2369

Short Communication

Microliter Volume Determination of Cosmetic Mercury with a Partially Crosslinked Poly(4-vinylpyridine) Modified Screen-Printed Three-Electrode Portable Assembly Jyh-Myng Zen,b Annamalai Senthil Kumar,b Sing-Chuan Lee,a Ying Shih a* a

Department of Applied Cosmetology, Hung Kuang University, Taichung 433, Taiwan *e-mail: [email protected] b Department of Chemistry, National Chung Hsing University, Taichung 402, Taiwan Received: June 18, 2007 Accepted: July 30, 2007 Abstract We successfully demonstrated microliter (mL) volume determination of Mercury (Hg) using an in-built screen-printed three electrodes containing partially crosslinked poly(4-vinlylpyridine) (designated as pcPVP) modified carbonworking, carbon-counter, and Agþ-quasireference electrodes (SPE/pcPVP) in a pH 4 acetate buffer solution with 2 M KCl by using the square wave anodic stripping voltammetric (SWASV) technique. Instrumental and solution phase conditions were systematically optimized. Experiments were carried out by simply placing a 500 mL-droplet of Hg containing real sample mixed with the base electrolyte on the SPE/pcPVP surface. The SPE/Agþ quasi-reference system shifted the Hg-SWASV detection potential ca. 250 mV positive, but the quantitative current values were appreciably similar to that of a standard Ag/AgCl reference electrode. Under optimal condition, the calibration graph is linear in the window of 100 – 1000 ppb of the Hg droplet system with a detection limit of 69.5 ppb (S/N ¼ 3). Finally real sample assays were demonstrated for prohibited cosmetic Hg containing skin-lightening agents in parallel with ICP-OES measurements. Keywords: Mercury, Poly(4-vinylpyridine), Screen-printed electrode, Microanalysis DOI: 10.1002/elan.200703990

Sensitive and selective detection of the neurotoxic heavy metal mercury (Hg) is important research, due to unknown pathways of Hg into food materials and other usable goods [1 – 2]. For example, illegally formulated Hg species (inorganic Hg) has been detected in cosmetic products (both branded and unbranded), especially with skin-lightening reagents (up to 40 mg/g of the [Hg]) [3]. It was noted that the Hg presence and its concentration, [Hg], were particularly hidden in the labels of the goods. Simple, easy and low cost Hg detection assays are thus important for Hg pollution and prevention. Most of the [Hg] detection methods were based on UVvis, fluorescence [4, 5], cold vapor-atomic absorbance (CVAAS), -atomic fluorescence (CV-AFS) [6 – 8], and ICP-MS spectroscopic approaches [9]. Those conventional methodologies always require tedious off-line sample preparation procedures and extensive instrumentation with skilled technician. For instance, recently, 2-mercaptobenzimidazol loaded silica gel column in couple with the CV-AFS detection has been introduced for efficient and selective Hg pre-concentration and detection [6]. In that procedure, 0.05 M KCN þ 2 M HCl solution was used as an eluent to desorbs the preconcentrated Hg. In further, intense UV radiation was applied to decompose the organic mercury to inorganic form in presence of strong acid þ SnCl2 mixture solution. The assay was limited to low concentrations of Electroanalysis 19, 2007, No. 22, 2369 – 2374

halide (X), due to serious interference of the [HgX4]2 complex ions. Note that using of KCN, UV irradiation and strong acid are critical and even more dangerous than the Hg. Apart from that KCN in strong acid solution produce deadly toxic HCN vapor. Hence such risky methods are not secure for routine analytical measurements. On the other hand, recent times, electrochemical techniques rank first in the analytical chemistry as a simple detection methodology, and preferential extension to portable single use device suitable for unskilled persons. In this regards, screen-printed electrode (designated as SPE) based electrochemical detection offers safer, low-cost, flexible design, mass-producible and disposable approach [10 – 12]. Common electrochemical Hg-preconcentration methods are based on ion-exchange materials including polymeric resins, chelating and/or complexation ligands and amalgamation etc. [13 – 19]. It is note worthy that almost all of the electrochemical detections worked with conventional electrodes; glassy carbon (GCE) and carbon paste (CPE) working, Ag/AgCl (or Saturated calomel electrode (SCE)) reference systems with a ca. 10 mL working cell. Meanwhile, non-conventional electrodes like boron doped diamond [20] and Gold ultramicroelectrode (Au-UME) were also reported for the trace Hg detection [21]. Nevertheless those electrodes are expensive and some difficulty in the construction. So far, few reports with low cost SPE based Hg G 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

