Liquid chromatographic simultaneous determination of peroxycarboxylic acids using postcolumn derivatization

Article abstract of DOI:10.1021/ac980256b

The first liquid chromatographic method with postcolumn derivatization for the simultaneous determination of peroxycarboxylic acids is described. Aliphatic peracids with chain lengths from C₂ to C₁₂ are separated by HPLC on a reversed-phase C₁₈ column with acetonitrile/water gradient elution. For improved peak shape, tetrahydrofuran and acetic acid are added to the aqueous eluent. After chromatographic separation, the peroxycarboxylic acids react with 2,2′-azino-bis(3-ethylbenzothiazoline)-6-sulfonate, a popular substrate for the enzyme peroxidase. Iodide traces are added as catalyst. The oxidation product, a green radical cation, is determined using a UV/ visible detector in four characteristic regions of the visible and near-infrared spectrum in the range 405-815 nm. The advantages of the new method are detection limits in the low micromolar range, negligible matrix interferences, high reproducibility, and the possibility for simultaneous determination of several peroxycarboxylic acids.

Full text of DOI:10.1021/ac980256b

Anal. Chem. 1998, 70, 3857-3862
Liquid Chromatographic Simultaneous
Determination of Peroxycarboxylic Acids Using
Postcolumn Derivatization
S. Effkemann,† U. Pinkernell,† R. Neumu1ller,‡ F. Schwan,‡ H. Engelhardt,‡ and U. Karst*,†
Abteilung Analytische Chemie, Anorganisch-Chemisches Institut, Westfa¨lische Wilhelms-Universita¨t, Wilhelm-Klemm-Strasse
8, D-48149 Mu¨nster, Germany, and Instrumentelle Analytik/Umweltanalytik, Universita¨t des Saarlandes, Postfach 151 150,
D-66041 Saarbru¨cken, Germany
solvent for carboxylic acids with chain lengths between C6 and
C18. On one hand, concentrated sulfuric acid serves as a solvent
for the carboxylic acids; on the other hand, it removes water from
the right side of eq 1 to shift the equilibrium to the side of the
peroxycarboxylic acid.
A method for the in situ preparation of peroxycarboxylic acids
in laundry detergents is the reaction of hydrogen peroxide with
a bleach activator. In most cases, carboxylic acid esters or amides
are used for this purpose.4 Significant differences concerning the
nature of the formed peroxycarboxylic acids are observed between
North American and European laundry detergents.5 In most
European laundry detergents, peroxyacetic acid is formed as an
active species. In contrast, long-chain peroxycarboxylic acids in
the range between C7 and C12 are generated in many North
American laundry detergents.
Most analytical methods for the determination of peroxycar-
boxylic acids are based on the redox properties of these peroxides.
An early approach to peroxyacetic acid (PAA) determination
involves a two-step titration.6,7 D’Ans and Frey6 oxidized hydrogen
peroxide with potassium permanganate in the first step. After
addition of an excess of iodide and the oxidative formation of
iodine by PAA, a thiosulfate titration provides indirect information
on the PAA content in solution. This method is a convenient way
to determine the concentration of both analytes quasi-simulta-
neously with no need of external calibration. However, its
application is limited by high limits of detection and reproducibility
problems.7
The first liquid chromatographic method with postcolumn
derivatization for the simultaneous determination of per-
oxycarboxylic acids is described. Aliphatic peracids with
chain lengths from C2 to C1 2 are separated by HP LC on a
reversed-phase C1 8 column with acetonitrile/ water gradi-
ent elution. For improved peak shape, tetrahydrofuran
and acetic acid are added to the aqueous eluent. After
chromatographic separation, the peroxycarboxylic acids
react with 2 ,2 ′-azino-bis(3 -ethylbenzothiazoline)-6 -sul-
fonate, a popular substrate for the enzyme peroxidase.
Iodide traces are added as catalyst. The oxidation prod-
uct, a green radical cation, is determined using a UV/
visible detector in four characteristic regions of the visible
and near-infrared spectrum in the range 4 0 5 -8 1 5 nm.
The advantages of the new method are detection limits in
the low micromolar range, negligible matrix interferences,
high reproducibility, and the possibility for simultaneous
determination of several peroxycarboxylic acids.
