Sequential Injection Analysis for Automation of the ...



Sequential injection analysis for automation of the Winkler methodology, with real-time SIMPLEX optimization and shipboard application

Burkhard Horstkotte1,*, Antonio Tovar1, Carlos M. Duarte1, Víctor Cerdà2

1 Department of Global Change Research. IMEDEA (CSIC-UIB) Institut Mediterráni d'Estudis Avançats, Miquel Marques 21, 07190 Esporles, Spain

2 University of the Balearic Islands, Department of Chemistry

Carreterra de Valldemossa km 7,5, 07011 Palma de Mallorca, Spain

Abstract:

A multipurpose analyzer system based on sequential injection analysis (SIA) for the determination of dissolved oxygen (DO) in seawater is presented. Three operation modes were established and successfully applied onboard during a research cruise in the Southern ocean: 1st, in-line execution of the entire Winkler method including precipitation of manganese(II) hydroxide, fixation of DO, precipitate dissolution by confluent acidification, and spectrophotometric quantification of the generated iodine/tri-iodide (I2/I3-), 2nd, spectrophotometric quantification of I2/I3- in samples prepared according the classical Winkler protocol, and 3rd, accurate batch-wise titration of I2/I3- with thiosulfate using one syringe pump of the analyzer as automatic burette.

In the first mode, the zone stacking principle was applied to achieve high dispersion of the reagent solutions in the sample zone. Spectrophotometric detection was done at the isobestic wavelength 466 nm of I2/I3-. Highly reduced consumption of reagents and sample compared to the classical Winkler protocol, linear response up to 16 mg L-1 DO, and an injection frequency of 30 per hour were achieved. It is noteworthy that for the offline protocol, sample metering and quantification with a potentiometric titrator lasts in general over 5 min without counting sample fixation, incubation, and glassware cleaning. The modified SIMPLEX methodology was used for the simultaneous optimization of four volumetric and two chemical variables. Vertex calculation and consequent application including in-line preparation of one reagent was carried out in real-time using the software AutoAnalysis. The analytical system featured high signal stability, robustness, and a repeatability of 3 % RSD (1st mode) and 0.8 % (2nd mode) during shipboard application.

Keywords:

Sequential injection analysis, Dissolved Oxygen, Winkler protocol, Seawater Monitoring, modified SIMPLEX method, AutoAnalysis

Fax: + 34 971 173 426, email: Burkhard.Horstkotte@uib.es

1 Introduction

The concentration of dissolved oxygen (DO) in seawater is an essential property used to characterize water masses and derive ocean circulation [1] and ecosystem metabolism [2], and assess the risk of hypoxia of the various water masses [3].

To this aim, the Winkler method [4] is routinely used in oceanographic research, specially where high analytical precision and independence from ambient pressure, temperature, or ion strength of the sample are required [5-7].

Although Clark-type electrodes and fluorescence-based optochemical oxygen sensors (oxygen optrodes) are available to measure oxygen concentration in seawater, their performance is only comparable to that of high-precision Winkler measurements. By example, the both approaches are generally affected by membrane / sensor bio-fouling, the requirement of a constant approach flow to the membrane, temperature and pressure dependency, and considerable delay times to reach steady state signal. Non-linearity of the calibration function, costs, photo-bleaching of the sensor layer, and limited lifetime are typical shortcuts of oxygen optrodes [8,9] but can be overcome by adequate protective layers on the sensor [10]. Use of cyclic voltametry for DO quantification renders high robustness but lengthens also the measurement procedure [11]. Both sensor types are adaptable to in-situ measurements and DO depth profiling.

However, amperiometric sensors are inadequate for incubation experiments due to the consumption of dissolved oxygen by the sensor itself. On the other hand, invasive optodes have to be used during the entire incubation experiment although only the integral consumed DO is of interest mostly. In consequence the number of processed samples is limited by the number of purchased optodes.

