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Introduction

Since the middle of the 19th century, Nessler’s reagent has been widely applied for the detection as well as for the determination of ammonia. The sensitivity offered by this method is comparable with those reported in the analytical literature.1 Contrary to many other approaches this method is simple (one reagent only, no need of heating), inexpensive and fast. Only the reagent toxicity is considered as a drawback. In spite of this disadvantage, the Nessler’s reagent method is still widely applied in biochemical2 and biological3 research, environmental studies (soil3,4 and water5–7 analysis) and mineralogy8 and is also used as a reference method for newly developed sensors9 and methods10,11 for ammonia determination.

Nessler’s reagent is composed of potassium tetraiodomercurate (II) dissolved in an alkaline solution of potassium hydroxide. The reagent allows ammonia detection in a very short time. In a few seconds it forms an orange soluble product that slowly precipitates as a brown deposit. The reaction can be summarized by the following equation:

NH3 + 2HgI42– + 3OH– → OHg2NH2I + 7I– + 2H2O (1)

The Nessler’s method of ammonia determination was adapted to the flow injection analysis (FIA) format,12,13 although the problem of deposition of the reaction product inside the FIA manifolds was still troublesome. In this short communication we present a modern, miniaturized and economic flow system based on the current trend of multicommutation/multipumping in analytical spectrometry.14 The system is based on low-cost

microsolenoid devices14–16 and extremely low-cost flow-through optoelectronic detector fabricated according to the paired light emitting diodes concept.17,18 In the developed flow system, the above mentioned problem of precipitate deposition has been eliminated.

Experimental

Nessler’s reagent (product No. 72190) was obtained from Fluka Analytical (Sigma-Aldrich). For all experiments reported in this work, the commercially available reagent was 20- to 200-fold diluted with water. All other reagents of analytical grade were obtained from POCh. Double-distilled water was used throughout.

The developed detector was operated according to paired emitted detector diode (PEDD) principle.17,18 Each combination of 395, 400, 405, 430, 470 and 475 nm LEDs (5 mm diameter, transparent lens, flat front, 40° view angle) from the same manufacturer (Optosupply, Hong Kong) was tested. In agreement with earlier findings,12,13 that for detection of the initially formed soluble orange product a wavelength of near 400 nm is recommended, the highest sensitivity was obtained for the pair: 395 nm LED and 405 nm LED, used as a light emitter and a detector, respectively. These two LEDs were integrated in the compact form of a flow cell (70 μL total volume and 10 mm optical pathlength). The fabrication details of flow-through photometric PEDDs are given elsewhere.19 The LED emitter was powered with stable currents using a low-voltage circuit based on L272 chip obtained from TME (Poland). The analytical signal (the electromotive force generated by illuminated LED-based detector)18 was measured and recorded with UNI-T multimeter (Model UT70B, China) operating in the voltmeter mode and connected with a data

2014 © The Japan Society for Analytical Chemistry

† To whom correspondence should be addressed.E-mail: rkoncki@chem.uw.edu.pl

A Novel Optoelectronic Detector and Improved Flow Analysis Procedure for Ammonia Determination with Nessler’s Reagent

Kamila KOŁACINSKA*,** and Robert KONCKI*†

* University of Warsaw, Department of Chemistry, Pasteura 1, 02-093 Warsaw, Poland ** Institute of Nuclear Chemistry and Technology, Dorodna 16, 03-195 Warsaw, Poland

This paper presents a simple, rapid and effective method for ammonia determination in a flow analysis regime using Nessler’s reagent. The proposed modification of the common flow procedure results in the total elimination of the problem of precipitate deposition inside the flow manifold. The improved procedure has been adapted to a flow analysis system based on microsolenoid pumps combined with a dedicated optoelectronic detector fabricated specifically for this purpose. This photometric device has been constructed in the form of a compact flow-through cell (70 μL total volume and 1 cm optical pathlength) integrated with 395 nm LED emitter and 405 nm LED-based detector. The presented analytical system is capable of ammonia determination in the submillimolar concentration range with a detection limit below 0.1 mM and high throughput (over 20 injections per hour).

