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Analytica Chimica Acta 522 (2004) 275280

Simultaneous spectra-kinetic determination of peracetic acid


and hydrogen peroxide in a brewery cleaning-in-place
disinfection process
I.A. Pettas , M.I. Karayannis
Laboratory of Analytical Chemistry, Department of Chemistry, University of Ioannina, 45110 Ioannina, Greece
Received 27 April 2004; received in revised form 7 July 2004; accepted 7 July 2004
Available online 9 August 2004

Abstract
A spectra-kinetic approach was applied for the simultaneous determination of peroxyacetic acid (PAA) and hydrogen peroxide with multiple
linear regression (MLR) method, using the initial rates of their reaction with diphenylamine (DPA). The predictive ability of the MLR method
is based on the slight kinetic differences of these analytes occur at two wavelengths when react with DPA as a common ligand. A stopped flow
apparatus was used, and the time-resolved UVvis spectra were measured with a coupled charge device (CCD) spectrophotometer. This novel
instrumentation allowed to obtain high quality kinetic data at a maximum of many wavelengths simultaneously. The method was successfully
applied to the simultaneous determination of these peroxides in residues from a disinfection process in a beer brewery.
2004 Elsevier B.V. All rights reserved.
Keywords: Peracetic acid; Peroxides; Disinfectant determination; Chemometric spectra-kinetic analysis

1. Introduction
Peroxides, such as hydrogen peroxide and peroxyacetic
acid (PAA) find increasing use in industry for disinfection
and bleaching purposes due to their ecologically beneficial
properties. PAA is rapidly cidal at low concentrations
against a broad spectrum of microorganisms, including
gram-positive and gram-negative bacteria, yeasts, molds,
and algae under a wide variety of conditions. It is also
effective against anaerobic and spore-forming bacteria. PAA
is effective at killing biofilm microorganisms at low concentrations and short contact time [1]. It is also well suited as a
biocide in industrial cooling water and papermaking systems
[2]. Although PAA is a potent biocide, it is unique in that
it does not produce toxic byproducts and its decomposition
products (acetic acid, water, and oxygen) are innocuous and
environmentally acceptable. The technical synthesis of PAA
Corresponding author. Tel.: +30651 98406; fax: +30651 98407.
E-mail address: ipettas@cc.uoi.gr (I.A. Pettas).

0003-2670/$ see front matter 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.aca.2004.07.010

comprises the reaction of acetic acid with hydrogen peroxide,


often in the presence of a strong acid as catalyst. Therefore,
technical PAA solutions contain significant amounts of
hydrogen peroxide as a residue or equilibrium concentration.
Mixtures of PAA (2.510 vol.%), acetic acid (2.510 vol.%)
and hydrogen peroxide (2.510 vol.%) have found a wide
application in food industry as sanitizers, with various trade
names (OXONIA ACTIVE by Henkel-Ecolab, Oxysanitizer
by OAKITE). The determination of the contained peroxides
requires a reliable distinction between the two substances,
which are commonly analyzed using their redox properties.
Both peroxides are strong oxidizers, and their technical application is mainly based on oxidation chemistry. However,
in the presence of strong acids hydrogen peroxide serves
as a reducing agent. This difference in redox properties has
been used in titration techniques [3] for resolving mixtures
of both analytes quasi-simultaneously. Data reduction
for curve analysis methods has also been applied for the
determination of peroxides [4]. These techniques include
various operation modes to obtain measurement curves: the

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I.A. Pettas, M.I. Karayannis / Analytica Chimica Acta 522 (2004) 275280

direct temperature variation and the sinusoidal temperature


modulation, for the evaluation of voltammograms. Further
methods for the determination of PAA include photometry
[5], electrochemistry [6] gas chromatography [7] and direct
liquid chromatography [8]. However, none of these methods
is suited to determine both PAA and hydrogen peroxide
selectively and simultaneously. Pinkernell et al. [9] has
developed an HPLC method which performs well for the
analysis of real samples, but it requires a sample pretreatment
with triphenylphosphine and phosphine oxide.
In this paper, a multiple linear regression (MLR) method
was developed for the simultaneous selective determination
of PAA and hydrogen peroxide in residues from a disinfection
process in a beer brewery without prior sample preparation.
A univariate method was utilized to build calibration curves,
investigate the experimental conditions, and examine the actual applicability of the MLR spectroscopic method in such
situations.

