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Anal. Chem. 1882, 64,3187-3190 a i 8 7

Determination of Mercury( I ) in Dithizone- Impregnated LatexMicroparticles by Photochromism- Induced PhotoacousticSpectroscopyV. A. VanderNoot and E. P. C. La?Centre for Analytical and Environmental Chemistry, Ottawa-Carleton Chemistry Institute, Department of Chemistry,Carleton University, Ottawa, Ontario, Canada KlS 5B6

The determination of trace levels of toxic heavy metalscontinues to represent an important area of analyticalchemistry. Mercu ry has been determined over th e years intrace levels by reaction w ith th e ligand phenylazothioformicacid 2-phenylhydrazide, more comm only known asdithizone.'Dithizone forms complexes with m any heavy m etals of whicha num ber are photochromic. Photoch romism refers to th ereversible color change that the compounds undergo onexposure to visible light. Analysis of these photochrom icspecies by photoacoustic spectroscopy (PAS) has been q uitesuccessful. The method is sensitive an d is quite specific formercury if samples are prepared in solid films; no oth er heavymeta l complexes with dithizone exhibit photochromism inthe solid state.2 Frequ ently however, th e preparatio n of solidsamples can be somewhat time-consuming. In this work, anovel technique is presented in which aqueous Hg(I1) issequestered in to small uniform polystyrene latex micropar-ticles which are previously impregn ated with dithizone. Th eparticles can then be easily urned into a solid sample suitablefor photochrom ism-induced photoacoustic spectroscopy (PC -PAS) analysis by simple fitr atio non to commercially availablesmall-pore membrane filters. Th e method is fast, simple,and has th e potential for simultaneous quantification andclean-up of Hg(I1) in env ironmen tal waters.

Much work has been carried out to make use of the veryregular s urfaces of latex m icroparticles.3 Th e particles haverecently been used as a su pp ort on which silver needles weregrown for surface-enhanced raman spe ctro ~co py .~ anysubstances have been adsorbed o nto th e surfaces of micro-particles, not th e least of which being immunoglobulins forth e now well-established latex immunoassay a gglutinationtests.5 Th e technique for dyeing microparticles has been wellestablished, and p articles are available in a variety of colors,usually toaid in visual determination of agglutination assays.Fluorescent dyes have also been used for a number ofspectroscopic techniques. In these cases th e dye has beenshown to be deep in t he interior of th e particle and does notalter the su rface chemistry.6 A numb er of iron(II1)complexeshave been recently incorporated into sodium dodecyl sulfate

* Author to whom all correspondence should be addressed.(1)Sandell, E. B.; Onishi, H. Colorimetric Determination of Tracesof Metals, 4th ed.; Wiley-Interscience: New York, 1978; p 391.(2) Chen, N.; Guo, R.; Lai, E. P. C. A w l . Chem. 1988 ,60, 2435-39.(3) Pelton, R. NATO ASZ Series, Ser. C 303 (Scientific Methods forthe Study of Polymer Colloids and Their Applications)1990, 493-516.(4 ) Wachter, E. A.; Moore, A. K.; Haas, J. W., 111. Vibr. Spectrosc.(6) Masson,P.L.; ambiaso, C. L.; ollet-Cassart, D.; Magnusson , C.G. M.; Richards, C. B.; Sindic, C. J. M. Methods Enzymol. 1981, 74 ,106-139.(6) Bangs, L.B. Uniform Latex Microparticles; Seradyn Inc.: Indi-anapolis, 1984; Chapter VII.

