Environmental Science Water Research & Technology This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online PAPER View Journal | View Issue Highly versatile and automated total ammonia nitrogen compact analyzer suitable for different Open Access Article. Published on 19 October 2023. Downloaded on 12/4/2023 10:28:02 AM. Cite this: Environ. Sci.: Water Res. Technol., 2023, 9, 3366 types of water samples Antonio Calvo-López, Julián Alonso-Chamarro and Mar Puyol * Continuous monitoring of total ammonia nitrogen (TAN) currently needs to improve further not only to meet optimal analytical features but also to reduce equipment maintenance needs and avoid manual intervention. The development, evaluation in different aqueous samples (fresh and wastewater) and validation of a fully automated and adaptable miniaturized potentiometric analytical instrument with this purpose are presented. The main goals accomplished are a wider and adaptable working range (0.08 to Received 12th May 2023, 1000 mg L−1 NH4+) with a detection limit of 0.03 mg L−1 and a significant reduction of reagent and sample Accepted 7th October 2023 consumption, as well as waste generation, compared to current commercial potentiometric flow analyzers. DOI: 10.1039/d3ew00348e Moreover, different programmable calibration and sample analysis protocols have been successfully applied. Finally, suitable analytical features in terms of accuracy and precision are achieved even with rsc.li/es-water samples with very complex matrices without any pretreatment required. Water impact Total ammonia nitrogen (TAN) needs to be monitored to assess water quality with the goal of sustainable development. We present a highly portable and versatile analyzer for continuous monitoring of TAN, which is applicable to different water bodies. It features autonomy, automation, low sample and reagent consumption, interference-free, a wide linear range, low maintenance and minimal waste generation. Introduction L−1 NH4+, while concentrations above 1.5 and 35 mg L−1 NH4+ cause odor and taste problems, respectively. In drinking Total ammonia nitrogen (TAN), expressed as the sum of NH3 water, high levels of TAN are related to ineffective chlorinated and NH4+ due to the pH dependent equilibrium of both disinfection processes,2,7 in which it is combined with compounds,1 is an important parameter to monitor in water chlorine and can produce di- and trichloramines causing and aqueous ecosystems because it is involved in many taste and odor problems. About human health, acute applications and natural processes.2 Regarding flora and poisoning due to ammonium or ammonia ingestion could fauna, TAN is a growth limiting nutrient for algae, so its lead to lung edema, nervous system dysfunction, acidosis, concentration is related to water eutrophication.2,3 Moreover, kidney damage and even death.2,8,9 whereas ammonium is harmless, ammonia is a toxic product Thus, EU legislation establishes a TAN limit concentration for fishes because it limits the proper elimination of their of 0.5 mg L−1 (expressed as mg L−1 NH4+) in drinking water metabolized ammonia.4 In both industrial and urban and 10–50 mg L−1 NH4+ in effluents discharged into natural wastewater treatment plants, monitoring TAN is important to water bodies from wastewater treatment plants. However, the verify the proper operation of nitrification processes (where concentration of their effluents could range 10–100 mg L−1 under aerobic conditions, ammonium/ammonia is oxidized and 10–1000 mg L−1 NH4+ in urban and industrial wastewater to nitrite and finally to nitrate) and to assure that its treatment plants, respectively.5,10–14 In this context and in concentration in the effluents to natural water bodies is order to fulfill the required analytical features of all the within the allowed range by legislation.3,5,6 The natural TAN scenarios, miniaturized analyzers, suitable for in-field concentration in surface and groundwater is below 0.2 mg monitoring of a wide range of concentrations of TAN in a wide variety of complex matrices, with higher autonomy and Group of Sensors and Biosensors, Department of Chemistry, Autonomous University automation, are necessary. of Barcelona, Edifici Cn, 08193 Bellaterra, Barcelona, Spain. There are currently different analytical methods to determine E-mail: mariadelmar.puyol@uab.es; Tel: +34935818296 TAN in aqueous samples such as spectrophotometry,10,15 3366 | Environ. Sci.: Water Res. Technol., 2023, 9, 3366–3376 This journal is © The Royal Society of Chemistry 2023 View Article Online Environmental Science: Water Research & Technology Paper conductimetry16,17 and potentiometry.18–20 In particular, ion were used to fabricate the microfluidic platform: Topas 8007 selective electrodes (ISEs) are preferred for continuous foil of 25 and 50 μm thick and Topas 5013 layers of 300 μm monitoring thanks to the possibility to be integrated into flow and 1 mm thick. A graphite–epoxy composite, resulting from techniques, which allows achieving a high level of automation mixing graphite powder of 50 μm particle size (Merck, This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. and autonomy and high speed of analysis, as well as Germany), Araldite-M epoxy-resin and HR hardener (Ciba- simplicity, versatility, robustness, selectivity and a wide Geigy, Switzerland), was used for the ISE conductive support. working range.4,15,21,22 On the other hand, TAN analyzers that A screen-printable Ag/AgCl paste (C2030812D3, Gwent, take advantage of the separation of TAN in the ammonia form Pontypool, UK) was used for the Ag/AgCl reference electrode from the sample matrix through a gas-diffusion membrane fabrication. A 0.45 μm pore diameter polyvinylidene fluoride exhibit the highest selectivity towards this analyte.17,23,24 (PVDF) hydrophobic membrane (Millipore, USA) was used to Open Access Article. Published on 19 October 2023. Downloaded on 12/4/2023 10:28:02 AM. However, the ones that combine potentiometric detection separate, by gas diffusion, the analyte from the sample systems with gas-diffusion stages and continuous flow matrix. In order to prepare the ammonium sensor techniques20,25–27 still lack enough autonomy to be adapted membrane, nonactin, bis(1-butylpentyl)adipate (BBPA) and to any type of water source and concrete requirements polyvinyl chloride (PVC), all dissolved in tetrahydrofuran without any sample pretreatment. Therefore, the desired (THF), were used (obtained all from Fluka, Barcelona, Spain). operational requirements for a suitable TAN analyzer are high All reagents used for the development and portability for in situ measurements, autonomy, automation characterization of the analytical system were of analytical and low sample and reagent consumption for continuous grade. Milli-Q water was used for the preparation of all monitoring with low maintenance requirements, a wide or solutions. Two ammonium chloride (Panreac, Spain) stock adaptable working range to face TAN determination in solutions containing 1 and 1000 mg L−1 NH4+ were made to different types of water sources, and high capability to operate be used in the automatic calibration process. As a reference with complex matrices, providing reliable results in real time. solution to set at a constant value the reference electrode For all these reasons, a low cost, compact and portable full potential, a 100 mM KCl solution (Sigma Aldrich, Germany) analytical instrument to determine total ammonia nitrogen in was employed. A 10 mM tris(hydroxymethyl)aminomethane the ammonium form in aqueous samples, based on the flow (Tris) solution (Sigma Aldrich, Germany) set with injection analysis (FIA) technique, is proposed in this work. It hydrochloric acid (Merck, Germany) to pH 7.4 was employed is composed of four main parts: 1) a monolithic and compact to transform ammonia into the ammonium ion. This microfluidic platform fabricated in cyclic olefin copolymer solution is called acceptor solution throughout the text. As a (COC) similar in size to a credit card, which integrates a gas- basic solution, a 100 mM NaOH solution (Sigma Aldrich, diffusion membrane and the detection system consisting of Germany) containing 1 mM ethylenediamine tetraacetic acid an ammonium ion selective electrode and a Ag/AgCl reference (EDTA) (Panreac, Spain) was employed. The obtained solution electrode; 2) a miniaturized liquid management system, with from mixing the sample/standard solution and this basic flow elements such as microvalves and micropumps; 3) an solution is called donor solution throughout the text. analytical data acquisition and processing system; 4) a computer to manage the whole system that enables an automatic and unattended operation by means of versatile Fabrication of the microfluidic platform processes of autocalibration and sample analysis. The COC-based microanalyzer was fabricated following a COC has been selected to fabricate the microfluidic multilayer microfabrication procedure described in detail platform because it allows easy monolithic integration of gas- elsewhere.27,32 The process consists of the lamination of diffusion polymeric membranes, besides showing other different COC layers presenting two different glass transition interesting advantages such as good mechanical resistance, temperatures (Tg): Topas 8007 foil with Tg of 75 °C (used as chemical inertia against most acids and alkalis, easy sealing layers) and 5013 layers with Tg of 130 °C (used as microfabrication of hermetically sealed three-dimensional structural layers containing the microfluidic structures), so structures and the possibility to integrate conductive that once the process is finished, complete sealed 3D tracks.28–31 The whole system is comparable in size to a shoe microfluidic structures are obtained in one substrate. The box and its portability, wide adaptable working range, low following four steps are required to fabricate the maintenance and low waste generation and high accuracy microanalyzer: layer design, structure micromachining, and precision without any pretreatment required constitute integration of the separation membrane and electrodes and the differential features with respect to existing commercial the final lamination of the layers. equipment for TAN determination. CAD software was used to design the microsystem prototype (Fig. 1). It consisted of four layers: two COC 5013 layers of 1 Experimental mm (a and c), a 300 μm-COC 5013 layer (d) and 50 μm-COC 8007 foil (b). The microchannel dimensions were 0.4 mm × 0.3 Materials and reagents