Enhancing the Concentration Capability of Nonsupported Electrically Driven Liquid-Phase Microextraction through Programmable Flow Using an All-In-One 3D-Printed Optosensor: A Proof of Concept.

A versatile millifluidic 3D-printed inverted Y-shaped unit (3D-YSU) was prototyped to ameliorate the concentration capability of nonsupported microelectromembrane extraction (µ-EME), exploiting optosensing detection for real-time monitoring of the enriched acceptor phase (AP). Continuous forward-flow and stop-and-go flow modes of the donor phase (DP) were implemented via an automatic programmable-flow system to disrupt the electrical double layer generated at the DP/organic phase (OP) interface while replenishing the potentially depleted layers of analyte in DP. To further improve the enrichment factor (EF), the organic holding section of the OP/AP channel was bifurcated to increase the interfacial contact area between the DP and the OP. Exploiting the synergistic assets of (i) the continuous forward-flow of DP (1050 µL), (ii) the unique 3D-printed cone-shaped pentagon cross-sectional geometry of the OP/AP channel, (iii) the bifurcation of the OP that creates an inverted Y-shape configuration, and (iv) the in situ optosensing of the AP, a ca. 24 EF was obtained for a 20 min extraction using methylene blue (MB) as a model analyte. The 3D-YSU was leveraged for the unsupervised µ-EME and the determination of MB in textile dye and urban wastewater samples, with relative recoveries ≥88%. This is the first work toward analyte preconcentration in µ-EME with in situ optosensing of the resulting extracts using 3D-printed millifluidic platforms.

Lab-In-Syringe automation of deep eutectic solvent-based direct immersion single drop microextraction coupled online to high-performance liquid chromatography for the determination of fluoroquinolones.

Lab-In-Syringe direct immersion single drop microextraction is proposed as an automated sample pretreatment methodology and coupled online to HPLC with fluorescence detection for the determination of fluoroquinolones in environmental waters. For the first time, a drop of a natural deep eutectic solvent (NADES), synthesized from hexanoic acid and thymol, has been used as an extractant in automated single-drop microextraction. The extraction procedure was carried out within the 5 mL void of an automatic syringe pump. A 9-position head valve served the aspiration of all required solutions, air, waste disposal, and hyphenation with the HPLC instrument. Sample mixing during extraction was done by a magnetic stirring bar placed inside the syringe. Only 60 µL of NADES were required omitting toxic classical solvents and improving the greenness of the proposed methodology. By direct injection, linear working ranges between 0.1 and 5 µg L-1 were achieved for all fluoroquinolones. The limit of quantification values and enrichment factors ranged from 20 ng L-1 to 30 ng L-1 and 35 to 45, respectively. Accuracies obtained from the analysis of spiked surface water and wastewater treatment plant effluent analysis at two concentration levels (0.5 and 4 µg L-1) ranged from 84.6% to 119.7%, with RSD values typically <3%.

Sample preparation under turbulent flow with renewable sorbent.

Turbulent flow chromatography is an online solid phase extraction mode that achieves the extraordinary effect of proxying an upper molecular weight cutoff for the retained molecules, based on loading the sample at high linear velocities. Despite the potential of being a universal sample preparation technique prior to inductively coupled plasma mass spectrometry and liquid chromatography mass spectrometry, it employs specific hardware and expensive consumables. In the present work we apply this technique using off-the-shelf fluidic components and the niche "bead injection" methodology. For the first time, this procedure has been executed with a pressure of approximately 20 bar, compared to the low pressure of the classic setup, achieving a sample throughput >285 h-1 for the SPE/TFC procedure, or 20 h-1 if the procedure involves renewing the sorbent, using no more than 4 mg of sorbent for every µ-SPE. Another novelty is that sorbent packing and unpacking has been controlled with a smart method using real-time pressure feedback as quality control for truly unattended operation. Finally, the turbulent flow chromatography principle has been comprehensively characterized, providing similar performance to that demonstrated in earlier literature, and the ancillary sample preparation capabilities, e.g., in-valve acidification, have been demonstrated by the fractionation of gadolinium in surface waters prior to ICP-MS, an element of increasing surface water concern due to its use as a magnetic resonance contrast agent.

