Technical Notes Anal. Chem. 1994,66, 2599-2602
Determination of Peroxyacetic Acid Using High-Performance Liquid Chromatography with External Calibration U. Plnkernell, U. Karst,' and K. Cammann Institut fur Chemo- und Biosensorik and Lehrstuhl fur Analytische Chemie, WestiPIlische Wilhelms-Universitat Minster, Wilhelm-Klemm-Strasse 8, 0-48 149 Minster, Germany
A new method for the determinationof peroxyaceticacid (PAA) in aqueous solutionsis described. Methyl ptolyl sullide (MTS) is oxidized by PAA, forming the corresponding methyl ptolyl sulfoxide (MTSO), which can be separated easily by highperformance liquid chromatographyon a reversed-phasecolumn and detected by ultraviolet spectroscopy at a wavelength of 230 nm. MTSO is a commercially available solid substance which can be used as an external standard for calibration. Therefore, calibration with the unstable PAA can be avoided. This method is characterized by a high reproducibility, low detection limits at 4 X lo-' moVL (30 pg/L), an applicable concentration range of more than two decades, and negligible cross reactivity toward hydrogen peroxide. Peroxyacetic acid (PAA) is a widely used disinfectant in the food and beverage industry. Because its application does not lead to toxic halogenated organic compounds, PAA is ideal for disinfection in cleaning-in-place (CIP) systems in breweries and dairies. Typical concentrations range between 6.6 X IO4 and 1.3 X mol/L (50-1000 mg/L) for disinfection purposes. Accurate determination of PAA is required to monitor PAA levels in the disinfectant solutions and control adjustment of the concentration through addition of concentrated PAA solutions. Therefore, the development of a fast and reliable method for the determination of PAA in aqueous solutions is d great interest. Generally, PAA is determined by means of conductivity measurement, photometry,14 t i t r a t i ~ n ,or~ chromatographic ?~ determination^.^-^ Conductivity measurements are rapid and convenient, but their common disadvantage is their low selectivity. Every ion in the solution contributes to the total conductivity which might, for instance, lead to false positive results caused by the presence of small amounts of acids or bases from previous cleaning steps in the CIP system. (1) Davies, D. M.; Deary, M.E. Analyst 1988, 113, 1477-1479. (2) Interox Chemicals Ltd. EP 0 150 123 B1, 1990. (3) Frew, J. E.;Jones, P.; Scholes, G. Anal. Chim. Acta 1983, 155, 139-150. (4) Merck Patent GmbH DE 37 43 224 AI, 1989. (5) Greenspan, F. P.; McKellar, D. G. Anal. Chem. 1948, 20, 1061-1063. (6) Sully, B. D.; Williams, P. L. Analysr 1962,87, 653-657. (7) Di Furia, F.; Prato, M.; Quintily, U.; Salvagno, S.; Scorrano, G. Analyst 1984, 109, 985-987. (8) Cairns, G. T.;Ruiz Diaz, R.; Selby, K.; Waddington, D. J. J . Chromarogr. 1975, 103, 381-384. (9) Kirk, 0.;Damhus, T.; Christensen, M. W. J . Chromatogr. 1992,606,49-53. 0003-2700/94/0386-2599$04.50/0 Cm 1994 American Chemical Societv
As with conductivity measurements, photometric methods require a calibration using PAA standards. This is a serious problem because diluted aqueous solutions of PAA are very unstable; they have to be recalibrated by titration several times a day. Another difficulty is caused by the presence of varying amounts of hydrogen peroxide in the disinfection solutions, which is a result of the synthesis of PAA from acetic acid and hydrogen peroxide and the decomposition of PAA via the reverse reaction. As some of these methods show a considerable cross reactivity toward hydrogen peroxide, the accuracy of the determination is not satisfactory. For this reason, the most popular titration method uses a two-step procedure. First, hydrogen peroxide is consumed by addition of potassium permanganate solutions (eq 1) and then PAA is titrated with iodine/thiosulfate (eqs 2a and 2b): 5H,O,
- + - +
+ 2Mn0; + 6H'
CH,C(O)OOH
+ 21- + 2H+ I,
+ 2s,032-
50,
2Mn2++ 8H,O
(1)
+ 1, + H,O
(2a)
CH,COOH 21-
s,o,z-
(2b)
The accuracy of this method decreases rapidly with a decrease in PAA concentration. Additionally, the first equation demonstrates that two molecules of potassium permanganate react with five molecules of hydrogen peroxide. As the concentration of potassium permanganate should not be too low because of the time dependence of this reaction,5 a small overtitration in the first step leads to a considerable inaccuracy in determination of PAA. Most chromatographic methods published use the direct separation of the peroxides without previous derivatization.8~9 These methods are very rapid, but require a calibration with PAA standards. Therefore, a second method, e.g., titration, has to be used for determining the concentration of the PAA standards to obtain reliable results. Furthermore, all determination methods mentioned above suffer from the fact that the samples have to be analyzed directly after sampling and can neither be stored nor be shipped due to the instability of the analyte. In 1984, Di Furia et al. described a method for the determination of PAA using the reaction of PAA and methyl AnaiyticalChemIstry, Vol. 88, No. 15, August 1, 1994 2599
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p-tolyl sulfide (MTS) forming the correspondingmethylp-tolyl sulfoxide (MTSO):l0 CH,C(O)OOH
+ CH,C,H,SCH,
+
CH,COOH
+ CH,C,H,S(O)CH,
(3)
The oxidation of sulfides by PAA is described to be fast and quantitative. For the oxidation to the sulfoxide, an equimolar amount of PAA is required and the sulfones are synthesized with a 1:2 molar ratio only at higher temperatures." The amount of MTSO formed in this reaction was determined by packed column gas chromatography (GC). After addition of MTS, aqueous solutions of disinfectants first have to be extracted with chloroform to obtain a solution \ extraction :step iso suitable for gas chromatography. As any time consuming and a source for errors, it was our main objective to improve the method of Di Furia et al. for the direct analysis of aqueous disinfectant solutions. Therefore, we adopted the reaction of MTS with PAA for HPLC analysis using reversed-phase columns with water and methanol mixtures as mobile phases. This method combines the advantages of a direct determination of PAA in aqueous solutions without any extraction step and a reliable calibration using external standards.
EXPERI MENTAL SECT1ON Chemicals. All chemicals were purchased from Aldrich Chemie GmbH (Steinheim) in the highest quality available. Peroxyacetic acid is sold as a solution in dilute acetic acid and contains hydrogen peroxide in considerable concentrations. As PAA is a strong oxidizer, concentrated solutions should not be mixed with organic solvents. Therefore, the new technique described in this paper can only be applied for concentrations of PAA of 2000 mg/L or after dilution of more concentrated samples with water. HPLC Apparatus. The high-performance liquid chromatograph consisted of the following components: pump H P 1050 (Hewlett Packard (HP)), injector Model 7010A, 20-pL sample loop (Rheodyne), column Supelcosil LC 18,250 X 4.6 mm (Supelco), detector Model 1040M (HP), software HPLC Chemstation (HP), and mobile phase 75% methanol/25% water (v/v). Photometer. The UV/VIS spectrometer Lambda 2 (Perkin Elmer) with software PECSS V4.1 (Perkin Elmer) was used. GC/MS. The GC/MS apparatus consisted of the following: gas chromatograph 5890 Series I1 (HP); column H P Ultra 2, 50 m, 0.2 mm (inner diameter), 0.33 pm (layer thickness) (HP); 5971A mass selective detector (HP); and CHEM PC (HP) software. Thin-Layer Chromatography. The TLC system consisted of the following: TLC plates reversed-phase C- 18 material, RP-18 F254S, precoated, layer thickness 0.25 mm (Merck); UV lamp, Fluotest 406 AC (Heraeus); and mobile phase, 75% methanol/25% water (v/v). To optimize the conditions for HPLC, MTS, and MTSO were dissolved in methanol and TLC chromatograms were recorded using TLC plates with C-18 stationary phase (with fluorescence indicator FZ5,) as (10) Di Furia, F.; Prato,
M.;Scorrano, G.; Stivanello, M.Analyst
1988, 113,
793-795.
2600
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AnelyticalChemistry, Vol. 66,No. 15, August 1, 1994
.
o
i'
'-'
200
220
240
260
280
300
320
340
1 [nml Flgurr 1. UV absorption spectra of MTS and MTSO substances as dlluted samples in methanol: [MTS] = le5mol/L; [MTSO] = lo4 mol/L.
stationary and methanol/water (75/25 v/v) as the mobile phase. The substances were detected at a wavelength of 254 nm. UV Absorption Measurements. To determine the optimum wavelength for UV detection in HPLC, MTS and MTSO were dissolvedin methanol (concentration, mol/L [MTS] and 10-5 mol/L [MTSO]) and UV spectra were recorded in the range from 190 to 350 nm. Sampling Procedure. For the measurements and calibration of PAA, 1 mL of aqueous solutions of PAA was mixed with 1 mL of a solution of MTS in methanol. The concentration of the MTS was 2 times higher than the highest PAA concentration in the calibration measurements. After the mixture was shaken well, it was diluted with the 9-fold volume of methanol. The calibration of MTS and MTSO has been done similarly,including the mixing of the methanolic solutions with HzO. HPLC Analysis. The diluted samples were directly injected into the sample loop. After separation on the reversed-phase column, MTSO and MTS were detected by UV absorption at 230 nm.
RESULTS TLC experiments proved that reversed-phase C-18 materials as stationary phases in combination with binary mobile phases from methanol and water are generally suitable for the separation of MTS and MTSO. A ratio of 75% methanol and 25% water (v/v) shows excellent separation properties for the relevant compounds: RAMTS) = 0.4; . RAMTSO) = 0.6. For UV detection in HPLC, a suitable wavelength (230 nm) for the determination of MTS and MTSO was obtained by recording the UV absorption spectra, which are presented in Figure 1. The HPLC separation of an excess of MTS and its reaction product with PAA is presented in Figure 2. (1 1) Sawaki, Y.In Organic Peroxides; Ando, W., Ed.;John Wiley Br Sons: York, 1992; pp 4 2 5 4 7 7 .
New
336
A
10000 r
249
2 w
f
J
0
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162 1
75
-121
1
,
I
1
,
,
I
1
,
Lf
104
I0 3
10'
102
~(PAA)[mol/ll
Flgure 9. Calibration curve for aqueous solutionsof PAA by detection of MTSO. 1000
The identity of the reaction product as MTSO was verified by comparison of the retention times with the MTSO standard and by collecting the substance from 20 HPLC runs with following analysis via GC/MS. In GC, the retention time of the collected substance was identical with the retention time of the MTSO standard, and the corresponding mass spectra of sample and standard coincided. To determine the minimum time required for quantitative reaction of MTS and PAA, a solution of MTS was analyzed by HPLC/UV at several different times (2, 16, 30, 44, and 74 min) after addition of PAA. Any of these chromatograms indicated the same amount of formed product. Therefore, the reaction can be assumed to be quantitative after 2 min. This could be verified by adding a known amount of PAA to the MTS solution, calibrating the HPLC system with MTSO standards, and comparing the calculated amount of MTSO with the determined. Additional support for this fast reaction is the fact that reacting various excess amounts of MTS with a constant amount of PAA for 2 min forms an identical peak area of MTSO in all cases. A calibration curve of PAA is presented in Figure 3. The range of the single values was approximately f0.5% about the mean for repetitive measurements (n = 3) at all concentration levels. A linear calibration curve in the range from 1.3 X lo.-" to 2.6 X mol/L PAA (10-2000 mg/L) wasobtained. It can bevaried by changing the levelofdilution. In a further calibration without the usual dilution, the limit of detection (LOD) was determined to 4 X lk7mol/L (30 cLg/L). The calibration data of MTSO standards are determined to (309 f 3) unit~/lO-~ mo1.L-I for a concentration range of 2.7 X 10-4-2.7X 10-2mol/L MTSO. The range of the single values was approximately &OS% of the mean for repetitive measurements (n = 3) at all concentration levels. Additional information can be obtained by calibrating via the amount of remaining MTS. The corresponding calibration data for the determination of MTS in methanolic solution are determined to (182 f 1) units/lk3 mo1.L-I for a concentration range of 2.7 X 104-2.7 X 10-I mol/L MTS. The range of the single
I
I
800
Y
I 200 -
Flgure 4. Mean peak areas (n = 3) for the determination of the cross reactivity toward hydrogen peroxide.
values was again approximately &0.5% of the mean for repetitive measurements (n = 3) at all concentration levels. This way, PAA can be determined using two different external calibrations. Generally, calibrationvia MTSO is be preferred because of direct calibration with aqueous solutions of the standard. Using the calibrationvia MTS, higher concentrated solutions of the standard have to be prepared in methanol due to the low solubilityof MTS in water. In addition, the accuracy of this calibration technique is not as high as in calibration with MTSO especially for the determination of small amounts of PAA with a large excess of MTS, because any result is the difference of two measurements. The cross reactivity toward hydrogen peroxide has been examined by adding solutions of MTS to aqueous solutions of H2Oz as previously described for the determination of PAA. Analysis of the samples was performed according to the procedure described above. The mean peak areas (n = 3) of the detected sulfoxide are presented in Figure 4. Based upon the peak areas from calibration in Figure 3, the cross reactivity of this method toward hydrogen peroxide is about 0.1%. We obtained the same peak area from a 1.3 Ana&ticalChemistry, Vol. 66, No. 15, August 1, 1994
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X mol/L solution of PAA and a 1.3 mol/L solution of hydrogen peroxide. Other peroxycarboxylic acids, such as m-chloroperoxybenzoic acid, react in a way similar to PAA. Nevertheless, as disinfectant solutions usually do not contain mixtures of peroxycarboxylic acids, false positive results will not be expected. Other oxidizing agents, e.g., chlorine, hypochlorite, bromine, hypobromite, or permanganate, might interfere with this determination. Since the qualitative composition of the applied disinfectant solution is known, no serious interferences by other oxidizing agents should occur in determination of PAA in disinfectant solutions.
CONCLUSIONS The new chromatographic method for determination of PAA in aqueous solutions is characterized by its high reproducibility, its low limit of detection, and its high
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selectivity. The cross reactivity toward hydrogen peroxide is negligiblein those concentration ranges of PAA usually applied for disinfection of CIP systems. The method requires standard instrumentation with an isocratic HPLC system and a reversed-phase C-18 column. Therefore, it can be applied in most analytical laboratories without needing any special equipment. The analysis time for a single sample is less than 15 min including sampling. With automation and by using a shorter column and a binary gradient elution for HPLC it could be further reduced. The high stability of the sulfoxide formed warrants the possibilityof storing and shipping any sample after the reaction with MTS within a time span of at least 2 days. Received for review January 18, 1994. Accepted April 20, 1994.' Abstract published in Aduance ACS Absrracfs.June 1, 1994.
