Simultaneous HPLC Determination of PeroxyaceticAcid and Hydrogen Peroxide

Ulrich Pinkernell, Stefan Effkemann, and Uwe Karst*

Abteilung Lehrstuhl fur Analytische Chemie, Anorganisch-Chemisches Institut, Westfalische Wilhelms-Universitat Munster,Wilhelm-Klemm-Strasse 8, D-48149 Munster, Germany

The first instrumental method for simultaneous determi-nation of peroxyacetic acid (PAA) and hydrogen peroxidehas been developed. The successive quantitative reactionof PAA with methyl p-tolyl sulfide (MTS) and hydrogenperoxide with triphenylphosphine (TPP) yields the cor-responding sulfoxide MTSO and phosphine oxide TPPO.The reagents and their oxides are separated by HPLC onreversed-phase columns with acetonitrile/water gradientelution within 5 min. External calibration with the solidstandards of MTSO and TPPO leads to a very accurateand reliable method. Samples are stable and can bestored after derivatization for several days prior to analy-sis. Real samples from brewery disinfection were ana-lyzed in comparison to titration with excellent correlation.

Peroxides such as peroxyacetic acid (PAA) and hydrogenperoxide find increasing use in industry for disinfection andbleaching purposes due to their ecologically beneficial properties.The technical synthesis of PAA comprises the reaction of aceticacid with hydrogen peroxide, often in the presence of a strongacid as catalyst. Therefore, technical PAA solutions containsignificant amounts of hydrogen peroxide as a residue or equi-librium concentration.

The determination of these peroxides requires a reliabledistinction between the two substances, which are commonlyanalyzed using their redox properties. Both peroxides are strongoxidizers, and their technical application is mainly based onoxidation chemistry. Hydrogen peroxide, however, may also serveas a reducing agent toward other strong oxidizers in the presenceof strong acids.

Different titration techniques use this difference in redoxproperties to determine hydrogen peroxide by permanganate1 orcerium (IV)2 oxidation in the first step. In a second step, iodideis added and converted to iodine by PAA.1,2 Titration withthiosulfate is used to determine the concentration of iodine and,indirectly, the concentration of PAA. On the one hand, thistitration is a very convenient way to determine the concentrationof both analytes quasi-simultaneously with no need for calibrationusing the instable peroxide solutions themselves. On the otherhand, it is known that Mn(II) ions as a reaction product of thefirst step may catalyze the decomposition of PAA.1 This resultsin a severe time dependence of the two-step titration. Further-more, the titration methods can be applied only for relatively highconcentrations and are, therefore, not suitable for the monitoringof peroxide residues. Additionally, titration has to be performed

immediately after sampling to avoid decomposition of the perox-ides during storage or transportation of the sample.

Further methods for the determination of PAA includephotometry,3-9 electrochemical sensing,10-13 gas chromatogra-phy,14,15 and direct liquid chromatography.16,17 None of thesemethods, however, is suited to determine both PAA and hydrogenperoxide selectively and simultaneously.

Recently, we have described a method for liquid chromato-graphic determination of PAA using the oxidation of methyl p-tolylsulfide (MTS) to the corresponding sulfoxide (MTSO):18,19

Di Furia et al. have introduced this reaction with successiveextraction of the product into chloroform to determine the PAAconcentration by gas chromatography.15

Although both the liquid and the gas chromatographic ap-proaches perform well for the analysis of most real samples,problems may occur in strongly acidic solutions with very highconcentrations of hydrogen peroxide.19 This results in a limitedstorability of the samples. Analysis has to be done within a shorttime after derivatization to avoid a steadily increasing MTSO peakdue to oxidation of the MTS by excess hydrogen peroxide. Theseproblems have been overcome by the addition of manganesedioxide to the sample after derivatization to ensure stability andstorability.19 However, more effort has to be put into sample

(1) D’Ans, J.; Frey, W. Chem. Ber. 1912, 45, 1845.(2) Greenspan, F. P.; McKellar, D. G. Anal. Chem. 1948, 20, 1061-1063.