3D printed fluidic platform with in-situ covalently immobilized polymer monolithic column for automatic solid-phase extraction.

In this work, 3D stereolithographic printing is proposed for the first time for the fabrication of fluidic devices aimed at in-situ covalent immobilization of polymer monolithic columns. Integration in advanced flow injection systems capitalized upon programmable flow was realized for fully automatic solid-phase extraction (SPE) and clean-up procedures as a 'front-end' to on-line liquid chromatography. The as-fabricated 3D-printed extraction column devices were designed to tolerate the pressure drop of forward-flow fluidic systems when handling large sample volumes as demonstrated by the determination of anti-microbial agents, plastic additives and monomers as models of emerging contaminants (4-hydroxybenzoic acid, methylparaben, phenylparaben, bisphenol A and triclosan). Decoration of the monolithic phase with gold nanoparticles (AuNPs) was proven most appropriate for the enrichment of phenolic-type target compounds. In particular, the absolute recoveries for the tested analytes ranged from 73 to 92% both in water and saliva samples. The 3D printed composite monolith showed remarkable analytical features in terms of loading capacity (2 mg g-1), breakthrough volume (10 mL), satisfactory batch-to-batch reproducibility (<9% RSD), and easy on-line coupling of the SPE device to HPLC systems. The fully automatic 3D-printed SPE-HPLC hyphenated system was also exploited for the on-line extraction, matrix clean-up and determination of triclosan in 200 µL of real saliva samples.

In quest of effect directed analysis in the smart laboratory: Automated system for flow-through evaluation of membranotropic effects of emerging contaminants.

The rate-determining step of the human exposome workflow is the acquisition of physiologically relevant data (e.g., effect directed analysis), which can be performed retrospectively or with ad hoc experiments. In this contribution, an automated system is proposed for evaluating potential interaction mechanisms of xenobiotics across cell membranes, the so-called membranotropic effects, using liposomes as a mimicry of biological membranes, and fluorescent membrane probes. The smart fluidic method features real-time acquisition of fluorescence readouts, data processing and feedback in a fully unsupervised mode. As a proof of concept applicability, the behavior of newly synthesized cholesterol-laden biomimetic liposomes, and the in-vitro potential toxicant action of bisphenol A and diclofenac as model of emerging contaminants on cell membrane surrogates were investigated in a flow-through format. Unattended operation resulted in excellent intermediate precision (<1.5%) and unveiled that diclofenac affected the liposomal bilayer order very slightly, regardless of the cholesterol concentration, because it accumulates at a superficial level, while the membranotropic effect of bisphenol A was more pronounced at low concentration levels of cholesterol because at increased levels, the membrane reduces its permeability.

Membrane Enhanced Bioaccessibility Extraction (MEBE) of hydrophobic soil pollutants - Using a semipermeable membrane for separating desorption medium and acceptor solvent.

Bioaccessibility extractions are increasingly applied to measure the fraction of pollutants in soil, sediment and biochar, which can be released under environmentally or physiologically relevant conditions. However, the bioaccessibility of hydrophobic organic chemicals (HOCs) can be markedly underestimated when the sink capacity of the extraction medium is insufficient. Here, a novel method called "Membrane Enhanced Bioaccessibility Extraction" (MEBE) applies a semipermeable membrane to physically separate an aqueous desorption medium that sets the desorption conditions from an organic medium that serves as acceptor phase and infinite sink. The specific MEBE method combines HOC (1) desorption into a 2-hydroxypropyl-ß-cyclodextrin solution, (2) transfer through a low-density polyethylene (LDPE) membrane and (3) release into ethanol, serving as analytical acceptor phase. The surface to volume ratio within the LDPE membrane is maximized for rapid depletion of desorbed molecules, and the capacity ratio between the acceptor phase and the environmental sample is maximized to achieve infinite sink conditions. Several experiments were conducted for developing, optimizing and pre-testing the method, which was then applied to four soils polluted with polycyclic aromatic hydrocarbons. MEBE minimized sample preparation and yielded a solvent extract readily analyzable by HPLC. This study focused on the proof-of-principle testing of the MEBE concept, which now can be extended and applied to other samples and desorption media.