The Winkler method is based on the quantitative oxidation of manganese (II) hydroxide precipitate by DO in alkaline media followed by the reduction in presence of iodide in acidic media. The coupled reduction of iodide to iodine can be determined by titration with thiosulfate either using starch as visual end-point indicator or assisted by potentiometry. Alternatively, iodine can be quantified directly by spectrophotometry with improvements reported in respect of manual labor, time of analysis, and reliability [12,13]. With a surplus of iodide, iodine (I2) forms the tri-iodide ion (I3-) [14], which has led to the consideration to perform spectrophotometric quantification of I2/I3- at their isosbestic wavelength [15] for an improved reliability. Main disadvantages of the Winkler method are lengthy times for reaction and determination, considerable reagent consumption, and a large and precisely metered sample volume.

Analytical flow techniques present an elegant way for the application of wet-chemistry procedures for monitoring, i.e. semi-continuous, automated proceeding by simultaneous reduction of reagent consumption and sample handling. Therefore, they are widely used where fast, economic, reliable, and automated analysis of a large number of similar samples is demanded.

In spite of these potential advantages, mainly the quantification of I2/I3- originated from the classical Winkler method has been automated applying by example spectrophotometry [16], amperometry [17], or indirect spectrofluorimetry [18]. Only in two works, fully [19] and semi-automated [20] implementation of the entire Winkler chemistry in complex, multichannel flow injection analyzers (FIA) [21] has been described. In both analyzers, peristaltic pumps were used as liquid drivers implying the well-known shortcomings of flow rate drift, high solution throughput, and laborious optimization by manual manipulation of the manifold.

Among the distinct flow techniques extensively described in recent reviews [22-25], Sequential Injection Analysis (SIA) proposed by Ruzicka and Marshall in 1990 [26] ranks as one of the most simple, economic, and versatile flow techniques, given by the multitude of analytical applications [27-28]. It uses only one single, pressure-robust syringe pump module for the precise handling of minute volumes of the required solutions, aspirated from the lateral ports of a selection valve and introduced into a flow detection cell. Any required procedures including mixing of sample and reagents and incubation are carried out following a software-based instruction protocol, which can easily be adapted using distinct operation schemes for each analytical parameter.

In this work, we describe a SIA automation of the entire Winkler reaction (precipitation of manganese (II) hydroxide, reaction with dissolved oxygen in the sample, precipitate dissolution, and generation and spectrophotometric detection of I2/I3-). Determination of I2/I3- in samples prepared according the classical Winkler method was further enabled by either batch titration with thiosulfate or by spectrophotometry obtaining an efficient, multipurpose analyzer. Due to the linear operation of SIA, zone stacking of reagents and sample is imperative, which is in contrast to FIA, where merging flows of sample and reagents is a usual concept. To enable acid addition after the oxidation of the precipitate, the SIA was extended by an additional syringe using a multisyringe device as liquid propelling device described in detail elsewhere [29-31]. To achieve the maximal reaction volume of sample with the precipitate formed in-line by the mixture of the used reagents manganese sulfate and alkaline iodide solution, thorough optimization of especially the volumetric variables affecting the precipitate formation and sample penetration was required.

Computer assisted optimization such as by the modified SIMPLEX method [32,33] provides a highly useful tool to find an optimum of the variables effecting the performance of an analytical instrument. This potential has been demonstrated for flow technique-based analyzers before [34-36], however, considerable user's work has still been required for SIMPLEX optimization such as preparation of reagents and manual modification of the tubing manifold. In contrast, Gine et al. presented for the first time in 1998 [37] real-time SIMPLEX optimization of a multicommutated FIA system [38,39]. Here, the calculated variables of each vertex were directly applied to the operation scheme of the solenoid valves implemented in the analyzer manifold.

There is nevertheless a paucity of real-time application of SIMPLEX to flow technique systems, one reason being the availability of versatile control programs able to control instruments typically used in flow techniques such as pumps, valves, and detectors, of on-line data evaluation, and perform the required calculations. A second bottleneck is that the variables of interest need to show interdependence. Finally, the information deficit about the robustness of the optimum and the presence of other optima has to be acknowledged as a main drawback of SIMPLEX optimization, which requires a posterior univariant study of each variable for the validation of the found optimum.

