Solid-Phase Microextraction Coupled withHigh-Performance Liquid Chromatography for theDetermination of Aromatic Amines

Yu-Chao Wu and Shang-Da Huang*

Department of Chemistry, National Tsing Hua University, Hsinchu, Taiwan 300

Solid-phase microextraction coupled with HPLC for theanalysis of the aromatic amines is described. Four kindsof fiber [Carbowax/templated resin (CW/TPR), Carbowax/divinylbenzene, poly(dimethylsiloxane)/ divinylbenzene(PDMS/DVB), and polyacrylate] were evaluated for ex-traction of the aromatic amines. CW/TPR and PDMS/DVB were selected for further study. The parameters ofthe desorption procedure [such as desorption mode, thecomposition of the solvent for desorption, the period thatthe fiber was flushed by the mobile phase (desorptionperiod), the duration of fiber soaking, and the flow rateof the mobile phase during the desorption period] werestudied and optimized. The effects of the properties ofanalytes and fiber coatings, carryover, duration and tem-perature of absorption, pH, ionic strength, and elutropicstrength, of samples were also investigated. The methodwas applied to environmental samples (lake water).

The carcinogenic activity of benzidine and several aromaticamines (e.g., 3,3′-dimethylbenzidine (DMBz), 4-aminobiphenyl (4-ABP), 3,3′-dichlorobenzidine (DCBz), 2-naphthylamine (2-NA),and 4-aminoazobenzene (4-AAB)) is well-known.1 These com-pounds were widely used as intermediates in the production ofdyes, pesticides, pharmaceuticals, etc.

Aromatic amines are typically analyzed by liquid/liquid2 orsolid-phase3 extraction followed by determination with high-performance liquid chromatography (HPLC) or gas chromatog-raphy (GC) determination after derivatization.4 Liquid/liquidextraction is time-consuming and tedious and requires largequantities of toxic and environmentally unfriendly solvents. Solid-phase extraction requires less solvent, but the presence ofparticulate matter in the samples can cause plugging of thecartridges, breakthrough is sometimes experienced with highlyconcentrated samples, and a large volume of sample is generallyrequired for trace analysis. Derivatization prolongs the analyticalprocedure, and is susceptible to contamination and loss of analytes.

Solid-phase microextraction (SPME), a relatively new extrac-tion technique, was introduced by Pawliszyn and co-workers.5,6

Recent trends in SPME were reviewed by Eisert and Pawliszyn.7

The SPME technique integrates sampling, extraction, concentra-tion, and sample introduction into a single step.8 Until recently,the extensive applications of SPME were based almost exclusivelyon separation and analysis by GC.9-16 However, many classes oforganic compounds widely used today [such as pharmaceuticalproducts, drugs, peptides and proteins, some pesticides, andpolycyclic aromatic hydrocarbons (PAHs)] are semi- or nonvolatileand are best analyzed by HPLC. SPME coupled with HPLC wasrecently reported by Chen and Pawliszyn;17 it retains the advan-tages of SPME as a fast, portable, and inexpensive samplingtechnique. The extraction process used for SPME/HPLC isexactly the same as that described for GC analysis; only thedesorption technique must be modified for HPLC analysis. Theinterface for SPME and HPLC is recently commercially availablefrom Supelco. It is based on the initial design of Chen andPawliszyn for coupling SPME with HPLC.17 The SPME/HPLCinterface enables the mobile phase to make contact with the SPMEfiber, remove the adsorbed analytes, and deliver them to thecolumn for separation. Analytes can be removed in a movingstream of mobile phase (dynamic desorption), or when analytesare more strongly adsorbed onto the fiber, the fiber can be soakedin the mobile phase or another stronger solvent for a specificperiod of time before the material is injected onto the column(static desorption). Several applications of SPME coupled withHPLC, such as the analysis of polyaromatic hydrocarbons,17

alkylphenol ethoxylate surfactants,18 proteins,19 pesticides,20,21 andcorticosteriods,22 were found in the literature. The results were

(1) Ferber, K. H. Benzidine and Related Diaminobiphenyls. In Encyclopedia ofChemical Technology, 3rd ed.; Wiley: New York, 1978; Vol. 3, p 772.

(2) Bailey, J. E. Anal. Chem. 1985, 57, 189.(3) Trippel-Schulte, P.; Zeiske, J.; Kettrup, A. Chromatographia 1986, 22, 138.(4) Concialini, V.; Chiavari, G.; Vitali, P. J. Chromatogr. 1983, 258, 244.(5) Belardi, R. P.; Pawliszyn, J. J. Water Pollut. Res. Can. 1989, 24, 179.(6) Arthur, C. L.; Pawliszyn, J. Anal. Chem. 1990, 62, 2145.

(7) Eisert, R.; Pawliszyn, J. Crit. Rev. Anal. Chem. 1997, 27, 103.(8) Zhang, Z.; Yang, M. J.; Pawliszyn, J. Anal. Chem. 1994, 66, 884A.(9) Louch, D.; Motlagh, S.; Pawliszyn, J. Anal. Chem. 1992, 64, 1187.