2370

Y. Shih et al.

detection approach. Wang and Baomin reported an Au plated SPE, Ugo et al. reported a chelating resin, Sumichelate Q10R (contains dithiocarbamate groups) modified SPE and Augelli et al. reported gold CDs as disposable working electrode for the Hg detections [22 – 24]. Recently our group demonstrated a 1,5-dibromopentane partially cross-linked poly (4-vinlypyridine) modified SPE (designated as SPE/ pcPVP, where pcPVP ¼ partially cross-linked (4-vinylpyridine)) for the selective and sensitive Hg detection with a detection limit of 3.15 ppb (i.e., 15.7 nm), through SPE/ pcPVPþ – 1/2[HgCl4]2 complexation (formation constant ¼ 1.2  1015) as a preconcentration mechanism in excess chloride media [3]. There are advances for the preconcentration of the anionic form of the Hg, [HgCl4]2, since most of the interference species exist as cationic form in the real samples, which can easily excluded by the cationic pcPVP sites. Detected values found to be parallel with ICPMS measurements [3]. Hence in this work, we further extend the technique to microliter (mL) volume analysis with portable in-built three SPE device containing chemically modified carbon-working and -counter and Agþ-reference systems as in Scheme 1. XPS was used in this work in order to characterize the surface feature at few Angstrçm (M) depth of the pcPVP film on the SPE. Figure 1 shows typical survey scan response of unmodified SPE and SPE/pcPVP surfaces. The unmodified SPE shows three predominant peaks corresponding to O 1 s, C 1 s, Cl 2 p core energy levels (Fig. 1a). Resulted SPE/ pcPVPNs response qualitatively similar to the earlier case, but an additional peak for N 1 s energy level corresponding to the pyridine moieties of the pcPVP (Fig. 1b) was observed. The O 1 s peak count response of the modified

Scheme 1. A) Cartoon for an in-built three screen-printed electrode assembly (SPE), constructed with carbon disc (3 mm diameter) working (WE), carbon-ring counter (CE) and Ag reference (RE) electrode units. B) Typical SPE fixing model into a holding device. C) Picture of [Hg] containing 500 mL droplet electrolyte on a SPE/pcPVP surface. Silica plates used to protect the SPE assembly and to avoid experimental shocks. Electroanalysis 19, 2007, No. 22, 2369 – 2374

Fig. 1. XPS survey scan responses for the unmodified SPE (a) and SPE/pcPVP (b) surfaces.

film was relatively weaker than that of the modified electrode. Note that the Cl 2 p peak (due to the organic binder in the carbon screen-printing ink) was considerably retained even after the pcPVP modification on the SPE. This observation indicated that the SPE/pcPVP modified film is thin layer characteristic with few monolayer thickness with porous in nature. Initial experiments were carried out with conventional 10 mL working cell along with a standard Ag/AgCl reference electrode. Figure 2A is a comparative cyclic voltammetric (CV) response of unmodified SPE with SPE/pcPVP for 10 ppm Hg at a scan rate (v) 50 mV/s in potential window of 1.0 to þ 1.0 V vs. Ag/AgCl. As seen in the figure, both the unmodified and SPE/pcPVP yield a sharp anodic peak corresponds to an oxidative striping of the Hg0 ! Hg2þ (Hg0 deposited from [HgCl4]2 complex during cathodic negative region at about 0.5 V vs. Ag/AgCl) [3]. A negative shift in the Hg stripping peak potential was observed with the SPE/ pcPVP. This indicates selective adsorption through a complexation mechanism (Fig. 2B). The SPE/pcPVPNs stripping peak current is about twice higher over the unmodified SPE, and the modified electrode response is stable and reproducible. Possible structure for the formation of {SPE/ pcPVPþ  1/2[HgCl]2} intermediate complex during the initial cathodic CV scan is sketched in the Figure 2B. This complex expects to further deposits as Hg0 in final stage of the cathodic scan. In our previous work unbuffered mixture solution made up of 0.02 M H2SO4 þ 0.01 M KCl was used as an electrolyte with a conventional setup [3]. But working with quasireference system, SPE/Agþ, the base electrolyte pH must be fixed, in order to avoid variation in the peak potential and the current values during the real measurements. Hence in this work a fixed buffer solution along with a KCl salt system (to generate the [HgCl4]2 species) was used. Further