In this work, we applied the modified SIMPLEX methodology for the real-time optimization of four volumetric variables and two concentrations of one in-line prepared reagent of a SIA system using the software AutoAnalysis. The optimized and characterized analyzer was used for the determination of DO in seawater samples during a research cruise in the Southern Ocean

2 Materials and methods

2.1 Reagents and standards

All chemicals were of analytical-reagent grade (Scharlab, Barcelona, Spain) and for the preparation of the overall solutions distilled water was used for all laboratory experiments and MilliQ water during shipboard application. A solution of 0.3 mol L-1 MnSO4 was used as a reagent 1 (R1). A solution of 0.6 mol L-1 NaI and 0.2 mol L-1 NaOH was used as a reagent 2 (R2). Reagent 3 (R3) being 0.3 mol L-1 H2SO4 was propelled by syringe 2 and a carrier being 0.05 mol L-1 H2SO4 was propelled by syringe 1. The reagents were not sparged with nitrogen prior to use for practical reasons.

For the classical Winkler protocol the following reagents were prepared and used according the standard protocol [6]: 3 mol L-1 MnSO4 (R1c), 4 mol L-1 NaI and 8 mol L-1 NaOH (R2c), and 5 mol L-1 H2SO4 (R3c). KIO3 was dried at 105°C overnight and used to prepare solutions of 0.023 mol L-1 and 0.0023 mol L-1. A 0.2 mol L-1 Na2S2O3 solution was prepared as a titrant of I2/I3- and standardized against 10 mL of 0.0023 mol L-1 KIO3, previously mixed with 1 mL of R3c and R2c. A 30 w/v % starch solution in glycerol was used as visible I2/I3- end-point indicator for titration. For modified SIMPLEX optimization, stock solutions of NaI and NaOH of 2 mol L-1 each were used.

Artificial seawater (ASW) as detailed elsewhere [5] by dissolving the following compounds in distilled water in the given order 3 mg L-1 NaF, 20 mg L-1 SrCl2·6 H2O, 30 mg L-1 H3BO3, 100 mg L-1 KBr, 700 mg L-1 KCl, 1,470 mg L-1 CaCl2·2H2O, 4,000 mg L-1 Na2SO4, 10,780 mg L-1 MgCl2·6 H2O, 23,500 mg L-1 NaCl, 20 mg L-1 Na2SiO3·9 H2O, and 200 mg L-1 NaHCO3.

For calibration of the in-line Winkler method, about 280 mL ASW solutions of different DO contents were measured on the proposed analyzer. Standards were prepared by sparging of ASW with nitrogen, air, or oxygen for at least 15 min, which were found to yield steady-state gas saturation. Intermediate contents of DO were obtained by mixing of saturated ASW in a Winkler bottle, which was closed and shaken vigorously hereafter. The objective was not to prepare DO standard of known but different concentrations to be quantified by the classical method. Oxygen was generated in the laboratory by the catalytic decomposition of H2O2 (about 15 %v/v) continuously added to an initial volume of KMnO4. After in-line analysis, the unknown contents of DO of each standard were determined by application of the classical Winkler titration protocol [6]. For this, Winkler bottles of approximately 120 mL were filled carefully with the standards and 0.5 mL of R1c and R2c were added. The bottles were sealed, shaken vigorously, and incubated for at least 1 h in the darkness. Afterwards, 0.5 mL of R3c was added and the formed I2/I3- was quantified by titration.

For the spectrophotometric determination of I2/I3-, calibration standards of 100-800 µmol L-1 DO were prepared by appropriate addition of 0.023 mol L-1 KIO3 to about 280 mL ASW, where 1 µmol of KIO3 corresponds to 1.5 µmol DO. Afterwards, 1 mL of R2c and 1 mL of R3c were added, by which iodate is reduced quantitatively to I2/I3- [6,16].

