Monitoring of aromatic amines by hplc with electrochemical detection

Talanta, Vol. 32, No. 4, pp. 279-283, 1985 Printed in Great Britain. All rights reserved

Copyright

MONITORING OF AROMATIC ELECTROCHEMICAL

0

0039-9140/85 %3.W + 0.00 1985 Pergamon Press Ltd

AMINES BY HPLC WITH DETECTION

COMPARISON OF METHODS FOR DESTRUCTION CARCINOGENIC AROMATIC AMINES IN LABORATORY

OF WASTES

JIG BAREK, VERA PAC~KOVA, KAREL &UL~K* and J~rii ZIMA Department of Analytical Chemistry, Charles University, Albertov 2030, 128 40 Prague 2, Czechoslovakia (Received 21 May 1984. Revised 31 October 1984. Accepted 21 November 1984) Summary-A new chemical method for destruction of carcinogenic aromatic amines in laboratory wastes has been developed. The method is based on enzymatic oxidation of the amines in solution (with hydrogen peroxide and horseradish peroxidase), followed by oxidation of the solid residues with permanganate in sulphuric acid medium. To monitor the efficiency of destruction, a reversed-phase HPLC system has been developed, with voltammettic detection with a carbon-fibre detector, which is substantially more sensitive (detection limits from a few ng down to a few pg of amine) than the commonly used ultraviolet photometric detection. It has been demonstrated that the proposed method of destruction is highly efficient (> 99.8% destruction).

Many aromatic arnines are known or suspected to be carcinogenic towards humans,’ and therefore must be effectively destroyed in industrial and laboratory wastes before these are disposed of. Solution of this problem requires first a sufficiently effective, simple, cheap and rapid procedure for conversion of the carcinogens into harmless products, and secondly a reliable analytical method to check the completeness of destruction. Several methods have been proposed’ for the destruction. The most useful involves oxidation with permanganate in sulphuric acid medium,3 but is unsuitable for wastes containing oxidizable solvents, such as methanol or ethanol, or large amounts of other oxidizable substances. Decontamination of large volumes of aqueous solution is also inconvenient, as large amounts of permanganate are required. A very promising method has been proposed by Kalibanov and Morris,4 based on oxidation of the aromatic amines by hydrogen peroxide, catalysed by horseradish peroxidase, to give the corresponding aromatic amine free radicals, which diffuse from the enzyme active site into the solution, where they polymerize. In contrast to the monomers the polymers are virtually insoluble in water, and can be separated by sedimentation, filtration or centrifugation. Unfortunately, the solid residues have been found to be mutagenic.5 The present work deals with the combination of these two methods, the enzymatic oxidation to preconcentrate the carcinogens from large volumes of

*Author for correspondence. 219

wastes, and the permanganate treatment to destroy the solid residues. Representative aromatic amines were selected for the study (see Table 1). Various methods can be used to determine trace concentrations of aromatic amines, and hence to monitor the effectiveness of their destruction.6 Titration and spectrophotometric methods are insufficiently selective for analysis of complex samples, and better results are obtained by combining spectrofluorimetric determination with TLC separation.’ Gas chromatography has been widely used6 but mostly requires use of derivatives, which complicates the determination and increases the error. HPLC is therefore most suitable, as direct determination of the aromatic amines is then possib1e.6*“‘5With ultraviolet photometric detection, a detection limit of 5 x lo-“M has been achieved.3 The sensitivity can be considerably improved by use of electrochemical detection.“’ In view of the polar character of amines, non-polar stationary phases (e.g., C,s bonded phases) are suitable in combination with aqueous solutions of methanol or acetonitrile; to increase the electrical conductivity of the mobile phase, aqueous buffers or salt solutions are used. Amines are readily oxidized at solid electrodes, monoamines at potentials close to + 1.OV (us. SCE), diamines at around +0.5 V (us. SCE). In the present work, the conditions for HPLC determination of selected aromatic amines in a reversed-phase system with a C,, chemically bonded stationary phase and a methanol-aqueous buffer mobile phase, with combined ultraviolet photometric and voltammetric detection were studied. The method was then applied to monitor the effectiveness of degradation of some amines.