Keywords Ammonia, optoelectronic detector, flow analysis, microsolenoid pumps

(Received July 22, 2014; Accepted August 29, 2014; Published October 10, 2014)

Notes

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storage PC using RS232 interface.The preliminary experiments were performed using a common

double-channel FIA system consisting of a peristaltic pump (Model Minipuls 3 from Gilson, France), a manual rotary injection valve (Model 5020 from Rheodyne, USA) and 0.8 mm internal diameter Teflon tubing (Cole-Palmer, USA). For further research, the flow system based on microsolenoid pumps (operating with 2 Hz frequency and indicated stroke volume of 10 μL, product No. 120SP1210-4TE), and microsolenoid valve (product No. 100T3MP12-62-5) has been developed. These devices were purchased from Bio-Chem Fluidics (Boonton, USA). The operating of the microsolenoid manifold was programmed and controlled by a PC using the KSP Measuring System (Poland). As previously, the flow manifold was arranged using of a PTFE Microbore tubing (0.8 mm internal diameter).

Results and Discussion

In the course of preliminary experiments, the detector was tested in the conventional double-channel FIA system (Fig. 1A) based on a peristaltic pump (applied flow rate 1.9 mL min–1). The injection valve (125 μL) was located in water line. The second line was accounted for delivering 50-fold diluted Nessler’s reagent. The ammonia standard was injected into a flowing carrier stream that moved it downstream and mixed with the reagent after a Y-shaped connector in a 70 cm mixing coil located before the detector. The recording for consecutive injections of 0.5 mM ammonia standard is shown in Fig. 1B. The obtained peaks were very high (the signals exceeded 1000 millivolts and were limited by almost total discharge of LED-based detector).18 Unfortunately, the stepwise changes of the baseline after each injection were distinctly observed. They were caused by the deposition of the reaction product on the inner walls of the FIA system (particularly inside of the window

of the PEDD detector). The same problem has been reported in earlier communications.12,13 To solve the problem of reaction product accumulation inside the system, the use of poly(vinyl alcohol)12 or potassium sodium tartrate13 in a carrier as agents preventing precipitate deposition has been recommended. Unfortunately, these attempts were unsuccessful. The protectives added to the water carrier only reduced and did not eliminated changes of the baseline after consecutive standard injections, as it is shown in Fig. 1B.

The precipitate can be easily removed from the FIA manifold by its washing with acidic solutions. For such purpose, the basic flow system was extended to include a second valve, located in Nessler’s reagent carrier (Fig. 1A), which introduced the segment of 0.1 M HCl. The flow procedure was modified so that after each peak registration, the acidic washing with HCl segment using the second valve was performed. As shown in Fig. 1C (where the first two injections were recorded according to the above procedure and the drift of the baseline is observed and further injections were preceded by washing with HCl) this approach has brought the expected results. The registration can be clearly seen throughout the procedure: firstly, the ammonia is injected and the sample flows through the detector causing peak formation. After this peak the signal does not return to the initial baseline. Then the second valve is switched to begin the washing step with the segment of acid and the signal value will rise. After this washing segment, Nessler’s reagent is flowing and therefore the signal is slightly changed. At this moment, the real baseline is defined by the reagent absorbance and the next injection of analyte is possible. The sequence of absorbance changes is illustrated in Fig. 1D. Hereby, the baseline in the course of consecutive injections is stable over time.

The improved flow analysis procedure can be effectively performed in the systems based on modern, economic and independently PC-controlled microsolenoid devices.14–16 The developed multipumping flow analysis (MPFA) manifold is

Fig. 1　FIA manifold based on peristaltic pump (A) and recordings of analytical signal obtained in the course of consecutive injections of 0.5 mM ammonia standard without (B) and with (C, D) washing of FIA system with 0.1 M hydrochloric acid between analyte injections.