(10) Arthur, C. L.; Killam, L. M.; Buchholz, K. D.; Pawliszyn, J. Anal. Chem. 1992,64, 1960.

(11) Potter, D. W.; Pawliszyn, J. J. Chromatogr. 1992, 625, 247.(12) Gorechi, J.; Pawliszyn, J. Anal. Chem. 1995, 67, 3265.(13) Buchholz, K. D.; Pawliszyn, J. Environ. Sci. Technol. 1993, 27, 2844.(14) Horng, J. Y.; Huang, S. D. J. Chromatogr., A 1994, 678, 313.(15) Huang, S. D.; Ting, C. Y.; Lin, C. S. J. Chromatogr., A 1997, 769, 239.(16) Huang, S. D.; Cheng, C. P.; Sung, Y. H. Anal. Chim. Acta 1997, 343, 101.(17) Chen, J.; Pawliszyn, J. B. Anal. Chem. 1995, 67, 2530.(18) Boyd-Boland, A. A.; Pawliszyn, J. B. Anal. Chem. 1996, 68, 1521.(19) Liao, J. L.; Zeng, C. M.; Hjerten, S.; Pawliszyn, J. J. Microcolumn Sep. 1996,

8, 1.(20) Jinno, K.; Muramatsu, T.; Saito, Y.; Kiso, Y.; Magdic, S.; Pawliszyn, J. J.

Chromatogr., A 1996, 754, 137.(21) Eisert, R.; Pawliszyn, J. Anal. Chem. 1997, 69, 3140.(22) Volmer, D. A.; Hui, J. P. M. Rapid Commun. Mass Spectrom. 1997, 11,

1926.

Anal. Chem. 1999, 71, 310-318

310 Analytical Chemistry, Vol. 71, No. 2, January 15, 1999 10.1021/ac980614a CCC: $18.00 © 1999 American Chemical SocietyPublished on Web 12/04/1998

summarized in Table 1 in which desorption procedures wasemphasized. Chen and Pawliszyn17 determined PAHs using PDMSfiber (7 µm) and SPME/HPLC interface with dynamic desorptionmode. The effect of solvent composition (mixture of acetonitrileand water) on desorption was studied by connecting the interfacedirectly to the detector cell. “A very small desorption volume, lessthan 0.2 µL was found when 90:10 acetonitrile/water was used assolvent. However, the desorption volume increased drasticallywhen a solvent mixture with a higher concentration of water wasused, due to the lower solubility of benzo[a] pyrene.”17 Boyd-Boland and Pawliszyn18 determined Triton X-100 and othernonionic surfactant using fibers coated with various phases.Dynamic desorption mode was used. The desorption time profilefor Triton X-100 using Carbowax/templated resin (CW/TPR) fibershowed that most of the analytes was desorbed in the first min.The bandwidths of the peaks in the chromatogram for 1- and 50-min desorption are the same. The effect of solvent compositionon desorption was tested by injection of a small amount (between20 and 40 µL) of polar solvent (methanol, acetone, dichlo-romethane) into the desorption chamber via the injection portusing a 1-min desorption time. “No significant improvement indesorption was observed by the addition of the modifier.”18 Liaoet al.19 demonstrated the extraction of protein with a polyacrylate(PA) fiber, followed by off-line desorption of the protein in a vialcontaining sodium chloride and sodium phosphate aqueoussolution, which was then analyzed by HPLC. No interface ofSPME/HPLC was used for this work. Jinno et al.20 analyzed 10pesticides (insecticide, herbicide, fumigant) using a PA fiber anda SPME/HPLC interface. The design of the interface they usedis different from that used by the other investigators17,18,22 and byus. The PA fiber with adsorbed pesticides was soaked in thesolvent (acetonitrile, 80 µL) in the desorption device. Not allanalytes desorbed from the fiber are introduced to the LC columnto avoid peak broadening. The solvent containing analytes des-orbed from the fiber was carried out into the HPLC injector bymanually flushing a certain amount of solvent (∼20 µL) using amicrosyringe. The solvent containing analytes was injected to thecolumn using a standard LC injector. The effect of the time thefiber soaked in the solvent for desorption on carryover wasstudied. “For most pesticides, carry-over did not decrease after30 min, although most still had a few percent of carry-over.”20