mm, except for the meander-shaped microchannels inside the Different grades and thicknesses of COC layers and foil, gas-diffusion module, which were 1 mm × 0.1 mm in order to obtained from Topas Advanced Polymers (Florence, KY, USA), increase the contact area between the solution and the This journal is © The Royal Society of Chemistry 2023 Environ. Sci.: Water Res. Technol., 2023, 9, 3366–3376 | 3367 View Article Online Paper Environmental Science: Water Research & Technology This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Open Access Article. Published on 19 October 2023. Downloaded on 12/4/2023 10:28:02 AM. Fig. 1 A: Layered CAD design of the microfluidic platform. Layers “a” and “c” have fluidic structures on both sides, depicted in black in front and gray in back; B: image of the final microanalyzer. hydrophobic PVDF membrane and thus, promote ammonia THF evaporation. Finally, liquid connectors were set over the diffusion. The detection chamber, where the ammonium corresponding inlets or outlets using a bracket and screws, selective electrode was placed, was 2 mm in diameter. The final and the detection chamber and microchannels exposed to dimensions of the microfluidic platform were 50 × 65 × 3 mm the surface were sealed with an adhesive film (Fig. 1). with a total weight of 10 g. The detection chamber and total microsystem dead volumes were 2 and 97 μL, respectively. A computer controlled Protomat C100/HF micromilling Experimental setup machine (LPKF, Germany) was used for the microfluidic Fig. 2 shows a drawing of the whole equipment developed. It is structure machining (holes, microchannels and bas-relief for composed of four main parts: the microfluidic platform or the ISE conductive support integration) onto the COC layers. microanalyzer, the liquid management system, the analytical For the conductive support for the ammonium selective signal acquisition and processing system, and the global electrode, a mixture of Araldite-M, HR hardener and graphite management system. The flow system setup consists of three powder at 36, 14 and 50% in weight, respectively, was solenoid micropumps, one performing (Fig. 2, SP3) 4 μL per prepared. The composite mixture was placed inside the pulse and two of them (Fig. 2, SP1 and SP2) pumping 10 μL per corresponding bas-relief (Fig. 1) and was cured for 24 h at 40 pulse (BiochemValve Inc., Montluçon Cedex, France), one °C. In order to obtain a flat surface facilitating a proper final peristaltic micropump (Fig. 2, PP) (Kamoer KP-S10DGC0, lamination process, the electrode was polished. For the Shanghai, China), which integrates a customized rpm reference electrode, a Ag/AgCl paste was screen-printed using controller (TMI, Barcelona, Spain) and operates with tubing of a DEK 248 screen-printer machine (DEK, Spain), being cured 0.19 mm internal diameter (Ismatec, Wertheim, Germany) for 30 min at 80 °C. For the monolithic PVDF membrane made of Tygon®, and four three-way solenoid valves (Fig. 2, Vx) integration, first it was cut, washed with Milli-Q water and (NResearch, Switzerland). Tubing of 0.8 mm internal diameter dried with air and then, placed (layer b in Fig. 1A) with the (Scharlab, S.L., Sentmenat, Spain) made of Teflon is used to more hydrophobic side oriented towards the donor solution. join the microfluidic platform with all the flow elements. A As has been reported previously,27,33 some holes were made controller for flow elements (Fig. 2, Flow controller) on the PVDF membrane in order to favor the correct sealing (Flowtest™, Biotray, France) is used to actuate the flow during the final lamination. elements. A customized and miniaturized potentiometer In order to obtain a fully sealed monolithic microfluidic (Fig. 2, P) (TMI, Barcelona, Spain) is used for the signal platform, final lamination was performed using a acquisition and processing. Both the flow controller and the temperature-controlled customized press (Francisco Camps, potentiometer are managed using software driven by a Granollers, Spain) set at 4 atm pressure and 102 °C. personal computer that allows customized programming to Regarding the formulation of the ammonium selective automate the entire analytical procedure (calibration and polymeric membrane, it was previously optimized27 and sample analysis) without user interaction. Simple signal prepared with 1% nonactin, 65.5% BBPA and 33.5% PVC in 3 acquisition and processing were carried out using LabVIEW- mL THF. The resulting membrane cocktail was deposited based software, in which the raw potential signal provided by inside the corresponding cavity and over the graphite the potentiometric cell was taken and averaged to obtain 10 conductive support until it was completely filled with measurements per second. It was subsequently used to successive dosages of 2 μL using a micropipette every five calculate the peak heights of each transient signal generated minutes to avoid the formation of bubbles due to the rapid when the ammonium ion is detected, corresponding to each 3368 | Environ. Sci.: Water Res. Technol., 2023, 9, 3366–3376 This journal is © The Royal Society of Chemistry 2023 View Article Online Environmental Science: Water Research & Technology Paper This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Open Access