Analytica Chimica Acta 373 (1998) 197206

Cloud point preconcentration and liquid chromatographic determination of aromatic amines in dyestuffs
Yu-Chao Wu, Shang-Da Huang*
Department of Chemistry, National Tsing Hua University, Hsinchu, Taiwan 300, ROC, Taiwan Received 16 February 1998; received in revised form 4 June 1998; accepted 6 June 1998

Abstract Aromatic amines at mg l1 levels in water were determined with cloud point preconcentration followed by liquid chromatography (LC) with UV absorption detection. This method was applied to the determination of aromatic amines at 10 mg g1 levels in commercial dyestuffs. The dyestuff was dissolved in water and precleaned with a SAX cartridge packed with an anion-exchange resin; the efuent was then analyzed using the proposed cloud point preconcentration, with subsequent determination by LC. # 1998 Elsevier Science B.V. All rights reserved.
Keywords: Aromatic amines; Dyestuffs; Liquid chromatography; Cloud point preconcentration

1. Introduction The carcinogenic activity of benzidine and several aromatic amines (e.g. 3,3H -dimethylbenzidine (DMBz), 4-aminobiphenyl (4-ABP), 3,3H -dichlorobenzidine (DCBz), 2-naphthylamine (2-NA) and 4aminoazobenzene (4-AAB)) is well-known [1]. These compounds were widely used as intermediates in the production of azo dyes, pesticides and pharmaceuticals. Over 200 dyes based on benzidine appear in the Colour Index or are in commercial use [2]. Sunset Yellow FCF, Tartrazine and Amaranth were important synthetic food dyestuffs. Amaranth is now prohibited in food due to its potential carcinogenic activity, but is currently used to dye wool and silk. Both for user and worker protection, accurate but simple analytical
*Corresponding author. Fax: +886-3573-6969; e-mail: sdhuang@chem.nthu.edu.tw 0003-2670/98/$19.00 # 1998 Elsevier Science B.V. All rights reserved. PII S0003-2670(98)00393-6

methods are demanded to detect carcinogenic aromatic amines in these dyestuffs. Several methods have been developed for the determination of benzidine and related congener. Colorimetric [3,4] and spectrophotouorimetric methods [5] are sensitive, but not highly specic in general. The polar nature of aromatic amines and their involatility make their analysis by gas chromatography difcult, because the amino groups may be adsorbed on the chromatographic support resulting in severe tailing or losses of components [6]. Better separation of aromatic amines is obtained with derivatives rather than with free analytes [7,8]; however, preparing derivatives prolongs the analytical procedure. Therefore liquid chromatography (LC) is generally regarded as the best technique for determination of aromatic amines. Since the concentration levels of interest for environmental analysis are very low, a preconcentration step is needed. Usually, isolation and

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enrichment of aromatic amines are carried out by liquidliquid extraction (LLE) [912] or solid-phase extraction (SPE) [13,14]. A method frequently employed to determine aromatic amines in watersoluble dyes involves extraction of amines with chloroform, followed by diazotization of amines and coupling of diazonium salts with a reagent (Rsalt or pyrazolone-T) to form a mixture of colored products [1012]. The products are then separated by LC and determined with UV absorption at 254 and 510 nm. However, the lengthy and complicated procedures are not only tedious, which limited the number of samples that can be analyzed, but also susceptible to contamination and loss of aromatic amines. Hence a simple and specic method is needed to determine aromatic amines in dyestuffs. Aqueous solutions of nonionic surfactants became turbid when they are heated above the temperature known as the cloud point [15,16]. The solution is then separated into two isotropic phases, i.e. a surfactantrich phase and a bulk aqueous phase. The hydrophobic solutes can be enriched into the surfactant-rich phase. The small volume of the surfactant-rich phase obtained with this methodology permits the design of extraction schemes that are simple and cheap, and have lower toxicity than extraction with organic solvents. They can provide results comparable to those obtained by other separation techniques. The comprehensive reviews of the theory and applications of surfactant-mediated separations in analytical chemistry are available [17,18]. The cloud point methodology has recently been applied to the preconcentration of a wide range of analytes as a step prior to their determination by LC [1926]. When cloud point preconcentration prior to LC analysis is used, the signicant limitations are the high background absorbance in the UV region and the lengthy operating time required for total elution of the surfactant injected. Several ways to overcome this problem have been proposed: Hinze et al. [19] used the zwitterionic surfactants 3-(nonyldimethyl ammonium) propyl sulfate (C9APSO4) and 3-(decyldimethyl ammonium) propyl sulfate (C10APSO4) that do not absorb at the customary working wavelengths in LC, Cordero et al. [20,23] used the electrochemical detection due to the electrochemical inactivity of commercially available surfactants such as Triton X-114, and Guiteras et al. [26] used a clean up step

