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Automated Determination of HydrogenPeroxide at the Micro-Molar Level inRainwater and Snow Using a Stopped-Flow Approach in a Hybrid SequentialInjection/Flow Injection ManifoldDr. Paraskevas D. Tzanavaras a & Euridiki Boulimari aa Department of Chemistry, Laboratory of Analytical Chemistry ,Aristotelian University of Thessaloniki , Thessaloniki , GreeceAccepted author version posted online: 19 Mar 2012.Publishedonline: 11 Jun 2012.

To cite this article: Dr. Paraskevas D. Tzanavaras & Euridiki Boulimari (2012) AutomatedDetermination of Hydrogen Peroxide at the Micro-Molar Level in Rainwater and Snow Using a Stopped-Flow Approach in a Hybrid Sequential Injection/Flow Injection Manifold, Analytical Letters, 45:9,1086-1097, DOI: 10.1080/00032719.2012.670798

To link to this article: http://dx.doi.org/10.1080/00032719.2012.670798

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Flow Injection Analysis

AUTOMATED DETERMINATION OF HYDROGENPEROXIDE AT THE MICRO-MOLAR LEVEL INRAINWATER AND SNOW USING A STOPPED-FLOWAPPROACH IN A HYBRID SEQUENTIAL INJECTION/FLOW INJECTION MANIFOLD

Paraskevas D. Tzanavaras and Euridiki BoulimariDepartment of Chemistry, Laboratory of Analytical Chemistry, AristotelianUniversity of Thessaloniki, Thessaloniki, Greece

A new automated method is reported for the determination of H2O2 in real samples. The

method is based on the quenching effect of the analyte on the reaction between

tris(2-carboxyethyl)phosphine (TCEP) and Ellman’s reagent (DTNB). All necessary

steps were accomplished under flow conditions using a hybrid sequential injection (SI)/flow

injection (FI) setup. The sensitivity was enhanced by applying a stopped-flow step (120 s)

in order to promote the reaction between H2O2 and TCEP. The proposed analytical proto-

col was validated for linearity (10–75lmolL�1), limits of detection (cL¼ 1.0lmolL�1),

quantitation (cQ¼ 3.3lmolL�1), precision (sr¼ 1.3–1.7%), accuracy, and selectivity. It

was then applied successfully to the analysis of H2O2 in spiked rainwater and snow samples.

Keywords: Ellman’s reagent; Hybrid sequential injection=flow injection; Hydrogen peroxide;

Stopped-flow; Tris(2-carboxyethyl) phosphine

INTRODUCTION

Hydrogen peroxide (H2O2) is one of the most important oxidants of the tropo-sphere contributing to the control of the chemical composition of the atmosphere attrace gas levels (Sakugawa et al. 1990). Acidity of rainwater has become one of themain environmental problems and topic for several studies. Themain factors contribu-ting to the acidity of rainwater is the concentration of anions such as nitrates and,more significantly, sulfates (Bravo et al. 2000). Sulfates in the atmosphere can be cre-ated by a reaction of SO2 with H2O2 in the gas phase particularly at pH values lowerthan 4.5–5.0 (Goncalves et al. 2010). The monitoring of H2O2 in environmental=atmospheric samples is therefore important as it can be directly connected to criticalpollution phenomena. The levels of H2O2 vary significantly as can be derived from

Received 8 July 2011; accepted 25 October 2011.

Address correspondence to Dr. Paraskevas D. Tzanavaras, Laboratory of Analytical Chemistry,

Department of Chemistry, Aristotelian University of Thessaloniki, GR-54124, Thessaloniki, Greece.

E-mail: ptzanava@chem.auth.gr

Analytical Letters, 45: 1086–1097, 2012

Copyright # Taylor & Francis Group, LLC

ISSN: 0003-2719 print=1532-236X online

DOI: 10.1080/00032719.2012.670798

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numerous reports from around the world; levels up to as high as 70–80mmolL�1 havebeen measured with average values being 40mmolL�1 (Gulf of Mexico), 13mmolL�1

(Western Atlantic Ocean), 28mmolL�1 (Florida), and 26mmolL�1 (Central AtlanticOcean) while similar or even higher averages were observed in Brazil (Deng andZuo 1999; Yuan and Shiller 2000; Goncalves et al. 2010; Jacob et al. 1990).

