Analytica Chimica Acta 401 (1999) 191–196

Workplace monitoring of gas phase hydrogen peroxide by means offluorescence spectroscopy

Jörg Meyer, Uwe Karst∗Westfälische Wilhelms-Universität Münster, Anorganisch-Chemisches Institut, Abteilung Analytische Chemie, Wilhelm-Klemm-Str. 8,

D-48149 Münster, Germany

Received 5 February 1999; received in revised form 31 May 1999; accepted 13 June 1999

Abstract

A novel method for workplace monitoring of gas phase hydrogen peroxide is described. It is based on the dimerization ofp-hydroxyphenylacetic acid (p-HPA) by hydrogen peroxide in the presence of peroxidase as catalyst. The fluorescent dimerof p-HPA is quantified by means of fluorescence spectroscopy. The use of a microplate fluorophotometer allows for highsample throughput. Gas phase sampling was carried out with impingers containing different types of sampling media. Thenew method meets easily with the required sensitivity for gas phase monitoring of H2O2 in an industrial environment. Fora 10 l air sample, the linear range of calibration covers the concentrations from 0.04 to 6 times the maximum allowableconcentration (MAK) value of 1 ppmv (1.4 mg/m3), thus allowing for workplace monitoring of the analyte. This method isvalidated with a photometric method using 2,2′-azino-bis(3-ethylbenzothiazoline)-6-sulfonate diammonium salt) (ABTS) asreagent. ©1999 Elsevier Science B.V. All rights reserved.

Keywords:Hydrogen peroxide; Fluorescence; Workplace monitoring

1. Introduction

Hydrogen peroxide has gained an increasing im-portance as disinfectant or bleaching agent in variouscommercial, industrial or clinical applications due toits environmentally beneficial properties. The high ox-idation potential of this substance which is exploitedhere implements also its hazards with respect to humanhealth. Therefore, consequent monitoring of work-place exposure to gaseous hydrogen peroxide is obvi-ously needed. This is especially true for H2O2 produc-

∗ Corresponding author. Tel.: +49-251-8333-182; fax: +49-251-8333-169E-mail address:uwe.karst@uni-muenster.de (U. Karst)

ers or in places where high concentrations of H2O2are used for disinfection or cleaning purposes.

Although a large number of methods for the de-termination of hydrogen peroxide in aqueous solu-tions has been described in literature, only compara-bly few methods for gas phase analysis of the analyteare known.

Methods for the determination of hydrogen perox-ide frequently make use of the oxidative propertiesof the substance as well as of selective formation ofmetal peroxo complexes. For gas phase determinationof hydrogen peroxide, several methods are reported.For industrial workplace monitoring, metal complexformation with subsequent photometric quantifica-tion is the most common procedure up-to-date [1–3].The formation of binary and ternary complexes of

0003-2670/99/$ – see front matter ©1999 Elsevier Science B.V. All rights reserved.PII: S0003-2670(99)00488-2

192 J. Meyer, U. Karst / Analytica Chimica Acta 401 (1999) 191–196

titanium and vanadium species has been describedand has found widespread analytical use in combi-nation with photometric detection of the colouredcomplexes. The major drawback of photometricmethods is their lack of sensitivity, especially whenshort sampling periods are required to obtain a goodtemporal resolution of the data. The advantages ofthe photometric methods are robustness and simpleinstrumentation.

In atmospheric chemistry, thep-hydroxyphenylaceticacid (p-HPA) method is established for ultratracedetermination of various peroxides [4–7].p-HPA isdimerized in the presence of the enzyme peroxidaseby peroxides with concentrations in the lower ppbrange. The peroxides are first separated under cool-ing on a liquid chromatography (LC) system, beforefluorescence detection is performed after post col-umn derivatisation. Alternatively, some authors use aFIA set-up with less resolution of the peroxo species.However, the procedures are laborious and requireinstrumentation which is not readily available in thetypical analytical laboratory of industrial hydrogenperoxide applicants.

Recently, an amperometric method has been pro-posed by Dasgupta et al. [8]. Gaseous H2O2 is sam-pled with a nafion membrane diffusion scrubber andanalysed by oxidative pulse amperometry in a minia-ture flow-over sensor [8]. The reported LOD for thismethod is 0.11 ppbv hydrogen peroxide. However, theinstrumentation is too complex for ready to use anal-ysis and the applications for this method conferringto the analytical parameters are rather atmosphericthan indoor measurements at typical workplaceconcentrations.