Reliable Sensing Platform for Plasmonic Enzyme-Linked Immunosorbent Assays Based on Automatic Flow-Based Methodology.

Plasmonic enzyme-linked immunosorbent assays (ELISA) using the localized surface plasmon resonance (LSPR) of metal nanoparticles has emerged as an appealing alternative to conventional ELISA counterparts for ultrasensitive naked-eye detection of biomolecules and small contaminants. However, batchwise plasmonic ELISA involving end-point detection lacks ruggedness inasmuch as the generation or etching of NP is greatly dependent on every experimental parameter of the analytical workflow. To tackle the above shortcomings, this paper reports on an automatic flow methodology as a reliable detection scheme of hydrogen peroxide related enzymatic bioassays for ultrasensitive detection of small molecules. Here, a competitive ELISA is combined with the in-line generation of plasmonic gold nanoparticles (AuNPs) followed by the real-time monitoring of the NP nucleation and growth rates and size distribution using a USB miniaturized photometer. Glucose oxidase was labeled to the secondary antibody and yielded hydrogen peroxide that acted as the measurand and the reducing agent of the Au(III)/citrate system in the flow network. High-throughput plasmonic assays were feasible by assembling a hybrid flow system composed of two microsyringe pumps, a perfluoroalkoxy alkane reaction coil, and a 26-port multiposition valve and operated under computer-controllable flow conditions. The ultratrace determination of diclofenac in high matrix samples, e.g., seawater, without any prior sample treatment was selected as a proof-of-concept application of the flow-based platform for determination of emerging contaminants via plasmonic ELISA. The detection limit (0.001 µg L-1) was 1 order of magnitude lower than that endorsed by the first EU Watch List for diclofenac as a potentially emerging contaminant in seawater and also than that of a conventional colorimetric ELISA, which in turn is inappropriate for determination of diclofenac in seawater at the levels endorsed by the EU regulation. The proposed automatic fluidic approach is characterized by the reproducible timing in AuNPs nucleation and growth along with the unsupervised LSPR absorbance detection of AuNPs with a dynamic range for diclofenac spanning from 0.01 to 10 µg L-1. Repeatability and intermediate precision (given as normalized signal readouts) in seawater were <4% and <14%, respectively, as compared to RSDs as high as 30% as obtained with the batchwise plasmonic ELISA counterpart.

A flow-based platform hyphenated to on-line liquid chromatography for automatic leaching tests of chemical additives from microplastics into seawater.

An automatic flow-based system as a front end to liquid chromatography (LC) for on-line dynamic leaching of microplastic materials (polyethylene of medium density and poly(vinyl chloride)) with incurred phthalates and bisphenol A is herein presented. The microplastic particles were packed in a metal column holder, through which seawater was pumped continuously by resorting to advanced flow methodology. Each milliliter of the leachable (bioaccessible) fraction of chemical additives was preconcentrated on-line using a 10 mm-long octadecyl monolithic silica column placed in the sampling loop of the injection valve of a HPLC system that served concomitantly for analyte uptake and removal of the seawater matrix. After loading of the leachate fraction, the LC valve was switched to the inject position and the analytes were eluted and separated by a monolithic column (Onyx C18HD 100 × 4.6 mm) using an optimized acetonitrile/water gradient with UV detection at 240 nm. The automatic flow method including dynamic flow-through extraction, on-line sorptive preconcentration, and matrix clean-up was synchronized with the HPLC separation, which lasted ca. 9 min. The only two currently available multi-component certified reference materials (CRM) of microplastics (CRM-PE002 and CRM-PVC001) were used for method development and validation. Out of the eight regulated phthalates contained in the two CRMs, only the 2 most polar species, namely, dimethyl phthalate and diethyl phthalate as well as bisphenol A, were leached significantly by the seawater in less than 2 h, with bioaccessibility percentages of 51-100%. The leaching profiles were monitored and modeled with a first-order kinetic equation so as to determine the rate constants for desorption in a risk assessment scenario. Intermediate precision values of bioaccessibility data for three batches of CRMs were for the suite of targeted compounds ≤22%. This work for the first time reports a fully automatic flow method with infinite sink capacity (i.e., using a surplus of extracting solution) for the target species able to mimic the leaching of additives from plastic debris across the water body in marine settings under worst-case extraction conditions.

