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Mercury is one of the most harmful pollutant present on Earth and shows some peculiar characteristics which make this element relevant to study. Hg undergoes to complex biogeochemical transformations in the environment and shows different isotopic fractionation phenomena of both mass dependant (MDF) and mass independent (MIF) nature. The study of the isotopic mass fractionation, seems to be a powerful tool to trace and identify the sources of pollution and the behavior of this element in natural systems and organisms. In order to determine the isotope ratios of mercury an analytical procedure was set up by using a multicollector ICP/MS coupled with an on-line reduction apparatus as sample introduction system. Instrumental mass bias was corrected by using the 205Tl/203Tl isotopic reference coupled to a standard-sample bracketing approach to compensate any instrumental drift. Due to the high mass of the element, the fractionation phenomena are very small thus, the measure precision must be good enough to be able to significantly detect the differences in the isotopic composition. Because of the high number of instrumental parameters which must be set and which may influence the measurements, the instrumental conditions were optimized in order to obtain the suitable internal precision even for sample with low amount of mercury. The optimization step of the instrumental determination was conducted by means of the most advanced and innovative chemometric techniques of Design of Experiments (DoE). The multivariate approach is suitable for optimization purposes and in particular when many parameters must be varied at …