1992,3,73-78.

micelles an d stud ied spectroscopically,7 bu t the se were limitedto th e case of th e continuo us phase being aqueous. Thisworkis th e firstexample reported in th e literature, to the authors'knowledge, of an analytical reagent being pu t inside a latexmicroparticle. Th e reagent-impreg nated microparticlea arestable in aqueous suspension and can be removed from th eaqueous phase along with the Hg(I1) for analysis underamb ient conditions. Additionally, th e poten tial for avarietyof organic complexing agents being incorporated in micro-spheres to create tailor-made reagents for toxic clean-up isgreat.E X P E R I M E N T A L S E C T I O N

Latex M icroparticles a nd Dithizone. Polystyrene latexmicroparticles were obtained from Seradyn (Indianapo lis, N )in a range of particle diam eters from 0.204 to 0.944 pm. Thepartic les were of uniform size distrib ution and very regular inappearance. Stan dard suspensions were prepared by simpledilution of the 15% (w/w) stocks with 18-MQdeionized water.These suspensions were stored at room temperature. Unlessotherwise specified,allanalyseswere carried ou tusing he smalleatdiameter, 0.204 pm. Dithizone was purified by Irving's method,and the purified dithizone solution in spectroscopic grade CC4was stored under a layer of 1 N H2SO4 in the dark. So lutionsstored this way keep very weLSReagent Impregnation and Hg(I1) Ext ractio n. In a smallvial,50p L of 0.01% (w/v) dithiz one (H2Dz) solution in CCL wasadded to 900 p L of deionized water. The H2Dz was extractedinto the aqueous phase by addition of 10 pL of 25% (v/v) NH4-OH followed by gentle shaking. Th e CC4 layer was carefullyremoved after all traces of th e green HzDz were gone. A 100-pLaliqu ot of 0.2% (w/w) latex microparticle suspension was added,and the suspension, a s oft orange-yellow color, was acidified with10 p L of 3% (v/v) HzSO4 as the pH needed to drive HzDz intothe microparticles is below 6. Th e resu lting green suspension ofHzDz-impregnated atex microparticles (see Figure 1)was addedto 1.0-mL Hg(I1) samples in 100-pL aliquots. Th is volumecontained s ufficie nt H2Dz to com pletely complex the m ostconcentrated standard plus a modest excess. The samples werefiltered through 0.1-pm pore size filters (Nuclepore, Toronto,ON, Canad a) which were then removed from the filte r housingand allowed to air dry before analysis.PCPAS M easure men t. The schematic diagram of th ePCPAS system is illustrated in Figure 2. The excitation sourcewas an argon ion-pumped dye laser (Spectra-Physica 164 and375B,Mountainview,CA). The o utpu t beam from the dye laser,605 m in wavelength and typically90 mW in pow er, was directedthrough a window into the photoacoustic cell after beingmechanically chopped a t200Hz. Th e samples (membrane fiiterasupporting latex microparticles) were placed directly into th e

~ ~~ ~(7 ) Mazumdar, S.J. Chem. SOC.alton Trans. 1991,2091-6.(8) Irving, H. M. N. H. Dithizone; T he Chemical Society: London,1977; Chapter 8.0003-2700/92/0364-3187$03.00/0 0 1992 Amerlcan Chemlcal Soclety

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5188 ANALYTICAL CKM ISTRY. VOL. 64 . NO. 24, DECEMBER 15 ,b)

H'H D i

Hg" Hg"m . Schema(ic showing the Impregnalbn and exIra&p r o c l g ~ ~ ~ .a)Repmentatkf~ f a hlex miaopartlde:open chclesare SOa- iunclbnalgroups from lhe swfactanl used in lhe &glnalproduclbn of the atex microparticlea: shaded circles are SO: groupsresumng from the tenninatbn of Ihe styrene polymerlzalbn steps inIhe mlnopartlcb me. (b) Representation showing dkhkone in Iheprocess of transferring into the mlcropatilcle cenler at solullon pH 50. (c)Representation showing Hg2+n Iheprocess of complexlngwk hH P z in tim interior of timmicroparticle.