Peroxycarboxylic acids are important reagents in industrial and
household bleaching, in disinfection in the food and beverages
industries, and as oxidants in organic synthesis.1 The peroxy-
carboxylic acids are synthesized technically in aqueous solution
by the reaction of carboxylic acids with hydrogen peroxide in the
presence of sulfuric acid:2
In the literature, several methods for the photometric deter-
mination of peroxycarboxylic acids have been described.8-15
(4) Hauthal, H. G.; Schmidt, H.; Scholz, H. J.; Hofmann, J.; Pritzkow, W. Tenside,
Surfactants, Detergents 1 9 9 0 , 27, 187-193.
(5) Kirk, O.; Damhus, T.; Christensen, M. W. J. Chromatogr. 1 9 9 2 , 606, 49-
53.
Sulfuric acid serves as a catalyst to accelerate the formation of
the peroxycarboxylic acids in aqueous solutions. Problems may
arise in case of carboxylic acids with chain lengths of more than
five C atoms. The very low solubility of these peroxycarboxylic
acids in aqueous media decreases the reaction rate considerably.
In ref 3, concentrated sulfuric acid is suggested as a suitable
(6) D’Ans, J.; Frey, W. Chem. Ber. 1 9 1 2 , 45, 1845.
(7) Greenspan, F. P.; McKellar, D. G. Anal. Chem. 1 9 4 8 , 20, 1061-1063.
(8) Frew, J. E.; Jones, P.; Scholes, G. Anal. Chim. Acta 1 9 8 3 , 155, 139-150.
(9) Davies, D. M.; Deary, M. E. Analyst 1 9 8 8 , 113, 1477-1479.
(10) Christner, J. E.; Lucchese, L. J. (Serim Research Corp.). WO 92/ 22806, 1992.
(11) Kru¨ ssmann, H.; Bohnen, J. Tenside, Surfactants, Detergents 1 9 9 4 , 31 (4),
229-232.
† Westfa¨lische Wilhelms-Universita¨t.
‡ Universita¨t des Saarlandes.
(1) Swern, D. Organic Peroxides; Wiley-Interscience: New York, 1970; p 360.
(2) Boullion, G.; Lick, C.; Schank, K. In The Chemistry of Functional Groups,
Peroxides; Patai, S., Ed.; John Wiley & Sons: London, 1983; pp 287-298.
(3) Parker, W. E.; Ricciuti, C.; Ogg, C. L.; Swern, D. J. Am. Chem. Soc. 1 9 5 5 ,
77, 4037-4042.
(12) Mallard de la Varende, J.; Crisinel P. (L’Air Liquide S.A.). US 5 438 002,
1995.
(13) Williams J. (Interox Chemicals Ltd.). EP 0 150 123, 1985.
(14) Fischer, W.; Arlt, E.; Braba¨nder, B. (Merck Patent GmbH). DE 37 43 224,
1987; EP 322 631, 1988; US 4 900 682, 1988.
S0003-2700(98)00256-X CCC: $15.00 © 1998 American Chemical Society
Published on Web 08/14/1998
Analytical Chemistry, Vol. 70, No. 18, September 15, 1998 3857

Table 1. Preparation of the Peroxycarboxylic Acids in the Range C3-C12
C3
C4
C5
C6
C7
C8
C9
C10
C12
n(carboxylic acid) (mmol)
m(carboxylic acid) (mg)
V(sulfuric acid) (µL)
n(hydrogen peroxide) (mmol)
V(hydrogen peroxide) (µL)
1
74
125
1.5
146
1
88
125
1.5
146
1
102
125
1.5
146
2
2
1.5
1.5
1
172
430
1.5
146
1
200
600
1.5
146
232
464
3
260
464
3
216
324
2.25
219
237
300
2.25
219
291
291
Direct photometric determination of the peracids is not possible
due to their UV absorption maxima at low wavelengths and small
extinction coefficients.
The photometric method described in ref 15 is based on the
iodide-catalyzed selective oxidation of 2,2′-azino-bis(3-ethylben-
zothiazoline)-6-sulfonate diammonium salt (ABTS) to a green
radical cation by PAA or m-chloroperoxybenzoic acid (mCPBA).