Eisert and Pawliszyn21 described the first approach to develop anautomated SPME/HPLC system. Instead of using a fiber, a pieceof ordinary capillary GC column with its coating (e.g., Omegawax250) was used for the extraction of analytes (phenylurea orcarbamate pesticides) from the aqueous sample (in-tube SPME).A sample of 25 µL was aspirated and dispensed several times fromthe sample into the capillary using a syringe. After the extractionthe absorbed analytes were released from the coating by aspiringmethanol into the HPLC injector loop. “Compared to the manualversion this automated SPME/HPLC system could increaseproductivity and reproducibility.”21 Volmer and Hui22 determinedcorticosteroids in urine using the SPME/HPLC interface fromSupelco (Bellefonte, PA). The analytes were determined usingan internal standard method and mass spectrometry. The analytedesorption was achieved in the static mode for a desorption timeof 5 min. The fiber was continuously exposed to the mobile-phaseflow during analyses. To avoid peak broadening, the mobile-phase

flow was kept isocratic for an additional 2 min before the start ofthe gradient program, while the flow rate was linearly increasedfrom 0.2 mL/min to the regular 1 mL/min during the initial 2min. Four types of fiber [poly(dimethylsiloxane)/divinylbenzene(PDMS/DVB), PA, CW/DVB, and CW/TRP) were evaluated.“The polar CW fibers exhibited superior performance as comparedto the less polar PA and PDMS/DVB fiber. The least polarcompound, deoxycorticosterone was recovered equally well byall four fibers, indicating strong hydrophobic interactions with thePA and the PDMS/DVB phases.” Only CW/TPR was selectedfor further investigation.

As noted by Chen and Pawliszyn,17 “the desorption procedureis extremely important in SPME/HPLC and needs to be optimizedfor each application with different solvent compositions adjustedfor solubility of target analytes in the mobile phase.” However, asshown in Table 1 and the text, the performance of the desorptionmode (static or dynamic) had never been compared, and thoseworks were run almost exclusively under the specific desorptionconditions, and the parameters for the desorption procedure [thecomposition of the solution for desorption, the duration of fibersoaking before the solution is injected to the column, the flowrate for delivering the solution in the desorption chamber to thecolumn, and the duration of the desorption time (the time thatthe fiber was washed by the mobile phase)] had not beenthoroughly studied or optimized. This is partly due to the factthat the desorption of the analytes from the fiber is a fast process(the desorption is nearly complete within 1 min) for the targetanalytes and fibers studied. For instance, the desorption of PAHsfrom a fiber coated with a thin layer of PDMS (7 µm) is very fast;“the peaks of PAHs desorbed from the fiber in the desorptionchamber are all very sharp, with no differences from the peakshapes produced by loop injection, and the desorption chambergeometry has very little effect on the dead volume of the wholesystem.”17 The desorption of the surfactants (e.g., Triton-X 100)from the CW/TPR fiber is also fast; most of the surfactants weredesorbed in the first minute.18 However, when one needs to desorbpolar analytes from a fiber with a polar coating, since the analytesare more strongly adsorbed onto the fiber, the parameters of thedesorption procedure need to be carefully optimized. Note thatno SPME/HPLC interface was used for the protein analysiswork,19 and the designs of the interfaces (see text) for the pesticideanalysis work20,21 were different from what we used. We expectthat if the commercially supplied SPME/HPLC interface (fromSupelco) and a fiber with polar coating (e.g., CW/TPR) were usedto analyze polar pesticides, the optimization of desorption proce-dure is relevant.

In this paper, we present the first application of SPME/HPLCto the analysis of aromatic amines. Four kinds of fiber (50-µmCW/TPR, 65-µm CW/DVB, 60-µm PDMS/DVB, and 85-µm PA)were compared for the extraction efficiency of the aromaticamines. CW/TPR and PDMS/DVB were selected for furtherstudy. The parameters of the desorption procedure are studiedand optimized. The effects of the properties of the analytes andfiber coatings, carryover, duration and temperature of absorption,pH, ionic strength, and elutropic strength of the sample solutionwere also investigated. This method was applied to environmentalsamples (lake water).

Analytical Chemistry, Vol. 71, No. 2, January 15, 1999 311

Ta

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312 Analytical Chemistry, Vol. 71, No. 2, January 15, 1999

EXPERIMENTAL SECTIONReagents. Analytical-reagent grade benzidine (Bz), DMBz,

4-ABP, DCBz, and 2-NA were purchased from Sigma (St. Louis,MO) and 4-AAB was obtained from Tokyo Chemical Industry(Tokyo, Japan). LC-grade sodium acetate was obtained fromFisher Scientific, and acetonitrile was obtained from Tedia. Stockstandard solutions were prepared by weighing the aromaticamines and dissolving them in methanol (Tedia). A workingcomposite standard solution was prepared by combining an aliquotof each of the stock standard solutions and diluting the mixturewith water. Methanol and sodium sulfate (Merck) were used toprepare the sample solution. Deionized water was purified in aMilli-Q purification system (Millipore). Lake water from theNational Tsing Hua University served as the environmentalsample.

