns of cysteine and cystine was found that there is a good correlation between the area and the concentration of the standards, having been an R2 = 0.9981 for cysteine and R2 = 0.9978 to cystine. The detection limit for registered cysteine showed the value of 0.040 mM, while for cystine value was 0.021 mM. The limit of quantification was verified pair cysteine and 0.120 mM cystine was 0.064 mM. xi The results of validation applied to the linearity, limits of detection and quantification and accuracy of the samples demonstrated the applicability of both methods in the detection and quantification of cysteine and cystine in hair samples. However making the comparison of the two methods, the results presented by the spectrophotometry method allowed to detect a larger number of residues present in hair samples cysteine.
Description
Keywords
Cisteína Cistina Cabelo Espetrofotometria HPLC Validação