Fully Automated Electric-Field-Driven Liquid Phase Microextraction System with Renewable Organic Membrane As a Front End to High Performance Liquid Chromatography.

This article reports for the first time a programmable-flow-based mesofluidic platform that accommodates electric-field-driven liquid phase microextraction (µ-EME) in a fully automated mode. The miniaturized system is composed of a computer-controlled microsyringe pump and a multiposition rotary valve for handling aqueous and organic solutions at a low microliter volume and acts as a front-end to online liquid chromatographic separation. The organic membrane is automatically renewed and disposed of in every analytical cycle, thus minimizing analyte carry-over effects while avoiding analyst intervention. The proof-of-concept applicability of the automated mesofluidic device is demonstrated by the liquid chromatographic determination of nonsteriodal anti-inflammatory drugs in µ-EME processed complex samples (such as urine and influent wastewater) using online heart-cut approaches. Using 5 µL of 1-octanol, 7.5 µL of untreated sample and 7.5 µL of acceptor solution (25 mM NaOH), and 250 V for only 10 min in a stopped-flow mode, the extraction recoveries for the µ-EME of ibuprofen, ketoprofen, naproxen, and diclofenac exceed 40% in real samples. The flow-through system features moderately selective extraction regardless of the sample matrix constituents with repeatability values better than 13%.

3D Printing: The Second Dawn of Lab-On-Valve Fluidic Platforms for Automatic (Bio)Chemical Assays.

In this work, inexpensive manufacturing of unibody transparent mesofluidic platforms for pressure-driven Lab-On-a-Valve (LOV) methodologies is accomplished via rapid one-step 3D prototyping from digital models by user-friendly freeware. Multichannel architecture having 800-1800 µm cross-sectional features with unconventional 3D conduit structures and integrating optical and electrochemical detection facilities is for the first time reported. User-defined flow-programming capitalizing upon software control for automatic liquid handling is synergistically combined with additive manufacturing based on stereolithographic 3D printing so as to launch the so-called fourth generation of microflow analysis (3D-µFIA). Using an affordable consumer-grade 3D printer dedicated LOV platforms are 3D printed at will and prints are characterized in terms of solvent compatibility, optical and mechanical properties, and sorption of inorganic and organic species to prospect potentialities for the unfettered choice of chemistries. The unique versatility of the 3D-printed LOV device that is attached to a multiposition rotary valve as a central design unit is demonstrated by (i) online handling of biological materials followed by on-chip photometric detection, (ii) flow-through bioaccessibility tests in exposome studies of contaminated soils with miniaturized voltammetric detection, (iii) online phospholipid removal by TiO2-incorporated microextraction approaches using on-chip disposable sorbents, and (iv) automatic dynamic permeation tests mimicking transdermal measurements in Franz-cell configurations. A multipurpose LOV fluidic platform can be fabricated for less than 11 Euros.

In-line carbon nanofiber reinforced hollow fiber-mediated liquid phase microextraction using a 3D printed extraction platform as a front end to liquid chromatography for automatic sample preparation and analysis: A proof of concept study.