During the last decade there have been several methods reporting the determi-nation of H2O2 in rainwater and=or snow samples. The main analytical figures ofmerit of these methods (mode, principle, detection, LOD, range, and sampling rate)are included in more detail in Table 1S of the supplementary material. From a detec-tion point of view the previously reported analytical protocols for the determinationof H2O2 can be divided in UV-vis (Mathew, Pillai, and Gupta, 2009; Chang et al.2009; Luo et al. 2008; Zhuang et al. 2008; Matos et al. 2006; Zheng et al. 2006),fluorimetric (FL) (Luo et al. 2010; Tang, Zhang, and Xu 2006), amperometric(Paixao and Bertotti 2008; Matos, Pedrotti, and Angnes 2001), and chemilumino-metric (CL) (Yang, Guo, and Mei 2009; Tahirovic et al. 2007; Marle and Greenway2005; Kiba et al. 2003; Li, Zhang, and Zhao 2001). A popular approach seems to bethe use of peroxidases that catalyze enzymatically the reaction of H2O2 with varioussubstrates such as 4-aminoantipyrine (Matos et al. 2006), tricarbochlorocyanine dye(Tang et al. 2006), and the luminol-H2O2 CL system (Kiba et al. 2003; Yang et al.2009). The use of enzymes in solutions increases the cost of the analysis, while immo-bilization on solid supports (e.g., on magnetic beads (Yang et al. 2009) or ionexchange resins (Matos et al. 2006)) increases the complexity of the methods by bothinvolving additional preparation steps and by raising reactor-stability issues. Aninteresting alternative to enzymatic procedures for the determination of H2O2 mightbe the use of substances that mimic the catalytic activity of peroxidases. BiFeO3 andFe3O4 nanoparticles (Luo et al. 2010; Chang et al. 2009; Zhuang et al. 2008) andmyoglobin (Zheng et al. 2006) were reported to have such behavior. It seems thatthe main disadvantage of these methods is the slow reaction rate—compared toenzymes—requiring several minutes for completion [10min at 40�C (Zheng et al.2006)–60min at 37�C (Zhuang et al. 2008)]. The same drawback stands fornon-catalytic batch methods based on the oxidation of leucocrystal violet (Mathewet al. 2009) and methyl orange (Luo et al. 2008). Although quite sensitive, theyrequire manual handling of the analytical procedure and several minutes for colordevelopment. Batch CL sensors are rather unattractive for routine analysis mainlydue to the difficulties of reproducible handling and monitoring fast CL reactionsunder non-flow conditions (Tahirovic et al. 2007). Finally, when it comes to theapplication of electroanalytical methods to the analysis of real samples the foulingof the active surface of the electrodes is always a concern. For example, electrodesmodified with ruthenium oxide hexacyanoferrate were reported to be stable for only48 h despite the operation under flow conditions (Paixao and Bertotti 2008). Practi-cal applicability concerns might become even more evident when electroanalyticalapproaches are combined with immobilized enzymes (Matos et al. 2001).

Sequential injection analysis (SI) is considered as the second generation ofautomated flow-injection techniques and its advantages over traditional FI arewell-documented: (1) single-channeled manifolds, (2) minimization of reagentsconsumption, (3) computer-controlled operation, and (4) enhanced reproducibilityduring stopped-flow experiments. However, SI suffers on the automation of multistep

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reactions due to inadequate mixing of the zones in the holding coil and competitiveactions upon stacking. A viable solution is the development of hybrid FI=SImanifolds, where, for example, the first step of a process is carried out using SI andthe second proceeds under FI conditions, combining in this way the advantages ofboth techniques. Representative examples of the effective implementation of suchconfigurations are the determination of glucose by CL (Panoutsou and Economou2005) and of sulfite in wines by gas-diffusion (Tzanavaras, Thiakouli, and Themelis2009).

In the present study we propose an automated flow method for the determi-nation of H2O2 at the micro-molar level. The method is based on an alternative‘‘chemistry,’’ that is, on the quenching effect of the analyte on the cleavage of theS-S disulfide bond of the Ellman’s reagent (DTNB, Fig. 1S in the supplementarymaterial) by tris(2-carboxyethyl)phosphine (TCEP, Fig. 2S in the supplementarymaterial) (Han et al. 1996). The developed protocol is automated, avoids the use ofenzymes and other type of catalysts, is simple, straightforward, and uses commer-cially available reagents. Especially, TCEP is considered to be one of the most prom-ising reducing agents of S-S bonds as it offers critical advantages in terms of rapidityunder wide pH range, stability in aqueous solutions, and safe handling (odorless)(Thermo Scientific 2011). The two-step chemical process of the developed analyticalprotocol was automated effectively by using a hybrid SI=FI system. Reaction betweenH2O2 and TCEP is carried out in SI under stopped flow conditions achieving repro-ducible timing and minimizing the consumption of the reducing agent; reactionbetween the remaining TCEP and DTNB was accomplished under FI conditionsachieving adequate on-line mixing. The applicability of the analytical procedurewas demonstrated by analysis of real rainwater and snow samples.