The first application for the simultaneous determination of
several peroxycarboxylic acids in laundry detergents using am-
perometric detection was published by Kirk et al.5 In this work,
no gradient elution was used due to the amperometric detection
system. It was not possible to separate the peroxycarboxylic acids
in the range between C2 and C12 within one chromatogram
because different mobile phases had to be used for the separation
and detection of different peroxycarboxylic acids. Here, 100 mM
phosphate buffer (adjusted to pH 6) for the detection of peroxy-
acetic acid and 15 mM phosphate buffer (adjusted to pH 6)/
methanol (30:70 v/ v) for the separation and detection of perox-
ycarboxylic acids in the range between C8 and C12 were used.
Therefore, the simultaneous determination of the complete
series of peroxycarboxylic acids in the range between C2 and C12
with gradient elution was the major objective of the present work.
As stated above, the analytical chemistry of the peroxycarboxylic
acids is based on their oxidation properties. This leads to the
formation of the same oxidation product in the case of all peracids.
Therefore, postcolumn derivatization is best suited to determine
several peroxycarboxylic acids in one sample.
The detection of the product can be performed at 405, 415, 649,
732, and 815 nm.15
Further methods for the determination of PAA include the use
of electrochemical sensors16-19 and chromatographic methods.5,20-27
Recently, we have developed a method for the simultaneous
determination of peroxyacetic acid and hydrogen peroxide.26 This
method is based on the selective oxidation of methyl p-tolylsulfide
(MTS) to the corresponding sulfoxide (MTSO) by PAA. In a
second step, triphenylphosphine is oxidized to the corresponding
phosphine oxide by hydrogen peroxide. All four compounds can
be separated easily using HPLC with UV detection. MTSO and
triphenylphosphine oxide are commercially available and can be
used for external calibration.
EXPERIMENTAL SECTION
Sa fety Note: Peroxycarboxylic acids are strong oxidizers and
may react with organic substances. Pure peroxycarboxylic acids
or their concentrated solutions may cause severe explosions. They
should never be mixed with organic substances. The procedures
mentioned within this publication have been investigated for
peroxycarboxylic acid concentrations up to 100 mM.
Chemicals. Hydrogen peroxide, PAA, mCPBA, all carboxylic
acids, MTS, MTSO, and ABTS were purchased from Aldrich
Chemie (Steinheim, Germany) in the highest quality available.
Potassium iodide was obtained from Merck (Darmstadt, Ger-
many). Solvents for HPLC were Merck (Darmstadt, Germany)
LiChroSolv gradient grade. Ultra Biz is a laundry detergent which
is distributed in the United States by Procter & Gamble (Cincin-
nati, OH). Persil Megaperls is a laundry detergent which is
distributed in Germany by Henkel (Du¨ sseldorf, Germany).
P reparation of the P eroxycarboxylic Acids. All peroxycar-
boxylic acids with the exception of PAA and mCPBA were
prepared according to ref 3. PAA and mCPBA were purchased
from Aldrich Chemie. In ref 3, only the preparation of the
peroxycarboxylic acids in the range from C6 to C18 is described.
The method has been adapted for the preparation of the peracids
C3-C5. Some modifications have been made: the carboxylic acid
was dissolved in concentrated sulfuric acid, the solution was
cooled to -5 to -10 °C, and hydrogen peroxide (35%) was added
dropwise and very slowly to the stirred solution. In Table 1, the
composition of the reaction mixtures for the preparation of all used
peroxycarboxylic acids is illustrated.
Unfortunately, the HPLC and photometric methods described
above do not allow a specification of the peroxycarboxylic acids
in the range from C2 to C12. If several peroxycarboxylic acids
occur in combination, only a sum parameter for these substances
is obtained.
(15) Pinkernell, U.; Lu¨ ke, H.-J.; Karst, U. Analyst 1 9 9 7 , 122, 567-571.
(16) Birch, B. J.; Marshman C. E. (Unilever NV). EP 0 333 246, 1989.
(17) Pinkowski, A. (ProMinent Dosiertechnik GmbH). US 5 395 493, 1995.
(18) Teske, G. (Dr. Thiedig & Co.). US 5 503 720, 1996.
(19) Kaden, H.; Herrmann S. (Forschungsinstitut “Kurt Schwabe” Meinsberg).