A novel concept for automation of nanostructured hollow-fiber supported microextraction, combining the principles of liquid-phase microextraction (LPME) and sorbent microextraction synergically, using mesofluidic platforms is proposed herein for the first time, and demonstrated with the determination of acidic drugs (namely, ketoprofen, ibuprofen, diclofenac and naproxen) in urine as a proof-of-concept applicability. Dispersed carbon nanofibers (CNF) are immobilized in the pores of a single-stranded polypropylene hollow fiber (CNF@HF) membrane, which is thereafter accommodated in a stereolithographic 3D-printed extraction chamber without glued components for ease of assembly. The analytical method involves continuous-flow extraction of the acidic drugs from a flowing stream donor (pH 1.7) into an alkaline stagnant acceptor (20 mmol L-1 NaOH) containing 10% MeOH (v/v) across a dihexyl ether impregnated CNF@HF membrane. The flow setup features entire automation of the microextraction process including regeneration of the organic film and on-line injection of the analyte-laden acceptor phase after downstream neutralization into a liquid chromatograph (LC) for reversed-phase core-shell column-based separation. Using a 12-cm long CNF@HF and a sample volume of 6.4 mL, linear dynamic ranges of ketoprofen, naproxen, diclofenac and ibuprofen, taken as models of non-steroidal anti-inflammatory drugs, spanned from ca. 5-15 µg L-1 to 500 µg L-1 with enhancement factors of 43-97 (against a direct injection of 10 µL standards into LC), and limits of detection from 1.6 to 4.3 µg L-1. Relative recoveries in real urine samples ranged from 97% to 105%, thus demonstrating the reliability of the automatic CNF@HF-LPME method for in-line matrix clean-up and determination of drugs in urine at therapeutically relevant concentrations.

3D-Printed Microflow Injection Analysis Platform for Online Magnetic Nanoparticle Sorptive Extraction of Antimicrobials in Biological Specimens as a Front End to Liquid Chromatographic Assays.

In this work, the concept of 3D-printed microflow injection (3D-µFI) embodying a dedicated multifunctional 3D-printed stator onto a rotary microvalve along with a mesofluidic sample preparation platform is proposed for the first time. A transparent 3D-printed stereolithographic mesofluidic chip device accommodating polyaniline (PANI) decorated magnetic nanoparticles (32.5 ± 3.8 mg) is harnessed to in-line sorptive microextraction as a front end to liquid chromatography with peak focusing. As a proof-of-concept application, the 3D-µFI assembly was resorted to matrix cleanup and automatic programmable-flow determination of organic emerging contaminants (4-hydroxybenzoate analogues and triclosan as antimicrobial model analytes) in human saliva and urine samples. By using a sample volume of 1.0 mL with a loading flow rate of 200 µL min-1, an eluent volume of 120 µL at 80 µL min-1, and online HPLC injection of 300 µL of the mixture of eluate and Milli-Q water (in a 1:2 ratio) to prevent band broadening effects of the most polar analytes, the limits of detection (3σ criterion) ranged from 1.1 to 4.5 ng mL-1 for methylparaben (MP), ethylparaben (EP), propylparaben (PrP), phenylparaben (PhP), butylparaben (BP), and triclosan (TCS). Enhancement factors of 16-25 were obtained for the target analytes. Spike recoveries ranged from 84 to 117% for both saliva and urine samples. The online 3D-µFI hyphenated method is synchronized with the chromatographic separation and features a chip lifetime of more than 20 injections with minimal losses of moderately nonpolar compounds on the walls of the mesofluidic device.

Towards an automatic lab-on-valve-ion mobility spectrometric system for detection of cocaine abuse.

A lab-on-valve miniaturized system integrating on-line disposable micro-solid phase extraction has been interfaced with ion mobility spectrometry for the accurate and sensitive determination of cocaine and ecgonine methyl ester in oral fluids. The method is based on the automatic loading of 500µL of oral fluid along with the retention of target analytes and matrix clean-up by mixed-mode cationic/reversed-phase solid phase beads, followed by elution with 100µL of 2-propanol containing (3% v/v) ammonia, which are online injected into the IMS. The sorptive particles are automatically discarded after every individual assay inasmuch as the sorptive capacity of the sorbent material is proven to be dramatically deteriorated with reuse. The method provided a limit of detection of 0.3 and 0.14µgL-1 for cocaine and ecgonine methyl ester, respectively, with relative standard deviation values from 8 till 14% with a total analysis time per sample of 7.5min. Method trueness was evaluated by analyzing oral fluid samples spiked with cocaine at different concentration levels (1, 5 and 25µgL-1) affording relative recoveries within the range of 85±24%. Fifteen saliva samples were collected from volunteers and analysed following the proposed automatic procedure, showing a 40% cocaine occurrence with concentrations ranging from 1.3 to 97µgL-1. Field saliva samples were also analysed by reference methods based on lateral flow immunoassay and gas chromatography-mass spectrometry. The application of this procedure to the control of oral fluids of cocaine consumers represents a step forward towards the development of a point-of-care cocaine abuse sensing system.