Apparatus and Procedure. The SPME fiber assembly andSPME/HPLC interface were purchased from Supelco (Bellefonte,PA). The SPME/HPLC interface consists of a six-port injectionvalve and a desorption chamber (chamber volume, 200 µL) whichreplaces the injection loop in the HPLC system. After sampleextraction, the SPME fiber is introduced into the desorptionchamber under ambient pressure when the injection valve is inthe load position. For static desorption, the fiber was soaked inthe mobile phase (or other solvent) for 2 min, and then the valvewas switched to the inject position and integration was begun.During the first 2 min, the analytes were delivered to the columnat a lower flow rate (0.2 mL/min) to minimize band broadeningand peak tailing on the column. After 2 min, the valve was returnedto the load position and the mobile phase was introduced intothe column without passing through the desorption chamber atan increased rate (1 mL/min). The fiber was held in the desorptionchamber for 5 min, flushed twice with 500-µL portions of mobilephase to minimize the possibility of analyte carry-over, and thenthe SPME fiber was removed. For dynamic desorption, theanalytes were removed in a moving stream of mobile phase at alower flow rate (0.2 mL/min) during the first 2 min, and then thevalve was returned to the load position and the flow rate wasincreased to 1 mL/min. The microextraction fibers (from Supelco)were coated with CW/TPR (50 µm), CW/DVB (65 µm), PDMS/DVB (60 µm), or PA (85 µm). The polar series of these fibercoatings are as follows: polar f CW/TPR f CW/DVB f PDMS/DVB f PA f PDMS f nonpolar. The fiber should be conditionedwith the mobile phase, until a very stable baseline is obtained.

Aliquots of 3 mL of standard solutions or samples wereextracted from 4-mL vials sealed with hole caps and Teflon septa.The sample solution is stirred with a stirring bar and controlledby a Digital/magnetic stirrer (Electrothermal HS 4000/5000). Thespeed of rotation of the stirring bar was 550 ( 10 rpm and thetemperature of the sample solution was 25 ( 2 °C, unlessotherwise specified. After extraction for 20 (PDMS/DVB fiber)or 30 min (CW/TPR fiber), the fiber is placed in the desorptionchamber of the SPME/HPLC interface.

The HPLC system, assembled from modular components(Waters), consisted of a model 600E pump and a model 486 UVdetector. A Millennium workstation (Waters) was utilized tocontrol the system and for acquisition and analysis of data. A 4-µmNova-Pak C18 column (15 cm × 3.9 mm, Waters) was used; the

mobile phase was a mixture of acetonitrile and acetate buffer (0.1M, pH 4.66) (40:60) and UV detection was at 280 nm.

Safety. The carcinogenic activity of benzidine and severalaromatic amines (e.g., DMBz, 4-ABP, DCBz, 2-NA, and 4-AAB)used in this study is well-known. Exposure to these compoundsshould be as low as reasonably achievable. Minimum protectionof gloves and safety glasses should be worn to prevent samplecontact with the skin and eyes. Use these reagents in a hoodwhenever possible. A reference file of material data handlingsheets should be available to all personnel involved in the chemicalanalysis. Waste should be collected and sent for further treatmentby a qualified agent.

RESULTS AND DISCUSSIONFiber Evaluation. The relative extraction efficiencies of the

aromatic amines (expressed by peak areas in the chromatograms)with various fiber coatings are shown in Figure 1. Both aromaticamine molecules and coating of PA contain aromatic groups;according to the rule of “like dissolves like”, PA fiber is a goodcandidate for extraction of aromatic amines. However, the lesspolar PA fiber exhibited the lowest extraction efficiencies for allanalytes. The 65-µm CW/DVB fiber provided good extractionefficiencies for all analytes. However, although this fiber is oneof the fibers recommended by Supelco to be used for the SPME/HPLC, this fiber was not suitable for SPME/HPLC using aceto-nitrile/acetate buffer mixture as the desorption solvent; afterseveral analyses the coating was stripped off the fiber in theSPME/HPLC interface after desorption due to swelling of thephase in the desorption solvent. Volmer and Hui 22 also observedswelling of the CW/DVB phase in the water/methanol mixture,and this fiber could be used for only one or two analyses. Themost polar CW/TPR fiber exhibited better extracting efficiencyfor the more polar analytes Bz and DMBz than the other fibers.For the other less polar analytes, the less polar PDMS/DVB fiberexhibited better or equal extraction efficiency as compared to theCW/TPR fiber. CW/TPR and PDMS/DVB fibers were thereforeused for further investigation.

Desorption Mode and Composition of Solvent for Desorp-tion. After immersion of the fiber in the sample solutions for 30(CW/TPR fiber) or 20 min (PDMS/DVB fiber), the fiber wasintroduced into the SPME/HPLC for desorption. Table 2 showsthe effect of the desorption mode and the composition of thesolvent used for static desorption with a CW/TPR fiber. Most ofthe differences between the dynamic and static modes arestatistically insignificant, with the exception of DCBz and 4-ABP.Improvements in sensitivity (peak area) of 36 (DCBz) and 15%(4-ABP) were achieved using static mode as compared to that fordynamic mode using mobile phase (mixture of acetonitrile andacetate buffer) as the desorption solvent. This indicates that themore polar analytes (DCBz, 4-ABP) are adsorbed onto the morepolar fiber (CW/TPR) strongly, such that the static mode canimprove desorption of the polar analytes from the fiber. Increasingthe proportion of acetonitrile in the desorption solvent (mixtureof acetonitrile and water) decreased the desorption of the analytes.The static mode with the mobile phase as the desorption solventwas chosen for subsequent experiments using CW/TPR fiber toachieve the best sensitivity. Table 3 shows the effect of thedesorption mode and composition of the desorption solvent witha PDMS/DVB fiber. No improvement in sensitivity is achieved