with a silica-gel column to remove the surfactant before sample injection. In this work, we rst investigated the enrichment of six aromatic amine at mg l1 levels in water by cloud point preconcentration, with subsequent determination by LC with UV absorption detection. The chromatographic conditions were optimized to separate the analyzed compounds from the surfactant and to shorten the separation time. A simple method to determine aromatic amines at 10 mg g1 levels in dyestuff samples, based on clean-up with an SAX cartridge packed with anion-exchange resin [27], followed by cloud point preconcentration and determination by LC was developed. 2. Experimental 2.1. Reagents Analytical reagent grade benzidine (Bz), 3,3H dimethybenzidine (DMBz), 4-aminobiphenyl (4ABP), 3,3H -dichlorobenzidine (DCBz) and 2naphthaylamine (2-NA) were purchased from Sigma (St. Louis, MO, USA) and 4-aminoazobenzene (4AAB) was obtained from Tokyo Chemical (Tokyo, Japan). The nonionic surfactant Triton X-114 was obtained from Fluka and used without further purication. LC-grade sodium acetate was obtained from Fisher Scientic (Fairlawn, NJ, USA) and acetonitrile was obtained from Tedia (Faireld, OH, USA). Deionized water was puried in a Milli-Q ltration system (Millipore, Bedford, MA, USA) to obtain LC-grade water for preparing mobile phases and standard solutions. 2.2. Apparatus The LC system, assembled from modular components (Waters), consisted of a Model 600E pump, a Model 486 UV detector and a Model 715 automatic sampler. The wavelength of the UV detector was set at 280 nm. A 4 mm Nova-Pak C18 column (15 cm3.9 mm, Waters) was used. A Millennium workstation (Waters) was utilized to control the system, and for acquisition and analysis of data. Cloud point preconcentration was carried out in a YSC P610 water bath (Yeong Shin, ROC.).

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A Hettich universal centrifuge (Hettich Gmbh, Germany) was used to separate the surfactant-rich phase from the aqueous phase during cloud point preconcentration. 2.3. Standards and samples Stock standard solutions were prepared by weighing the aromatic amines and dissolving them in methanol. A working composite standard solution was prepared by combining an aliquot of each of the stock standard solutions and diluting the mixture with water. These solutions were stored in dark glass bottles at 48C. Dyestuff samples of three kinds (Amaranth, Sunset Yellow FCF and Tartrazine) were used (Tokyo Chemical). Dyestuff (0.1 g) was weighed into a volumetric ask (100 ml), water (ca. 50 ml) was added and the solid was dissolved completely with ultrasonic vibration. For recovery tests, a suitable aliquot of the standard solution of the aromatic amines was added to the dyestuff solution. 2.4. Procedure 2.4.1. Cloud point determination and ratio of phase The procedure for the determination of the phase diagram of Triton X-114 and calculation of the phase relationships are similar to that reported by Cordero et al. [21] and Martinez et al. [24], except that the sample volume we used is 10 ml instead of 15 ml (due to the limitation of the capacity of the centrifuge we used). The cloud point of Triton X-114 was determined by observing the appearance of the two phases on heating cold aqueous solutions of surfactant in a water bath. The ratio of phases was measured in tubes calibrated for different amounts of surfactant under the same experimental conditions as those used for phase separation (heating at 408C and centrifuging at 3500 rpm). 2.4.2. Cloud point preconcentration Aliquots of 10.0 ml of the cold solutions containing the analytes in 0.203.0% Triton X-114 were kept for 5 min in a water bath at 408C; the two phases were separated by centrifugation for 5 min at 3500 rpm. The supernatant aqueous phase was removed with a Pasteur pipette, and the surfactant-rich phase was

