Beyond equations: Experimental insights into detection limits in electroanalysis

The limit of detection (LOD) is a key parameter for determining the lowest measurable analyte concentration. Despite the widespread use of theoretical equations to estimate LOD, significant discrepancies are often observed between calculated and empirically achievable values. This study critically examines these discrepancies by applying multiple calculation approaches - including blank signal statistics, error propagation, and graphical methods - to square-wave voltammetry (SWV) and differential pulse voltammetry (DPV) measurements of [Fe(CN)₆]^3-/4- at a gold electrode. The results reveal that commonly used equations can yield LOD values that differ from experimental observations by up to an order of magnitude, depending on the technique and calculation method. Critically, we demonstrate that numerous literature reports incorrectly equate the limit of detection with the limit of quantification (LOQ), leading to overoptimistic sensitivity claims. Furthermore, we revisit the mechanistic understanding of the [Fe(CN)₆]^3-/4-/Au system, showing that the gold electrode is not inert but participates via Au(CN)₂^- formation, which contributes to signal instability at low concentrations. Based on these findings, we propose a statistically rigorous experimental framework for empirical LOD determination that accounts for matrix effects and electrode stability, thereby improving the reliability of electroanalytical methods for real-world applications.