Analytical Chemistry, Vol. 71, No. 2, January 15, 1999 313

with the static mode compared to that with the dynamic mode.This indicates that the analytes desorb more rapidly from thePDMS/DVB fiber than from the CW/TPR fiber. The diffusion ofthe analyte in the desorption chamber (for the static mode) maydecrease the peak areas of the analytes somewhat. Increasing theproportion of acetonitrile in the desorption solvent (a mixture ofacetonitrile and water) increased the analyte desorption from theless polar PDMS/DVB fiber, which contradicted the tendencyfound for the more polar CW/TPR fiber. The mobile phase (amixture of acetonitrile and acetate buffer) is a better solvent todesorb the analytes from the fibers (PDMS/DVB and CW/TPR)than the other solvent (mixture of acetonitrile and H2O). Thedynamic mode was used for further study with the PDMS/DVBfiber.

Desorption Period. The effects of the desorption period (theperiod during which the fiber is washed by the mobile phase)were studied. The chromatograms for desorption periods of 2 and5 min using either CW/TPR or PDMS/DVB fibers are shown in

Figure 2. Note that the flow of the mobile phase was maintainedat a slower rate (0.2 mL/min) for different periods of time (1-5min) during the desorption process; therefore, the retention timeschanged with desorption period. For the CW/TPR fiber (the morepolar fiber coating), from 80 to 82% as much Bz and DMBz (themore polar analytes) were desorbed in 2 min as were desorbedin 5 min. From 85 to 100% as much of the other analytes weredesorbed in 2 min as were desorbed in 5 min. The peak area ofthe analytes increased with increasing desorption period (espe-cially when the desorption period is increased from 1 to 2 min),which indicates that the desorption of analytes from the CW/TPR fiber is a slow process. A total of 92-97% of the analyteswere desorbed from PDMS/DVB fiber in a desorption period of2 min, except for Bz (74%) and 2-NA (79%). Significant peakbroadening and tailing appear for a desorption period of 3 or 5min with either fiber; these cause shifting of the baseline andoverlapping of the peaks (see Figure 2). Therefore, a desorptionperiod of 2 min was chosen as the optimum.

Figure 1. Relative extraction efficiencies of aromatic amines with various fiber coatings: concentration, 100 ng/mL; absorption time, 20 min;desorption mode: static, 2 min.

Table 2. Peak Areas of Analytes in the Chromatograma

Showing the Effect of Desorption Mode andCompositionb of the Solution Used for StaticDesorption by a CW/TPR Fiber

static

compds dynamic mobile 40:60 60:40 80:20

Bz 56 125 57 491 59 482 32 940 21 699DMBz 72 878 74 599 77 808 48 684 31 0612-NA 21 905 21 928 19 523 18 647 17 9394-ABP 138 889 159 334 135 452 126 636 115 832DCBz 87 056 118 273 98 382 83 770 70 1054-AAB 56 096 56 428 42 643 42 752 37 618

a Concentration, 100 ng/mL; absorption time, 20 min; desorptionmode, dynamic or static 2 min. b Composition of desorption solventCH3CN/H2O, and mobile phase, CH3CN/0.1 M pH 4.66 acetate buffer(40:60).

Table 3. Peak Areas of Analytes in the Chromatograma

Showing the Effect of Desorption Mode andCompositionb of the Solution Used for StaticDesorption from a PDMS/DVB Fiber

static

compds dynamicc mobilec 40:60 60:40 80:20

Bz 32 095 ( 1416 28 502 ( 542 15 481 21 353 26 875DMBz 60 963 ( 2968 54 418 ( 506 29 636 42 078 51 8092-NA 42 932 ( 1372 40 736 ( 1766 28 584 33 304 36 3694-ABP 174 665 ( 5134 162 408 ( 10321 115 240 128 666 142 829DCBz 92 863 ( 3622 78 110 ( 4851 58 539 62 448 64 4564-AAB 39 210 ( 1183 37 256 ( 2893 25 648 28 179 30 666

a Concentration, 100 ng/mL; absorption time, 20 min; desorptionmode, dynamic or static 2 min. b Composition of desorption solvent,CH3CN/H2O, and mobile phase, CH3CN/0.1 M pH 4.66 acetate buffer(40:60). c Average and standard deviation of the peak area of triplicateruns.

314 Analytical Chemistry, Vol. 71, No. 2, January 15, 1999

Flow Rate during Desorption. During the desorption period,the analytes were delivered to the column at a lower flow rate(0.2 mL/min) to minimize band broadening and peak tailing onthe column. Figure 3 shows that serious band broadening andpeak tailing were observed if a higher flow rate (1 mL/min) wasused during the desorption period.