Up-to-now, the fluorimetric determination usingp-HPA has not found access to workplace monitoringdue to complex procedures required for sampling andfluorescence measurement. The goal of this work wasto merge the advantages of the known photometricand fluorimetric methods to provide high sensitivityand reasonably short sampling times using simple andreliable instrumentation, thus providing the industrialuser of H2O2 with a readily available and easy to usemethod for rapid determination of the substance. Theconcentration range of interest which is considerablyhigher than in atmospheric chemistry, for this purposeis between a few hundred ppbv and the low ppmvregion.

2. Experimental section

2.1. Chemicals

p-HPA was purchased from Fluka (Deisenhofen,Germany) in the highest purity available. It wasrecrystallised twice from water. When dissolvedfor the first time, activated carbon was addedand the hot suspension was filtered. The resultingwhite crystals were stored under nitrogen at 4◦C.2,2′-Azino-bis(3-ethylbenzothiazoline)-6-sulfonatediammonium salt (ABTS) (98%) was from Sigma(Deisenhofen, Germany) and used without furtherpurification. It was stored in a freezer at−18◦C.Horseradish peroxidase (HRP) Type I (EC 1.11.1.7,RZ 1,3, 116 purpurogallin units/mg) was purchasedfrom Sigma. Hydrogen peroxide with a concentrationof 35% and peroxyacetic acid (PAA, 32%) were fromAldrich Chemie (Steinheim, Germany). Acetonitrilewas LiChroSolv gradient grade from Merck (Darm-stadt, Germany). All other reagents were from Merckin the highest quality available.

2.2. Microplate spectrophotometer

For photometric determinations, the microplatespectrophotometer Spectra Max 250 and software SoftMax Pro, version 1.1 from Molecular Devices (Sun-nyvale, CA, USA.) were used. The absorbance wasread at wavelengths of 405 and 732 nm, respectively.

2.3. Microplate fluorophotometer

For measurements using fluorophotometry, themicroplate fluorophotometer Fluostar from BMGLabTechnologies (Offenburg, Germany) with software2.10-0 was used. Excitation filter: 320 nm (bandwidth10 nm), emission filter: 405 nm (bandwidth 6 nm).

2.4. Spectrofluorophotometer

Fluorescence spectra were recorded with a Shi-madzu (Duisburg, Germany) RF-5301 PC spectro-fluorophotometer with software version 1.10.

J. Meyer, U. Karst / Analytica Chimica Acta 401 (1999) 191–196 193

Fig. 1. Schematic set-up of the gas sampling device.

2.5. Reagent solutions

The p-HPA reagent was prepared by dissolv-ing 7.6 mg p-HPA and 2.5 mg HRP in 50 ml ofNH3/NH4Cl buffer (pH 9.5; 0.01 M). For the ABTSreagent, 5.5 mg ABTS and 0.5 mg HRP were dis-solved in 10 ml of phosphate buffer (pH 5.8; 0.01 M).Reagent solutions were prepared freshly prior tomeasurement and if necessary stored cold and dark.

2.6. Determination of H2O2

Hydrogen peroxide standards of the desired con-centrations (97, 9.7, 0.97 mM) were prepared by suc-cessive dilutions of a 10 M stock solution in acetoni-trile. Acetonitrile was used to achieve optimum evap-oration of the samples. For all gas phase measure-ments, impingers were used as sampling devices. Twoimpingers were connected in series as collecting im-pinger to absorb gas phase H2O2 and as backup im-pinger to detect incomplete recovery in the first im-pinger. The respective set-up is depicted in Fig. 1. Gasphase hydrogen peroxide was prepared as follows: asmall plug of quartz wool was fitted into a glass tubeof approximately 5 mm diameter. The glass tube wasconnected to the impingers by means of PVC tubing.The respective amount of H2O2 standard was pipet-ted onto the glass wool and evaporated by an air flowof 1 l/min. Two separate sets of measurements wereperformed. In the first experiment, the impingers werefilled with deionised water to dissolve hydrogen per-oxide. The derivatisation was then performed on themicroplate. The second experiment comprised sam-pling in impingers filled with the reagent solution. The

amounts of hydrogen peroxide were selected to 0.33,1, 3.3, 10, 33 or 100mg for sampling in water. Halfof this amount was used for sampling in the reagentsolution to ensure intercomparability of the data.

When sampling was performed in 25 ml of water,the impinger solution was filled up to 50 ml after sam-pling. 50ml of this solution was pipetted into a mi-croplate well, and 50ml of the respective reagent wasadded. After a reaction time of 15 min, the fluores-cence or extinction of the samples was determined. Ascontrol, and for the determination of recovery rates,equal amounts of hydrogen peroxide were diluted in50 ml deionised water.