J~rii BAREK et al.

280 Table

1. Characteristics

of the substances

studied,

and their detection

limits Detection

No.

Substance

pK$

1 Benzidine (4,4’-diaminobiphenyl) 2 o-Dianisidine (3,3’-dimethoxybenzidine) 3 3,3’-Dichlorobenzidine 4 3,3’-Diaminobenzidine 5 o-Tolidine (3,3’-dimethylbenzidine) 6 CAminobiphenyl 7 4Nitrobiphenyl 8 I-Naphthylamine 9 2-Naphthylamine 10 2,5-Diaminotoluene 11 MOCA [4,4’-methylenebis(o-chloroaniline)]

1,,,,a,,‘s nm

pK:

Et,* (K,), v

0.003

4

0.05

(+0.51) +0.17 +0.33

16

0.45 0.05 0.03

(+0.58) - 0.73 (DME) (f0.51) (f0.58) +0.45 +0.29 (+0.63)

10 12 11.4 11.4 12.2 5

3.7

268

+0.36

4.7

3.6

212; 303

+0.29

3.7

285t 211; 224; 278 282

3.3 3.0

278 222; 243; 237; 294 247:

4.2 4.0 4.2 5.1 3.5

305 318; 328 259; 291; 337 298

(+ 1.23)

4

*pK, values for the conjugate acids of the bases; pK,, refers to the singly protonated base. tThe value recommended in Castegnar0.r

EXPERIMENTAL

Apparatus The polarization curves of the substances were obtained with a PA-3 polarographic analyser (Laboratomi Piistroje, Czechoslovakia) with a glassy-carbon rotating disk electrode or a dropping mercury electrode. For determination of the conditional dissociation constants of the amines, an ABU13/TTT60/REC61 automatic titrator (Radiometer) was used. The pH values in mixed water-methanol media were not corrected. The chromatographic system included a Pye Unicam LC-XP liquid chromatograph with a TZ4200 dual-line recorder (Laboratomi Piistroje), a stainless-steel column 25 cm long, 3 mm bore, packed with 10 pm Partisil ODS (Pye Unicam), an LC-UV variable-wavelength photometric detector (Pye Unicam), and a carbon-fibre voltammetric detector constructed by the authors and described elsewhere.” The two detectors were connected in series by a short (5 cm long, 0.2 mm bore) stainless-steel capillary. On the basis of published data, 2,‘*the photometric detector was operated at 280 nm, and the voltammetric detector (see Table 1) at a potential of f0.6 V for a mobile-phase pH of co. 7 and a potential of +0.9V for a PH of cc. 3.5. The samples were injected through a 20-~1 ioop. The mobile phase was deaerated by continuous passage of helium. The measurements were made at laboratory temperature and all the potentials were measured vs. the SCE. Reagents The aromatic amines were of analytical purity and obtained from Fluka (benzidine), Merck (3,3’-dimethoxybenzidine and 3,3’-dichlorobenzidine), Sigma (2-naphthylamine, 1-naphthylamine, 4-aminobiphenyl, 2,5-diaminotoluene and- Cnitrobiphenyl), Lachkma,- Czechoslovakia (3.3’-diaminobenzidine) and Serlabo. France _ 14.4’-methvldnebis(o-chloroaniline)]. The substances were dissolved -in O.lM hydrochloric acid to give 5 x 10m3M concentration. If necessary, the dissolution was hastened by sonication. Horseradish peroxidase (hydrogen peroxidase oxidoreductase), from Sigma, was used as a salt-free. powder with a specific activity of 175 purpurogalhn units per mg. Ah other solvents and chemicals used were of analytical purity. The mobile phase consisted of O.lM ammonium acetate containing various amounts of methanol; the pH was adjusted with perchloric acid and sodium hydroxide.

