A double-layered paper-based analytical device for speciation and simultaneous determination of iron in waters within single-shot analysis

• The newly developed double-layered PAD enables the simultaneous determination of Fe(II) and Fe(III) within a single-shot analysis under the same paper device. • Fe(II) specifically bounds to bathophenanthroline while Fe(III) selectively chelates with the novel class of the “in-lab” synthesized fluorescein derivative on the topside and underside layers, respectively, without cross-reactivity. • Successive validation and application to real waters of various matrices with high precision and high accuracy. This work demonstrates the use of a paper-based analytical device (PAD) for the speciation and the simultaneous determination of iron (Fe) in waters within a single-shot analysis. The PAD is designed as having stacked, double layers viz. , the topside and underside layers. Each layer (25 × 25 mm 2 ) consists of a defined circular hydrophilic reservoir (10 mm Ø), situated in the adhesive glue-fabricated hydrophobic zone. Both layers are assembled by a two-sided mounting tape (3.0 mm thickness). The sample is introduced onto the topside reservoir and only Fe (II) reacts specifically with bathophenanthroline (Bphen) to form the stable, red-colored product. Consecutively, the sample flows vertically and exposes the underside reservoir. Then, the PAD is flipped, and the new class of the fluorophore derivative is transferred to the underside reservoir. Fe (III) selectively forms the complex with the fluorophore, and this results in quenching of the green, fluorescent light under UV irradiation. The optical image of each reservoir is captured via a smart phone camera under a light control studio and their color intensities are evaluated by ImageJ™ software. Under the optimal conditions, the calibrations, ranging from 0–0.5 mg L -1 Fe (II) and 0–10 mg L -1 Fe (III) are observed with good linearity ( r 2 > 0.99). Detection limits (3SD) of 0.02 mg Fe (II) L -1 and 0.21 mg Fe (III) L -1 are achieved. The developed PAD offers good recoveries (94.8–107 %) and high precision (RSD < 4 %). It is also successfully validated against the UV–visible spectrophotometric and the ICP-OES methods. The Fe contents are not significant differences at 95 % confidence under the statistical paired t -test (Fe(II): t cal = -0.28, t cri = 2.45 and Fe (III): t cal = 1.77, t cri = 2.45). Remarkedly, The PAD enables the simultaneous determination of Fe(II) and Fe(III) without cross- reactivity effect.