DE 43 19 002, 1995.
(20) Cairns, G. T.; Ruiz Diaz, R.; Selby, K.; Waddington, D. J. J. Chromatogr.
1 9 7 5 , 103, 381-384.
(21) Di Furia, F.; Prato, M.; Scorrano, G.; Stivanello, M. Analyst 1 9 8 8 , 113, 793-
795.
(22) Di Furia, F.; Prato, M.; Quintly, U.; Salvagno, S.; Scorrano G. Analyst 1 9 8 4 ,
109, 985-987.
(23) Baj, S. Fresenius J. Anal. Chem. 1 9 9 4 , 350, 159-161.
(24) Pinkernell, U.; Karst, U.; Cammann, K. Anal. Chem. 1 9 9 4 , 66, 2599-2602.
(25) Pinkernell, U.; Effkemann, S.; Nitzsche, F.; Karst, U. J. Chromatogr. A 1 9 9 6 ,
730, 203-208.
(26) Pinkernell, U.; Effkemann, S.; Karst, U. Anal. Chem. 1 9 9 7 , 69, 3623-3627.
(27) Effkemann, S.; Pinkernell, U.; Karst, U. Anal. Chim. Acta 1 9 9 8 , 363, 97-
103.
3858 Analytical Chemistry, Vol. 70, No. 18, September 15, 1998

Figure 1. Schematic assembly of the HPLC system with postcolumn derivatization.
The reaction mixtures were stirred for an additional 3 h at room
temperature. The pure peracids were not isolated from their
solutions to avoid an explosion hazard.28 The reaction mixture
was diluted with 25 mL of acetonitrile/ water mixture (80:20 v/ v)
to a concentration range between 10 and 100 mM. These
solutions were stored at -20 °C. No significant decomposition
of the peroxide solutions was observed under these storage
conditions.
P ostcolumn Derivatization. The separations were per-
formed with a modular HPLC system consisting of a Merck-
Hitachi (Darmstadt, Germany) gradient pump L6200 as eluent
pump, a Knauer (Berlin, Germany) HPLC pump 64 as reagent
pump, a Waters 484 tunable absorbance detector, and a Rheodyne
injection valve with a 5-µL sample loop. The reversed-phase
column was Merck LiChroSorb RP18 (125 × 4 mm), 5 µM particle
size. Gradient elution was carried out using binary mixtures of
acetonitrile (eluent A) and water plus additives (eluent B) as the
mobile phase at a flow rate of 1.4 mL/ min. To optimize the peak
shape, water with 2% acetic acid and 1% tetrahydrofuran (THF)
(v/ v) as additives was used as eluent B. The linear gradient used
was from 25% eluent A up to 100% A within 4 min, 2.5 min isocratic
at 100% eluent A, and from 100% eluent A down to 25% A within
0.5 min.
Six milliliters of acetic acid, 2 mg of potassium iodide, and 50
mg of ABTS15 were dissolved in water and diluted to 300 mL. This
reagent solution was stored under protection from light at 4 °C
in a refrigerator. The reagent solution with 0.3 mL/ min was
mixed with the HPLC effluent using a 1 µL turbo mixing
chamber.29,30 A 10-m knitted Teflon capillary30,31 with 0.3-mm
diameter and an internal volume of approximately 0.7 mL was
integrated in an oven and was used as a manifold for the
postcolumn derivatization. Under these conditions, 24 s is
available for the reaction in the oven. An oven temperature of
110 °C was selected unless stated otherwise. An additional 1-m
knitted capillary was used as a heat-exchanger before entering
the UV/ visible detector. Detection was performed at the UV/
visible maximum of the ABTS radical cation at 415 nm. In Figure
1, the schematic assembly of the HPLC instrumentation is shown.
Gradient profiles displayed in the chromatograms recorded
without a column are the effective profiles determined by using
methanol with 200 ppm toluene instead of eluent A and methanol
instead of eluent B and the reagent solution. The increasing
toluene concentration was used to monitor the gradient profile
(λ ) 254 nm). It should be noted that these gradient profiles
report the composition of the mobile phase at the head of the
column. The resulting time delay between the chromatogram and
the gradient profile is due to the void volume of the respective
column.