2. Theory
2.1. Multiple linear regression (MLR) method
It involves the application of MLR to the inverse expression of Beers law of spectroscopy [10]:


1
C=
(1)
An
n b
where C is the concentration of the constituent of interest,
the molar absorptivity, b the pathlength of the radiation in the
cell and A, the respective absorbance at a given wavelength,
n.
For the initial rate method [11,12], the initial rate of the reaction dAn /dt at wavelength n, replaced the absorbance value
An at Eq. (1), according to the equation:
 
1 dAn
(2)
C=
k
dt
where k is the reaction rate constant.
The matrix of concentrations of the analytes (C) are treated
as the dependent variables, whereas the responses of the detector (X; either absorbances or initial rates) are the independent variables during calibration (P) according to the model
equation:
C = PX

(3)

The MLR method requires only one matrix inversion for


developing the calibration model:
1

P = CXT (XXT )

(4)

For the prediction of the unknowns only one matrix multiplication is required:
Cunkwown = PXunknown

(5)

Examination of the method included fitting of the developed model to the calibration data, and the results were plotted and inspected visually.

3. Experimental
3.1. Apparatus
Spectra-kinetic measurements were made using an S2000
spectrophotometer with CCD detector (Optronics Inc.,
Florida) equipped with fiber optics. A stopped-flow module
supplied by Tri-tech Dynamic Instruments (Cantech Scientific Ltd., Canada), with an observation cell of 3 mm path
length, was fitted to the spectrophotometer. A MP-1035D
tungsten-halogen lamp (model MP-1035D), attached with the
aid of optical fibers, was used as light source. The solutions
in the module and the cell were kept at a constant temperature
of 25 C, by circulating water from a thermostated tank. The
data were acquired and edited using a Pentium III 750 MHz
personal computer with 256 MB of RAM. The initial rates
of the reactions were calculated, with a homemade algorithm
[11], after the data acquisition of the kinetic curves to the
PC. All algorithms were written in MATLAB version 5.x
environment, or used from PLS 2.11 Toolbox for MATLAB.
3.2. Reagents
All reagents were of analytical-reagent grade. A hydrogen
peroxide solution 30% (Merck) was utilized for the preparation of standard dilute solutions. An aqueous stock solution
of 1 mM of peroxyacetic acid (PAA) was prepared (Merck)
in distilled water. To minimize any analytical errors, spectra
were collected immediately after the preparation of all peroxide solutions. A stock solution of 10 mM of DPA was also
prepared in a mixture of 90% formic acid and 10% distilled
water (Riedel-De Haen). Copper(II) solution with a concentration of 0.1 mM was obtained by diluting CuCl2 , purchased
by Merck, in distilled water.
3.3. Safety considerations
3.3.1. Hazard identication
Strong oxidizer: stabilized peracetic acid under fire conditions intensifies the fire. The liquid phase and mist of peracetic and formic acid are corrosive (causing burns); direct
contact could cause irreversible damage to eyes. It will irritate nose, throat, and lungs but will usually subside when
exposure ceases.
3.3.2. Handling and storage of peroxyacetic and
formic acid
Transfer products from drums to process in closed
system. Use exhaust ventilation. Empty drum/container
as thoroughly as possible, rinse before disposal. Avoid
contamination. Never return to original container. Use