EXPERIMENTAL

Instrumentation

The hybrid SI=FI setup is depicted schematically in Fig. 1A. It is comprised ofthe following parts: a micro-electrically actuated 10-port valve (Valco, Switzerland);a Gilson (Minipuls3, France) peristaltic pump equipped with Tygon tubes (for the SIpart); a milliGAT (GlobalFIA, U.S.) bidirectional pump (for the FI part); aSPD-10AV UV-Vis detector (kmax¼ 412 nm) (Shimadzu, Japan); a FIAStar 5101(Tecator, Sweden) thermostat was employed to control the temperature of the reac-tion coil (RC); all necessary connections including the holding (HC) and reactioncoils (RC) were made of PTFE tubing (0.5mm i.d.).

The control of the system was performed through a program developed inhouse using LabVIEW 5.1.1 (National Instruments, U.S.), while the Clarity software(DataApex, Czech Republic) was used for data acquisition (1mV¼ 1mA.U.).

Reagents and Solutions

All reagents were of analytical grade, while de-ionized water produced by aMillipore system was used throughout this study.

Tris(2-carboxy ethyl)phosphine (TCEP) was supplied by Sigma. The stocksolution of the reducing reagent was prepared in water at a concentration of

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10mmol L�1 and was found to be stable for at least one month if kept refrigeratedand protected from the light. Working solutions were prepared daily either in wateror buffer.

The stock solution of the Ellman’s reagent (DTNB, Sigma) was prepared at aconcentration of 1mmol L�1 by dissolution in a suitable buffer (10mmol L�1 TRIS,pH¼ 7.5) and was also stable for at least one month. Working solutions wereprepared daily by appropriate dilutions of the stock in water.

The concentrated hydrogen peroxide solution (30% w=w, Merck) was standar-dized weekly by potassium permanganate titration. Working solutions were pre-pared daily in water by serial dilutions of the stock.

The pH of the buffer solution (75mmol L�1 TRIS, Sigma) was regulated bysuitable amounts of 1.0mol L�1 HCl or NaOH.

Synthetic rainwater was prepared according to the literature by analyticalgrade reagents (Merck or Sigma) (Quevauviller et al. 1998), filtered through0.45-mm membrane filters and kept refrigerated. A table with the detailed compo-sition of the synthetic matrix can be found in the supplementary material (Table 2S).

Figure 1. Schematic depiction of the hybrid SI=FI setup: C¼ carrier (water); PP¼ peristaltic pump;

HC¼holding coil; MPV¼multiposition valve (low pressure); R=TCEP (250mmolL�1)=buffer

(75mmolL�1 TRIS, pH¼ 9.0); S¼ sample; W¼waste; MGP¼miliGAT pump; DTNB¼ c(DTNB)¼200mmolL�1; RC1¼ reaction coil (100 cm=0.5mm i.d.); TS¼ thermostat; RC2¼ reaction coil (30 cm=

0.5mm i.d.) D=UV-Vis detector (kmax¼ 412nm).

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Procedure for Aqueous Solutions

The typical analytical cycle can be described by the following steps (a detailedtable can be found in the supplementary material as Table 3S):

1. 25 mL of the reducing reagent (c(TCEP)¼ 250 mmolL�1) is ‘‘sandwiched’’between two zones of sample (V¼ 100 mLþ 50 mL) in the HC of the SI setup.

2. The reaction mixture is propelled (QV¼ 0.6mLmin�1) to the first reaction coil(RC1: 100 cm=0.5mm i.d.) and is left to react for 120 s at 40�C under stopped-flowconditions.

3. The reaction mixture is subsequently merged with a continuous flowing of DTNB(c¼ 200 mmolL�1, QV¼ 0.2mL min�1) and the remaining TCEP reduces DTNBon passage through a second reaction coil (RC2: 30 cm=0.5mm i.d.).

4. UV-Vis detection was carried out downstream at 412 nm (see Fig. 3S insupplementary material).

Based on a 300 s long cycle, the sampling rate was estimated to be 12 h�1. Threereplicates were made in all instances, while peak height was used for data acquisition(representative peaks can be found in the supplementary material as Fig. 4S).