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shown in Fig. 2A. The MPFA system contains an additional valve (V) coupled with pump P1 applied as a module allowing calibration with the use of a single standard. The operation principle of this V-P1 module is reported in detail elsewhere.20 As the micro-pumps can operate independently, the MPFA system allows saving of Nessler’s reagent, which is pumped only when a segment of calibrant (or sample) is introduced. After each simultaneous injection of reagent and calibrant, 0.1 M HCl follows prevent precipitate deposition. Consequently, contrary to the former FIA system (Fig. 1), in this case the baseline is defined by the absorbance of hydrochloric acid solution. Therefore, in the absence of ammonia, a blank signal (peaks) for the reagent is registered. Real value of the blank signal is marked in Fig. 2B with red lines.

The calibrations performed at different PEDD currents and Nessler’s reagent concentrations (Fig. 2B) confirm that the problem of baseline drift in flow systems has been solved. The corresponding calibration graphs are shown in Fig. 2C. The lower detection limit is defined by the absorbance of Nessler’s reagent, whereas, the upper detection limit is caused by a total discharging of weakly illuminated LED-detector. The observed shift of calibration graph with PEDD supplying current stays in agreement with previous findings and is discussed in detail elsewhere.18 The analysis of the obtained results (Fig. 2C) leads to the conclusion that the most applicable choice is to use 2% Nessler’s reagent and 20 mA PEDD current. Under such conditions, the MPFA system allows determination of ammonia in the submillimolar range of concentration with a detection limit of about 0.1 mM. For higher concentrations of Nessler’s reagent, a lower detection limit is observed (Fig. 2C) althought

the blank peaks are higher. The sample throughput offered by this flow system is 22 injections per hour. Although the analytical application is not the main goal of this contribution, the developed PEDD-MPFA system has been applied for urinary ammonia determination. Ammoniuria21 is a strong predictor of liver cirrhosis, kidney damage, acidosis and urinary tract infection, thus urinary ammonia level is an important marker in clinical diagnostics. The obtained results (Table 1) clearly indicate the utility of the developed system for real analytical applications.

Fig. 2　Multipumping flow manifold (A), the readings of calibrations (B) preformed at different concentrations of Nessler’s reagent and currents powering the detector and the corresponding calibration graphs (C). Upper envelope of the signal corresponds to the baseline.

Table 1　Results of urine analysis using the developed system and reference method (manual Berthelot method)22

Sample No.Urinary ammonia/mM

(developed method)Urinary ammonia/mM

(reference method)

1234567

3.9 ± 0.2 5.8 ± 0.2 5.9 ± 0.2 9.2 ± 0.315.4 ± 0.329.5 ± 0.638.6 ± 0.4

4.4 ± 0.3 5.7 ± 0.4 5.6 ± 0.311.9 ± 0.915.8 ± 0.629.2 ± 0.838.3 ± 0.8

The urine samples were 10- or 100-times diluted with water depending on analyte concentration.

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Conclusions

In this short communication, we present a simple flow system for ammonia determination based on computer-controlled economic microsolenoid pumps, an ordinary voltmeter and an extremely low-cost optoelectronic detector (the total cost of device is lower than one Euro). The presented PEDD-MPFA system is robust and compact and requires low currents for operation, thus it can be applied as a portable analyzer. This dedicated flow system is applicable for the determination of ammonia in the submillimolar range with a high sample throughput and a low consumption of inexpensive reagents. Worth noting is that the analytical parameters offered by the developed flow analysis system meet the requirements of several specific biomedical measurements. A study is currently underway on the real bioanalytical application of the developed detector and system for clinical needs (namely for the control of hemodialysis therapy).23

Acknowledgements

Helpful comments on this work from Prof. Marek Trojanowicz are kindly acknowledged. Kamila Kołacinska, a Ph.D. student, acknowledges the support from the Institute of Nuclear Chemistry and Technology. This study was supported by the Polish National Science Center (Project Opus NCN No. 2011/01/B/NZ5/00934).

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