Article. Published on 19 October 2023. Downloaded on 12/4/2023 10:28:02 AM. Fig. 2 Schematic illustration of the complete analytical equipment, where: 1, 2, 3 and 6 are inlets; 4 and 5 are outlets to waste; a) micromixer; b) PVDF gas-diffusion membrane; c) ammonium selective electrode; d) reference electrode; e) reference electrode electric connector; f) ISE electric connector; P: customized potentiometer; PP: peristaltic micropump; SPx: solenoid micropump; Vx: three-way solenoid microvalve. standard solution injected for calibration purposes and each value, a 100 mM KCl solution is pumped at 100 μL min−1 sample analyzed. Afterwards, the concentration of TAN present through the fourth inlet port (6) flowing through the in the sample was obtained by interpolation of the peak height microchannel where the reference electrode is located. This generated by the sample in the Nernst equation, previously joins also the microchannel of the acceptor solution after the obtained in the calibration procedure. One calibration per day detection chamber.27 was performed in order to ensure reliable results. Sample analysis procedure Analysis principle In order to validate the performance of the analytical The microfluidic platform includes four liquid inlet and two microsystem and verify that the proposed method does not outlet ports (Fig. 2). The channels of the first two input ports present significant differences compared to a reference (1 and 2) converge at a junction point, where the injected method, real samples from different origin, matrices and samples or standard solutions into the carrier solution (1) get TAN concentrations were analyzed off-line. They were mixed with the NaOH solution (2), thus becoming the donor obtained from three different places: 1) water from different solution. This mixed stream is carried towards the gas- stages of a wastewater treatment plant of a surfactant diffusion module, where the generated ammonia gas diffuses manufacturing industry, placed in Granollers (Barcelona, through the PVDF membrane along a meander-shaped Spain), 2) water from different stages of the pilot plant of the microchannel to the acceptor solution (3) (Tris buffer Micro-Ecological Life Support System Alternative (MELiSSA), adjusted to pH 7.4), where ammonia is converted back to the which is a project carried out by the European Space Agency ammonium ion. The remains of the donor solution reach the (ESA) at the Autonomous University of Barcelona (Spain),34 waste outlet (4), while the acceptor solution is directed and 3) tap water from the city of Cerdanyola del Vallès towards the detection chamber, where the analyte is detected (Barcelona, Spain). In the first case, biomass was allowed to by the ammonium selective electrode. Finally, liquid exits precipitate and water was decanted and analyzed without through the second outlet port (5). With the aim of filtration before the analysis, simulating real sampling maintaining the reference electrode potential at a constant procedures. In the second case, biomass was filtered before This journal is © The Royal Society of Chemistry 2023 Environ. Sci.: Water Res. Technol., 2023, 9, 3366–3376 | 3369 View Article Online Paper Environmental Science: Water Research & Technology analysis because of the current specific protocol for this EDTA was used to avoid possible metal hydroxide application. Finally, tap water was analyzed without any precipitation, thus preventing damage or blockage inside filtration after being spiked with the ammonium ion. microfluidics or on the gas-diffusion membrane. Optimum Additionally, the simulation of real-time and in situ solution compositions for basic and acceptor solutions were This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. continuous monitoring of TAN in wastewater of a reactor finally 100 mM NaOH with 1 mM EDTA and 10 mM Tris, with biomass from a surfactant manufacturing industry was respectively. carried out to verify the applicability of the developed Regarding hydrodynamic variables, the injection volume of prototype to real work environments. Results obtained from the sample was tested between 25 and 1000 μL while the flow the analysis of water from treatment plants were validated by rate of each stream (carrier, basic and acceptor solutions) was comparison with those obtained with the indophenol blue studied between 50 and 800 μL min−1. The 100 mM KCl Open Access Article. Published on 19 October 2023. Downloaded on 12/4/2023 10:28:02 AM. standard method.15 Recovery rates were calculated with tap solution to keep constant the reference electrode potential was water to verify results. pumped at a flow rate of 100 μL min−1. As results show, hydrodynamic parameters can be selected in order to fit the Results and discussion linear range and limit of detection to the expected TAN concentration in samples to be analyzed, so this can be Fabrication and optimization of the microfluidic platform automatically modulated depending on the final application. The main objective of the present work was to develop a Moreover, the analyte is separated from the matrix due to compact, versatile, low cost and portable analytical the gas-diffusion stage, so only those compounds able to instrument for TAN monitoring in different aqueous