Samples Preparation

Rainwater and snow samples were collected from the campus of the AristotelianUniversity of Thessaloniki and the center of the city of Thessaloniki during December2010–January 2011 and September–October 2011. After collection, the samples werefiltered through 0.45 membrane filters and kept refrigerated at �18�C until analysis(carried out as soon as possible within 2–3 h from collection (Ceron, Muriel, andCeron 2004)). After spiking with suitable amounts of H2O2 the samples were analyzedby the proposed automated method without any other pretreatment.

RESULTS AND DISCUSSION

Preliminary Studies

First, a series of preliminary experiments was carried out in order to investigatethe linearity of the reaction between DTNB and TCEP under SI conditions. In atypical three-zone SI experiment, 50 mL each of various concentrations of TCEP(5–100 mmolL�1), buffer (pH¼ 7.0 and 8.0), and DTNB (100 and 200 mmolL�1)were aspirated in this order into the HC. The reaction mixture was propelled towardsthe detector at a flow rate of 0.6mL min�1 through a 100-cm long reaction coil (aschematic depiction of the SI setup used in these series of experiments can be foundin the supplementary material as Fig. 5S). The best linearity (r¼ 0.9999) and highestsensitivity (slope¼ 10.764mV�1) was achieved up to 75 mmolL�1 TCEP using200 mmolL�1 DTNB at pH¼ 8.0 (supplementary material Fig. 6S).

A second series of experiments was carried out in order to investigate the quench-ing effect of H2O2 on the TCEP-DTNB reaction (Han et al. 1996). Using the same SIconfiguration mentioned in the previous paragraph [c(TCEP)¼ 75mmolL�1,c(DTNB)¼ 200mmolL�1, pH¼ 8.0), elevated concentrations of the analyte were

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mixed off-line with TCEP and were left to react for 10min. The experiments confirmedthat the reaction between TCEP and H2O2 was fast and practically stoichiometric.Excellent linearity (r¼ 0.9999) was obeyed up to 70mmolL�1 H2O2 with a negativeslope of �10.451mV�1 (supplementary material Fig. 7S).

Based on the aforementioned promising results, the next step was to try toautomate both reactions—namely between TCEP–H2O2 and TCEP–DTNB—underflow conditions. Unfortunately, our initial efforts failed to produce satisfactoryresults in terms of sensitivity. This phenomenon was attributed to insufficient mixingbetween the zones of the sample and TCEP in the holding coil and to limited reactiontime before mixing with the third zone (DTNB) that caused initiation of a competi-tive reaction. After several trials with various configurations, the most viable sol-ution to the effective automation of this two-step chemical process proved to bethe modification of the typical SI setup and the development of a hybrid SI=FI alter-native (Fig. 1A). The main idea was to carry out the reactions independently, that isto use SI for the first step in order to take advantage of its unique characteristics interms of handling and strictly controlled operations (e.g., stopped-flow) and to carryout the second step under FI conditions by continuous flow of DTNB.

Compared to our initial experiments, the effective mixing of the H2O2 andTCEP zones was achieved by: (1) adaptation of a ‘‘sandwich’’ approach, that is, bybracketing the zone of the reducing reagent (25 mL) by two zones of H2O2 (2� 50 mL)and (2) the forward movement of the reaction mixture toward the first reaction coil(RC1) that promotes overlapping compared to stacking of the zones in the holdingcoil (Fig. 1B). Using this alternative design the reaction between TCEP and DTNB(200 mmolL�1, QV¼ 0.2mLmin�1) was linear up to 250 mmolL�1 TCEP. This con-centration was selected as a starting point for further studies. It should be noted thatanother modification was the preparation of the reducing reagent and the buffer(75mmol L�1 TRIS, pH¼ 8.0) in the same vial. The stability of TCEP in the bufferfor at least a full working day was confirmed experimentally.

Method Development

In order to come up with the finalized conditions, the effect of several instru-mental and chemical parameters was investigated. These included the stopped-flowtime, the pH, the concentrations of TCEP and DTNB, the sample injection volume,the temperature of the first reaction coil (RC1), and the length of the second reactioncoil (RC2). During all experiments the volume of TCEP was kept at 25 mL, the flowrate of DTNB at 0.2mLmin�1 and the length of RC1 at 100 cm. The investigatedparameters, the studied range and the final selected values can be found in Table 1.