Soaking Period. The effect of duration of the soaking of theCW/TPR fiber in the desorption solvent in the desorption chamberwas studied. No significant variation in the recovery of the analyteswas observed for soaking periods from 1 to 5 min. This is to beexpected as SPME is a partitioning process and the volume ofsolvent in the desorption chamber is small (200 µL). Therefore,increasing the soaking time cannot desorb more analytes fromthe fiber. A soaking period of 2 min was chosen for further studyas it offered somewhat better sensitivity.

Carry-Over. For the runs using CW/TPR fiber, no carry-overof DMBz, 2-NA, and 4-AAB was found, and the carry-over of Bz,4-ABP, and DCBz were 1.7, 0.28, and 0.79%, respectively. For the

runs using PDMS/DVB, no carry-over of the analytes wasobserved, except for 4-ABP (2.7%) and DCBz (1.5%). All com-pounds have no carry-over by the second desorption. However,carry-over in SPME is not of such a big concern as in many othermethods because SPME is an equilibration method. Carry-overmay become a problem only when the concentration of the analytein the following sample is so low that the equilibrium concentrationin the coating is lower than the concentration caused by carry-over from previous analysis.12 When samples of widely differingconcentrations are analyzed in sequence, it is recommended thatthe fiber be conditioned by a second desorption step.

Absorption/Time Profile. The absorption-time profile usinga CW/TPR fiber is shown in Figure 4. The equilibration periodwas 20-30 min for Bz, DMBz, and 2-NA and 40 min for 4-ABP.The absorption of DCBz and 4-AAB on the fiber had not reachedequilibrium even after 150 min (data not shown). The much longer

Figure 2. Chromatograms of aromatic amines for desorptionperiods of (a) 2 and (b) 5 min with CW/TPR fiber and (c) 2 and (d) 5min with PDMS/DVB fiber. Peak assignment: 1, Bz; 2, DMBz; 3,2-NA; 4, 4-ABP; 5, DCBz; 6, 4-AAB. Concentration, 100 ng/mL. Seetext for the other experimental conditions.

Figure 3. Chromatograms of aromatic amines for 1 mL/min flowrate during the desorption period with (a) CW/TPR and (b) PDMS/DVB fibers. Peak assignment and concentration as in Figure 2.

Figure 4. Absorption/time profile for CW/TPR fiber: Concentration,100 ng/mL; desorption mode, static 2 min. Peak notation: (O) Bz,(b) DMBz, (0) 2-NA, (9) 4-ABP, (∆) DCBz, and (2) 4-AAB.

Analytical Chemistry, Vol. 71, No. 2, January 15, 1999 315

equilibration periods of DCBz and 4-AAB are mainly due to thelarger distribution constants of these analytes. It is interestingthat the peak areas of Bz and DMBz decrease when the absorptiontime is above 40 min. This is probably caused by overadsorptionof the more polar analytes (Bz, DMBz) on the polar fiber due tothe stronger polar/polar interaction force between the analyte andfiber at absorption periods of 20-30 min. These analytes (Bz,DMBz) on the fiber are then displaced by analytes (such as DCBzand 4-AAB) with larger distribution constants during the longerequilibration period. Factors that influence the equilibration periodwere investigated by Pawliszyn and co-workers.9-11 The equilibra-tion rate is limited by the mass-transfer rate of the analytesthrough a thin static aqueous layer at the fiber/solution interface.The equilibration period increased with increasing distributionconstant of the analyte and with increasing thickness of the fibercoating. An extraction period of 30 min was chosen for subsequentexperiments using the CW/TPR fiber. The absorption/time profileobtained using a PDMS/DVB fiber is shown in Figure 5. Theequilibration period required is longer than 100 min. An extractionperiod of 20 min was chosen for subsequent experiments withthe PDMS/DVB fiber, since this time was approximately equiva-lent to the time required to run the HPLC chromatogram. It isnot essential for equilibrium to be reached; shorter times can beused as long as the extractions are timed carefully and the mixingconditions remain constant.13 Table 4 shows the percent recoveriesof analytes [the percent ratios of the amounts of analytes desorbedfrom the fibers to the amounts of analytes in the sample solution

(100 ng/mL)]. The greater extraction efficiencies of DCBz, 4-AAB,and 4-ABP as compared to those of other aromatic amines arehighly significant.

Effect of pH. It was found that the pH of the sample solutionin the range pH 5-11 does not have a significant effect on theextraction efficiency of the aromatic amines using CW/TPR orPDMS/DVB fiber. When the pH of the solutions was adjusted to3, the absorption of analytes on the fibers decreased, due to theprotonation of the weak bases in the acidic solutions.