www.electroanalysis.wiley-vch.de

G 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

2371

Microliter Volume Determination of Cosmetic Mercury

Fig. 2. A) Cyclic voltammetric response of SPE and SPE/pcPVP with 10 ppm of Hg in pH 4 ABS þ 2 M KCl containing solution at a scan rate of 50 mV/s in a conventional 10 mL containing working system. B) Conceptional representation for the preconcentration Hg as [HgCl4]2 complex on SPE/pcPVPþ.

Fig. 3. A) Cyclic voltammetric response of a SPE/pcPVP for the 10 ppm of Hg in a pH 4 ABS containing solution with various [KCl] (0.1 – 3 M) against Ag/AgCl or in-built quasi-SPE/Agþ reference electrodes at a scan rate of 50 mV/s (conventional 10 mL working system). B) and C) are plots of peak potential or peak current against chloride ion concentration respectively.

experiments are optimization of [KCl] (with a SPE/Agþ) against two different reference systems. Figure 3A is typical 100 ppm HgNs stripping peak current responses in the pH 4 ABS mixed with various [KCl] (0.1 – 3 M) using SPE/Agþ and Ag/AgCl references. As can be in the plot Figure 3B, Ag/AgCl reference shows a peak at 0.31 V (ipa ¼ 0.23 mA, independent of [KCl]), while the SPE/Agþ reference result at ca. 0.25 V negative shift in the peak potentials with variable peak currents values (Fig. 3A – C). For example, in presence of 3 M KCl; ipa ¼ 0.07 mA and Epa ¼ 0.03 V vs. SPE/ Agþ, while with 2 M KCl respective values are 0.23 mA and 0.041 V vs. SPE/Agþ. The pH 4 ABS þ [KCl] ¼ 2 M electrolyte mixture solution found to yield identical peak current values with that of the Ag/AgCl and SPE/Agþ reference systems and hence it is chosen for further experiments. Electroanalysis 19, 2007, No. 22, 2369 – 2374

Using high concentration of the chloride expect to eliminate current-deviation caused by trace amount of other halide species presence in real sample. Further experiments are optimization of the SWASV parameters. Interrelated instrumental parameters like amplitude, frequency and potential step were individually optimized for 300 ppb of Hg containing an electrolyte droplet on the three electrodes SPE/pcPVP. The optimal SWASV values are; SW frequency, 40 Hz; SW amplitude, 45 mV; step height, 4 mV. Meanwhile, PE and preconcentration time (tp) were also optimized as 0.5 V vs. SPE/Agþ and 60 s respectively. Figure 4 shows a calibration SWASV response for increasing [Hg] in the window 100 – 1000 ppb under the optimal working condition. As can be seen, the peak currents systematically increase with increasing in the

www.electroanalysis.wiley-vch.de

G 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

2372

Y. Shih et al.

Fig. 4. SWASV calibration response for [Hg] within a 500 mL droplet containing pH 4 ABS þ 2 M KCl electrolyte at three electrode SPE/pcPVP surface. Insert plot is a calibration plot of the peak current (base-line corrected) against [Hg]. SWASV conditions are: frequency ¼ 40 Hz, amplitude ¼ 45 mV and a step height ¼ 4 mV. Preconcentration potential ¼ 0.5 V vs. SPE/Agþ for 60 s.