For sampling in reagent solution, 25 ml of the re-spective reagent solution (see Section 2) was trans-ferred into each impinger. The solutions were filled upto 50 ml after sampling. 100ml was pipetted into a mi-croplate well. Controls were prepared by direct mixingof the respective amount of H2O2 standard with 25 mlreagent solution and subsequent dilution to 50 ml. Af-ter sampling, all samples were analysed immediately.

All measurements (control solutions, gas samplesand backup solutions for all six concentrations) werecarried out four-fold.

3. Results and discussion

In this study, the suitability of two different peroxi-dase substrates for gas phase determination of hydro-gen peroxide is assessed. The results are summarisedin Table 1. The values were calculated for a flow rateof 1 l/min and 10 min sampling time, i.e. a total vol-ume of the gas sample of 10 l. LODs and LOQs aregiven as 3 or 10 times standard deviation of the blank,respectively. The relative standard deviation is givenas an average over the indicated concentration range(with n= 4 for each concentration).

Best sensitivity was achieved with the fluorimetricmethod usingp-HPA as a reagent and water as sam-pling medium (for sampling in reagent solution, seebelow). The fluorescence spectrum of a derivatisedH2O2 solution is shown in Fig. 2. The maximum exci-tation wavelength is 323 nm and the maximum emis-sion wavelength is 403 nm, therefore excitation andemission filters were selected as 320 and 405 nm, re-spectively. The linear range comprises more than twodecades with a possible extension to higher concen-

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Table 1Analytical parameters of the tested methods

Method Sample medium Linear range (ppbv–ppmv) LOD (ppbv) LOQ (ppbv) Mean RSDa (%) Mean recoverya (%) R

p-HPA water 36–6.6 36 110 0.9 85 0.999reagent 66–6.6 66 200 2.7 34 0.996

ABTS405b water 220–6.6 170 510 9.2 90 0.999

reagent 660–6.6 470 1410 6.5 54 0.986ABTS732

b water 220–6.6 190 570 12.5 90 0.999reagent 660–6.6 550 1650 8.9 53 0.987

aAverage for the concentration range 220 ppbv–6.6 ppmv or the linear range, respectively.bThe wavelength used for measurement is indexed.

Fig. 2. Fluorescence spectrum of dimerisedp-HPA (emission spectrum recorded with excitation wavelength 320 nm, excitation spectrumrecorded for emission wavelength 405 nm).

Fig. 3. Calibration functions for gas phase sampling and control solutions.

J. Meyer, U. Karst / Analytica Chimica Acta 401 (1999) 191–196 195

trations which were not tested. The average standarddeviation within the linear range is very low and theaverage recovery with 85% is good, taking into ac-count the low concentrations of hydrogen peroxidemeasured and the possible degradation of the analytedue to reductive or decomposition processes duringevaporation and sampling. In Fig. 3, the calibrationgraphs for gas phase sampling and the control are de-picted. The linear regression functions are almost par-allel and show similar values for their slope, thus sug-gesting agreement of the data. Recovery gets betterwith higher concentrations of hydrogen peroxide inthe gas phase, which can be explained by relativelyless degradation of the analyte during the collectionprocess.

For photometric determinations with ABTS, sam-pling in water and measurement at a wavelength of405 nm provides slightly better sensitivity comparedto 732 nm. This is not surprising due to the molar ab-sorptivity for ABTS•+ at 405 nm compared to 732 nm[9]. However, the difference is small, thus giving theopportunity to use the longer wavelength in case ofcoloured matrix constituents to achieve higher selec-tivity. The tested photometric method is robust withan average recovery of 90% and it requires only sim-ple and readily available instrumentation. The higherrecovery rates as compared to thep-HPA method maybe a result of faster reaction rates and the resultingreduced degradation of the analyte. The relative stan-dard deviation is reasonably good at concentrationsclose to the LOD and gets even better with higherconcentrations.

The reagent solutions as substitute for water in thesampling devices seem to be disadvantageous in allcases considered. This may be explained by the poorstability of the reaction products with respect to lightexposure and ambient air, both of which lead to adecrease of the detectable signal in case of prolongedstorage prior to measurement. An additional reasonmay be given by impurities of the peroxidase, e.g.residual catalase, which might decompose the analytein part before the reaction is completed.

Horseradish peroxidase shows only low selectiv-ity for hydrogen peroxide. This fact is exploited inatmospheric chemistry where hydroperoxides andhydrogen peroxide are separated by HPLC with sub-sequent postcolumn derivatisation using HRP andp-HPA [7].