Effect of Temperature of Extraction. The absorption/temperature profile obtained using a CW/TPR fiber is shown inFigure 6. The amount of Bz, DMBz, and 2-NA absorbed decreaseddramatically with increasing temperature of extraction from 20to 60 °C. The decrease in absorption with increasing temperatureis due to the decrease of the distribution constant with increasingtemperature. Because absorption is generally an exothermicprocess, the amount of analyte absorbed decreases with increasingtemperature.10 The amounts of DCBz absorbed increased withtemperature in the range 10-40 °C and then decreased substan-tially with increasing temperature of extraction. The decreasingabsorption of analytes with decreasing temperature below 40 °Cwas due to the decreased rate of diffusion of the analytes at lowertemperatures, so more analyte is absorbed at a higher temperatureif equilibrium has not been reached. The decreasing absorptionwith increasing temperature above 40 °C is presumably due to adistribution constant which decreases with increasing tempera-ture.10 The profiles of 4-ABP and 4-AAB were similar to that ofDCBz’s, although the optimum absorption temperature was notthe same.

The absorption/temperature profile obtained with a PDMS/DVB fiber indicates that the amounts of analytes absorbedincrease with increasing temperature in the range 10-40 °C anddecrease above 40 °C. Note that equilibrium has not been reachedfor an absorption time of 20 min at 25 °C (Figure 5). Both theeffects of absorption temperature on the rate of diffusion of theanalyte species and distribution constant of coating/water playan important role on the absorption of analytes on the fiber.

Effect of Elutropic Strength. The effect of elutropic strengthof the sample on absorption was studied by preparing a series of

Figure 5. Absorption/time profile for PDMS/DVB fiber: concentra-tion, 100 ng/mL; desorption mode, dynamic. Peak notation as inFigure 4.

Table 4. Percent Recovery of Aromatic Amines

fiber

compds CW/TPR PDMS/DVB

Bz 2.2 1.4DMBz 4.0 3.62-NA 3.5 6.94-ABP 8.8 9.6DCBz 22.8 14.74-AAB 13.8 9.8

Figure 6. Absorption/temperature profile for CW/TPR fiber. Condi-tions and peak notation as in Figure 4.

316 Analytical Chemistry, Vol. 71, No. 2, January 15, 1999

samples that contained methanol at concentrations from 0 to 20%(w/v). As methanol concentration increases, less analyte wasabsorbed onto the CW/TPR fiber. The decreases in absorptionof the analytes in 2% methanol are in the range 2.3 (DCBz) to13.2% (DMBz and 2-NA). An increased proportion of methanol inaqueous solution decreases the polarity of the aqueous sample,so the distribution constant decreases.10 A similar effect ofmethanol on the absorption of analytes was observed with aPDMS/DVB fiber. The decreases in absorption of the analytesin 2% methanol are in the range 7.4 (2-NA) to 14.2% (DMBz).

Effect of Ionic Strength. The effect of ionic strength on theabsorption of aromatic amines by a CW/TPR fiber (Figure 7) anda PDMS/DVB fiber was studied by preparing standards with Na2-SO4 concentrations ranging from 0 to 20% (w/v). The increase inabsorption of 2-NA and 4-ABP, resulting from the “salting-outeffect” was highly significant with either fiber. The amount of Bzand DMBz absorbed decreased at 2% Na2SO4 and then increasedsubstantially with increasing Na2SO4 concentration above 2%. Thelower absorption of analytes at 2% Na2SO4 is due to the zwitterionsof the resonance forms of these analytes being stabilized in 2%Na2SO4, but the salting-out effect dominates above 2% Na2SO4. Asimilar effect of ionic strength was observed for the absorptionof 4-chlorophenyl phenyl ether and 4-bromophenyl phenyl etheron PDMS (100 or 7 µm) and PA (85 µm) fibers.15 The effect ofNa2SO4 on the absorption of DCBz and 4-AAB on the fibers isprobably due to three factors. The first is the salting-out effect,which decreases the solubility of analytes and thus increases theabsorption. Second, salt dissolved in the solution may change thephysical properties of the static aqueous layer on the fiber andreduce the rate of diffusion of the analyte through the staticaqueous layer to the fiber. Third, the zwitterions of the resonanceforms of DCBz and 4-AAB molecules are more stable in thesolutions with higher ionic strength, so that DCBz and 4-AABbecome more soluble in water due to the increased contributionof zwitterion from to the resonance structure. These effectscompensate each other, so that the absorption of DCBz and 4-AABon the CW/TPR fiber increases with Na2SO4 in the range 0-5%and decreases above 5% Na2SO4. A similar effect of ionic strength

was observed for the absorption of aromatic amines on the PDMS/DVB fiber.

Detection Limits, Precision, and Linearity. The limits ofdetection (LODs) and precision (RSDs) are shown in Table 5.Limits of detection are calculated as 3 times the standard deviationof seven replicate runs. The detection limits are 0.66-1.5 (CW/TPR fiber) and 0.33-2.4 ng/mL (PDMS/DVB fiber). The preci-sion of the method was investigated for a set of seven replicates.The RSD range from 3 to 8% (CW/TPR fiber) and from 2 to 8%(PDMS/DVB fiber). The linearity of the method for analyzing thearomatic amines has been investigated over the range 1000-10ng/mL for a CW/TPR fiber and 500-10 ng/mL for a PDMS/DVB fiber. The correlation coefficients were better than 0.995(Table 6).