Online coupling of fully automatic in-syringe dispersive liquid-liquid microextraction with oxidative back-extraction to inductively coupled plasma spectrometry for sample clean-up in elemental analysis: A proof of concept.

A proof of concept of a novel automatic sample cleanup approach for metal assays in troublesome matrixes as a front-end sample pre-treatment to inductively coupled plasma optical emission spectroscopy - ICP-OES - is herein presented. Target metals, namely, copper, lead, and cadmium were complexed in-system quantitatively using ammonium pyrrolidine dithiocarbamate (APDC) and transferred into a minute volume of toluene as extractant employing lab-in-syringe magnetic stirring-assisted dispersive liquid-liquid microextraction (LIS-MSA-DLLME). After discharge of the sample, the analytes were back-extracted into nitric acid and injected on-line into ICP-OES. To promote and expedite this process in-syringe, advantage was taken from oxidative decomposition of the chelate by potassium iodate, reported in this article for the first time. Experimental conditions for LIS-MSA-DLLME were optimized by Box-Benkhen multivariate analysis using the geometric mean of analyte recoveries as the desirability function. Times of extraction and back-extraction of 300s and 100s, respectively, pH 5.5 at 30mmol/L acetate, 300µL of extraction solvent, and 600µmol/L of APDC were finally applied. Online interfacing to ICP-OES for back-extract analysis yielded average repeatabilities for Cd, Cu, and Pb of 2.9%, 3.5%, and 3.5% with limits of detections (3s) of 1.9, 1.4, and 5.6ng/mL, respectively. Oxidative back-extraction was proven reliable for the determination of metal species in coastal seawater, surrogate digestive fluids and soil leachates with recovery values for Cd, Cu, and Pb ranging from 90% to 118%, 68% to 104%, and 86% to 112%, respectively.

On-line monitoring of in-vitro oral bioaccessibility tests as front-end to liquid chromatography for determination of chlorogenic acid isomers in dietary supplements.

A novel fully automated in-vitro oral dissolution test assay as a front-end to liquid chromatography has been developed and validated for on-line chemical profiling and monitoring of temporal release profiles of three caffeoylquinic acid (CQA) isomers, namely, 3-CQA,4-CQA and 5-CQA, known as chlorogenic acids, in dietary supplements. Tangential-flow filtration is harnessed as a sample processing approach for on-line handling of CQA containing extracts of hard gelatin capsules and introduction of protein-free samples into the liquid chromatograph. Oral bioaccessibility/dissolution test assays were performed at 37.0±0.5°C as per US Pharmacopeia recommendations using pepsin with activity of ca. 749,000 USP units/L in 0.1mol/L HCl as the extraction medium and a paddle apparatus stirred at 50rpm. CQA release rates and steady-state dissolution conditions were determined accurately by fitting the chromatographic datasets, namely, the average cumulative concentrations of bioaccessible pools of every individual isomer monitored during 200min, with temporal resolutions of ≥10min, to a first-order dissolution kinetic model. Distinct solid-to-liquid phase ratios in the mimicry of physiological extraction conditions were assessed. Relative standard deviations for intra-day repeatability and inter-day intermediate precision of 5-CQA within the 5-40µg/mL concentration range were <3.4% and <5.5%, respectively. Trueness of the automatic flow method for determination of 5-CQA released from dietary supplements in gastric fluid surrogate was demonstrated by spike recoveries, spanning from 91.5-104.0%, upon completion of the dissolution process. The proposed hyphenated setup was resorted for evaluating potential differences in dissolution profiles and content of the three most abundant chlorogenic acid isomers in dietary supplements from varied manufacturers.

Rapid estimation of readily leachable triazine residues in soils using automatic kinetic bioaccessibility assays followed by on-line sorptive clean-up as a front-end to liquid chromatography.