I.A. Pettas, M.I. Karayannis / Analytica Chimica Acta 522 (2004) 275280

airless spray to minimize mist generation. Store in a cool,


dry, well-ventilated area away from alkalies and metals. Do
not store in direct sunlight. Do not stack drums, use first in,
first out storage system. Expected shelf life 1 year.
3.3.3. First aid measures
Immediately flush with plenty of water for at least 15 min.
Immediately remove contaminated clothing and wash with
soap and water. If irritation persists, see a physician. Remove
to fresh air. If breathing discomfort occurs, call a physician.
Do not induce vomiting. Give copious amounts of water to
cause dilution in stomach. Probable mucosal damage may
contraindicate the use of gastric lavage.
3.4. Optimization of experimental conditions
The MLR method was used for the analysis of mixtures of
hydrogen peroxide and PAA. This method utilizes the difference in the initial rates of the reactions of the two compounds
with DPA, in the presence of Cu(II) as catalyst. Both peroxides react with DPA to give coloured products with the same
UVvis spectra, but with different formation rates [13]. The
reaction was monitored simultaneously in two wavelengths,
which correspond to the maximum peaks (350 and 582 nm)
of the visible area of the spectrum. The oxidation of DPA in
the presence of Cu(II) in strong acidic media occurs firstly
by an irreversible process to the colourless diphenylbenzidine, which then, is oxidized reversibly in two steps to a blue
violet diquinonediimine [14]. The absorption of the last compound was finally measured in short time, because it is unstable, and the measured color is destroyed irreversibly (Fig. 1).
The kinetic data are acquired from the absorbance values of
diquinonediimine that appear after two previous steps and

277

later it is destroyed irreversibly, suggesting the necessity of


determining the time zero. However, the rate dependent step
of the formation of the diquinonediimine upon the other (instantaneous) steps of the reaction eliminate the implications
in the determination of the time zero, and only the instrumental limitations determined a dead time of 3 ms for all collected
kinetic curves.
The reaction rate of PAA with DPA had duration of a few
seconds, whereas the ratio of initial rates of the reactions of
10-fold hydrogen peroxide to PAA was 9.7:1, respectively.
All optimized experimental conditions were kept constant
during calibration and prediction experiments, assuring the
extraction of kinetic information which is based exclusively
on the changes of both analyte concentrations.
The effect of pH on the decomposition of peroxyacetic
acid in an aqueous solution has been studied. It is known
from the literature that three potential reactions, namely (i)
the spontaneous decomposition, (ii) the hydrolysis and (iii)
the transition metal catalyzed decomposition, are responsible
for the consumption of peroxyacetic acid [15]. The spontaneous decomposition reaches its maximum at pH 8.2, while
both the hydrolysis and metal ion catalyzed decomposition
increase as the pH increases. At pH 10.5 and higher, the hydrolysis becomes dominant. The kinetics of the peracetic acid
hydrolysis was developed, which can predict satisfactory the
development of peracetic acid and hydrogen peroxide during the decomposition reaction. The oxidation of DPA takes
place very slowly (practically not observed) at a mild acidic
media. It was found that the use of formic acid as the dilutant, had the advantage of being a very good solvent of DPA
(better than the water), and at the same time acted as buffer
solution, keeping the pH values practically constant in low
levels. From the other hand, the water is a better solvent for

Fig. 1. Oxidation of DPA in strong acidic media.

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Fig. 2. The initial rate of the reaction H2 O2 DPA at 350 nm, for mixtures
of formic acidwater of various proportions ([DPA] = 0.1 mM, [H2 O2 ] =
0.2 mM, [Cu(II)] = 10 mM, temp = 25 C).

the salt of copper, the molecular absorptivity of the colored


product is higher, and the peroxides have a more stable chemical behavior in water solutions. For these reasons, the initial
rate of the reaction hydrogen peroxideDPA with Cu(II) as
catalyst, was monitored at 350 nm, for mixtures of formic
acidwater of various proportions (Fig. 2). A mixture of 90%
formic acid and 10% water was selected as the optimum solvent for the reactions to take place in maximum rate and the
highest sensitivity.
The maximum working temperature was 25 C, because
further increase led to the degradation of the peroxides and
the formation of bubbles of oxygen gas in the stopped-flow
syringes and the cell.
Fig. 3 demonstrates the effect of concentration of the catalyst on the reaction rate at 582 nm. The highest of the five
tested concentrations was chosen as the optimum one for the
calibration and the determination of the peroxides. Using the
specific concentration of 10 mM Cu(II), the mechanism of
DPA oxidation followed a pseudo-first order route in respect
to PAA and hydrogen peroxide concentrations. In addition,
the incubation time of the reaction was minimized.