C h o p p e r

Argon Ion Laser. -I ;L

Lock.," Amplifier I Prolector pand Computer Optical FibreBundlem . Schsmatlc &@'am of IheW A S apparatus.to o I

I0 .o 80 120 l l D ZOO

Tlmelra. ~yplcalW A S signal for ~ I I )Hh!zonate in htaxmlcropsrllcb. The slgnal rlses abruptly when exposed to vlslbleIllumination from lheprojeclw sowco due lo htgher absorpavnyof Iheblue excltebstate oomplex a1 Ihe wavalengm of 605 nm.gas-tightcell which was fitled to a condenser microphone anda sound m eter which contained a p reamplifier and an octavefdter set (BriielandKjaer4144and2209,Pte.Claire,PQ,Canada).The baseline PAS signal from the sample (see Figure 3)was firs trecorded using a lock-in amplifier (Stanford Research SR510,Sunnyvale, CA). The n the sample was illuminated by the lightof an 80-W projector lam p focused into an optical fiber bundle.The optical fiber bundle was brought closeto he photoacoustic

, 1992cell and directed onto the sample through the back window. ThePAS signal was again reeorded for sufficient time to achieve asteady-statesignal. The absolutedifference nth e signal voltages,the PCPAS signal amplitude, was taken as an indication of theamount of Hg(I1) present in the sample.Interference from other heavy metals was examined bypreparation of mixed metal standards. The dithizone present inthe 100-pL liquot remained in excess of the total amoun t ofextractable metals. These samples were analyzed in the sameway as describe d above.

RESULTSAND DISCUSSIONTh e chemistry of the H pz-im preg nate d latex micropar-ticles preparation and Hg(I1) ex trad ion is very interesting.It in not clear yet how HzDz, and th en Hg(II), in transportedinto the m icroparticle center. Thes e solid particles ofpolystyrene,witharigid struc tur edo win gth em to he filteredwhile retaining their shape, possess a negatively chargedsurface due to residual surfactant from their production. Itin unlikely, then, that HzDz adsorbs onto th e surface a t anytime tobe followed by seepage into them icroparticle interior.It may be that th e presence of residual CC4 facilitates thetransfer ofHzDzin aman nersimilarto the procedure involvedwith d yeing the particles. In dyeing particles, th e particlesare initially swollen with a dye solution in a solvent forpolystyrene, suchasC C4. Thes olven t is then carefullyboiledoff, trappin g th e dye inside the latex.6 Although very littleCC4 (50 pL) was involved in the HZDz-impregnation step,the transport process might he analogous since the latexvolume itself was quite small. The particles were henceslightly swollen by the organic solvent, allowing enoughfluidity o r flexibility to the structure to allow transfer of Hz-Dz across th e interface.The p H of the extraction in generally imp ortant toachievequantitative results. As illustrated in eq 1,a Large HzDzconcentration will tend to drive the reaction toward com-pletion. Sigmoid m e a of the percentage extraction of

primary dithizonates into a solution of 10-5 M HzDz in asuitable organic solvent reach 100%above pH 4 while th eextraction drops to zero helow p H 3. Lower concentrationsof HzDz n th e organic phase exaggerate themea and pushthem to higher pH regions; a 10-8 M solution will require apH of 8to chieve quan titative extraction.9 In th is work, th econcentration of HzDz was essentially constant a t approxi-mately 2 % (w/w) in the latex. This represented a largerconcentration of HzDz n the latex phase mmpared tosolution-phase ex tractions which are typically carried out in t he lW 7to 10-5M range. Hence, the control of pH was less criticalan d a pH of ca.6 was maintained in th e extraction vessel toensure quantification. Th e extraction was assumed to hecomplete after 15 min of occasional shaking, although t hereaction was seen to he much faster in those samplesconcentrated enough to observe the color change visually.Thequ antification w asascertained by reanalyzing the filtratefrom aqueous samplea after the primary reaction and filtra-tion. T he da ta verified tha t extraction efficiency waa greaterthan99% for 50 an d 100ng Hg(I1) n aqu eous samples underthe given experimental conditions.Photoacoustic analysis of Hg(I1) dithm nate-im pregn atedlatex microparticlea was straightforward. Th e presence ofessentially nonahaorbing latex polystyrene surrounding t heanalyte species did not complicate the issue of signalgeneration appreciably a t a chopping frequency of 200 Hz.

(9)Irving, H. . .H. i thizom; The C h e m i d Society: London,1911;chapter 5.