(3) Frew, J. E.; Jones, P.; Scholes, G. Anal. Chim. Acta 1983, 155, 139-150.(4) Davies, D. M.; Deary, M. E. Analyst 1983, 113, 1477-1479.(5) Christner, J. E.; Lucchese, L. J. (Serim Research Corp.). WO 92/22806,

1992.(6) Krussmann, H.; Bohnen, J. Tenside Surf. Deterg. 1994, 31 (4), 229-232.(7) Mallard de la Varende, J.; Crisinel, P. (L’Air Liquide S.A.). US 5 438 002,

1995.(8) Williams, J. (Interox Chemicals Ltd.). EP 0 150 123, 1985.(9) Fischer, W.; Arlt, E.; Brabander, B. (Merck Patent GmbH). US 4 900 682,

1988.(10) Birch, B. J.; Marshman, C. E. (Unilever NV). EP 0 333 246, 1989.(11) Pinkowski, A. (ProMinent Dosiertechnik GmbH). US 5 395 493, 1995.(12) Teske, G. (Dr. Thiedig & Co.). US 5 503 720, 1996.(13) Kaden, H.; Herrmann, S. (Forschungsinstitut “Kurt Schwabe” Meinsberg).

DE 43 19 002, 1995.(14) Cairns, G. T.; Ruiz Diaz, R.; Selby, K.; Waddington, D. J. J. Chromatogr.

1975, 103, 381-384.(15) Di Furia, F.; Prato, M.; Quintly, U.; Salvagno, S.; Scorrano, G. Analyst 1984,

109, 985-987.(16) Kirk, O.; Damhus, T.; Christensen, M. W. J. Chromatogr. 1992, 606, 49-

53.(17) Baj, S. Fresenius J. Anal. Chem. 1994, 350, 159-161.(18) Pinkernell, U.; Karst, U.; Cammann, K. Anal. Chem. 1994, 66, 2599-2602.(19) Pinkernell, U.; Effkemann, S.; Nitzsche, F.; Karst, U. J. Chromatogr. A 1996,

730, 203-208.

Anal. Chem. 1997, 69, 3623-3627

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preparation, as the manganese dioxide has to be removed bycentrifugation prior to HPLC analysis.

To overcome these problems and to obtain informationabout both peroxide concentrations over a wide concentra-tion range, we have developed a method for quasi-simultaneousHPLC determination of PAA and hydrogen peroxide based on atwo-step oxidation which requires no additional sample pretreat-ment.

EXPERIMENTAL SECTIONSafety Note: PAA and hydrogen peroxide are strong oxidiz-

ers, and their concentrated solutions should neither be mixed withreducing agents nor with organic substances including solvents.Concentrated peroxide solutions should, therefore, be diluted withwater prior to the derivatization reaction. The proceduresdescribed here have only been tested with peroxide concentrationsup to 2000 mg/L each.

Chemicals. All chemicals were purchased from AldrichChemie (Steinheim, Germany) in the highest quality available.Organic solvents for HPLC were Merck (Darmstadt, Germany)gradient grade. Peroxide solution I was obtained from Aldrichwith a concentration of 37% PAA and 5% hydrogen peroxide, whileperoxide solution II was P3 Oxonia Aktiv (Henkel-Ecolab, Dus-seldorf, Germany) with a concentration of 4.5% PAA and 25%hydrogen peroxide. Both solutions were diluted down to thetypical industrial disinfection application concentration of 400mg/L PAA.

