Embedded microdrops configuration for diffusion-based colorimetric reactions: a novel approach for thiol determination by the Ellman reaction

Abstract

A new variant of micro-colorimetry, called Extractive Reactions in Embedded Drops (EXRED), utilizes an aqueous drop of a reagent (2 μL) surrounded by a liquid film of isooctane : octanol (1 : 1, v/v; 2.5 μL) and supported by a microsyringe placed immersed into the aqueous sample solution. This configuration conducted all events of the reaction occurring in a single step, viz., microextraction, matrix cleanup and preconcentration of the analyte by diffusion into the reagent drop, and the specific colorimetric reaction. The method has been verified for the determination of thiols by the Ellman reagent (5,5′-dithio-bis(2-nitrobenzoic acid), disodium salt). The embedded drops were constructed by sequentially withdrawing measured aliquots of the Ellman reagent and the organic solvent into a microsyringe and mounting the needle tip with a polytetrafluoroethylene (PTFE) sleeve terminating into a microfunnel. Then, while protruding the sleeve tip into the acidified aqueous thiol sample, the syringe plunger was slowly pressed to form a composite reagent drop. The embedded drop configuration allowed the diffusion of unionic thiols through the organic solvent film from the acidified donor phase (the sample) into the acceptor Ellman reagent drop. The intense yellow colour of 2-nitro-5-thiobenzoate formed was measured by fiber optic CCD array NanoDrop micro-spectrophotometry. The thiol migration rate was a function of the molecular geometry of individual thiols, attaining molar absorptivities in the range from 3.73 × 10⁴ to 1.33 × 10⁵ L M⁻¹ cm⁻¹ that were 3 to 10 times the reported value of the classical Ellman reaction, establishing the calibration range of 5–700 μM of thiol. In the second method, ionic and unionic thiols were prereacted with the Ellman reagent, and the reaction product 2-nitro-5-thiobenzoate, after acidification, was made to diffuse through the solvent film into an acceptor drop of pH 8 phosphate buffer. Here, the average molar absorptivity obtained (3.85 × 10⁵ L M⁻¹ cm⁻¹) was 28 times the value of the classical reaction, producing the calibration range of 2–60 μM of thiol. The averaged LOD/LOQ values for the two methods were 0.048/0.16 μM. The response surface methodology-optimized parameters were (a) Ellman reagent (25 mM), (b) extraction time (17 min) and (c) sodium sulphate (500 mg). The EXRED methods obeyed the green analytical chemistry criteria, viz., AGREE (0.65), AGREEprep (0.83), ComplexGAPI, BAGI (65.0) and metric of sustainability (7.79).