Electrochemical

3

4.1

4.7

Photometric

limit, ng

base, pK,, to the doubly

1.1 1.4 9.0 0.06 4.6 protonated

Procedures Determination of the conditional dissociation constants of the conjugate acids. A 20-ml portion of a lo-‘M solution of amine in a 1: 1 mixture of methanol and 0.2M aqueous sodium perchlorate was potentiometrically titrated with O.OlM hydrochloric acid standardized with sodium tetraborate. The pK, values were taken as the pH corresponding to half-neutralization. The values given in Table 1 are averages of two determinations. Determination of the half-wave (peak) potentials. The polarization curves were obtained by using a three-electrode circuit with a 1: 1 v/v mixture of methanol and aqueous O.lM ammonium acetate, with an SCE (aqueous inner solution) as reference; no correction was made for liquidjunction potential. The solution was deaerated by passage of purified nitrogen. Decontamination

of laboratory

wastes

Simple permanganate method. About 9 mg of test amine was dissolved in 10 ml of 0. 1M hydrochloric acid (10 ml of glacial acetic acid for substances Nos. 6 and 1 l), then 5 ml of 0.2M potassium permanganate and 5 ml of 2M sulphuric acid were added and the mixture was allowed to react overnight. The solution was then subjected to HPLC analysis to check the completeness of degradation. Combined enzymatic andpermanganate oxidation. The pH of the waste solution (containing up to 100 mg of an aromatic amine per litre and up to 20% v/v methanol or ethanol) was adjusted to pH 5-7 with sodium hydroxide or sulphuric acid, and 3.5 ml of 3% hydrogen peroxide solution and 1000 units of horseradish peroxidase (i.e., ca. 6mg) were added per litre of waste solution. After 3 hr, the precipitate formed was filtered off with a porosity-4 frit. The frit and precipitate were then immersed in a mixture of 50 ml of 0.2M potassium permanganate and 50 ml of 2M sulphuric acid and stirred magnetically overnight. The precipitate was thus completely dissolved, and the solution was then subjected to HPLC analysis. HPLC

analysis

Solid ascorbic acid was gradually added to an aliquot of the solution from the permanganate oxidation until the solution became colourless, then the pH was adjusted to about 8 with IOM sodium hydroxide and the solution was centrifuged. One ml of the supernatant liquid was diluted with 3 ml of methanol and centrifuged, and 20~1 of the supernatant solution were injected into the column. The

Monitoring of aromatic amines by HPLC separation was done with a mobile phase consisting of 40% methanol and 60% aqueous O.lM ammonium acetate, pH 354.0, at a flow-rate of 1 ml/mitt. The detectors were operated at 280 nm and +0.9 V respectively and the amine concentration was determined from the voltammetric peak height by the standard-addition method (standard amine solution added to the sample immediately after the ascorbic 0&i) RESULTS AND

281

0

10

3 11

DISCUSSION 0

HPLC of aromatic amines The substances studied are listed in Table 1, and the conjugate acids had very similar conditional dissociation constants in water-methanol mixtures.

The pK, values of substances Nos. 3 and 4 could not be measured by the technique used, because the substances were available only as the hydrochlorides. The absorption maxima*,‘* given in Table 1 indicate that the optimal wavelength for detection of the studied substances is around 280 nm. All the substances studied, except 4-nitrobiphenyl, are readily oxidized at a glassy-carbon electrode. The values given in Table 1 were obtained for a 1: 1 mixture of methanol and aqueous O.lM ammonium acetate, pH 7.1. With some substances (Nos. 3, 6, 8 and 9) there is strong adsorption of the oxidation products on the electrode surface, leading to pronounced drops in the limiting currents; the polarization curves then appear as peaks rather than normal waves and in these cases the peak potentials are given in Table 1 instead of the half-wave potentials. It follows from Table 1 that a potential of ca. +0.6 V is sufficient for the detection of all the substances (except No. 7) at pH 7.1. However, the anodic waves shift to more positive potentials with decreasing pH, by about 80mV/pH. Therefore, the working electrode potential of the detector must be made more positive than +0.6 V when working at lower pH. To maximize the signal-to-noise ratio, the working electrode potential should be as low as possible (to suppress the background current); the best compromise is a potential of f0.9V and a mobile-phase pH of 3.5. AS 4-nitrobiphenyl is not oxidized at carbon electrodes, it cannot be voltammetrically determined. However, as seen from Table 1, it can be reduced at a dropping mercury electrode and thus can be detected polarographically. With the mobile phase used, methanol/aqueous ammonium acetate, the separation efficiency and resolution can in principle be modified by varying the methanol content and the pH. The dependence of the capacity factors on the methanol content of the mobile phase is given in Fig. 1. The dependences are similar for all the substances studied and thus it is impossible to attain a substantial improvement in the separation by changing the methanol content. To optimize the absolute values of the capacity factors (between ca. 1.0 and lO.O), a methanol content of 40% v/v is most suitable. With a 40% v/v methanol mobile phase, the