[Hg]. Plot of stripping peak currents (i.e., ipa) against [Hg]Ns is linear up to 1000 ppb with a slope and regression value of

0.0241 mA/ppb and 0.9929 respectively. Note that earlier conventional approach with 0.02 M H2SO4 þ 0.01 M KCl base electrolyte showed a current sensitivity value of 0.4572 mA/ppb, which is about 20 times higher over the present approach. The reason could be solely due to the change in the base electrolyte near to neutral pH, this in turn lowering the formation of the cationic pcPVPþ species (since pKa  5). Nevertheless repetitive measurement of a 100 ppb of Hg containing base electrolyte droplet yielded a RSD value of 4.55% (n ¼ 7), indicates appreciable reproducibility of the detection method in this work. Calculated detection limit (S/N ¼ 3) is 69.5 ppb. The SPE/pcPVP is tolerable to commonly co-existing metals like ZnII, NiII, SnII, PbII, CoII, CuII, CrVI and BiIII up to 100 fold excess (with respect to 100 ppb of Hg in the a droplet) as like previous conventional approach. Figure 5A and B are SWASV responses of the real samples using the SPE/pcPVP at PE ¼  0.5 V vs. SPE/Agþ for tp ¼ 60 s. Table 1 provides quantitative information about the real sample analysis. Detected [Hg] are 373.63 and 350.46 ppb respectively for the sample #1 and #2. Parallel inductively coupled plasma-optical emission spectroscopic (ICP-OES) measurements showed respective values of 387.70 and 373.30 ppb. Calculated recovery values are 94.29 and 98.07% respectively for the above samples. It is surprising that both the real samples containing significant Hg concentration, where there is no label about the Hg compounds. Overall, the analytical methodology intro-

Fig. 5. Typical SWASV microliter volume analysis for two different skin-lightening cosmetic real samples at SPE/pcPVP surface. First and second spike corresponds to without and with standard 300 ppb [Hg]. Other experimental conditions as in the Figure 4. For the Sample #1 and #2 dilution factor ¼ 100. Table 1. Microliter volume analysis for Hg containing cosmetic real samples using SPE/pcPVP system. Sample [a]

[ Hg2þ] (ppb) ICP-OES

1st detected

Std. injected

2nd detected

Recovery (%)

#1 #2

387.70 373.30

373.63  8.11 350.46  6.70

300 300

656.51  5.93 644.68  8.21

94.29  3.35 98.07  3.53

[a] No. of assays ¼ 3.