To establish the most effective conditions in terms of reaction time betweenH2O2 and TCEP two stopped-flow protocols were tested and compared. Thefirst approached involved stopped-flow in the holding coil (HC) immediately afteraspiration of the zones and the second involved stopped-flow in the first reaction coil(RC1). The experimental results are shown in Fig. 2. As can be clearly seen in Fig. 2,stopping the flow in the reaction coil resulted in enhanced sensitivity in terms of DAin all cases. This phenomenon is explained by the more efficient overlapping of theH2O2 and TCEP zones upon propulsion to the reaction coil. Regarding the effect ofthe stopped-flow time, an almost linear increase in sensitivity was observed. A ca.

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80% increase in DA was achieved in the range of 0–120 s. The latter time was selectedfor further studies making a compromise between sensitivity and sampling rate.

The pH also had a critical effect on the sensitivity of the determination. As canbe seen in Fig. 3, it was studied in the range of 7.0 to 10.0 by regulating the pH of thereducing reagent with suitable TRIS buffers (75mmol L�1). Although the values ofthe blank signals remained practically unaffected in the range of 8.0–9.0, higherquenching (DA) was achieved at pH¼ 9.0. The latter value was therefore selectedfor subsequent experiments.

The effect of the concentrations of TCEP and DTNB were investigated simul-taneously. Four concentrations of TCEP (100, 150, 200, 250 mmolL�1) were com-bined with four concentrations of DTNB (100, 200, 300, 400 mmolL�1), for a totalof sixteen experiments. The concentration of H2O2 was 50 mmolL�1 in all cases. Tak-ing into account the sensitivity and the consumption of the reagents—especially ofDTNB that flows continuously—the best results were obtained with the combinationof 250 mmolL�1 TCEP=200 mmolL�1 DTNB.

Table 1. Overview of the study of instrumental and chemical variables

Variable Studied range Selected value

Instrumental

qV(C)=mLmin�1 — 0.6

qV(DTNB)=mLmin�1 — 0.2

tSF=s 20–180 120

V(H2O2)=mL 50–200 150

V(TCEP)=mL — 25

l(RC1)=cm — 100

l(RC2)=cm 30–90 30

Chemical

T=oC 25–60 40

pH 7.0–10.0 9.0

c(TCEP)=mmol L�1 100–250 250

c(DTNB)=mmol L�1 100–400 200

Figure 2. Effect of the stopped-flow time on the sensitivity of the determination (DA): RC1¼ stopped-flow

in the reaction coil (RC1); HC¼ stopped flow in the holding coil.

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The sample volume is an important parameter that has significant impact onthe sensitivity of flow-based methods. On this basis the effect of the analyte volumewas studied in the range of 50–200 mL using 50 mmolL�1 H2O2. In all cases, the‘‘sandwich’’ approach depicted in Fig. 1B was followed. The sensitivity (DA)increased almost linearly in the range of 50–150 mL and remained leveled-off there-after. A sample injection volume of 150 mL (100þ 50) was therefore selected.

The effect of the length of the second reaction coil (RC2) was investigated inthe range of 30–90 cm (0.5mm i.d.). This coil determines the reaction time betweenTCEP and DTNB. No significant effect was observed in the studied range indicatingfast reaction kinetics. The value of 30 cm was therefore selected. It should be notedthat the length of the first reaction coil (RC1) was not studied and was set at 100 cm(0.5mm i.d.), since the reaction time between H2O2 and TCEP is determined by thestopped-flow time.

Finally, the effect of the temperature of the first reaction coil (RC1) was inves-tigated in the range of 25–60�C. The experiments showed minimal effect of the tem-perature on the blank signals. On the other hand, the sensitivity (DA) was improvedby ca. 20% in the range of 25–40�C. The temperature of 40�C was therefore selectedfor further experiments.

Analytical Figures of Merit

The proposed SI=FI method was validated in terms of linearity, limits of detec-tion (cL) and quantitation (cQ), precision, and accuracy=selectivity.

Linearity was found in accordance in the range of 10–75 mmolL�1 H2O2. Theregression equation was:

A ¼ �7:076 ð�0:059Þ � cðH2O2Þ þ 712:1 ð�2:5Þ

where A is the absorbance as measured by the detector and c(H2O2) is theconcentration of the analyte in mmolL�1. The regression coefficient was >0.999and the percent residuals ranged between �0.8% and þ1.2%, and as can be seenin Fig. 4 were distributed randomly along the ‘‘zero’’ axis.