An automatic batchwise bioaccessibility test was proposed for on-line monitoring of readily mobile pools of ametryn and atrazine residues in agricultural soils with different physicochemical properties. A 0.01molL(-1) CaCl2 solution mimicking rainwater percolation through the soil profiles was used for the herbicide extractions. The extract aliquots were successively sampled at regular time intervals in order to investigate the extraction kinetics. For extract clean-up and retention of freely dissolved target species, 30mg of restricted-access like copolymer were used as in-line sorptive material followed by elution with methanol and on-line heart-cut injection towards a C18 silica reversed-phase monolithic column (100×4.6mm) in a liquid chromatographic system. A mathematical model emphasized that the readily available pools vs time can be in most instances described by a first-order exponential equation, thus an asymptotical value is approached. Consequently, the leaching assays can be performed without attaining chemical equilibrium. Enhancement factors and detection limits were 10.2 and 18.8, and 0.40 and 0.37mgkg(-1) for ametryn and atrazine, respectively. The automatic method features good repeatability for leaching tests (r.s.d.: 11.8-10.2% for sandy and 3.7-6.2% for clayey soil). Reliable data, demonstrated with relative recoveries in the soil leachates ranging from 86 to 104%, were achieved in less than 35min, thus avoiding the need for up to 24h as recommended by standard leaching methods.

Hybrid flow system integrating a miniaturized optoelectronic detector for on-line dynamic fractionation and fluorometric determination of bioaccessible orthophosphate in soils.

An integrated Sequential Injection (SI)/Flow Injection (FI) system furnished with a miniaturized LED-based fluorometric detector is presented in this work for expedient bioaccessibility tests of orthophosphate in soils. Equipped with a microcolumn of conical shape containing 50 mg of soil, the hybrid flow system was resorted to on-line dynamic leaching and real-time quantification of pools of mobilizable orthophosphate using a bi-directional syringe pump and multiposition valve. The flexibility of the flow manifold was harnessed to explore both bi-directional and uni-directional flow extraction modes with the added degree of freedom of on-line dilution of extracts whenever needed. Bioaccessible orthophosphate was split in three fractions, the so-called NH4Cl fraction containing labile exchangeable phosphates, the alkaline fraction with Fe and Al-bound phosphates and the acidic fraction containing Ca-bound phosphates. The prevailing molybdenum blue photometric detection method is replaced by spectrofluorometric detection based on the ion pair formation between the phosphomolybdate heteropolyacid and rhodamine B with the subsequent quenching of the dye fluorescence. The dedicated optoelectronic detector was integrated in a secondary FI manifold and operated according to the fluorometric paired emitter-detector diode (FPEDD) principle involving two light emitting diodes as fluorescence inductors and one as detector of LED-induced fluorescence. Demonstrated with the analysis of a standard reference material (SRM 2711) and a real agricultural soil, the developed FI/SI fractionation system with FPEDD detection is proven reliable against the standard molybdenum blue method (p>0.05), and useful for investigation of the leaching kinetics of orthophosphate in bioaccessibility tests through in-line recording of the extraction profiles.

Automatic kinetic bioaccessibility assay of lead in soil environments using flow-through microdialysis as a front end to electrothermal atomic absorption spectrometry.

In-line microdialysis is in this work hyphenated to electrothermal atomic absorption spectrometry via a dedicated flow-based interface for monitoring the batchwise leaching test endorsed by the Standards, Measurements and Testing Program (SM&T) of the European Commission. The bioaccessible pool of lead in soils is measured using 0.43 mol/L AcOH as extractant. The proposed method allows to gain knowledge of leaching kinetics at real-time, simplify the overall procedure by accurate detection of steady-state conditions and overcome sample filtration or centrifugation. Soil leachates were automatically sampled at specified timeframes (e.g, every 20 or 80 min), processed in an external container (where dilution can be applied at will) and further injected into the atomizer. The method was experimentally validated by comparison of in situ microdialysis sampling results with in-line microfiltration in two soils of varying physicochemical properties. A mathematical framework was used for discrimination of different metal fractions (that is, readily mobilizable against slowly mobilizable lead) and also for estimating the total extractable lead under actual steady-state conditions. We have demonstrated that bioaccessibility tests lasting 16 h as endorsed by SM&T might not suffice for ascertainment of maximum (steady-state) bioaccessibility of lead in terrestrial environments as demanded in risk assessment programs.