Electroanalysis 19, 2007, No. 22, 2369 – 2374

www.electroanalysis.wiley-vch.de

G 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

2373

Microliter Volume Determination of Cosmetic Mercury

duced in the work is quite suitable for rapid and easy single use Hg detection in the cosmetic product. In conclusion, 500 mL volume working electroanalysis of Hg on an in-built all-in-one three-electrode SPE/pcPVP system was successfully demonstrated. The SPE/Agþ unit shows quantitatively similar SWASV stripping current values with that of a standard Ag/AgCl reference system under the optimal condition regardless of its detection potential. The method was effective to detect the illegally formulated [Hg] in the cosmetic skin-lightening real samples with the results comparable to that of ICP-OES measurements. Since the approach is simple, easy, low-cost with portability, it can be extendable to other Hg containing real samples also. Experimental Poly(4-vinylpyridine) (PVP, MW ¼ 50 000) solution in methanol containing ca. 20 wt% polymer, was purchased from Aldrich. The standard solutions (1000 ppm) of HgII, and other metal solutions were obtained from Merck (Darmstadt, Germany). Supporting electrolyte solutions were also prepared from Merck reagents. Other compounds (ACScertified reagent grade) were used without any further purification. Aqueous solutions were prepared with doubly distilled deionized water by reverse osmosis technique. Unless authorize stated the base electrolyte solution used in this work was 0.1 M pH 4 acetate buffer solution (ABS) þ 2 M KCl (optimal). Electrochemistry was performed on a CHI 620A electrochemical workstation (Austin, TX, USA). A BAS VC-3 electrochemical cell (10 mL) was employed in the initial experiments. Conventional type electrode system consisted of unmodified or chemically modified screen-printed system as a working electrode (Zensor R&D, Taiwan) (3 mm diameter), an Ag/AgCl reference electrode (Model RE-5, BAS), and a platinum disc (3 mm diameter) auxiliary electrode. The three electrodes combined SPE assembly contains carbon printed disc as a working (3 mm diameter), an outer carbon-ring as a counter and an outer Ag printed layer as a reference electrode as like in Scheme 1A. Portable SPE holders used in the microliter volume analysis is also purchased from Zensor R & D under special design and order (Scheme 1B and C). Since dissolved oxygen did not interfere with the working potential window, no deaeration was performed. X-ray photoelectron spectroscopic (XPS) analysis (American Physical Electronics, Auger 670 PHI XI/ESCA PHI 1600) was performed with Mg Ka radiation source (1252.6 eV) with resolution of 0.1 eV. The pressure inside the analyzer was maintained at 109 Torr during the measurements. ICP-OES analyses were carried out using Perkin Elmer (PE optima 3000) instrument. Real samples were first pretreated by digesting with high-purity HNO3 þ H2O2 mixture and then subjected for the measurement under standard operation condition. Electroanalysis 19, 2007, No. 22, 2369 – 2374

Prior to the pcPVP modification, the SPE/Ag metallic layer part is oxidized by immersing the unit in a 30% H2O2 þ 0.1 M KCl solution for 20 min (referred as SPE/Agþ). The SPE/pcPVP modified system was prepared following the procedure mentioned previously [3]. In brief, a 4 mL mixture containing 0.25 wt% PVP þ 7% (vs. the pyridine moiety) 1,5-dibromopentane (cross-linking agent) in methanol was spin coated (at 3000 rpm/15 min) on a clean SPE. The electrode is subsequently heated at 90 8C for about 2 h, in order to fasten the cross-linking process and to prevent dissolution of the film in aqueous electrolyte. The SPE/ pcPVP finally exposed to UV light radiation for 30 min, in aim to rigid the polymeric structure and to activated the surface sites. Such chemical pretreaments results to highly reproducible and renewable surface features. The SPE holder device is first placed in stable surface as in the Scheme 1C. Prior to the regular electrochemical measurements, the SPE/pcPVP was equilibrated with the base electrolyte bath for about 120 s. 500 mL (optimal) of electrolyte solution is then dropped on the SPE, which cover all the three-electrode assembly (Scheme 1C). The modified surface is relatively hydrophilic over the unmodified electrode and hence a stable microdroplet formation on the SPE/pcPVP suitable for further electroanalytical detections. Square wave anodic stripping voltammetric (SWASV) technique was used for quantitative analysis by scanning the potential from 0.2 V to þ 0.6 V at a frequency 40 Hz, amplitude 45 mV and a step height 4 mV (optimal). Hg preconcentration was performed at a fixed applied potential (PE) of 0.5 V vs. Ag/AgCl for 60 s (optimal). Quantification was achieved by measuring the peak current of the oxidation process (ipa) after proper baseline correction. The SPE/pcPVP surface can be renewed by following successive pretreatments; (i) washing with blank base electrolyte, (ii) six continuous SWASVs with a droplet electrolyte and (iii) a constant potential treatment at 0.6 V for 60 s. Such a pretreatment procedure provides highly renewable SPE/ pcPVP surface suitable up to discrete 30 measurements. Two unbranded skin-lightening agents (#1 and #2) were purchased from local supermarket and night market respectively. There is no detail about the ingredients. The SPE/ pcPVPs were first screened by tested with a 300 ppb standard [Hg] droplet under the optimal SWASV condition. Electrodes yielding a current sensitivity 0.0241 mA/ppb within an error of 5% were further chosen for practical analysis. Real sample were prepared as follows: 500 mg of skin-lightening lotion samples dissolved in 1 mL of base electrolyte (dilution factor 0) or 5 mg the real samples in 500 mL base electrolyte without and with spiked standard [Hg] (dilution factor 100). There is no other sample pretreatments as like with conventional the ICP-OES measurements. Respective peak currents (y) were further fitted into a standard calibration equation, y ¼ 0.0241 x þ 6.6521 (optimal) in order to obtained the quantitative real sample [Hg] values (x). In addition, known [Hg] spiked (300 ppb) real samples were also discreetly tested under standard addition condition.