samples diffuse through the PVDF membrane in basic medium are based on the use of a potentiometric microfluidic platform, potential interfering compounds of the ammonium ISE. In previously optimized for other applications,27,35 and achieve this sense, only methylated amines could be an actual the best analytical features for ammonium determination in interfering compound, which is not expected to be found in water bodies regarding baseline signal stability, sensitivity, natural water but in industrial wastewater samples. the detection limit and the lifetime of the gas-diffusion hydrophobic membrane. The pursued TAN analyzer must also fulfill features such as simplicity, selectivity, robustness, Liquid management system optimization a wide working range, easy automation and low reagent and A miniaturized and fully automated computer-controlled sample consumption to minimize waste generation. (Flowtest™) liquid management system was applied In order to minimize analysis time, reagent and sample combining different solenoid micropumps and microvalves consumption and microanalyzer manufacturing costs, which and a peristaltic micropump. The operation is simple; the is key to further industrialization purposes, the microfluidic FlowTest™ provides a 12 V pulse to activate the solenoid platform was redesigned (microfluidics and detection devices (in the case of microvalves, to change the position; in chamber). For instance, the volume of the detection chamber the case of micropumps, to fill and empty the plunger with was reduced almost 8 times (15 to 2 μl).27 In this way, the the liquid to be dispensed). In the case of the peristaltic analysis time was optimized (2 minutes faster than using a micropump, during the operating period a potential between larger detection chamber) due to a faster solution renewal on 0 and 12 V is applied corresponding to the desired flow rate. the detection chamber. This not only allows speeding up the The FlowTest™ allows performing automatic signal baseline recovery but also saving reagents. multicommutation dilution sequences with the microvalves On the other hand, to provide robustness to the and modulating hydrodynamic parameters (injection volume microfluidic platform, an electrical connection system based and flow rate) to easily optimize the desired analytical on pin connections was implemented (Fig. 1B). It enables a features such as sensitivity, the detection limit, linear range more reliable electrical connection and noise reduction, thus and time of analysis, among others. The software interface is obtaining more reproducible results than previous very intuitive and very easy to use. In this sense, by simply analyzers.27 introducing the different operating parameters required The influence of chemical (reagent concentrations and (operation time of each microfluidic element, pH) and hydrodynamic (injection volume and flow rate) multicommutation time, etc.) the different sequences can be variables on the system performance was studied using a created in a very simple way and without the need for procedure of univariate optimization after programming advanced programming knowledge. automated operation sequences. The goal was to explore An automated calibration process can be performed using versatility to different water bodies by complying with the a multicommutation dilution procedure from a concentrated required linear ranges and each of the analytical features. stock solution, being able to prepare in real time adequate Tris buffer was selected as the acceptor solution due to standards.36,37 When carrying out this calibration process, the provided better analytical features compared to other different technical parameters must be taken into account buffers.20,21,27 Its concentration was tested from 5 to 100 such as the minimum microvalve operation time, the mM. NaOH concentration in basic solution was studied from injection time and the pumping system used. The accuracy of 0.1 to 1000 mM (with a fixed EDTA concentration of 1 mM). the dilution process and therefore the amplitude of the linear 3370 | Environ. Sci.: Water Res. Technol., 2023, 9, 3366–3376 This journal is © The Royal Society of Chemistry 2023 View Article Online Environmental Science: Water Research & Technology Paper range obtained will depend on the proper selection of the record for one calibration process using program 1. The found solenoid microvalve and the peristaltic micropump. Due to Nernst equation (using one calibration data, with triplicates for instrumental restrictions, the highest commutation speed each standard solution; n = 4 and 95% confidence) was E = 333 (on/off) of the solenoid microvalve used in this work was 100 (±4) + 57 (±1)log[NH4+] with r 2 = 0.9996. The linear range was This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. ms.37–39 Thus, dilution factors of 100 times the stock solution from 0.08 to 10 mg L−1 NH4+ with a limit of detection, concentration can be reached with an RSD less than 5%. according to IUPAC,40 of 0.03 mg L−1 NH4+. Fig. 3C and D show Therefore, in order to cover the desired range (0.08 to 1000 the signal record for one calibration process using program 2. mg L−1 NH4+–N), two stock standard solutions of 1 and 1000 The obtained Nernst equation (using