Figure 3. Effect of the pH on the determination of H2O2 by the proposed method.

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The limits of detection (cL) and quantitation (cQ) were estimated by the follow-ing equations (Hayashi et al. 2005):

cL ¼ 3� si=a and cQ ¼ 10� si=a

where si is the standard deviation of the intercept and a is the slope of the regressionline. The calculated values were cL¼ 1 mmolL�1 (34 mgL�1) and cQ¼ 3.3 mmolL�1

(110 mgL�1).The precision was evaluated by replicate analysis of both blank samples and

standard H2O2 solutions at the 20 mmolL�1 level (n¼ 8). The relative standarddeviations (sr) ranged between 1.3 and 1.7% in all cases.

The accuracy and selectivity of the proposed method were validated by prepar-ing a synthetic rainwater sample matrix according to (Quevauviller et al. 1998), thatis, also in agreement with other more recent literature reports (Salve et al. 2008).Analysis of the unspiked synthetic rainwater matrix produced signals equal to theaqueous blank ones, indicating absence of positive or negative interferences. The sel-ectivity was further verified by analysis of a pooled sample of real rainwater collectedat various time frames during September=October 2011 (n¼ 6). The blank sampleswere left standing at room temperature for at least 48–72 h to ensure completedecomposition of H2O2, mixed to create a pooled matrix and analyzed. No varia-tions from the aqueous blanks were observed. The accuracy was validated by analy-sis of spiked synthetic rainwater in the range of 10–50 mmolL�1 H2O2. The percentrecoveries were satisfactory in all cases, ranging between 97–105% (Table 4S,supplementary material).

Analysis of Snow and Rainwater Samples

The developed SI=FI automated method was applied to the analysis of realrainwater and snow samples collected in the area of Thessaloniki during winter2010=2011 and autumn 2011. All samples were analyzed by both the standardaddition technique (5 concentration levels in the range of 10–70 mmolL�1) and

Figure 4. Graphical depiction of the aqueous calibration curve and the distribution of the residuals

(insert).

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external aqueous calibration. As mentioned in the Experimental section, all sampleswere practically analyzed immediately after collection in order to avoid decompo-sition and loss of the analyte (Ceron et al. 2004). The experimental results are shownin Table 2.

For accuracy evaluation, all samples were spiked with elevated concentrationsof H2O2 in the range of 10–30 mmolL�1 and also were analyzed by both the standardaddition technique and external aqueous calibration. As can be seen in Table 3, forthe rainwater samples both external calibration and the standard additions techniqueproduced accurate results with the percent recoveries in the range of 95.9–104.6%.

Table 3. Recovery experiments from the analysis of H2O2 in real samples

Recovery (%)

H2O2 added (mmolL�1) External calibrationa Standard addition

Rainwater 1 (1=2011)

10 96.5 102.1

20 100.8 98.7

30 101.1 99.2

Rainwater 2 (9=2011)

10 102.5 98.5

20 100.6 102.9

30 104.7 102.4

Rainwater 3 (9=2011)

10 97.3 102.5

20 104.6 103.9

30 103.0 96.0

Rainwater 4 (10=2011)

10 98.4 95.9

20 99.6 100.8

30 101.9 102.1

Snow (12=2010)

10 — 95.3

20 — 97.7

30 — 96.9

aUsing aqueous solutions.

Table 2. Determination of H2O2 in real samples

H2O2 found (mmol L�1)

Sample External calibrationa Standard addition

Rainwater 1 (1=2011) n.d.b n.d.b

Rainwater 2 (9=2011) 13 (�2) 15 (�3)

Rainwater 3 (9=2011) 42 (�3) 38 (�3)

Rainwater 4 (10=2011) 19 (�2) 22 (�2)

Snow (12=2010) n.d.b n.d.b

aUsing aqueous solutions.bNot detected.

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On the other hand, snow samples were analyzed accurately only by the standardadditions approach due to matrix effect. The recoveries ranged between 95.3 and97.7%.

CONCLUSIONS

In the proposed analytical method we automated under flow conditions atwo-step reaction for the determination of H2O2 in real samples. The hybrid SI=FI setupenabled the development of a sensitive approach that allowed the analysis of H2O2 atthe low micro-molar level and at an acceptable sampling rate of 12h�1. The consump-tion of the reducing reagent (TCEP) was only 6.25 nmol per run, while the consumptionof the low-cost Ellman’s reagent was 250 nmol per run. The developed method wasapplied successfully to the determination of H2O2 in rainwater and snow samples.

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