However, in an industrial environment, interfer-ences caused by hydroperoxides are not likely. Thenatural concentration of these substances in the at-mosphere is in the ppt or at the most in the lowerppb range. Occurrence of hydroperoxides in higherconcentrations in common industrial sites has not tobe expected. Therefore, no investigations regardingthese substances have been made.

The same applies for interferences caused by ozone[10,11]. It is stated that for sampling in aqueous me-dia 100 ppb ozone will appear as 0.03 ppb H2O2 [10].In our case, typical H2O2 concentrations are to beexpected between 100 and 3 ppm (see above), withozone concentrations typically around 50 ppb, on hotsummer days up to 200 ppb. Therefore, even under theworst conditions, the error caused by ozone will notexceed 0.1% and ozone interferences may be excludedas well for this application.

We examined the reactivity of the reagent solutionstowards peroxyacetic acid (PAA), another powerfuldisinfectant and bleaching agent of growing impor-tance. All analysis steps remained unaltered, only theanalyte hydrogen peroxide was substituted by PAA.Sampling was carried out in water only. As control, amore selective method for PAA analysis reported byPinkernell et al. [9] using ABTS as reagent and KIas catalyst was used as well. In these experiments, allthree types of reagent solution were found to showsimilar sensitivity for PAA compared to hydrogen per-oxide. For the desired purpose of workplace monitor-ing, however, no significant interferences should beexpected for the following reasons. First, PAA is byfar a stronger oxidant compared to hydrogen peroxideand it is considered to be more harmful to the work-ers’ health than hydrogen peroxide [12]. Therefore,selective methods for PAA should be selected in casethat significant PAA concentrations may be expected.Secondly, the determination of a peroxide sum param-eter is possible with the methods described here. As athird aspect, most hydrogen peroxide applications forbleaching or disinfection are based on hydrogen per-oxide as single active ingredient, so that interferencesby PAA are unlikely to occur.

The p-HPA method in combination with a mi-croplate fluorophotometer can be successfully ap-plied to gas phase analysis of hydrogen peroxide inconcentrations relevant for workplace monitoring.Sample collection is rapid (10 min sampling time are

196 J. Meyer, U. Karst / Analytica Chimica Acta 401 (1999) 191–196

sufficient) and the overall procedure including cali-bration does not take more than 35 min. The methodshows a large dynamic range, low relative standarddeviation and good recovery. The german MAKvalue of 1 ppmv for H2O2 in the gas phase [12] iswell within the linear range (0.04–6.6 ppmv). Theinstrumental set-up for sampling and measurement issimple and readily accessible.

Acknowledgements

Financial support by the European Union (EU-ROPEROX project, SMT4-CT97-2153) is gratefullyacknowledged. J.M. thanks the Fonds der ChemischenIndustrie (Frankfurt, Germany) for a scholarship.

References

[1] DEV zur Wasser-, Abwasser- und Schlammuntersuchung:Bestimmung von Wasserstoffperoxid und seinen Addukten(H15), DIN 38409, Teil 15, DEV-18, Lieferung 1987.

[2] M. Possanzini, V. Di Palo, Anal. Chim. Acta 315 (1995) 225.[3] P.A. Tanner, A.Y.S. Wong, Anal. Chim. Acta 370 (1998) 279.[4] A.L. Lazrus, G.L. Kok, J.A. Lind, S.N. Gitlin, B.G. Heikes,

R.E. Shetter, Anal. Chem. 58 (1986) 594.[5] R.L. Tanner, G.Y. Markovits, E.M. Ferreri, T.J. Kelly, Anal.

Chem. 58 (1986) 1857.[6] A.L. Lazrus, G.L. Kok, S.N. Gitlin, J.A. Lind, Anal. Chem.

57 (1985) 917.[7] S. Gäb, E. Hellpointner, Nature 337 (1989) 631.[8] H. Huang, P.K. Dasgupta, Z. Genfa, Anal. Chem. 68 (1996)

2062.[9] U. Pinkernell, H.-J. Lüke, U. Karst, Analyst 122 (1997) 567.

[10] T. Gnauk, W. Rolle, G. Spindler, J. Atmos. Chem. 27 (1997)79.

[11] R.G. Zika, E.S. Saltzman, Geophys. Res. Lett. 9 (1982) 231.[12] Deutsche Forschungsgemeinschaft (Ed.), MAK-und BAT-

Werte Liste 1997, Wiley, VCH 1997.