one calibration data, with mg L−1 NH4+ were used and two different multicommutation triplicates for each standard solution; n = 4 and 95% dilution sequences were created, called programs 1 and 2. confidence) was E = 251 (±3) + 52 (±1)log[NH4+] with r 2 = Open Access Article. Published on 19 October 2023. Downloaded on 12/4/2023 10:28:02 AM. Program 1 was designed to determine low TAN 0.9993. The linear range was from 1 to 1000 mg L−1 NH4+ with concentrations (from 0.08 to 10 mg L−1 NH4+) so they would a limit of detection of 0.3 mg L−1 NH4+. These results be indicated for the analysis of drinking water or to monitor demonstrated the versatility, reliability and robustness of the water quality in natural water bodies such as rivers or lakes. complete analytical equipment. Analysis frequencies of 10 and On the other hand, program 2 would be adequate to 8 samples per hour using programs 1 and 2 were obtained, determine high TAN concentrations (from 1 to 1000 mg L−1 respectively. However, it is possible to shorten the analysis time NH4+) like the ones found in wastewater treatment plants. In by automating the injection of a new standard solution or order to optimize reagent consumption, waste generation sample once the baseline signal is recovered instead of waiting and analysis time, two different configurations of flow rate a specific time (6 min for program 1 and 8 min for program 2). and injection time were set: program 1 (flow rate: 350 μL Thus, the sample throughput could be easily doubled. min−1, injection time: 60 s, corresponding to 350 μL injection System repeatability was calculated from replicates of each volume) and program 2 (flow rate: 400 μL min−1, injection standard solution. The RSD of peak heights was less than time: 20 s, corresponding to 133 μL injection volume). 3%. Reproducibility was also tested during the first month Table 1 shows the different parameters related to the through 5 calibration experiments: a mean slope of the multicommutation dilution sequences used in each program calibration curve of 57 with a RSD value of 8% was obtained, to obtain the corresponding standard solutions. confirming the intra and inter-day precision. It should be noted that it would be also possible to Taking into account that the sample may have a complex combine automatically both programs in order to obtain a and highly aggressive matrix, the gas-diffusion membrane wider linear range (using two calibration curves avoids its contact with the surface of the sensor membrane simultaneously) or besides, to create a new program to fit the on the indicator electrode. In addition, the tangential flow expected TAN concentration present in samples. If extremely configuration in the gas-diffusion stage avoids the suspended high TAN concentrations were to be determined, it would be solids that may be introduced into the microfluidic platform even possible to integrate an automatic sample dilution by to obstruct or clog the separation membrane. Moreover, the including an extra microvalve. chosen strategy of the FIA technique allows the sample to be The operation of the dilution by multicommutation was in contact with the different parts of the measuring evaluated comparing the obtained signal peak height of equipment only for a short time, since only 133 or 350 μL of manually prepared standard solutions with the ones obtained sample is either injected, depending on the analysis program automatically. No significant differences were found between selected. For all this, the potentially negative effects caused them (RSD below 3%). by the sample matrix on each element of the analytical microsystem are greatly attenuated, improving the lifetime of the electrodes, gas-diffusion/separation membrane and Analytical features microfluidic platform, compared to other probe-type or flow- Two different calibration curves were obtained corresponding based commercial analytical systems. The lifetime of the to each established program. Fig. 3A and B show the signal microfluidic platform including the gas-diffusion membrane Table 1 Multicommutation parameters used to obtain each standard solution Program Standard solution Stock used Total injection Time on Time off number (mg L−1) (mg L−1) time (s) (stock solution) (s) (water) (s) Cycles Dilution factor 1 10 1000 60.00 0.10 9.90 6 1 : 100 1 1 1 60.00 60.00 0.00 1 — 1 0.1 1 60.00 0.10 0.90 60 1 : 10 1 0.08 1 60.00 0.10 1.15 48 1 : 12.5 2 1000 1000 20.00 20.00 0.00 1 — 2 100 1000 20.00 0.10 0.90 20 1 : 10 2 10 1000 20.00 0.10 9.90 2 1 : 100 2 1 1 20.00 20.00 0.00 1 — This journal is © The Royal Society of Chemistry 2023 Environ. Sci.: Water Res. Technol., 2023, 9, 3366–3376 | 3371 View Article Online Paper Environmental Science: Water Research & Technology This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Open Access Article. Published on 19 October 2023. Downloaded on 12/4/2023 10:28:02 AM. Fig. 3 Obtained recorded signal (A) and calibration plot (B) using program 1. TAN standard solutions of 0.08 (a), 0.1 (b), 1 (c) and 10 mg L−1 NH4+ (d). Obtained recorded signal (C) and calibration plot (D) using program 2. TAN standard solutions of 1 (1), 10 (2), 100 (3) and 1000 mg L−1 NH4+ (4). and ISE was at least 2 months. In addition, an active or stop in between after the