Derivatization Procedure. The following general procedureis applicable for PAA concentrations from 2.5 × 10-5 to 1.0 × 10-2

mol/L and for hydrogen peroxide concentrations from 7.5 × 10-5

to 3 × 10-3 mol/L in samples: 100 µL of a 20 mM solution ofMTS in acetonitrile and 300 µL of deionized water are added to100 µL of the sample solution. After a reaction time of 10 min,400 µL of acetonitrile and 100 µL of a 10 mM solution of TPP inacetonitrile are added to start the second derivatization step. Thesolution is stored in the dark (see below), left to react for 30min, and analyzed by HPLC subsequently. For sample concentra-tions exceeding the range stated above, the samples should bediluted, as higher concentrations of the reagents may lead totheir precipitation after addition of water. For lower concen-trations of analytes in the sample, the concentrations of thereagents may be reduced to at least a molar excess to meet theexpected concentrations. It should be noted that reagent con-centrations below 10-4 mol/L may result in significantly re-duced reaction rates. Reagent excess should not be too largefor both MTS and TPP to keep the blanks of the reaction prod-ucts as low as possible (see below). In general, the reagentconcentration should be at least twice as high as the expectedperoxide concentration in the sample. This procedure has beenapplied for all samples described in this work, with dilutions ofthe samples and reductions of the reagent concentrations when-ever required.

Photometer. The HP 8453 diode array spectrophotometer(Hewlett-Packard, Waldbronn, Germany) with HP Chem Station845x biochemical UV/VIS system software was used.

UV Absorption Measurements. To determine the optimumdetection wavelength for UV detection in HPLC, the spectra ofMTSO (10-5 M in acetonitrile/water 40/60) and TPPO (5 × 10-6

M in acetonitrile/water 40/60) were recorded in the wavelengthrange from 200 to 350 nm.

HPLC Instrumentation. The high-performance liquid chro-matograph consisted of the following components: two LC-10ASpumps (Shimadzu, Duisburg, Germany), a SPD-10AV detector(Shimadzu), an SIL-10A autosampler (Shimadzu), Class LC 10Version 1.4 software (Shimadzu), and a CBM-10A controller unit(Shimadzu). The injection volume was 10 µL. The columnconsisted of Nucleosil C8 reversed-phase material (Macherey-Nagel, Duren, Germany) in ChromCart cartridges (Macherey-Nagel): particle size, 5 µm; pore size, 100 Å; column dimensions,70 mm × 3 mm.

HPLC Analysis. The injection volume for all measurementswas 10 µL, and the UV detection wavelength was 225 nm. Thefollowing gradient of acetonitrile and water was selected to achieveseparation of MTS, MTSO, TPP, and TPPO:

The flow rate of the mobile phase was 1.0 mL/min. Note that, asMTSO and TPPO elute early under these conditions, the gradientascends steeply at 3 min to rinse the far less polar TPP off thecolumn and, therefore, reduce analysis time.

RESULTS AND DISCUSSIONIt is known from literature that hydrogen peroxide serves as

a popular reagent for the preparative-scale oxidation of phosphinesto the corresponding phosphine oxides.20 We have, therefore,investigated the suitability of triphenylphosphine (TPP) as areagent for hydrogen peroxide determination using HPLC withUV/visible detection:

This reaction was intended to be used as the second reaction stepafter the reaction of PAA with MTS (see above) to obtain a methodfor simultaneous HPLC determination of PAA and hydrogenperoxide. No oxidation of TPP by MTSO is observed, althoughit may occur under extreme conditions (T > 100 °C, in concen-trated acetic acid21 ) or in the presence of stoichiometric amountsof iodine/iodide.22

UV/visible spectra of MTSO and triphenylphosphine oxide(TPPO) were recorded to investigate the UV/visible detectionconditions for HPLC analysis. The spectra are presented in Figure1. The absorption spectra of both substances exhibit maxima at223 (TPPO) and 227 nm (MTSO). Therefore, λ ) 225 nm isselected as the detection wavelength for single-wavelength detec-

(20) Svara, J.; Weferling, N.; Hofmann, T. In Ullmann’s encyclopedia of industrialchemistry, 5th rev. ed.; Elvers, B., Ed.; VCH: Weinheim, 1991; Vol. A 19,pp 545-572.