2.2 Flow analyzer instrumentation

The implemented SIA system is depicted in Fig. 1-A wherein tubing dimensions are indicated. A valve module VA2 equipped with two rotary 8-port selection valves and a syringe module Bu4S [29-31] purchased from Crison Instruments S.A. (Allela, Barcelona, Spain) were used. The syringe module was equipped with two glass syringes of 5 mL (S1) and 1 mL (S2) total dispense volume (Hamilton Bonaduz AG, Bonaduz, Switzerland), driven simultaneously by the single step motor of the instrument (16000 steps, 24 s – 1024 s for total dispense). Solenoid head-valves allow the connection of the syringes either to the manifold (ON) or to the respective carrier reservoir (OFF) for re-filling. Both modules were connected in series via a RS232C serial interface to a PC for remote software control.

All liquid contacted parts were made of the chemical resistant polymers ETFE, PMMA, PEEK, and PTFE. The central port of the selection valve was connected via the holding coil HC1 to the position ON of the syringe head valve. About 6 cm from the selection valve, a 3-way connector was integrated into HC1 used as a confluence of R3 (in-line addition of acid).

A thermostatization coil (TC) was used to connect the selection valve and the detection flow cuvette. It was inserted into a homemade reactor flushed continuously with water provided from a miniature precision thermostat PT31 (Krüss Optoelectronic GmbH, Hamburg, Germany). The PT31 was further used for the thermostatization of the flow cuvette holder using a processor water-cooler copper shoe (Conrad, Hamburg, Germany).

An USB-2000 miniature spectrophotometer was used for detection and was directly connected to a cuvette support CUV-UV (both Ocean Optics Inc., Dunedin, USA) with a flow cuvette, type 75.15 SOG from Starna (Essex, UK). Dual wavelength detection was accomplished throughout for compensation of the schlieren effect [40] using the isosbestic wavelength 466 nm of I2/I3- [41] as detection wavelength and processed by subtracting the absorbance at a reference wavelength of 580 nm. A homemade light source was used for all measurements consisting of a halogen bulb and a 383UBC LED (390 nm - 510 nm, range < 20 % emission intensity) from Roithner Lasertechnik, Vienna, Austria. Both were arranged perpendicular such that the light emitted of the LED passed the bulb and both spectra were superposed achieving in approximation uniform emission spectra.

2.3 Software AutoAnalysis and Integration of SIMPLEX methodology

The software package AutoAnalysis 5.0 () [42] was used for instrumental control, data acquisition, and evaluation. The basic program is written in Delphi and provides tools for in-line data evaluation, use of variables, basic calculations, loops, conditional inquiries, and enable a modular setup of the instruction method by definition of procedures. Instrumental control was done via specific dynamic link libraries establishing the communication to the individual instrumental assembly.

The modified SIMPLEX method programmed on the AutoAnalysis 5.0 platform enabled the optimization of up to seven variables, six used in the present work. It enabled the calculation of the initial SIMPLEX from a given center point and SIMPLEX diameter. Three stop criteria were established: maximal optimization cycles, maximal repetition of the best vertex, and minimum threshold of the achieved improvement. It automatically calculated a new vertex either by normal reflection, expansion, external contraction, or internal contraction including a correction if one or more parameters of the new vertex passed prior defined working range limitations. It finally enabled the permutation of the second worst vertex if permutation of the worst vertex led to an even worse result.

2.4 Analytical protocols

The analytical protocol for the in-line execution of the Winkler method is given in the supplement materials, No 1. To achieve a high dispersion of the reagents in the sample zone and already in the holding coil, the zone stacking principle was applied by aspiration of three volumes of the same sample intercalating small volumes of R1 and R2 (VR1, VR2). The aspiration order was 1750 µL (VS1) of sample, 125 µL of R1, 100 µL of sample (VS2), 125 µL of R2, and 400 µL of sample (VS3). By this procedure, the penetration of VR1 and VR2 and consequently the formation of the manganese(II) hydroxide precipitate did mainly proceed during the aspiration of VS3 leading to enhanced dispersion. After 40 s, during which the precipitate could react with DO leading to the manganese oxyhydroxide and the syringes were refilled from the reservoirs in head-valve position OFF. Finally, the composite zone was dispensed towards the detector. At the confluence, the precipitate dissolves by the merging flow of R3 from syringe 2 under simultaneous formation of I2/I3-.