www.electroanalysis.wiley-vch.de

G 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

2374

Y. Shih et al.

Acknowledgement The authors gratefully acknowledge financial supports from the National Science Council of the Republic of China. References [1] B. Wheatley, Mercury as a Global Pollutant: Human Health Issues, Kluwer Academic Publishers, Boston, MA, 1997. [2] Food Products Association, Vol. 9, No. 27, Washington, DC 2005, Suite 300. [3] Y. Shih, A. S. Kumar, J.-M. Zen, J.-C. Hsu, Bull. Chem. Soc. Jpn. 2005, 78, 2130. [4] W. Langseth, Fres. J. Anal. Chem. 1996, 325, 267S. R.Segade, J. F. Tyson, Spectrochim. Acta B 2003, 58, 797. [5] Cs. Pa´ger, A. Ga´spa´r, Microchem. J. 2002, 73, 53. [6] H. Bagheri, A. Gholami, Talanta 2001, 55, 1141. [7] L.-J.Sha, W.-E.Gan, Q.-D.Su, Anal. Chim. Acta 2006, 562, 128. [8] E. Ramalhosa, S. R.Segade, E. Pereira, C. Vale, A. Duarte, Anal. Chim. Acta 2001, 448, 135. [9] C. F. Harrington, Trends Anal. Chem. 2000, 19, 167. [10] J. Wang, Acc. Chem. Res. 2002, 35, 811. [11] J. Wang, Analytical Electrochemistry, 2nd ed., Wiley, New York 2001.

Electroanalysis 19, 2007, No. 22, 2369 – 2374

[12] J.-M. Zen, A. S. Kumar, Screen-Printed Electrochemical Sensor, Encyclopaedia of Sensors (Eds. A. G. Craig, E. C. Elizabeth, V. P. Michael), American Scientific Publisher, California 2006, pp. 33 – 52. [13] P. Herna´ndez, E. Alda, L. Herna´ndez, Fresenius J. Anal. Chem. 1987, 327, 676. [14] W. Huang, C. Yang, S. Zhang, Anal. Bioanal. Chem. 2002, 374, 998. [15] J. Wang, M. Bonkadar, Talanta 1988, 35, 277. [16] J. Labuda, V. Plaskoni, Anal. Chim. Acta 1990, 228, 259. [17] Z. Navaratilova, Electroanalysis 1991, 3, 799. [18] M. D.Imisides, G. G.Wallace, J. Electroanal. Chem. 1988, 246, 181. [19] P. Ugo, L. M.Moretto, G. A.Mazzocchin, Anal. Chim. Acta 1995, 305, 74. [20] A. Manivannan, M. S. Seehra, A. Fujishima, Fuel Processing Tech. 2004, 85 513. [21] O. Ordeig, C. E. Banks, J. d. Campo, F. X. Mun˜oz, R. G. Compton, Electroanalysis 2006, 18, 573. [22] J. Wang, T. Baomin. Anal.Chim. Acta 1993, 274, 1. [23] P. Ugo, L. M. Moretto, P. Bertoncello, J. Wang, Electroanalysis 1998, 10, 1017. [24] M. A. Augelli, R. A. A. Munoz, E. D. Richter, A. G. Junior, L. Angnes, Electroanalysis 2005, 17, 755.

www.electroanalysis.wiley-vch.de

G 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim