cloud point pre concentration and liquid chromatographic

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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 lÀ1 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 gÀ1 levels in commercial dyestuffs. The dyestuff was dissolved in water and precleaned with a SAX cartridge packed

with an anion-exchange resin; the ef¯uent 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-dichloro-

benzidine (DCBz), 2-naphthylamine (2-NA) and 4-

aminoazobenzene (4-AAB)) is well-known [1]. These

compounds were widely used as intermediates in theproduction of azo dyes, pesticides and pharmaceuti-

cals. 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

methods are demanded to detect carcinogenic aro-

matic amines in these dyestuffs.

Several methods have been developed for the deter-

mination of benzidine and related congener. Colori-

metric [3,4] and spectrophoto¯uorimetric methods [5]

are sensitive, but not highly speci®c in general. The

polar nature of aromatic amines and their involatility

make their analysis by gas chromatography dif®cult,because the amino groups may be adsorbed on the

chromatographic support resulting in severe tailing or

losses of components [6]. Better separation of aro-

matic amines is obtained with derivatives rather than

with free analytes [7,8]; however, preparing deriva-

tives 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

Analytica Chimica Acta 373 (1998) 197±206

*Corresponding author. Fax: +886-3573-6969; e-mail:

[email protected]

0003-2670/98/$19.00 # 1998 Elsevier Science B.V. All rights reserved.

P I I S 0 0 0 3 - 2 6 7 0 ( 9 8 ) 0 0 3 9 3 - 6



enrichment of aromatic amines are carried out by

liquid±liquid extraction (LLE) [9±12] or solid-phase

extraction (SPE) [13,14]. A method frequently

employed to determine aromatic amines in water-soluble dyes involves extraction of amines with

chloroform, followed by diazotization of amines

and coupling of diazonium salts with a reagent (R-

salt or pyrazolone-T) to form a mixture of colored

products [10±12]. The products are then separated by

LC and determined with UV absorption at 254 and

510 nm. However, the lengthy and complicated pro-

cedures are not only tedious, which limited the num-

ber of samples that can be analyzed, but also

susceptible to contamination and loss of aromatic

amines. Hence a simple and speci®c method is neededto 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 surfactant-

rich 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 sol-vents. They can provide results comparable to those

obtained by other separation techniques. The compre-

hensive reviews of the theory and applications of

surfactant-mediated separations in analytical chemis-

try 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 deter-

mination by LC [19±26].

When cloud point preconcentration prior to LC

analysis is used, the signi®cant limitations are the

high background absorbance in the UV region andthe 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 ammo-

nium) propyl sulfate (C9APSO4) and 3-(decyl-

dimethyl 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 lÀ1

levels in water by cloudpoint preconcentration, with subsequent determina-

tion by LC with UV absorption detection. The chro-

matographic 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 gÀ1 levels in

dyestuff samples, based on clean-up with an SAX

cartridge packed with anion-exchange resin [27], fol-

lowed by cloud point preconcentration and determi-

nation by LC was developed.

2. Experimental

2.1. Reagents

Analytical reagent grade benzidine (Bz), 3,3H-

dimethybenzidine (DMBz), 4-aminobiphenyl (4-

ABP), 3,3H-dichlorobenzidine (DCBz) and 2-

naphthaylamine (2-NA) were purchased from Sigma

(St. Louis, MO, USA) and 4-aminoazobenzene (4-

AAB) was obtained from Tokyo Chemical (Tokyo,Japan). The nonionic surfactant Triton X-114 was

obtained from Fluka and used without further puri®-

cation. LC-grade sodium acetate was obtained from

Fisher Scienti®c (Fairlawn, NJ, USA) and acetonitrile

was obtained from Tedia (Fair®eld, OH, USA). Deio-

nized water was puri®ed in a Milli-Q ®ltration system

(Millipore, Bedford, MA, USA) to obtain LC-grade

water for preparing mobile phases and standard solu-

tions.

2.2. Apparatus

The LC system, assembled from modular compo-

nents (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 cmÂ3.9 mm, Waters) was used. A Millennium

workstation (Waters) was utilized to control the sys-

tem, and for acquisition and analysis of data. Cloud

point preconcentration was carried out in a YSC P610

water bath (Yeong Shin, ROC.).

198 Y.-C. Wu, S.-D. Huang / Analytica Chimica Acta 373 (1998) 197±206



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 weigh-

ing 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, SunsetYellow FCF and Tartrazine) were used (Tokyo Che-

mical). 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 vibra-

tion. 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 phaseThe 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 sameexperimental 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 0.20±3.0% 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 chromato-

graphic 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 minÀ1 using

the following gradient sequence: The column was

¯ushed with the mobile phase of acetonitrile±buffer

(40:60) for 6 min, followed by ¯ushing with pure

acetonitrile (100:0) for 9 min, and then the column

was ¯ushed with acetonitrile±buffer (40:60) for 5 min.

The gradient elution mode con®rmed that the analyteswere 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 andthe ef¯uent 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 (pH�5.66) and the eluent

was collected. The ef¯uent 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 (23±258C)

Y.-C. Wu, S.-D. Huang / Analytica Chimica Acta 373 (1998) 197±206 199



in the range of concentrations 0.2±5%, thus facilitat-

ing 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 volumeof 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 chromato-

graphic system. The theoretical preconcentration fac-

tor 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 reportedby 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 surfac-

tant rich phase.

3.2. Chromatographic behavior of the surfactant

Fig. 2(a) shows the chromatogram obtained by the

injection of an aliquot of the surfactant-rich phaseafter cloud point separation, and using an isocratic

elution with the acetonitrile/0.1 M acetate buffer (pH4.66) (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 thesurfactant eluted between 11 and 15 min.

3.3. Cloud point preconcentration and liquid

chromatographic analysis

The enhancement factor (the ratio of peak intensi-

ties with and without preconcentration) for six aro-

matic amines in solutions with three different

surfactant concentrations is shown in Table 1. The

phase ratios for solutions with three different surfac-

tant concentrations (1.0, 0.5, 0.2%) were 20, 40 and100, 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 extractionof aromatic amines was complete except for benzi-

dine. The enhancement factor for a few compounds is

greater than the phase ratio. This phenomenon was

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.

Table 1Enhancement factorsa for surfactant solutions with different

concentration

Compound Concentration of surfactant

1.0% 0.5% 0.2%

Bz 14 15 24

DMBz 20 26 52

2-NA 20 26 50

4-ABP 23 34 81

DCBz 24 36 100

4-AAB 33 50 135

aRatio of peak intensity with and without the preconcentration step.




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.




also observed by other investigators [22,23]. This

increase in sensitivity can be probably attributed tomodi®cations in the microenvironment by the surfac-

tant 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 ef®ciencies of the analytes

(expressed as percent recovery) as a function of

surfactant concentration are shown in Table 2. The

extraction ef®ciencies increase with increasing theconcentration 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 lÀ1, 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 lÀ1, 10 ml)

with 0.2% Triton X-114. The improvement in sensi-tivity after cloud point preconcentration was very

signi®cant.

Calibration graphs were constructed for 10 ml sam-

ples with 0.2% Triton X-114. This concentration of

surfactant ensures a suf®cient 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 detectionlimits were determined as the concentration equivalent

to three times the standard deviation of replicated

measurements (n�7). The detection limits can be

improved considerably by varying the volume of

sample and the amount of surfactant with the pre-

concentration 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 ef®ciency of the aromatic amines is practi-

cally negligible as the solution pH varied from 4 to 9.The aromatic amines are weak base (e.g. Bz, pK a14.66, pK a2 3.57; DMBz, pK a 4.44; 2-NA, pK a 4.16).

The peak intensity exhibited a drop as the solution pH

becomes lower than 3 (pH<pK a), because the weak base changed to ionic form in the acid solution. Cloud

point extraction of aromatic amines is thus performed

at solution pH 4±9.

Table 2

Percent recovery of the aromatic amines showing the effect of

surfactant concentration on the extraction efficiency

Compound Concentration of surfactant

1.0% 0.5% 0.2%

Bz 70 38 24

DMBz 100 65 52

2-NA 100 65 50

4-ABP 115 85 81

DCBz 120 90 100

4-AAB 165 125 135

Table 3Analytical characteristic of the methoda

Compound Intercept Slope R2 RSDb (%) LODc (mg lÀ1)

Bzd (À2Æ6)102 (3.84Æ0.02)103 0.9999 6.5 0.3

DMBz (1Æ1)103 (6.12Æ0.03)103 0.9999 6.0 0.3

2-NA (À2Æ1)103 (2.10Æ0.02)103 0.9999 6.8 0.1

4-ABP (À7Æ2)103 (7.47Æ0.04)103 0.9999 6.3 0.1

DCBz (5Æ1)103 (8.23Æ0.02)103 0.9999 6.3 1.1

4-AAB (À7Æ1)103 (3.32Æ0.04)103 0.9999 4.8 1.4

a Samples, 10 ml, with 0.2% Triton X-114; duplicate injection.b RSD: Relative standard deviation (n�6). Fortification level: 5 mg lÀ1.c LOD: Limit of detection (calculated as three times the standard deviation of replicated measurements ( n�7) of the analytes).d Calibration curves with concentration (5, 10, 20, 50 and 500 mg lÀ1).




Fig. 3. Chromatograms obtained for the 100 mg lÀ1 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.




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.




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 recov-

ery of aromatic amines from spiked dyestuffs was

excellent (89±115%) except that of DCBz (55±66%).

The low recovery of DCBz is probably due to the

stronger af®nity of the polar molecule of DCBz to the

SAX cartridge. The standard deviations of the meanswere 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, 4-

AAB 0.6 mg/g. The detection limits were measured

with a concentration equivalent to three times the

standard deviation of replicated measurements

(n�7) 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 speci®cation

[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 15.4Æ0.2 mg/g of

benzidine. These values were obtained from triplicate

determinations using a calibration curve. The concen-

tration 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-M-

007-042).

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Table 4

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Compound Recoveryb (%)

Sunset yellow FCF Tartrazine Amaranth

10 mg/g 100 mg/g 10 mg/g 100 mg/g 10 mg/g 100 mg/g

Bz 93Æ7 109Æ3 95Æ5 111Æ5 102Æ8 115Æ4

DMBz 103Æ4 110Æ3 100Æ4 111Æ5 91Æ5 109Æ3

2-NA 100Æ5 102Æ7 102Æ3 102Æ3 96Æ9 110Æ7

4-ABP 105Æ5 110Æ2 109Æ3 113Æ4 96Æ7 113Æ4

DCBz 63Æ2 66Æ3 56Æ3 60Æ4 55Æ3 66Æ4

4-AAB 93Æ3 96Æ2 95Æ3 95Æ4 89Æ1 97Æ6

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




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