Using the acidic carrier, an additional protocol enabled the quantification of I2/I3- in samples processed off-line by the classical Winkler method. For this, a volume of 800 µL of sample was aspirated, propelled through the thermostatization coil into the detection flow cell, and the absorbance value was measured in stopped flow over an averaging time of 5 s in order to compensate fluctuations of the light source intensity. During this time, the remaining sample in HC1 was discharged to waste. Finally, the detection cell was flushed with carrier for cleaning.

Syringe 1 could further be used as an automatic burette for the quantification of I2/I3- of off-line processed samples by titration with sodium thiosulfate. A software protocol was established for the dispense of user-defined volumes between 4 mL and 1 µL including the automated refilling of the syringe whenever required, counting the total consumed volume of the titration agent, and, if required, cleaning the syringe with the titrant and at the beginning and end of the procedure, respectively.

2.5 Real-time modified SIMPLEX optimization

For the in-line execution of the Winkler protocol, the five concentrations of reagents and carrier, five aspiration volumes, and the reaction time had to be optimized. Leaving apart the reaction time, these variables were interdependent. For example, a higher concentration of NaOH in R2 requires a higher concentration of R3 for dissolution of the precipitate; by a larger VS2, VS3 has to increase in order to enhance the dispersion and achieve penetration of R1 and R2. Due to the complexity of this interdependence, the modified SIMPLEX method was used for the optimization of VS2, VS3, VR1 and VR2 and the concentrations of NaOH and NaI in R2. For this, the volumes of interest were defined by variables, which values were calculated and directly applied by the used software.

In order to enable in-line preparation of R2, the analyzer system was expanded by a third glass syringe of 10 mL, using water as carrier (see figure 1-B). Syringe 3 was connected via a holding coil HC2 to the central port of the second selection valve with an open and continuously stirred mixing chamber of 4 mL volume as described elsewhere [43,44] and stock solutions of NaOH and NaI of 2 mol L-1 each positioned on its lateral ports. The mixing chamber was further connected to a lateral port of the first selection valve. For each optimization experiment, 1 mL of R2 was prepared automatically using the calculated volumes of the stock solutions (VOH, VI) and a replenishing volume of water. After application of the so-prepared reagent, the mixing chamber was emptied and cleaned. For this, the remaining reagent was aspirated by syringe 3 and dispensed to waste. Then the mixing chamber was filled with water from syringe 3 (carrier), which was again aspirated and discharged to waste.

Since simultaneous optimization of all influencing variables was not possible, the following preliminary considerations were made. To mimic the addition of manganese in the classical Winkler protocol [6] with a final concentration of about 30 mmol L-1, the concentration of R1 was affixed to 0.3 mol L-1 estimating a dispersion factor of at least 10 for R1 in HC1. Considering a similar dispersion factor for R2, a stochiometric ratio of Mn2+:OH-:I-:H+ of 1:2:3:4, and a merging flow ratio of sample and acid of 5:1, a concentration of 0.5 mol L-1 for R3 guaranteed a surplus of hydronium ion downstream the confluence. In order to complete the optimization within one day and evaluate the influence of the variables of interest on the reaction kinetic, a reaction time of only 5 s was applied. The sample volume was fixed by applying VS1 = 2.5 mL – (VS2 + VS3 + VR1 + VR2). The variation of VS1 in function of VR1 and VR2 was done to avoid the overfilling of the holding coil and to favor SIMPLEX progress to small reagent volumes. In order to suppress the formation of manganese precipitate in the carrier and by this to avoid the alteration of the signal height by the DO content of the carrier, 0.1 mol L-1 H2SO4 was used as sample carrier.

The SIMPLEX optimization started from the initial point 50, 200, 150, 250, 150, 300 [µL] (VOH-, VI-, VR2, VR1, VS2, VS3) and a SIMPLEX diameter of 80 %. For optimization, N2 saturated, continuously sparged ASW and air saturated ASW where measured in duplicate obtaining the average signal heights Abs(N2) and Abs(Air). To avoid sole optimization of the methods sensitivity and evolution to high blank values and reagent consumption, 20 % of the blank were subtracted from the sensitivity. Consequently, a lower blank value at constant sensitivity would be evaluated still as a better result. The used response function was “Abs(Air) – 1.2 x Abs(N2)”.