transferred to the samples vials of the autosampler with a 100 ml micro-transferpettor (Gilson), and then the sample (25 ml) was injected into the chromatographic system by the automatic sampler. 2.4.3. LC operating conditions The mobile phase was composed of acetonitrile and 0.1 M acetate buffer (pH 4.66). Separations were accomplished at a ow rate of 1.0 ml min1 using the following gradient sequence: The column was ushed with the mobile phase of acetonitrilebuffer (40:60) for 6 min, followed by ushing with pure acetonitrile (100:0) for 9 min, and then the column was ushed with acetonitrilebuffer (40:60) for 5 min. The gradient elution mode conrmed that the analytes were eluted and separated at the rst 8 min and the Triton X-114 was eluted after 10 min. 2.4.4. Determination of aromatic amines in dyestuff samples The large amount of co-existing dyestuff would interfere with the analysis. Therefore, a disposable SAX cartridge (3 ml volume tube containing 500 mg of SAX sorbent, Analytichem International) was used as a clean-up lter. The dyestuff solution (exactly 10 ml) was passed through the SAX cartridge and the efuent collected. By this means, while aromatic amines passed unretained through the SAX cartridge, dyestuff components were absorbed. To minimize losses of aromatic amines caused by partial absorption on the SAX cartridge and to maintain aromatic amines in the neutral form, the cartridge was then eluted with 4 ml of 0.5 M acetate buffer (pH5.66) and the eluent was collected. The efuent and eluent were combined and mixed. An aliquot of the mixed solution (10 ml) was then enriched with the cloud point method and analyzed in the manner described above. 3. Results and discussion 3.1. Phase ratio and diagrams The cloud point temperature of Triton X-114 varies between 228C and 308C for surfactant concentrations ranging between 0.1% and 10.0%, this result is similar to that reported by other investigators [21,26]. The cloud point temperature is roughly constant (23258C)

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3.2. Chromatographic behavior of the surfactant Fig. 2(a) shows the chromatogram obtained by the injection of an aliquot of the surfactant-rich phase after cloud point separation, and using an isocratic elution with the acetonitrile/0.1 M acetate buffer (pH 4.66) (40:60, v/v). The Triton X-114 does not show an appreciable signal during the rst 17 min when the aromatic amines studied are separated and detected; however, the time required for the chromatographic separation takes almost 1 h. Fig. 2(b) shows the chromatogram of the injection of an aliquot of the surfactant-rich phase after cloud point separation with a gradient elution. The aromatic amines are isolated during the rst 8 min and the surfactant eluted between 11 and 15 min. 3.3. Cloud point preconcentration and liquid chromatographic analysis The enhancement factor (the ratio of peak intensities with and without preconcentration) for six aromatic amines in solutions with three different surfactant concentrations is shown in Table 1. The phase ratios for solutions with three different surfactant concentrations (1.0, 0.5, 0.2%) were 20, 40 and 100, respectively. The amount of aromatic amines extracted into the surfactant-rich phase depends on the hydrophobility of the analyte and the amount of Triton X-114 used in the preconcentration step. It was found that by using 1.0% Triton X-114, the extraction of aromatic amines was complete except for benzidine. The enhancement factor for a few compounds is greater than the phase ratio. This phenomenon was
Table 1 Enhancement factorsa for surfactant solutions with different concentration Compound Concentration of surfactant 1.0% Bz DMBz 2-NA 4-ABP DCBz 4-AAB
a

Fig. 1. Variation of the preconcentration factor and the % of surfactant-rich phase obtained as a function of Triton X-114 concentration. Sample volume, 10 ml.