baseline stabilization. Therefore, the special pretreatment of the sample is not required prior to TAN analyzer works autonomously and without maintenance the analysis. until the required replacement of reagents and the microfluidic Thanks to the level of automation achieved both in the platform for at least two months. This autonomy is higher than calibration and measurement processes and to the that of most commercial equipment. miniaturization of the equipment, the consumption of reagents and the generation of waste are reduced. The reagent consumption was calculated to be 6 L per month, taking into Real sample analysis consideration that current automatic stations for water quality Fig. 4A shows the results from the analysis of 65 samples control perform one analysis of TAN per hour and that systems with a very complex matrix from different stages of the Fig. 4 Result comparison of the proposed analyzer and the indophenol blue reference method for the analysis of wastewater samples from a surfactant manufacturing industry (A) and MELiSSA (B). Each value is the result of an average of three measurements. 3372 | Environ. Sci.: Water Res. Technol., 2023, 9, 3366–3376 This journal is © The Royal Society of Chemistry 2023 View Article Online Environmental Science: Water Research & Technology Paper surfactant manufacturing industry wastewater treatment proposed prototype and those of different commercial plant, which includes nitrifying processes. The TAN equipment to measure TAN, classified into three groups: concentration was found between 1 and 124 mg L−1 NH4+, potentiometric probes, flow potentiometric equipment, and depending on the sample origin. No statistical differences spectrophotometric equipment. Broadly, it can be seen that This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. were found from the graphical representation (n = 65, 95% probe-type potentiometric devices show high portability and a confidence) of the results for each method: [NH4+]Ref = 1.01 wide working range with acceptable accuracy and repeatability. (±0.03) [NH4+]Analyzer + 1 (±2); R 2 = 0.9782, and in accordance However, in all cases, they require sample pretreatment, they with the paired t-test (tcalc = 1.934, ttab = 2.000, tcalc < ttab). have a low level of automation and autonomy, and in some Fig. 4B shows the results from the analysis of 25 samples cases, they present important interferences from other from different stages of the MELiSSA project pilot plant. The monovalent cations. Regarding continuous flow potentiometric Open Access Article. Published on 19 October 2023. Downloaded on 12/4/2023 10:28:02 AM. TAN concentration was found between 4 and 700 mg L−1 NH4+, equipment, it presents wide working ranges, good accuracy depending on the sample origin. No statistical differences were and repeatability and a high level of automation and found from the graphical representation (n = 25, 95% autonomy. However, it lacks portability because of its size and confidence) of the results for each method: [NH4+]Ref = 1.00 weight, it requires in most cases a sample pretreatment (mainly (±0.02) [NH4+]Analyzer −1 (±6); R 2 = 0.9972, and in accordance filtration), and in some cases there are interferences from other with the paired t-test (tcalc = 0.138, ttab = 2.064, tcalc < ttab). species. Interferences in potentiometric devices are usually Finally, 6 spiked tap water samples gave results shown in corrected by simultaneously measuring ammonium and Table 2. The obtained recoveries range from 98% to 106%. As interferent ions using an array of electrodes and applying the can be seen, the TAN concentration for the unspiked sample Nernst–Nikolsky equation. This considerably increases costs is under the ISE linear range (less than 0.075 mg L−1 NH4+); and the complexity of the system. Finally, spectrophotometric however, this concentration is below the maximum equipment shows narrower working ranges, which can be concentration allowed by the legislation in tap water (0.5 mg widened by the use of different kits of reagents, and both L−1 NH4+). Therefore, the system can be used to monitor TAN accuracy and repeatability are worse than those with in fresh or drinking water even below the maximum potentiometric equipment. These systems are not portable, concentration allowed by legislation. sample pretreatment (filtration or dilution) is required in In order to verify that the analyzer maintains its analytical general, and there are interferents, either from the sample features unaltered in real operation environments, a simulation color or other compounds. Regarding automation and of TAN monitoring in a wastewater treatment plant was autonomy, these depend on the equipment. performed. For this, successive analyses of a real sample The proposed prototype overcomes the presented provided by a surfactant manufacturing industry and kept drawbacks, showing the best balance in terms of cost, under constant agitation were carried out without any sample portability, linear range, accuracy and repeatability, without pretreatment (neither filtration nor biomass precipitation). the need for sample pretreatment, with the absence of After 7 hours of continuous monitoring, carrying out 4 analysis interfering compounds and showing a high level of