Finally, in order to avoid, that errors during the execution of the optimization experiments such as false peaks caused by air bubbles could affect the SIMPLEX evolution, the user had to confirm the use of each data set (four peaks) or else, order the repetition of the last experiment by binary input (Yes/No).

2.6 Shipboard application

The analyzer system was tested, under true operating conditions, on board of the Spanish oceanographic research vessel Hespérides during a research cruise in the Antarctic Peninsula sector (Belinghausen Sea, Bransfield Strait and Weddell Sea)of the Southern Ocean) in February 2009. Seawater was sampled, down to 200 to 1,000 m depth, depending on the studied area, from a SBE 32 carousel water sampler from Sea-Bird Electronics Inc. (Washington DC, USA) equipped with 24 Niskin bottles of 12 L each from OceanTest Equipment Inc. (Fort Lauderale, FL, USA) combined with a multiparameter SeaBird 9 CTD sensor registering, among others, temperature, pressure, salinity and dissolved oxygen. Surface water was sampled using a 30 L Niskin bottle from the same company. Glass Winkler bottles of about 280 mL and 110 mL were used for sampling for both the in-line and classical Winkler method. The bottles were filled from the Niskin bottles via a silicon tube (50 cm, 1 cm i.d.), were let overflow by about twice their volume, and were closed bubble-free. For the classical Winkler protocol, oxygen was fixed following the standard method (see section 2.1).

3 Results and Discussion

3.1 SIMPLEX optimization of in-line Winkler method

Optimization with the modified SIMPLEX method proceeded efficiently using the response function Abs(Air) – 1.2 x Abs(N2). Optimization of the sole sensitivity corresponding to the response function Abs(Air) – Abs(N2) showed to lead to increasing sensitivity but also to an unacceptably high blank value of > 1 AU. On the other side, increasing the weight of the blank value to 1.3 caused SIMPLEX evolution to decreasing sensitivity. Only twice, experiments had to be repeated by user demand due to the presence of air bubbles, which had caused false peak height evaluation. Initial and final vertex data including start and stop conditions are given in table 1.

The optimization stopped automatically after 7 initial and following 32 cycles due to repetition of the best vertex 10 times. During automation, all SIMPLEX progress modes had been successfully applied.

The parameter set of the vertex in cycle 26 was chosen as the highest sensitivity was achieved. The optima of the volumetric variables were verified by univariant study of each variable with results given in table 2. In 3 of 4 cases, the optima found with the modified SIMPLEX method could be confirmed, thus proving the efficiency of the procedure. However, a larger VS3 showed to improve the sensitivity due to enhanced dispersion of the manganese (II) hydroxide precipitate in the sample zone.

It is noteworthy that SIMPLEXS optimization allowed approaching an ideal starting parameter set for univariant study with a minimum of experiments. Performing the optimization in real-time was of high advantage since the labor and time for preparation of reagent 2 as well as for modification of the volumes of interest in the software protocol fall upon and the SIMPLEX optimization could be terminated in even less than 7 hours (about 10 min for reagent preparation, performance of four measurements, and cleaning of the mixing chamber afterwards).

3.2. Selection of chemical and physical variables for in-line Winkler method

Since higher sensitivity was achieved by increasing VS3, a further study of the remaining variables was done including the reaction time and the sulfuric acid concentrations of R3 and the carrier. The experiments were started from the former found optima 125 µL of R1, 190 µL of R2, 100 µL of VS2, 300 µL of VS3, and 0.2 mol L-1 NaOH and 0.4 mol L-1 NaI of R2.

The influence of the volume VR2 on the analytical response was studied over the range of 100 to 275 µL for concentrations of NaOH and NaI of 0.2 mol L-1 and 0.4 mol L-1, respectively. Experimental results are depicted in figure 2-A. Increasing VR2 led to higher peaks for both N2 and air saturated ASW but decreasing sensitivity. To use the entire absorbance range (up to 1 AU) for the DO concentration range of interest (0 – 400 µmol L-1), a volume of 125 µL R2 was chosen.

