Thin pellets, uncertain results? - optimizing pellet thickness for accurate EDXRF determination of trace elements in biological matrices

Determination of elemental concentrations in biological tissues is crucial for realizing both normal physiological functions and disease-related processes. Energy Dispersive X-ray Fluorescence (EDXRF) aided by the Fundamental Parameters (FP) method offers an accurate, reliable, non-destructive and multi-element approach for that purpose, nonetheless, its quantitative accuracy is known to be influenced by sample thickness and matrix effects. This study aims to assess the impact of sample's available mass, hence, pellet thickness on the quantification accuracy of FP method when applied to EDXRF spectra. This way, pressed pellets of NIST SRM 1566 Oyster Tissue were prepared at varying thicknesses (1.28–8.70 mm) and analyzed using an EDXRF system to systematically evaluate the impact of thickness on elemental intensities and calculated concentrations. Results show that for light and medium elements (P, S, Cl, K, Ca, Mn, Fe), both net intensities and concentrations remain largely independent of pellet thickness, indicating that saturation conditions are achieved even in relatively thin samples. In contrast, higher-Z elements (Cu, Zn, Br) exhibit clear thickness dependence at lower pellet thicknesses, with concentrations increasing progressively as saturation is approached. Residual deviations from certified values persist for most elements regardless of thickness, demonstrating that matrix assumptions in the FP model, rather than sample geometry, are the primary source of quantitative uncertainty. These findings demonstrate that pellet thickness is a critical factor for accurate quantification of heavier elements but less relevant for lighter elements, providing practical guidance for sample preparation in biomedical EDXRF studies using low available amounts of sample.