per hour, a mean concentration (n = 26, 95% confidence) of 54 automation and autonomy. ± 4 mg L−1 NH4+ was obtained, thus showing the analyzer robustness in a real operation environment. Conclusions All these results demonstrate the appropriateness of the developed analyzer for TAN determination in a very wide There are some commercial ammonium analyzers which range of sample matrices and concentrations under meet some specific requirements for TAN analysis in a unattended conditions fulfilling all the required analytical particular type of water (drinking water, fresh water or urban and operational features. or industrial wastewater). However they are expensive and still lack enough portability, automation, autonomy, versatility and selectivity as a whole to be adapted to any type Comparison with commercial equipment of water source, situation and TAN concentration, without There are not many commercial systems only dedicated to prior sample treatments. ammonium analysis. Table 3 shows the features of the We focused the present work on addressing these issues, procuring a low cost and highly versatile and portable analyzer prototype for the in situ measurement of TAN, which fulfils Table 2 Sample analysis from spiked tap water of Cerdanyola del Vallès. Concentration mean values in mg L−1 NH4+ (n = 3, 95%) features such as autonomy, automation and low sample and reagent consumption, thus enabling continuous monitoring Sample Added Found % Rec with low maintenance requirements. Miniaturization of the 1 0 <0.075 — equipment also aligns with a greener chemistry with the goal 2 0.1 0.11 ± 0.01 106 of generating less waste. Moreover, the system operates in a 3 0.2 0.20 ± 0.01 99 wide or adaptable working range to face different types of water 4 0.3 0.31 ± 0.02 104 5 0.4 0.39 ± 0.02 98 sources with even complex matrices, providing reliable results 6 0.5 0.51 ± 0.05 102 in real time. Through different automatic sequences of This journal is © The Royal Society of Chemistry 2023 Environ. Sci.: Water Res. Technol., 2023, 9, 3366–3376 | 3373 View Article Online Paper Environmental Science: Water Research & Technology Table 3 Features of different commercial equipment and the proposed analytical prototype. Information extracted from the commercial brochure of each equipment found on the website of each manufacturer Analytical device Mass LOD LR Sample Autom. Auton. (manufacturer) Technique (kg)/volume (dm3) (mg L−1 NH4+) (mg L−1 NH4+) Acc./repeat. pret. Int. level (month) This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Developed in this Potentiometry 5/19.6 0.03 0.08–1000 2%/2% No No High 2 work (flow) (at least) Aquaprobe (Aquaread) Potentiometry 2/2.4 — 0–9000 10%/− Yes Yes Low — (probe-type) (only probe) Ammonia probe 8002 Potentiometry −/0.3 — 0.1–1000 −/2% Yes No Low — (ABB) (probe-type) (only probe) Orion ammonia Potentiometry −/0.05 — 0.01–17 000 −/2% Yes No Low — Open Access Article. Published on 19 October 2023. Downloaded on 12/4/2023 10:28:02 AM. (ThermoFisher) (probe-type) (only probe) AN-ISE sc (HACH) Potentiometry 2.4/1.8 0.2 0–1000 5%/− Yes Yes Low — (probe-type) (only probe) Ammo::lyser™ Potentiometry 2.7/1 — 0.1–1000 3%/− Yes Yes Low — eco/pro (S::CAN) (probe-type) (only probe) AMTAX™ sc Potentiometry 31/151 0.02 0.02–1000 5%/2% Yes Yes High 3 ammonia (HACH) (flow) (different ranges) Aztec AAM631 (ABB) Potentiometry 15/43.7 — 0.05–1000 7.5%/2% No No High 3 (flow) (different ranges) PowerMon ionometer Potentiometry 60/234 — 0–1000 −/5% Yes Yes High 0.75 (Bran + Luebbe) (flow) DR6000 (HACH) Spectrophotometry 11/49.5 0.028 0.01–1800 −/8% Yes Yes Low — (discrete analysis) (different ranges) Aztec 600 (ABB) Spectrophotometry 15/43.3 — 0–0.529 5%/5% Yes Yes High 4 (flow) (undiluted range) WIZ probe (Systea) Spectrophotometry 8/8 0.001 0–0.5 10%/3% Yes Yes Medium 1 (probe-type) Ez series (HACH) Spectrophotometry 25/106 0.005 0.025–100 −/2% Yes Yes High — (flow) (different ranges) Acc.: accuracy, repeat.: repeatability, pret.: pretreatment, Int.: interferences, Autom.: automation, Auton.: autonomy. operation, a total linear range from 0.08 to 1000 mg L−1 NH4+ Conflicts of interest and a detection limit of 0.03 mg L−1 can be achieved, with high repeatability and reproducibility. In addition, by means of the There are no conflicts to declare. integration of an automatic gas-diffusion stage, all the potential interferences present in the sample matrix are Acknowledgements avoided and the lifetime of the electrode, and consequently that of the global system, is considerably extended because the The authors are grateful to Evonik Industries and the sample matrix is not in contact with the ISE membrane. MELiSSA project pilot plant for the collaboration in the The whole system is comparable in size to a shoe box and its analyzer validation process providing samples and contrast portability, along with the rest of the mentioned characteristics, analysis. The authors are grateful to the Spanish Ministry of constitutes the differential features and substantial Science and Innovation and the Catalan government for their improvement with respect to existing commercial equipment for financial support through the projects CTQ2012-36165, TAN determination. 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