Chromatographic MTS Method.24-26 One hundred micro-
liters of the peracid in the concentration range from 1 to 100 mM
was added to 1000 µL of 20 mM MTS solution in acetonitrile/
water (50:50 v/ v). After a reaction time of 15 min, 10 mL of
acetonitrile/ water (50:50 v/ v) was added to obtain a suitable
concentration range for HPLC analysis. The HPLC configuration
described above without the reagent pump and the postcolumn
detection system was used. Isocratic elution was carried out with
acetonitrile/ water (75:25 v/ v) as the mobile phase at a flow rate
of 1.5 mL/ min. Detection was performed by UV spectroscopy at
230 nm. Commercially available MTSO was used for external
calibration.
P hotometric ABTS Method.15 Prior to derivatization, the
prepared peracid solutions were diluted by a factor of 1000 using
acetonitrile/ water (50:50 v/ v). Photometric measurements were
carried out with an SQ 118 filter photometer (Merck, Darmstadt,
Germany) using quartz cuvettes with an optical pathway of 1 cm
and a volume of 3 mL. A filter with a wavelength of 405 nm is
used. The molar absortivity at this wavelength is ꢀ ) (3.16 (
0.02) × 104 L mol-1 cm-1 and was used for the quantitative
determination according to Pinkernell et al.15
(28) Swern, D. Organic Peroxides; Wiley-Interscience: New York, 1970; pp 476-
498.
(29) Engelhardt, H.; Lillig, B. Chromatographia 1 9 8 6 , 21, 136-142.
(30) Engelhardt, H.; Neue, U. D. Chromatographia 1 9 8 2 , 15, 403-408.
(31) Krull, I. S. Reaction Detection in Liquid Chromatography; Chromatography
Science Series 34; M. Dekker: New York, 1986; pp 1-61.
RESULTS AND DISCUSSION
First, the suitability of ABTS as a reagent for the photometric
determination of peroxycarboxylic acids with chain lengths
between C2 and C12 was subject to investigations. The content of
Analytical Chemistry, Vol. 70, No. 18, September 15, 1998 3859

Table 2. Comparative Measurements of Different
Peroxycarboxylic Acids
MTS
ABTS
peracid
c (mM) RSD (%) (n ) 3) c (mM) RSD (%) (n ) 3)
mCPBA
C2
C3
C4
C5
C6
C7
C8
C9
17.6
15.4
33.2
39.3
36.0
68.5
42.0
41.9
37.5
30.0
47.6
3.3
3.2
4.2
4.6
3.0
0.4
1.9
1.4
1.9
1.7
2.5
18.2
16.0
33.0
37.9
33.3
63.4
41.5
43.0
40.8
33.6
47.2
4.9
3.1
2.1
1.1
2.1
1.3
1.9
3.0
3.0
1.2
0.6
C10
C12
Figure 2. Chromatogram of the separation of several peroxycar-
boxylic acids using acetonitrile and water without any additives as
eluents (s, chromatogram; ‚‚‚, effective gradient).
peroxycarboxylic acids of several solutions which were prepared
according to the method stated above was determined using the
photometric ABTS and the liquid chromatographic MTS method.
These two methods have been especially adapted to the analyte
concentration range of the prepared solutions. In the case of lower
peracid contents, the RSD increases are caused by significant
blanks of the MTS and ABTS reagents, respectively. The results
of both methods are presented in Table 2.
In a quantitative reaction, a green radical ion is formed by the
iodide-catalyzed reaction between ABTS and the peroxycarboxylic
acids. Selectivity of ABTS toward all of these peracids in the
presence of hydrogen peroxide is observed. Both methods lead
to comparable results with excellent reproducibility.
Using the concentration data determined above, several
mixtures containing 1, 2.5, 5, 10, 25, 50, 100, 250, 500, and 1000
µM of each of the aliphatic peracids were prepared (dilution
matrix: acetonitrile/ water (50:50 v/ v)). Five microliters of the
respective solution was injected in the HPLC system using the
HPLC conditions stated above without additives in the mobile
phase. In the UV/ visible spectrum, a maximum at 206 nm is
observed for organic peracids.5 This wavelength was chosen as
the detection wavelength for the direct determination of these
peroxides. The limits of detection using direct UV/ visible
detection without postcolumn derivatization are in the range of
only 250 µM for all aliphatic peracids.