in the range of concentrations 0.25%, thus facilitating experimentation. The theoretical preconcentration factor and the percentage of the surfactant-rich phase obtained as a function of the concentration of Triton X-114 are shown in Fig. 1. The theoretical preconcentration factor was calculated as the ratio of the volume of solution used to the volume of the surfactant-rich phase. The percentage of the surfactant-rich phase was calculated as the ratio of its volume to the volume of solution used for the cloud point preconcentration. The theoretical preconcentration factor for the 0.2% Triton X-114 solution was 100, for which the volume of the surfactant-rich phase was 100 ml. This volume is easily handled with a micro-transferpettor and permits the injection of two 25 ml aliquots into the chromatographic system. The theoretical preconcentration factor as function of the concentration of Triton X-114 has been reported by Cordero et al. [21] and Martinez et al. [24]. The values of preconcentration factor shown in Fig. 1 are somewhat lower than that reported by Martinez et al. [24] and are much lower than that reported by Cordero et al. [21]. These differences are probably caused by the differences in the experimental conditions, such as sample volume (10 ml vs 15 ml) and the duration (5 min vs 15 min) the surfactant solution kept in the thermostatic bath. The theoretical preconcentration factor may increase using larger sample volume and/or using longer period of time to keep the surfactant solution in the thermostatic bath, due to their effect on the water content of the surfactant rich phase.

0.5% 15 26 26 34 36 50

0.2% 24 52 50 81 100 135

14 20 20 23 24 33

Ratio of peak intensity with and without the preconcentration step.

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Fig. 2. Chromatograms obtained for the injection of surfactant-rich phases after cloud point separation with (a) an isocratic elution and (b) a gradient elution. For other experimental conditions, see text. Peak assignment: (1) Bz; (2) DMBz; (3) 2-NA; (4) 4-ABP; (5) DCBz; (6) 4-AAB.

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Table 2 Percent recovery of the aromatic amines showing the effect of surfactant concentration on the extraction efficiency Compound Concentration of surfactant 1.0% Bz DMBz 2-NA 4-ABP DCBz 4-AAB 70 100 100 115 120 165 0.5% 38 65 65 85 90 125 0.2% 24 52 50 81 100 135

also observed by other investigators [22,23]. This increase in sensitivity can be probably attributed to modications in the microenvironment by the surfactant when the analytes reach the detector [22,23]. Although the analytes reach the detector before the main peak of the surfactant (aggregates of surfactant molecule with varied degree of aggregation), some of the surfactant molecules may be carried along with the analytes, and causing the increase in sensitivity of the detection. The extraction efciencies of the analytes (expressed as percent recovery) as a function of surfactant concentration are shown in Table 2. The extraction efciencies increase with increasing the concentration of surfactant, as expected. The values of percent recovery for some of the analytes are greater than 100 due to the same reason given above for the greater values of the enhancement factor for a few compounds than that of the phase ratios. Fig. 3 compares the chromatograms obtained by injecting aromatic amines standard (100 mg l1, 25 ml)

and by injecting the surfactant-rich phase (25 ml) which was obtained by cloud point preconcentration of the aromatic amines standard (100 mg l1, 10 ml) with 0.2% Triton X-114. The improvement in sensitivity after cloud point preconcentration was very signicant. Calibration graphs were constructed for 10 ml samples with 0.2% Triton X-114. This concentration of surfactant ensures a sufcient surfactant-rich phase volume to make two injections per sample. In all cases, linear relationships were obtained between peak area and concentration of the analytes studied. Table 3 shows the parameters of the least-squares ttings, the relative standard deviation for six samples to which the complete procedure was applied and the detection limits were determined as the concentration equivalent to three times the standard deviation of replicated measurements (n7). The detection limits can be improved considerably by varying the volume of sample and the amount of surfactant with the preconcentration step is carried out [23]. Solution pH is an important factor in those cloud point extractions involving analytes that possess an acidic or basic moiety [28]. The variation of the extraction efciency of the aromatic amines is practically negligible as the solution pH varied from 4 to 9. The aromatic amines are weak base (e.g. Bz, pKa1 4.66, pKa2 3.57; DMBz, pKa 4.44; 2-NA, pKa 4.16). The peak intensity exhibited a drop as the solution pH becomes lower than 3 (pH<pKa), because the weak base changed to ionic form in the acid solution. Cloud point extraction of aromatic amines is thus performed at solution pH 49.