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ANALYTICAL CHEMISTRY, VOL. 64, NO. 24, DECEMBER 15, 1992 8180F 3 0 n

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0 10 20 30 40 50Concentration of Hg(ii) (ng mL)

Flgure 4. Analytical calibration curve for H g(I1) dtthizonate in latexmicroparticles. The error bar shows the ypical standard deviation ofreplicate samples. Triangles represent data taken from samplesimpregnated into 0.2OCpm spheres while the circles are data from0.944-pm spheres.Calibration curves of latex microparticles dyed d ark blue withSudan Black (Seradyn , Inc.) showed a linear response withquantity as expected. Th e calibration curve for the dete r-mination of H g(I1) using 0.204-pm microparticles s shown inthe upp er trace of Figure 4. Th e current detection limit,determined a t twice the sta ndard deviation of the blan k, is500 pg (in a 1.0-mL aqueous sam ple) or 3 pmol of m ercury.It should be noted th at the ac tual amoun t of Hg(I1) beingsampled by the excitation beam, and thus generating thesignal, is abou t 1 % ,or 5 pg, of th e tota l am oun t of Hg(I1) inthe microp articles trapped on the filter surface. A 10-foldimprovem ent in the detection limit, from 500 to 50 pg, wasconveniently achievable simply by reducing the filtration areafrom the current comm ercially available size of 130 mm 2 to13 mmz. Samp les prepared in this man ner did generatemeasurable signals for 50-pg Hg(I1) stand ards, w ith a signal-to-noise ratio of 2.Given that HzDz does not exhibit any appreciable pho-tochromism a t the excitation wavelength of 605 nm, excessHzDz in the m icroparticles did no t add to he observed PCP ASsignal amplitude. Th e amoun t of residual HzDz in eachsample varied roughly inversely with the concentration ofthe standard. This meant t hat , since H2Dz absorbed at thiswavelength, he baseline signal amplitude did necessarilyvaryfrom sample to sample, but since it was only the photo-chromism-inducedsignal change th at was taken as a measureof mercury content, it did not represent a problem to th eanalysis. Nevertheless, the excess HzDz produced a back-ground PAS signal which, if large enough, could limit thesensitivity range used on the sound meter. It appearedpossible to remove some of th e excess dithizone from themicroparticles by addition of NH 40H t o bring the pH to 9after complexation with Hg(I1) was complete. In most cases,however, he excess of dithizon e was modest and d id not affectthe analysis to any significant extent.Th e lower trace of Figure 4 shows the resulting calibrationcurve when 0.944-pm m icrop articles were used for th eextraction. There did not appear to be any difference in theamou nt of HzDz tha t could be sequestered into equal volumesof particle s of differen t sizes. Sinc e he surface are ato volum eratio did not seem to affect the impregn ation, t was confirmedth at HzDz, and hence H g(II), was being transferred into theinterior of the latex and n ot being adsorbed on to the surface.The calibration results did show enhanced detection sensi-tivity when sma ller particles (0.204 pm) were used to collectHg(I1). This was undoubtedly due t o a comb ination of factors,including optical shielding of the interio r of the larger particles(0.944 pm) which inhibited the photochromic activity and

0 10 20 30 40 50Concentration of Hg(ll) (ng mL)