Fig. 4. The effect of concentration of the DPA on the reaction rate, at 582 nm:
(a) [DPA] = 0.05 mM, (b) [DPA] = 0.1 mM, (c) [DPA] = 0.2 mM, (d) [DPA]
= 0.5 mM, ([Cu(II)] = 10 mM, [H2 O2 ] = 0.2 mM, temperature = 25 C).

The optimization in respect to the DPA concentration was


also taken into consideration. The concentration of 0.1 mM
of DPA was selected by examination of the kinetic curves at
582 nm (Fig. 4). Further increase in the DPA concentration
did not result in a dramatic increase in the initial rate of the
reaction, and, moreover, the absorbance values exceeded 2,
where the Beers law is not valid.
Using the experimental conditions, optimized as mentioned above, the univariate calibration curves for H2 O2 and
PAA were separately constructed for the determination of
the linearity range and the detection limit for the given procedure. The calibration curves were constructed by plotting
the initial rate values at 582 and 350 nm, as a function of
each analyte concentration (Fig. 5). The linearity range for
PAA was 0.120.75 mM, whereas for H2 O2 the linearity
range was 0.030.1 mM. The detection limit, was found to
be 0.1 mM for PAA and 0.028 mM for H2 O2 , and was calculated according to IUPAC recommendation [16]. The biased calibration curve of PAA was due to its degradation,
giving acidic acid and H2 O2 . As a result, for the individual
determination of [PAA] the bias has been subtracted, and the
linearity range for the determined analyte was lower than
expected.

4. Results and discussion


4.1. MLR model

Fig. 3. The effect of concentration of the catalyst on the reaction rate, at


582 nm: (a) [Cu(II)] = 1 mM, (b) [Cu(II)] = 2 mM, (c) [Cu(II)] = 5 mM, (d)
[Cu(II)] = 7 mM, (e) [Cu(II)] = 10 mM ([DPA] = 0.1 mM, [H2 O2 ] = 0.2 mM,
temperature = 25 C).

MLR generates simple, easy to understand calibration


models, which relate directly the spectral features to the quantitative presence of the analytes to be determined. However,
in the presence of collinearity, estimates of MLR, are unstable and tend to lead to poor prediction. The absence of
synergistic effects ensured that the parameters obtained from
the kinetic curves for a mixture of the two analytes were

I.A. Pettas, M.I. Karayannis / Analytica Chimica Acta 522 (2004) 275280

279

Fig. 5. Univariate calibration curves for PAA and H2 O2 at: (a) 582 nm, [H2 O2 ] = 0 mM, temp = 25 C, and (b) 350 nm, [H2 O2 ] = 0 mM, temp = 25 C, and
PAA at: (c) 582 nm, [PAA] = 0 mM, temp = 25 C, and (d) 350 nm, [PAA] = 0 mM, temp = 25 C ([DPA] = 0.1 mM, [Cu(II)]= 10 mM).

the sums of the corresponding parameters obtained for each


analyte separately. Additivity was only fulfilled when the criterion of pseudo-first order in respect to the concentration of
both analytes was met. The presence of additivity can eliminate the collinearity problem, and prove the suitability of
MLR method for the specific situation.