(21) Szmant, H. H.; Cox, O. J. Org. Chem. 1996, 31, 1595-1598.(22) Olah, G. A.; Gupta, B. G. B.; Narang, S. C. Synth. Commun. 1978, 137-

138.

time (min:sec) cacetonitrile (%)

0:00 403:00 403:10 1004:00 1004:10 406:00 (stop) 40

3624 Analytical Chemistry, Vol. 69, No. 17, September 1, 1997

tion. If a dual-wavelength detector or a photodiode array detectoris available, both substances can be analyzed at their absorptionmaxima.

Chromatographic separation of the reagents and the productswas developed on reversed-phase C8 columns (conditions aregiven above). A chromatogram of a peroxide solution I sampleafter dilution and derivatization according to the general procedureas stated above is presented in Figure 2. As can be expectedfrom the polarity of the reagents and their reaction products,MTSO elutes first, followed by TPPO, MTS, and finally TPP. Thebaseline increase at t ) 4 min is due to a very rapid ascent inacetonitrile concentration, which was chosen to rinse the nonpolarTPP rapidly from the column to achieve short analysis times. Asonly MTSO and TPPO are quantified to obtain information onPAA and hydrogen peroxide concentrations, this shift in baselinedoes not have to be considered during evaluation of the chro-matogram.

Reaction conditions for PAA determination with MTS havebeen described in ref 18. A reaction time of 10 min is sufficientto achieve quantitative reaction of PAA in the presence of at leasta 2-fold excess of the reagent. Conditions for the reaction of TPPwith hydrogen peroxide were investigated as follows. Chromato-graphic analysis of the commercially available TPP proved thatthe reagent already contained approximately 1% of TPPO, thusproducing a significant blank which worsens the limit of detectionconsiderably. Recrystallization of TPP from ethanol was investi-gated as a means for reducing the blank and resulted in areduction of TPPO in the reagent to 0.5%. However, as the blankis rather constant in the absence of light and strong acids, and asthe concentration range for hydrogen peroxide is well known formost technical applications, it is easier to adapt the reactionconditions by adding only a double molar excess of reagent wherepossible. This reduces the blank to a tolerable value, andsubtraction of the blank yields reliable data on the hydrogenperoxide concentration.

The effects of pH and reaction time have also been investi-gated. In Figure 3, the concentration data obtained for hydro-gen peroxide using TPP as reagent are plotted depending on pHconditions and on two different reaction times. For comparison,the blanks for both reaction times and the complete pH rangeare given. It is obvious that the reaction occurs quantitativelyand without problems for a reaction time of 30 min within the pHrange from 2 to 10. On average, the TPPO concentrationsdetermined are only slightly lower when applying a reaction timeof 15 min in the same concentration range. Further investigationsup to a reaction time of 120 min do not result in significantdifferences to 30 min. At lower pH, more TPPO is detected dueto the increased oxidation of TPP by oxygen from ambient air.This effect depends on the reaction time and is observed for thesamples as well as for the blanks. Above pH 10, less TPPO isdetected due to either an increased decomposition rate ofhydrogen peroxide in alkaline media or a nonquantitative reactionof TPP with hydrogen peroxide under the selected conditions.For the analysis of disinfectant samples, pH was between 2 and 4due to the presence of acetic acid and sulfuric acid in the samples.

Early experiments resulted in a significant increase of theTPPO blank in TPP solutions with time. As this effect wasobserved exclusively for reagent solutions stored in the light, a

Figure 1. UV/visible spectra of MTSO and TPPO.

Figure 2. Chromatogram of a derivatized sample of the dilutedperoxide solution I. Final concentrations in chromatography: 0.53 mMMTSO, 0.18 mM TPPO, 0.47 mM MTS, 0.32 mM TPP.

Figure 3. Dependence of the oxidation of TPP by hydrogenperoxide on pH and reaction time.

Analytical Chemistry, Vol. 69, No. 17, September 1, 1997 3625

study about the storability of the samples after derivatizationover 1 week was conducted. The results are presented inFigure 4 for two different peroxide solutions (see above). Ingeneral, MTSO concentrations do not increase significantly during1 week of storage, neither in the light nor in the dark. TPPOconcentrations of those samples stored in the light, however,exhibit a strong increase during the period of investigation. Incontrast to this, TPPO increases are very moderate for thosesamples stored in the dark. Important information can be gainedfrom this experiment. First, the data obtained for PAA are veryreliable, even after a storage time of 1 week either in the light orin the dark. Storability of MTSO-containing solutions is evenbetter in this case compared with stabilization by addition ofmanganese dioxide. Second, storability for TPPO is at least 1week when the samples are stored in the dark. Although therelative increase of the TPPO concentration with time is observedin a similar way for both peroxide solutions when stored in thelight after derivatization, the absolute increase is stronger forperoxide solution 2.

In Figure 5, a logarithmic plot for the calibration of bothperoxides is presented. Concentrations for the reagents are

reduced by a factor of 10 compared to the general derivatizationprocedure (see above). Linearity for the calibration curves isachieved from 2.5 × 10-6 to 1 × 10-3 mol/L for PAA (r > 0.999)and from 7.5 × 10-6 to 3 × 10-4 mol/L for hydrogen peroxide (r> 0.999) after subtraction of the blanks. Limits of detection (LODS/N > 3) are 10-6 mol/L for PAA and 3 × 10-6 mol/L forhydrogen peroxide under these conditions. This problem is moreserious for TPP than for MTS, as TPP may contain rather highTPPO concentrations (see above). The RSD for 10-fold reactionand analysis of samples with concentrations in the linear rangeof the calibration curves is (1% for both analytes.

Commercially available disinfectant solutions and real samplesfrom the cleaning-in-place (CIP) process of a brewery have beenanalyzed using the simultaneous HPLC method in comparison tothe two-step titration according to refs 1 and 19. The data forPAA are presented in Figure 6, and those for hydrogen peroxideare presented in Figure 7. For both types of samples, goodagreement of the data using both methods has been achieved.This proves the applicability of the simultaneous HPLC determi-nation of PAA and hydrogen peroxide for real sample analysis inthe field of disinfection control. Reproducibility problems with

Figure 4. Storability of derivatized samples depending on lightduring storage. Times indicated in the figure are times after additionof TPP to the sample.

Figure 5. Logarithmic plot for the calibration of PAA and hydrogenperoxide.

Figure 6. Analysis of PAA in disinfectant solutions and breweryCIP samples using the simultaneous HPLC determination and thetwo-step titration.

Figure 7. Analysis of hydrogen peroxide in disinfectant solutionsand brewery CIP samples using the simultaneous HPLC determina-tion and the two-step titration.

3626 Analytical Chemistry, Vol. 69, No. 17, September 1, 1997

the titration are described in detail in ref 2 and may account forthe differences in the disinfectant solutions 1 and 2.

CONCLUSIONSThe simultaneous HPLC determination of PAA and hydrogen

peroxide is a valuable and reliable tool for peroxide analysis. Limitsof detection are very low for both peroxides, and the linear rangesfor determination can be adapted easily to varying samplingconditions by dilution of either the sample or the reagents.

This HPLC method is the first instrumental method which isable to simultaneously determine PAA and hydrogen peroxide.Results obtained for disinfection samples correlate well withtitration data. In comparison to titration, the new method ischaracterized by considerably lower limits of detection for both

analytes. Samples can be stored or shipped, after derivatizationbut prior to analysis, for at least 1 week. Only standard HPLCequipment is required to successfully perform this method.

ACKNOWLEDGMENTFinancial support by the DFG (Deutsche Forschungsgemein-

schaft, Bonn, Germany) and the Fonds der Chemischen Industrie(Frankfurt, Germany) is gratefully acknowledged. U.P. thanks theStiftung Industrieforschung (Koln, Germany) for a scholarship.

Received for review February 11, 1997. Accepted June 6,1997.X

AC9701750

X Abstract published in Advance ACS Abstracts, July 15, 1997.

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