8

L

E

0

5 1,6,7

1

2 9 4

-0

-0

8 t

I 30

I 50 %

I 70

methanol

Fig. 1.Dependence of log k’ on the methanol content in the mobile phase. Substance: l-0, 2-0, %x , 4-A, S--A, 6--V, 7--v, 8-0,9-H, lO--*, 1l-+. Conditions: O.lM aqueous ammonium acetate +xX v/v methanol, pH 7.1; flow-rate, 1 ml/min. For list of substances see Table 1.

dependences of log k’ on pH were then measured (Fig. 2). Although the pK, values of the studied substances are similar, the pH-dependence of the capacity factors varies more significantly than the methanol-dependence. The greatest changes occur between pH 3 and 5, corresponding to the pK, values of most of the substances. A decrease in the pH leads to a greater protonation of the amines, i.e., to an increase in their polarity (and solubility), and thus to a decrease in their retention times. With some substances the elution order is also changed (Nos. 1, 5, 6 and 7). As follows from Fig. 2, the optimal mobilephase pH for the separation is about 3.5-4.0. However, the detector electrode potential must be increased accordingly, see above. The optimal conditions for the separation and determination are: a mobile phase consisting of 40% methanol + 60% aqueous 0.l&f ammonium acetate, pH 3.5-4.0; photometric detector wavelength 280 nm; detector electrode potential, +0.9 V us. SCE. The response of the two detectors to the studied substances was measured under these conditions for various amounts injected. The detection limits (for a signal equal to twice the absolute noise value) are given in Table 1. It can be seen that for most of the substances the voltammetric detector is substantially more sensitive than the photometric detector and the lowest detection limit is a few picograms. The greatest difference in sensitivity between the two detectors is

282

Jrrii BAREK ez al.

I

‘08

11

3

04-

-t 7

-B

6

O-

-04-

-069 I

I 1

I

I

I

3

5

7'

PH

Fig. 2. Dependence of log k’ on the mobile phase pH. For list of symbols see Fig. 1. Conditions: O.lM aqueous ammonium acetate +40”% v/v methanol; flow-rate, 1 ml/min. For list of substances see Table 1. for substances Nos. 1, 2, 4, 5 and 10, for which the voltammetric detector is 2-3 orders of magnitude more sensitive than the photometric detector; the difference is cu. one order of magnitude for substances Nos. 3, 6 and 8, and the sensitivities are similar for substances Nos. 9 and 11. It follows that the voltammetric detector is clearly preferable for use in trace analyses. The calibration data show that the linear dynamic range of the voltammetric detector extends from the detection limit to cu. 50 ng. The calibration graphs exhibit good linearity (correlation coefficients ranging from 0.9996 for I-naphthylamine to 0.9978 for 2-naphthylamine) and satisfactory precision (the relative standard deviations of the peak height amount to only a few per cent and do not exceed l&12% (5 replicates, 95% limits) even at the lowest measured concentrations. Therefore, the method can be used to monitor the effectiveness of the degradation treatment. A chromatogram of the solution after degradation of 3,3’-diaminobenzidine is shown in Fig. 3. Chemical degradation

of the amines

Typical results for the degradation procedures, as monitored by HPLC with electrochemical detection, are summarized in Table 2. The degradation methods are highly efficient and lead to destruction of more than 99.95% of most of the substances tested. The efficiencies found by electrochemical detection are higher than those found by photometric detection3 because of the higher sensitivity. The apparently less

283

Monitoring of aromatic amines by HPLC

(a)

such degradation procedures, but also for trace analyses for these important substances in general.