4.2. Wavelength selection


The obtained results can further be improved by selecting
these variables which contain the most relevant analytical
information using stepwise regression. In order to find out
which of the wavelengths contribute significantly to the pre-

Table 1
The hydrogen peroxidePAA composition and the initial rates of 13 calibration mixtures used for multivariate calibration, at 350 and 582 nm
Sample number

[H2 O2 ] (mM)

[PAA] (mM)

Initial rate, A s1 (350 nm)

Initial rate, A s1 (586 nm)

1
2
3
4
5
6
7
8
9
10
11
12
13

0.20
0.74
0.34
0.24
0.34
0.39
0.20
0.15
0.29
0.55
0.15
0.20
0.24

0.04
0.04
0.05
0.03
0.03
0.05
0.06
0.06
0.07
0.07
0.08
0.05
0.08

0.0387
0.0793
0.0419
0.0301
0.0357
0.0401
0.0313
0.0319
0.0412
0.0610
0.0409
0.0325
0.0425

0.3197
0.8224
0.3290
0.2458
0.2551
0.2988
0.2490
0.2421
0.3146
0.6252
0.3064
0.2452
0.3221

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I.A. Pettas, M.I. Karayannis / Analytica Chimica Acta 522 (2004) 275280

Table 2
The spiking and the recoveries of 10 real samples used for prediction with MLR method
Number of mixtures

1
2
3
4
5
6
7
8
9
10

Spiked samples PAA:H2 O2

30:1
10:1.67
75:1
15:1
15:1
10:3.33

75:1
10:6.67

Estimated concentration

Predicted concentration

Recovery (%)

[H2 O2 ] (mM)

[PAA] (mM)

[H2 O2 ] (mM)

[PAA] (mM)

H 2 O2

0.30
0.30
0.75
0.75
0.15
0.15
0.30
0.00
0.75
0.15

0.01
0.05
0.01
0.05
0.01
0.05
0.00
0.01
0.01
0.10

0.3030
0.3060
0.7500
0.7950
0.1560
0.1485
0.2910
0.0000
0.7650
0.1530

0.0112
0.0490
0.0088
0.0520
0.0097
0.0425
0.0000
0.0104
0.0114
0.1000

101
102
100
106
104
99
97
106
102
102

PAA
112
98
88
104
97
85
98
104
114
100

diction model and which wavelengths can be left out without


reducing the effectiveness of the model, stepwise regression
was applied to the spectra-kinetic data. The stepwise regression technique starts with an empty model equation, with no
independent variables. As the procedure progresses, variates
are added one at a time, according to the minimization of both
the standard error of calibration (SEC) and the standard error
to the prediction (SEP). As the procedure increases, the number of variables in the equation at each step, the possibility of
deleting a previously included erroneous variable is considered. Thus, variable entered at a previous stage of selection
may be added at subsequent, later stages. The concurrence
of the selected wavelengths of 350 and 582 nm, with the two
absorption peaks of the mixture spectra is pure coincidence.
For the calibration, the initial rates of 13 kinetic curves at
350 nm, and 13 kinetic curves at 582 nm were calculated from
13 different mixtures with different combinations of analyte
concentrations (Table 1). Cross validation of the MLR model
took place using contiguous data blocks of the calibration
set without mean centering of the subsets, showing that with
MLR, the calibration and prediction data correlate well with
the conventional reference data.

97106%, and the recovery for PAA ranged from 85112%,


with satisfactory precision.

4.3. Application to real samples

[8]
[9]
[10]

To investigate the actual applicability of the developed


MLR method to a real life scenario, the hydrogen peroxide and PAA concentrations of brewery samples taken after
cleaning-in-place (CIP) were predicted. The CIP aimed to
disinfection of the brewery circuit, and the samples were dispatched for analysis after the passing of a couple of days.
Spiking of the samples with known hydrogen peroxide and
PAA concentrations and subsequent analysis are shown in
Table 2. The recovery for hydrogen peroxide ranged from

5. Conclusions
One of the most salient features of the proposed method
is speed. Since the time required to obtain analytical data
is a few seconds, the initial rate method in combination with
MLR can be implemented to an automated on-line procedure,
and it is a useful, simple alternative to routine procedures for
the determination of these analytes.

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