Test on Environmental Samples. The SPME/HPLC meth-ods were tested on a lake water sample. No aromatic amines werefound in the lake water sample. The slopes of calibration curvesof aromatic amines based on deionized water and lake water werecompared using a CW/TPR or a PDMS/DVB fiber (Table 6). Theslopes of the calibration curves were almost independent of thematrix of the sample solution (difference in slopes is less than5%) with a PDMS/DVB fiber. These indicated that the SPME/HPLC method based on a simple calibration curve could be usedto analyze aromatic amines in natural waters with a PDMS/DVB

Figure 7. Effect of ionic strength on absorption of aromatic aminesby CW/TPR fiber. Conditions and peak notation as in Figure 4.

Table 5. Limits of Detectiona and Relative StandardDeviationsb for the Analysis of Aromatic Aminesc

LOD (ng/mL) RSDs (%)

compd CW/TPR PDMS/DVB CW/TPR PDMS/DVB

Bz 1.5 2.4 8 8DMBz 0.66 1.4 4 52-NA 0.65 1.0 3 44-ABP 0.77 1.1 4 6DCBz 1.3 0.62 7 44-AAB 0.72 0.33 4 2

a Limits of detection are calculated as 3 times the standard deviationof seven replicate runs. b Data obtained by extraction in sevenreplicates. c Concentration of aromatic amines used for the run is 5ng/mL.

Table 6. Slopes and Correlation Coefficients ofCalibration Curvesa in Deionized Water (DIW) and LakeWater (LW)

slope corr coeff

compds matrix CW/TPR PDMS/DVB CW/TPR PDMS/DVB

Bz DIW 469 258 0.9979 0.9955LW 213 267 0.9999 0.9967

DMBz DIW 617 497 0.9969 0.9971LW 393 499 0.9988 0.9975

2-NA DIW 151 440 0.9988 0.9975LW 143 456 0.9994 0.9991

4-ABP DIW 1033 1529 0.9977 0.9996LW 983 1530 0.9994 0.9997

DCBz DIW 845 1016 0.9998 0.9999LW 823 1009 0.9990 0.9979

4-AAB DIW 436 364 0.9998 0.9999LW 429 365 0.9984 0.9992

a Calibration curves with the following concentrations: 10, 50, 100,500, 1000 ng/mL for CW/TPR and 10, 50, 100, 200, and 500 ng/mLfor PDMS/DVB.

Analytical Chemistry, Vol. 71, No. 2, January 15, 1999 317

fiber. The difference in slopes is very significant for Bz (54%) andDMBz (38%) with CW/TPR fiber. The method of standard additionshould be applied if these aromatic amines are analyzed with aCW/TPR fiber. The matrix of sample (such as dissolved inorganicsalts and organic compounds) may cause interference to theanalysis; this interference was observed in studies of the effect ofionic strength and elutropic strength. The distribution of theanalytes between the sample and the SPME fiber may be affectedif large quantities of particulate matter are present in the sample.23

CONCLUSIONSSPME coupled to the HPLC technique was successfully applied

to the analysis of aromatic amines in water. It is important to selectthe appropriate fibers and optimize the desorption procedure.When one needs to desorb the more polar analytes (such as DCBzand 4-ABP) from a fiber with a more polar coating (CW/TPR),the static mode can provide better sensitivity and/or sharper peakson the chromatogram then the dynamic mode, since the analytesstrongly adsorbed on the fiber can be desorbed more effectivelythrough static mode. However, if the desorption of the analytesfrom the fiber is rapid (volume of mobile phase or other solventrequired to desorb the nonpolar analytes from the fiber bydynamic mode is much less than the volumes of solvent in thedesorption chamber), the dynamic mode is a better choicebecause the diffusion of the analytes in the desorption chamber(for the static mode) may cause peak broadening and may

decrease the peak areas of the analytes somewhat. The desorptionperiod (the period during which the fiber is flushed by the mobilephase) should be carefully optimized for the determination ofaromatic amine. Severe lose in sensitivity was observed for adesorption period of 1 min, and significantly peak broadening andtailing appeared for a desorption period of 3 or 5 min (these causedshifting of baseline and overlapping of the peaks). A desorptionperiod of 2 min was chosen as the optimum for both fibers. Theflow rate of the mobile phase should be kept low (∼0.2 mL/min),otherwise, serious band broadening and peak tailing were ob-served. No significant variation in the recovery of the analyteswas observed for soaking periods from 1 to 5 min (static mode).A PDMS/DVB fiber is recommended for determination of thesearomatic amines in natural water, since a simple calibration curvemethod can be used. Detection limits are at the levels ofnanograms per milliliter. The RSDs for the analysis are in therange 2-8% (for a 5 ng/mL sample).

ACKNOWLEDGMENT

This work was supported by the National Science Council ofthe Republic of China (NSC 87-2113-M-007-042).

Received for review June 4, 1998. Accepted October 19,1998.

AC980614A(23) Potter, D. W.; Pawliszyn, J. Environ. Sci. Technol. 1994, 28, 298.

318 Analytical Chemistry, Vol. 71, No. 2, January 15, 1999