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Anal. Chem.

1882, 64,3187-3190


Determination of Mercury(I I) in Dithizone-Impregnated Latex Microparticles by Photochromism- Induced Photoacoustic Spectroscopy

V. 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 metals continues to represent an important area of analytical chemistry. Mercury has been determined over the years in trace levels by reaction with the ligand phenylazothioformic acid 2-phenylhydrazide, more commonly known as dithizone.' Dithizone forms complexes with many heavy metals of which a number are photochromic. Photochromism refers to the reversible color change that the compounds undergo on exposure to visible light. Analysis of these photochromic species by photoacoustic spectroscopy (PAS) has been quite successful. The method is sensitive and is quite specific for mercury if samples are prepared in solid films; no other heavy metal complexes with dithizone exhibit photochromism in the solid state.2 Frequently however, the preparation of solid samples can be somewhat time-consuming. In this work, a novel technique is presented in which aqueous Hg(I1) is sequestered into small uniform polystyrene latex microparticles which are previously impregnated with dithizone. The particles can then be easily turned into a solid sample suitable for photochromism-induced photoacoustic spectroscopy (PCPAS) analysisby simple fitration onto commercially available small-pore membrane filters. The method is fast, simple, and has the potential for simultaneous quantification and clean-up of Hg(I1) in environmental waters. Much work has been carried out to make use of the very regular surfaces of latex microparticles.3 The particles have recently been used as a support on which silver needles were grown for surface-enhanced raman spectro~copy.~ Many substances have been adsorbed onto the surfaces of microparticles, not the least of which being immunoglobulins for the now well-established latex immunoassay agglutination tests.5 The technique for dyeing microparticles has been well established, and particles are available in a variety of colors, usually to aid in visual determination of agglutination assays. Fluorescent dyes have also been used for a number of spectroscopic techniques. In these cases the dye has been shown to be deep in the interior of the particle and does not alter the surface chemistry.6 A number of iron(II1)complexes have been recently incorporated into sodium dodecyl sulfate
(1) Sandell, E. B.; Onishi, H. Colorimetric Determination of Traces of 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 for the Study of Polymer Colloids and Their Applications) 1990, 493-516. (4) Wachter, E. A.; Moore, A. K.; Haas, J. W., 1 1 Vibr. Spectrosc. 1. 1992,3,73-78. (6) Masson, P. L.; Cambiaso, C. L.;Collet-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.: Indianapolis, 1984; Chapter VII. 0003-2700/92/0364-3187$03.00/0

micelles and studied spectroscopically,7 but these were limited to the case of the continuous phase being aqueous. This work is the first example reported in the literature, to the authors' knowledge, of an analytical reagent being put inside a latex microparticle. The reagent-impregnated microparticlea are stable in aqueous suspension and can be removed from the aqueous phase along with the Hg(I1) for analysis under ambient conditions. Additionally, the potential for a variety of organic complexing agents being incorporated in microspheres to create tailor-made reagents for toxic clean-up is great. EXPERIMENTAL SECTION Latex Microparticles and Dithizone. Polystyrene latex microparticles were obtained from Seradyn (Indianapolis,IN) in a range of particle diameters from 0.204 to 0.944 pm. The particles were of uniform size distribution and very regular in appearance. Standard suspensions were prepared by simple dilution of the 15% (w/w) stocks with 18-MQdeionized water. These suspensions were stored at room temperature. Unless otherwisespecified,all analyseswere carriedout usingthe smalleat diameter, 0.204 pm. Dithizone was purified by Irving's method, and the purified dithizone solution in spectroscopic grade CC4 was stored under a layer of 1 N H2SO4 in the dark. Solutions stored this way keep very weLS Reagent Impregnation and Hg(I1) Extraction. In a small .1 vial, 50 p L of 0 0 % (w/v) dithizone (H2Dz) solution in CCL was added to 900 p L of deionized water. The H2Dz was extracted into the aqueous phase by addition of 10 pL of 25% (v/v) NH4OH followed by gentle shaking. The CC4 layer was carefully removed after all traces of the green HzDz were gone. A 100-pL aliquot of 0.2% (w/w) latex microparticle suspension was added, and the suspension, a soft orange-yellowcolor, was acidified with 10 p L of 3% (v/v) HzSO4 as the pH needed to drive HzDz into the microparticles is below 6. The resultinggreen suspension of HzDz-impregnatedlatex microparticles (see Figure 1)was added to 1.0-mL Hg(I1) samples in 100-pL aliquots. This volume contained sufficient H2Dz to completely complex the most concentrated standard plus a modest excess. The samples were filtered through 0.1-pm pore size filters (Nuclepore, Toronto, ON, Canada) which were then removed from the filter housing and allowed to air dry before analysis. PCPAS Measurement. The schematic diagram of the PCPAS system is illustrated in Figure 2. The excitation source was an argon ion-pumped dye laser (Spectra-Physica 164 and 375B,Mountainview,CA). The output beam from the dye laser, 605nm in wavelength and typically90 mW in power,was directed through a window into the photoacoustic cell after being mechanically chopped at 200Hz. The samples (membranefiitera supporting latex microparticles) were placed directly into the
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* Author to whom all correspondence should be addressed.