Flgure 5. Calibration for Hg(I1) (A)nd for mixed metal standardscontaining equal quantltles of Hg(I1 ) and Ag(1) (0 ) nd Zn(I1) (+).higher surface area of th e sm aller particles which lead tomoreefficien t transfe r of hea t to th e gas in the p hotoacou stic cell.In the present experimental setup, the projector illumi-nation entered th e PAS cell throu gh the back window. Wh ilethis simple design avoided interference with the incident laserbeam, it had a tendency to limit the intensity of projectorlight reaching the sample as he original filters were a m ixedesters/cellulose acetate typ e and were stron glyscattering. Theuseful dynamic range of PCPAS analysis was drasticallyimproved by changing to Nuclepore filters, which were apolycarbonate type an d were muchm ore transpa rent tovisiblelight (average absorbance in th e visible range = 0.75). Thenew filters allowed more than enough light to reach thesamples to ensure th at sm all variations in projector intensitydid n ot affect the generation of excited state Hg(II) dithizonate(Le. th e PAS signal vs light intensity is flat). Additionally,the switch served to improve th e ease of filtration. Th epolycarbonate pores are formed by nuclear bombardmentfollowed by etching leaving a clear channel; the celluloseacetate filters are of the tortuou s path typ e. Th e result wasless back-pressure allowing larger volumes of d ilute sam plesto be extracted for preconcentration of Hg(I1). Many tensof m illiliters of d ilute solutions can be incorporated in to verysmall quan tities of latex; a 50-mL volume of 50 ppb Hg(I1)will only require ca. 300 pg of dithizone-impregnated latex.Th e selectivity of the P CPA S determination for Hg(I1)was quite good using the latex microparticles as t is in othersolid sample preparations of Hg(HDz)2. Samples of Ag(1)an d Zn(I1) dithizonates showed only small PCP AS signale a tthe excitation wavelength of 605 nm even at very highconcentrations. Com parable quan tities of either Ag(1) orZn(I1) in a s tandard of Hg(I1) did n ot a dd app reciably to themeasured PCPAS signal amplitude for Hg(I1) as shown inFigure 5. Note that the slopes of these calibration curveswere esen tially the same; the variation from curve to curvewas due to slight variations in the preparation of dithizone-impregnated latex microparticles. This variation usually willnot affect a calibration series from th e sam e preparation.The presence of interfering heavy metals becomes aprob lem , however, if the total amou nt of extractable metalsis in excess of th e amo unt of HzDz introd uced to the sample.There will be competition between the metals for extractioninto the latex microparticles under this circumstance. Asmaller overall PCPAS signal for Hg(HDz)Zwill be prod ucedsince some Hg(I1) ions will remain in th e aq ueous phase a ndbe washed away with the filtrate. If large amou nts of otherheavy metals are suspected, a larger amount of HzDz-impregn ated latex microparticles will be needed. This maygenerate sm aller PCPAS signals for Hg(I1)asa result of theinner filter effect of the nonph otochrom ic, bu t absorbing ,interfering metal dithizonates. Again, the use of stronger

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9190 ANALYTICAL CHEMISTRY, VOL. 64, NO. 24, DECEMBER 15, 1992projector illumination to induce photochromism shouldsurmou nt the difficulty. Besides, intentionally swamping thelatex microparticles with heavy m etals will help remove anyexcess H2Dz after extraction of Hg (II) from a sam ple solutio n,resulting in a lower background signal, providing tha t th echoice of ad ded m etal does not interfere sp ectrally.

CONCLUSIONSA novel technique has been demonstrated for the deter-mination of m ercury at picomole levels in aqueous samples.

Th e extraction of Hg(I1) into H2Dz-impregnated latex mi-croparticles is both rapid an d quantitative. PCPA S detectionfor Hg(HDz)2 in these particles rem ains selective over othertoxic heavy metals, and the sensitivity of the technique isgood for environmental analysis.Since there is a noticeable color change of the HzDz-impregnated latex microparticles upon reaction with heavymetals (regardless of PC PAS detection ability), a packedcolumn of th e m icroparticles can be applied towater streams

aa a visual indicator for heavy metals contam ination. Ad-ditionally, the latex m icroparticles are a po tential tool forwater clean-up as hey can be collected easily on am em bran efiiter for safe disposal after qu antitative removal of th e heavymetals. It remains to be seen, however, whether i t will bepossible to successfully scale th e m ethod up to meet envi-ronmental needs.ACKNOWLEDGMENT

This work waa funded by th e Natu ral Sciences andEngineering Research Council of Canada. AGR-5 rant fromthe Faculty of Graduate Studies and Research, CarletonUniversity, is gratefully acknowledged.RECEIVEDor review June 29, 1992. Accepted September18, 1992.Registry No. Mercury, 7439-97-6;ithizone, 60-10-6;oly-sytrene, 9003-53-6;ater, 7732-18-5.