2llA

t

REFERENCES

1. IARC Monographs on the Evaluation of the Carcinogenic

I

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I

I

I

I

0

2

4

6

0

2

4

6

Time

2.

(mln)

Fig. 3. Chromatogram of 3,3’-diaminobenzidine after the decomposition of the waste solution (a) and the same with a standard addition corresponding to 7.65 x lo-‘,44 concentration (b). Conditions: O.lM aqueous ammonium acetate + 40% v/v methanol; pH 3.7; flow-rate 1 ml/mm, voltammetric detection at +0.9 V. favourable values for substances Nos. 9 and 11 are probably due to the poorer detection sensitivity (for substances Nos. 2, 3,9 and 11 the detection limit was higher than the residual amine concentration). The enzymatic cannot method for be used 2,5-diaminotoluene, as no precipitate is formed. For all the other amines tested, the permanganate oxidation is a highly efficient method for destruction of the solid mutagenic residues. It has been founds that the resulting solutions have no mutagenic effects and are thus environmentally harmless. It can be concluded that the method combining the enzymatic and chemical oxidation is suitable for large volumes of wastes containing small amounts of aromatic amines, and in the presence of oxidizable solvents and other oxidizable substances, where the simple permanganate method fails. The combined method is also somewhat more efficient than the simple permanganate method (see Table 2). The HPLC method with electrochemical detection is suitable not only for monitoring the effectiveness of

3. 4. 5. 6.

7. 8. 9. IO. 11. 12. 13. 14. 15. 16. 17. 18.

Risk of Chemicals to Humans. Supplement I, Chemicals and Industrial Processes Associated with Cancer in Humans, International Agency for Research on Cancer, Lyon, 1979. M. Castegnaro (ed.), Laboratory Decontamination and Destruction of Aromatic Amines in Laboratory Wastes, International Agency for Research on Cancer, Lyon, in the press. M. Castegnaro, Ch. Malaveille, I. Brouet and J. Barek, Am. Ind. Hyg. Assoc. J., in the press. A. M. Kalibanov and E. D. Morris, Enzyme Microbial. Technol., 1981, 3, 119. M. Casteganro, unpublished results. H. Egan (ed.), Environmental Carcinogens-Selected Methodr of Analysis, Vol. 4, Some Aromatic Amines and Azo Dyes in the General and Industrial Environment. International Agency for Research on Cancer, Lyon, 1981. I. M. Jakovljevic, J. Zynger and R. H. Bishara, Anal. Chem., 1975, 41, 2045. R. J. Passarelli and E. C. Jacobs, J. Chromatog. Sci., 1975, 13, 153. I. Mefford, R. W. Keller, R. N. Adams, C. A. Stemson and M. S. Yelo, Anal. Chem., 1977, 49, 683. J. R. Rice and P. T. Kissinger, J. Anal. Toxicol., 1979, 3, 64. D. N. Armentrout and J. Cutie, J. Chromatog. Sci., 1980, 18, 370. R. I. Riggin and C. C. Howard, Anal. Chem., 1979,51, 210. J. R. Rice and P. T. Kissinger, Environ. Sci. Technol., 1980, 16, 263. C. J. Pumell and C. J. Warwick, Analyst, 1980,105,861. V. Concialini, G. Chiavari and P. Vitali. J. Chromaton.. . , 1983, 258, 244. K. Stulik and V. Pacakova, CRC Crit. Rev. Anal. Chem., 1984, 14, 297. Idem, J. Chromatog., 1984, 298, 225. CRC Atlas of Spectral Duta and Physical Constants for Organic Compounds, 2nd Ed., CRC Press, Boca Raton, 1975.