(7) Mazumdar, S. J. Chem. SOC. Dalton Trans. 1991,2091-6. (8) Irving, H. M. N. H. Dithizone; The Chemical Society: London, 1977; Chapter 8.

0 1992 Amerlcan Chemlcal Soclety




cell and directed onto the sample through the back window. The PAS signal was again reeorded for sufficient time to achieve a steady-statesignal. The absolutedifferenceinthe signal voltages, the PCPAS signal amplitude, was taken as an indication of the amount of Hg(I1) present in the sample. Interference from other heavy metals was examined by preparation of mixed metal standards. The dithizone present in the 100-pL aliquot remained in excess of the total amount of extractable metals. These samples were analyzed in the same way as described above.


The chemistry of the Hpz-impregnated latex microparticles preparation and Hg(I1) extradion is very interesting. It in not clear yet how HzDz, and then Hg(II), in transported into the microparticle center. These solid particles of polystyrene,witharigid structuredowingthemto he filtered while retaining their shape, possess a negatively charged surface due to residual surfactant from their production. It in unlikely, then, that HzDz adsorbs onto the surface at any time to be followed by seepageinto themicroparticle interior. It may be that the presence of residual CC4 facilitates the transfer ofHzDzinamannersimilarto the procedure involved with dyeing the particles. In dyeing particles, the particles are initially swollen with a dye solution in a solvent for polystyrene, suchasCC4. Thesolvent is then carefullyboiled off, trapping the dye inside the latex.6 Although very little CC4 (50 pL) was involved in the HZDz-impregnation step, the transport process might he analogous since the latex volume itself was quite s a l The particles were hence ml. slightly swollen by the organic solvent, allowing enough fluidity or flexibility to the structure to allow transfer of HzDz across the interface. The pH of the extraction in generally important to achieve quantitative results. As illustrated in eq 1, a Large HzDz concentration will tend to drive the reaction toward completion. Sigmoid m e a of the percentage extraction of



resumng from the tenninatbn of Ihe styrene polymerlzalbn steps in Ihe mlnopartlcb m e . (b) Representation showing dkhkone in Ihe process of transferring into the mlcropatilcle cenler at solullon pH 5 0. (c)Representation showing Hg2+in Ihe process o complexlngwkh f H P z in tim interior of tim microparticle.

Schema(ic showing the Impregnalbn and exIra& p r o c l g ~ ~ ~ Repmentatkf~ a hlex miaopartlde:open chcles (a) . of are SOa- iunclbnal groups from lhe swfactanl used in lhe &glnal produclbnof the latex microparticlea: shaded circles are S : groups O

m 1.

Argon Ion Laser


Lock.," Amplifier and Computer


Optical Fibre



Schsmatlc &@'am o Ihe W A S apparatus. f









~yplcalW A S signal for ~ I I dHh!zonate in htax ) mlcropsrllcb. The slgnal rlses abruptly when exposed to vlslble Illumination from lhe projeclw sowco due lo htgher absorpavny of Ihe blue excltebstate oomplex a1 Ihe wavalengm of 605 nm.