Figure 3. Chromatogram of the separation of several peroxycar-
boxylic acids using acetonitrile and water with addition of 2% acetic
acid and 1% THF as eluents (s, chromatogram; ‚‚‚, effective
gradient).
equilibrium to the left in eq 3. Some 2% acetic acid was added to
the aqueous eluent to optimize the peak shape. Further addition
of 1% tetrahydrofuran inhibits secondary interactions between the
analytes and the free silanol groups of the stationary phase. Using
both additives, the same peroxycarboxylic acid solution was
injected again. In Figure 3, the resulting chromatogram is
presented.
Therefore, the use of ABTS as a reagent for postcolumn
derivatization of several peroxycarboxylic acids was investigated
in the following.
The chromatogram shows a strong improvement compared
to Figure 2. The peak shapes are significantly better. Hydrogen
peroxide passes the column unretained, and the peroxycarboxylic
acids elute as expected in the order of increasing chain lengths.
Peak identification was performed by injection of each peroxy-
carboxylic acid. The time discrepancy between the programmed
gradient and the real gradient is caused by the void volume of
the HPLC system.
Using the HPLC and derivatization conditions with acetonitrile/
water elution (without additives) as described above, 5 µL of a
solution containing 100 µM of the aliphatic peracids (dilution
matrix: acetonitrile/ water (50:50 v/ v)) was injected. Here, 415
nm was chosen as the wavelength for the detection of the ABTS
radical cation. In Figure 2, the resulting chromatogram and the
effective gradient are presented. All peroxycarboxylic acids can
be detected but with bad peak shape and high limits of detection.
The obvious reason for the peak shape is the equilibrium between
the peracids and their corresponding anions.
To maximize the peak height and peak area, the temperature
of the postcolumn oven with the integrated reaction capillary is
another important parameter. Increasing the temperature should
accelerate the derivatization reaction during the flow of the
reaction partners through the capillary. Five microliters of a
solution containing 100 µM of each aliphatic peracid (dilution
matrix: acetonitrile/ water (50:50 v/ v)) was injected using different
oven temperatures between 20 and 120 °C. The peak area of each
Addition of acetic acid to the aqueous eluent should shift the
3860 Analytical Chemistry, Vol. 70, No. 18, September 15, 1998

Table 3. Calibration Data of Several Peroxycarboxylic Acids
RSD (%)
tR
(min)
slope
linear
range (µM)
c ) 25
µM, n ) 5
c ) 100
µM, n ) 5
c ) 250
µM, n ) 5
peracid
r
(µM-1
)
LOD (µM)
mCPBA
C2
C3
C4
C5
C6
C7
C8
C9
4.85
1.36
1.64
2.35
3.93
4.87
5.49
5.97
6.40
6.79
7.55
>0.998
>0.999
>0.998
>0.998
>0.997
>0.998
>0.999
>0.999
>0.999
>0.999
>0.999
363
309
264
276
237
224
218
221
174
170
183
2.5
5
5
5
5
5
5
5
5
5-250
10-250
10-500
10-500
10-500
10-500
10-500
10-500
10-500
10-500
10-500
7.4
8.2
8.4
8.8
9.0
10.0
7.1
8.8
8.2
7.1
9.8
5.6
4.0
4.0
2.9
6.7
5.8
5.1
5.3
5.8
5.8
6.9
3.2
1.8
2.2
4.9
4.7
3.1
4.1
6.7
4.2
4.6
3.0
C10
C12
5
5
Figure 5. Chromatogram of the American laundry detergent Ultra
Biz (s, Ultra Biz sample; ‚‚‚, C8, C9, C10 peracid standards).
Figure 4. Dependence of the peak area from the postcolumn oven
temperature (9, C2; b, C3; 2, C4; 1, C5; [, C6; +, C7; ×, C8; *, C9; -,
C
10; |, C12
.
As shown in Table 3, the calibration curves of the highly
reactive peracids as PAA and mCPBA are linear only up to 250
µM. A further increase of the PAA or mCPBA concentration
exceeds the linear range of the method. In the case of the less
reactive peracids, linearity is observed up to a concentration of
500 µM. This is an additional indication of a lower reaction rate
in the case of the less reactive long-chain peroxycarboxylic acids.
mCPBA, the most reactive peracid of all the investigated com-
pounds, can be detected down to a concentration of 2.5 µM.