Table 3 Analytical characteristic of the methoda Compound Bzd DMBz 2-NA 4-ABP DCBz 4-AAB
a b

Intercept (26)102 (11)103 (21)103 (72)103 (51)103 (71)103

Slope (3.840.02)103 (6.120.03)103 (2.100.02)103 (7.470.04)103 (8.230.02)103 (3.320.04)103

R2 0.9999 0.9999 0.9999 0.9999 0.9999 0.9999

RSDb (%) 6.5 6.0 6.8 6.3 6.3 4.8

LODc (mg l1) 0.3 0.3 0.1 0.1 1.1 1.4

Samples, 10 ml, with 0.2% Triton X-114; duplicate injection. RSD: Relative standard deviation (n6). Fortification level: 5 mg l1. c LOD: Limit of detection (calculated as three times the standard deviation of replicated measurements (n7) of the analytes). d Calibration curves with concentration (5, 10, 20, 50 and 500 mg l1).

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Fig. 3. Chromatograms obtained for the 100 mg l1 of the aromatic amines standards: (a) without and (b) with cloud point preconcentration of 10 ml of the sample with 0.2% Triton X-114. Chromatographic conditions as described in Section 2. Peak assignment as in Fig. 2.

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Fig. 4. Chromatograms of (a) a 10 ng/ml aromatic amine standard and (b) a dye sample (Amaranth) after clean-up on a SAX cartridge, followed by the cloud point preconcentration with 0.2% Triton X-114. For other experimental conditions, see text. Peak assignment as in Fig. 2.

Y.-C. Wu, S.-D. Huang / Analytica Chimica Acta 373 (1998) 197206 Table 4 Recoveries of selected aromatic amines added to commercial dyesa Compound Recoveryb (%) Sunset yellow FCF 10 mg/g Bz DMBz 2-NA 4-ABP DCBz 4-AAB
a b

205

Tartrazine 100 mg/g 1093 1103 1027 1102 663 962 10 mg/g 955 1004 1023 1093 563 953 100 mg/g 1115 1115 1023 1134 604 954

Amaranth 10 mg/g 1028 915 969 967 553 891 100 mg/g 1154 1093 1107 1134 664 976

937 1034 1005 1055 632 933

The dyestuff solutions (0.1 g per 100 ml) were processed as described under Section 2. Average value and standard deviation of triplicate runs.

3.4. Determination of aromatic amines in dyestuff samples A disposable SAX cartridge was used as a clean-up lter, followed by cloud-point preconcentration and determination by LC. As shown in Table 4, the recovery of aromatic amines from spiked dyestuffs was excellent (89115%) except that of DCBz (5566%). The low recovery of DCBz is probably due to the stronger afnity of the polar molecule of DCBz to the SAX cartridge. The standard deviations of the means were less than 9%. The method detection limits of this produce were Bz 2.0, DMBz 0.5, 2-NA 1.2, 4-ABP 1.0, DCBz 0.9, 4AAB 0.6 mg/g. The detection limits were measured with a concentration equivalent to three times the standard deviation of replicated measurements (n7) of the analytes in the dyestuff sample (Sunset Yellow FCF). The Occupational Safety and Health Administration [29] states that the concentrations of these compounds in various matrices to which workers may be exposed must not exceed 0.1%. The content of aromatic amines in synthetic food dyestuffs is limited to 0.01% by European color additive specication [30]. Therefore, the proposed method is capable of determining aromatic amines at concentrations much lower than these two regulatory limits. The utility of the method was demonstrated by the analysis of Amaranth. Representative chromatograms resulting from analysis of the standard (10 mg/l) and sample are shown in Fig. 4(a) and (b), respectively. As the aromatic amines are well isolated from other dyestuff components, the presence of a peak with

the retention time of an aromatic amine gives a test for its possible presence. The result indicates that a certain amount of carcinogenic aromatic amine may be present in this dye, namely 15.40.2 mg/g of benzidine. These values were obtained from triplicate determinations using a calibration curve. The concentration of benzidine in this dye, determined by the method of standard additions, was 15.3 mg/g. Acknowledgements This work was supported by the National Science Council of the Republic of China (NSC 87-2113-M007-042). References
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