gas-tight cell which was fitled to a condenser microphone and a sound meter which contained a preamplifier and an octave fdter set (BriielandKjaer4144and2209,Pte.Claire,PQ,Canada). The baseline PAS signal from the sample (see Figure 3) was first recorded using a lock-in amplifier (Stanford Research SR510, Sunnyvale,CA). Then the sample was illuminated by the light of an 80-W projector lamp focused into an optical fiber bundle. The optical fiber bundle was brought close to the photoacoustic

primary dithizonates into a solution of 10-5 M HzDz in a suitable organic solvent reach 100%above pH 4 while the extraction drops to zero helow pH 3. Lower concentrations of HzDzin the organic phase exaggerate the m and push e a them to higher pH regions; a 10-8 M solution wl require a il pH of 8toachieve quantitative extraction.9 In this work, the concentration of HzDz was essentially constant at approximately 2% (w/w) in the latex. This represented a larger concentration of HzDzin the latex phase mmpared tosolutionphase extractions which are typically carried out in the l W 7 to 10-5 M range. Hence, the control of pH was less critical and a pH of ca.6 was maintained in the extraction vessel to ensure quantification. The extraction was assumed to he complete after 15 min of occasional shaking, although the reaction was seen to he much faster in those samples concentrated enough to observe the color change visually. Thequantification wasascertained by reanalyzing the filtrate from aqueous samplea after the primary reaction and filtration. The data verified that extraction efficiency waa greater than 99% for 50 and 100 ng Hg(I1)in aqueous samples under the given experimental conditions. Photoacoustic analysis of Hg(I1) dithmnate-impregnated latex microparticlea was straightforward. The presence of essentially nonahaorbing latex polystyrene surrounding the analyte species did not complicate the issue of signal generation appreciably at a chopping frequency of 200 Hz.
(9) Irving, H.M.N. H.Dithizom; The C h e m i d Society: London, 1911; chapter 5.





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Concentration of Hg(ii) (ng mL) Flgure 4. Analytical calibration curve for Hg(I1) dtthizonate in latex microparticles. The error bar shows the typical standard deviation of replicate samples. Triangles represent data taken from samples impregnated into 0.2OCpm spheres while the circles are data from 0.944-pm spheres.

Concentration of Hg(ll) (ng mL) Flgure 5. Calibration for Hg(I1) (A) and for mixed metal standards containing equal quantltles of Hg(I1) and Ag(1) (0)and Zn(I1) (+).

Calibrationcurves of latex microparticles dyed dark blue with Sudan Black (Seradyn, Inc.) showed a linear response with quantity as expected. The calibration curve for the determination of Hg(I1) using 0.204-pm microparticles is shown in the upper trace of Figure 4. The current detection limit, determined at twice the standard deviation of the blank, is 500 pg (in a 1.0-mL aqueous sample) or 3 pmol of mercury. It should be noted that the actual amount of Hg(I1) being sampled by the excitation beam, and thus generating the signal, is about 15% ,or 5 pg, of the total amount of Hg(I1) in the microparticles trapped on the filter surface. A 10-fold improvement in the detection limit, from 500 to 50 pg, was convenientlyachievable simply by reducing the filtration area from the current commercially available size of 130 mm2 to 13 mmz. Samples prepared in this manner did generate measurable signals for 50-pg Hg(I1) standards, with a signalto-noise ratio of 2. Given that HzDz does not exhibit any appreciable photochromism at the excitation wavelength of 605 nm, excess HzDz in the microparticlesdid not add to the observed PCPAS signal amplitude. The amount of residual HzDz in each sample varied roughly inversely with the concentration of the standard. This meant that, since H2Dz absorbed at this wavelength,the baseline signal amplitude did necessarilyvary from sample to sample, but since it was only the photochromism-inducedsignal change that was taken as a measure of mercury content, it did not represent a problem to the analysis. Nevertheless, the excess HzDz produced a background PAS signal which, if large enough, could limit the sensitivity range used on the sound meter. It appeared possible to remove some of the excess dithizone from the microparticles by addition of NH40H to bring the pH to 9 after complexation with Hg(I1)was complete. In most cases, however,the excess of dithizonewas modest and did not affect the analysis to any significant extent. The lower trace of Figure 4 shows the resulting calibration curve when 0.944-pm microparticles were used for the extraction. There did not appear to be any difference in the amount of HzDz that could be sequestered into equal volumes of particles of different sizes. Sincethe surface areato volume ratio did not seem to affect the impregnation,it was confirmed that HzDz, and hence Hg(II), was being transferred into the interior of the latex and not being adsorbed onto the surface. The calibration results did show enhanced detection sensitivity when smaller particles (0.204 pm) were used to collect Hg(I1). This was undoubtedly due to a combination of factors, includingopticalshielding of the interior of the larger particles (0.944 pm) which inhibited the photochromic activity and