This new method has been used to study the formation of
different peracids from commercially available laundry detergents.
One gram of Ultra Biz, a laundry detergent available in the United
States, was dissolved in 100 mL of water at a temperature of 20
°C using a magnetic stirrer. After 5 min, a sample of 100 µL was
diluted with 1000 µL of 0.1 M acetic acid. The diluted solution
was centrifuged for 5 min at 10 000 rpm to remove the turbidity.
Five microliters of this solution and a standard containing 100
µM of each peracid between C8 and C10 were injected into the
HPLC system using the standard parameters described above.
The resulting chromatogram is presented in Figure 5.
peracid was plotted versus the respective temperature. The
results are presented in Figure 4.
A sigmoid course of the plot is observed in the case of all
investigated peroxycarboxylic acids. Up to 70 °C, a strong
increase of the peak area of all peracids can be observed.
Temperatures higher than 70 °C lead to a further increase of the
peak area only in the case of the long-chain peracids. It is
noteworthy that the peak area decreases with increasing numbers
of C atoms of the respective peroxycarboxylic acids at each
temperature. This indicates that the reaction is obviously not
quantitative in the case of the long-chain peracids under the
reaction conditions used. A temperature of 110 °C was selected
for the postcolumn derivatization reaction. On one hand, a
suitable reaction rate is guaranteed at this temperature, and on
the other hand, the Teflon capillary will not yet be affected by
destruction.
The conditions stated above were used to record calibration
curves of the investigated aliphatic peroxycarboxylic acids and
mCPBA. Therefore, the peracid content of each prepared peracid
was determined by using the MTS method24-26 as stated above.
Each of these solutions was diluted with acetonitrile/ water (50:
50 v/ v) to the following concentrations: 1, 2.5, 5, 10, 25, 50, 100,
250, 500, and 1000 µM. The obtained calibration data are
presented in Table 3. Each solution was injected five times.
Additionally, the RSD (n ) 5) given in Table 3 was determined
for the peracid concentrations 25, 100, and 250 µM.
It is obvious that peroxynonanoic acid is formed in the aqueous
solution of the American detergent. To investigate the formation
of a peracid in a typical European laundry detergent, 1 g of Persil
Megaperls was dissolved in 100 mL of water at a temperature of
20 °C using a magnetic stirrer. After 15 min, a sample of 50 µL
was diluted with 1000 µL of 0.1 M acetic acid. The diluted solution
was centrifuged for 5 min at 10 000 rpm to remove the turbidity.
Analytical Chemistry, Vol. 70, No. 18, September 15, 1998 3861

In contrast to the American laundry detergents, peroxyacetic
acid is formed from most of the commercially available European
laundry detergents, as is the case in the investigated Persil
Megaperls sample. The developed method is, therefore, valuable
for investigations on American as well as European laundry
detergents.
CONCLUSIONS
ABTS is a well-suited reagent for postcolumn derivatization of
several peroxycarboxylic acids. The first method for the identi-
fication and the simultaneous quantitative determination of per-
oxycarboxylic acids in the range from C2 to C12 was developed.
All peroxycarboxylic acids can easily be separated within 8 min.
Addition of small amounts of acetic acid and tetrahydrofuran to
the aqueous eluent are required to obtain good peak shapes.
Figure 6. Chromatogram of the European laundry detergent Persil
Megaperls (s, Persil Megaperls sample; ‚‚‚, C2, C3, C4, C5 peracid
standards).
ACKNOWLEDGMENT
S.E. thanks the Hala´sz Foundation (Saarbru¨ cken, Germany)
for the Applied Physical Chemistry Award 1997 and the FAZIT-
Stiftung (Frankfurt, Germany) for a scholarship.
Five microliters of this solution and a standard containing 100
µM of each peracid between C2 and C5 were injected in the HPLC
system using the parameters described above. The resulting
chromatogram is presented in Figure 6.
Received for review March 6, 1998. Accepted June 30,
1998.
AC980256B
3862 Analytical Chemistry, Vol. 70, No. 18, September 15, 1998