Testing the linearity of the method up to 9 mg L-1, it turned out, that iodide was in limiting concentration. Therefore, the iodide concentration was heightened to 0.6 mol L-1, without any significant alteration of the method’s sensitivity but gaining linearity up to 16 mg L-1. Though the isosbestic wavelength of I2/I3- was chosen for detection, a higher iodide concentration further warranted the presence of iodine as tri-iodide and by this, offered the possibility of reliable detection at shorter, more sensitive wavelengths.

The influence of the sulfuric acid concentration of R3 on the analytical response was studied over the range of 0.2 to 1 mol L-1. Experimental results are depicted in figure 2-B. While no significant influence on the peak height of N2 saturated ASW was observed, the peak height of air saturated ASW decreased nearly linear with increasing acid concentration. To ensure the complete dissolution of the precipitates at a minimal consumption of acid, a concentration of 0.3 mol L-1 H2SO4 was chosen.

The influence of the reaction time on peak height was studied for 5, 10, 20, 40, and 80 s. Experimental results are depicted in figure 2-C. The peak heights of N2 saturated ASW increased nearly linear with time, whereas for air and oxygen saturated ASW, considerable decelerations of the increments were observed with increasing time. Since the observed improvement passing from 40 s to 80 s was low, a reaction time of 40 s was chosen as a compromise between time of analysis and sensitivity. The increase of the blank value was likely due to diffusion of oxygen through the PTFE tubing walls into the sample. So, a longer reaction time would mainly lead to a lower affection of the ambient temperature due to a more progressed reaction but not to a significant increase of sensitivity.

The influence of the sulfuric acid concentration of the carrier on the analytical signal was studied over the range of 0.025 to 0.3 mol L-1. Experimental results are depicted in figure 2-D. While no significant influence on the peak height of N2 saturated ASW was observed, the peak height of air saturated ASW clearly decreased slightly with higher acid concentration. As a compromise between sensitivity and the requirement to suppress the formation of manganese precipitate in the carrier, a concentration of 0.05 mol L-1 was chosen.

The influences of the volumes VS2 and VS3 on the analytical readout were studied over the range of 0 to 150 µL and 100 to 400 µL, respectively. Experimental results are depicted in figure 3. Increasing VS2 led to lower peak heights for N2 saturated ASW while for air saturated ASW a maximum was found for 50 µL. The highest sensitivity was achieved at a volume of 100 µL, which was therefore applied further on. A higher VS3 led to increasing sensitivity due to a higher dispersion of the manganese (II) hydroxide precipitate in the sample zone and consequently a larger reaction volume of sample. Therefore, a volume of 400 µL was chosen; larger volumes were not tested since the achieved sensitivity allowed to cope with the desired working range of 13 mg L-1.

As former reported [19], a brown coating of the inner tubing walls of the holding coil by manganese oxyhydroxide was observed. This for one proved the fully separation of the acidic carrier from the reaction composite zone but affected both reproducibility and the method's sensitivity. In order to proceed a cleaning of the holding coil prior to sample analysis, a small volume of R2 (50 µL) was aspirated prior to VS1. Applying this step, the precipitate dissolved immediately at the penetration zone of R2 and the acidic carrier. An aspiration step – former performed with the syringe head-valve in position OFF - was required nevertheless in order to overcome the backlash of the syringe module and improve the precision of the following aspiration step, so did require additional time.

3.3 Analytical performance of the in-line Winkler method

The entire analytical method required 120 s, where 40 s were for the oxidation time of the manganese precipitate. A sample frequency of 30 full analyses per hour is therefore achievable during monitoring but is slightly reduced by a cleaning protocol for the sample channel when discrete samples are measured. The high sample frequency was enabled by carrying out mixture of sample and reagents and precipitate oxidation already in the holding coil.

Linearity was proven up to 16 mg L-1 (480 µmol L-1) following a calibration function of 0.020 L mg-1  + 0.15 AU (see supplementary materials, No 3). The relative standard deviation (RSD) of the blank was generally ................
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