higher surface area of the smaller particles which lead to more efficient transfer of heat to the gas in the photoacoustic cell. In the present experimental setup, the projector illumination entered the PAS cell through the back window. While this simple design avoided interference with the incident laser beam, it had a tendency to limit the intensity of projector light reaching the sample as the original filters were a mixed esters/celluloseacetate type and were stronglyscattering. The useful dynamic range of PCPAS analysis was drastically improved by changing to Nuclepore filters, which were a polycarbonate type and were muchmore transparent tovisible light (average absorbance in the visible range = 0.75). The new filters allowed more than enough light to reach the samples to ensure that small variations in projector intensity did not affect the generation of excited state Hg(II) dithizonate (Le. the PAS signal vs light intensity is flat). Additionally, the switch served to improve the ease of filtration. The polycarbonate pores are formed by nuclear bombardment followed by etching leaving a clear channel; the cellulose acetate filters are of the tortuous path type. The result was less back-pressure allowing larger volumes of dilute samples to be extracted for preconcentration of Hg(I1). Many tens of milliliters of dilute solutions can be incorporated into very small quantities of latex; a 50-mL volume of 50 ppb Hg(I1) will only require ca. 300 pg of dithizone-impregnated latex. The selectivity of the PCPAS determination for Hg(I1) w s quite good using the latex microparticles as it is in other a solid sample preparations of Hg(HDz)2. Samples of Ag(1) and Zn(I1)dithizonates showed only small PCPAS signale at the excitation wavelength of 605 nm even at very high concentrations. Comparable quantities of either Ag(1) or Zn(I1) in a standard of Hg(I1) did not add appreciably to the measured PCPAS signal amplitude for Hg(I1) as shown in Figure 5. Note that the slopes of these calibration curves were esentially the same; the variation from curve to curve was due to slight variations in the preparation of dithizoneimpregnated latex microparticles. This variation usually will not affect a calibration series from the same preparation. The presence of interfering heavy metals becomes a problem, however, if the total amount of extractable metals is in excess of the amount of HzDz introduced to the sample. There will be competition between the metals for extraction into the latex microparticles under this circumstance. A smaller overall PCPAS signal for Hg(HDz)Zwill be produced since some Hg(I1) ions will remain in the aqueous phase and be washed away with the filtrate. If large amounts of other heavy metals are suspected, a larger amount of HzDzimpregnated latex microparticles will be needed. This may generate smaller PCPAS signals for Hg(I1) as a result of the inner filter effect of the nonphotochromic, but absorbing, interfering metal dithizonates. Again, the use of stronger



projector illumination to induce photochromism should surmount the difficulty. Besides, intentionally swamping the latex microparticles with heavy metals will help remove any excess H2Dz after extraction of Hg(II) from a samplesolution, resulting in a lower background signal, providing that the choice of added metal does not interfere spectrally.

aa a visual indicator for heavy metals contamination. Additionally, the latex microparticles are a potential tool for water clean-up as they can be collected easily on amembrane fiiter for safe disposal after quantitative removal of the heavy metals. It remains to be seen, however, whether it will be possible to successfully scale the method up to meet environmental needs.

A novel technique has been demonstrated for the determination of mercury at picomole levels in aqueous samples. The extraction of Hg(I1) into H2Dz-impregnated latex microparticles is both rapid and quantitative. PCPAS detection for Hg(HDz)2 in these particles remains selective over other toxic heavy metals, and the sensitivity of the technique is good for environmental analysis. Since there is a noticeable color change of the HzDzimpregnated latex microparticles upon reaction with heavy metals (regardless of PCPAS detection ability), a packed column of the microparticles can be applied to water streams

This work waa funded by the Natural Sciences and Engineering Research Councilof Canada. A GR-5 grant from the Faculty of Graduate Studies and Research, Carleton University, is gratefully acknowledged.

RECEIVED review June 29, 1992. Accepted September for

18, 1992.
Registry No. Mercury, 7439-97-6; dithizone, 60-10-6; polywater, 7732-18-5. sytrene, 9003-53-6;