Octadecyl Immobilized Surface for Precipitate Collection with a

The procedure was validated by analyzing cadmium in three certified reference materials, that is, river sediment (CRM 320), sea lettuce (CRM 279), and...
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Anal. Chem. 2005, 77, 5396-5401

Octadecyl Immobilized Surface for Precipitate Collection with a Renewable Microcolumn in a Lab-on-Valve Coupled to an Electrothermal atomic Absorption Spectrometer for Ultratrace Cadmium Determination Yang Wang, Jian-Hua Wang,* and Zhao-Lun Fang

Research Center for Analytical Sciences, Northeastern University, Box 332, Shenyang 110004, China

Octadecyl immobilized surface was, for the first time, proved to be a superb precipitate-collecting medium. Surface charge effect was assumed to dominate the adsorption of cadmium hydroxide precipitate, facilitated by electrostatic interaction between the negatively charged C18 bead surface and positively charged cadmium hydroxide clusters. Residual silanol groups on the C18immobilized silica surface did not contribute to precipitate adsorption. A novel procedure for ultratrace cadmium preconcentration was proposed by incorporating a renewable microcolumn in a lab-on-valve system. Cd(OH)2 precipitate was adsorbed onto the C18 surface, which was afterward eluted with 20 µL of nitric acid (1%) and quantified with detection by electrothermal atomic absorption spectrometry. An enrichment factor of 28 and a limit of detection of 1.7 ng L-1, along with a sampling frequency of 13 h-1 were obtained with a sample consumption of 600 µL within the concentration range of 0.01-0.2 µg L-1, achieving a precision of 2.1% RSD at the 0.05 µg L-1 level. The enrichment factor was further enhanced to 44 by increasing the sample volume to 1200 µL. The procedure was validated by analyzing cadmium in three certified reference materials, that is, river sediment (CRM 320), sea lettuce (CRM 279), and frozen cattle blood (GBW 09140). Good agreement between the obtained results and the certified values was achieved. The detrimental effects of toxic heavy metals are quite pronounced, even at very low concentrations. In the case of cadmium, it tends to accumulate in body tissues and thence leads to injury to kidney, liver, cardiovascular, and skeletal systems.1-3 Thus, novel, sensitive procedures for ultratrace cadmium screening in complex biological matrixes are highly desired because of its high toxicity and the strict limitations on the disposal of cadmium-containing wastes. Electrothermal atomic absorption * Corresponding author. Phone: +86 24 83687659. Fax: +86 24 83671628. E-mail: [email protected]. (1) Yaman, Y. J. Anal. At. Spectrom. 1999, 14, 275-278. (2) Vinas, P.; Pardo-Martinez, M.; Hernandez-Cordoba, M. Anal. Chim. Acta 2000, 412, 121-130. (3) Stalikas, G. D.; Pilidis, G. A.; Karayannis, M. I. J. Anal. At. Spectrom. 1996, 11, 595-599.

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spectrometry is among the most favorable techniques due to its high sensitivities for metal species.4,5 When applied to real sample analysis, that is, biological and environmental samples, however, matrix effects are very often problematic. The isolation of ultratrace cadmium is an ideal approach for eliminating matrix effects while achieving some extent of preconcentration. On-line protocols can partially avoid sample contamination, large sample and reagent consumption, labor intensiveness, and lengthy operations of manual operations.6,7 Precipitation is one of the most efficient approaches for cadmium isolation, and various means have been employed for precipitate collection, including PTFE knotted reactors (KRs),7-10 PTFE membranes,11,12 and a miniature filter.13 The open-ended feature of a KR entails the obvious advantage of low flow resistance and high sample throughput. In many cases, however, its relatively low retention efficiency, generally falling in the range of 30-60%,14 restricts further improving its enrichment capability, especially for ultratrace metal species in complex matrixes. Filter and membrane collectors offer higher collection efficiencies, but the analytical performance might frequently be deteriorated due to flow resistance during precipitate adsorption. Therefore, novel protocols with high efficiency of precipitate collection and minimum flow resistance are desired for the enrichment of ultratrace cadmium in complex matrixes. At this juncture, a microcolumn packed with appropriate sorbent beads should be an alternative, provided that the buildup of flow resistance could be avoided. The renewable microcolumn approach has proven to be a superb protocol for eliminating drawbacks associated with flow resistance and malfunctions of the sorbent surface properties in solid-phase (4) Wang, J.-H.; Hansen, E. H. TrAC, Trends Anal. Chem. 2005, 24, 1-8. (5) Yan, X.-P.; Li, Y.; Jiang, Y. Anal. Chem. 2003, 75, 2251-2255. (6) Fang, Z. Flow Injection Separation and Preconcentration; VCH Publishers: New York, 1993. (7) Wang, J.-H.; Hansen, E. H. TrAC, Trends Anal. Chem. 2003, 22, 836-846. (8) Fang, Z.; Dong, L. J. Anal. At. Spectrom. 1992, 7, 439-445. (9) Chen, H.-H.; Beauchemin, D. J. Anal. At. Spectrom. 2001, 16, 1356-1363. (10) Burguera, J. L.; Burguera, M.; Rivas, C.; Rondon, C.; Carrero, P.; Gallignani, M. Talanta 1999, 48, 885-893. (11) Eroglu, A. E.; McLeod, C. W.; Leonard, K. S.; McCubbin, D. Spectrochim. Acta, Part B 1998, 53, 1221-1233. (12) Shi, J.; Tang, Z.; Tan, C.; Chi, Q.; Jin, Z. Talanta 2002, 56, 711-716. (13) Sant’Ana, O. D.; Wagener, A. L. R.; Santelli, R. E.; Cassella, R. J.; Gallego, M.; Valcarcel, M. Talanta 2002, 56, 673-680. (14) Wang, J.-H.; Hansen, E. H. J. Anal. At. Spectrom. 2002, 17, 248-252. 10.1021/ac050638z CCC: $30.25

© 2005 American Chemical Society Published on Web 07/07/2005

extractions.15-18 Such benefits are expected to be equally valid for precipitate collection. Literature search indicated that the microcolumn approach has never been employed for precipitate collection; however, the key issue for realizing such a process is to find a sorbent surface that is capable of achieving high collection efficiency. Our investigations have revealed that precipitation of cadmium hydroxide shows promising potentials for ultratrace cadmium separation/preconcentration from complex matrixes. Further explorations have proved that octadecyl (C18)-bound surfaces provide an excellent collecting medium for this precipitate, although this approach has not yet been adopted for similar purposes. To the best of our knowledge, this is the first attempt to employ a C18 surface for precipitate collection, and it indeed opened a promising avenue for solid-phase separation. A novel procedure for ultratrace cadmium isolation was thus established on the basis of the collection of cadmium hydroxide on a microcolumn packed with C18 immobilized beads, with detection by electrothermal atomic absorption spectrometry (ETAAS). A renewable-surface approach was adopted by loading/discarding the microbeads in a sequential/bead injection lab-on-valve system to avoid the deterioration of analytical performance resulting from the buildup of flow resistance or malfunctions of the bead surface properties. EXPERIMENTAL SECTION Apparatus. A WFX-130A atomic absorption spectrometer (Rayleigh Analytical Instrument, Beijing, China) with deuterium background correction was employed. A cadmium hollow cathode lamp (Rayleigh Analytical Instrument) was used as light source at a wavelength of 228.8 nm and operated at 3.1 mA, with a 0.4nm spectral band-pass. Pyrolytically coated graphite tubes with L’vov platforms were used. Peak area values were adopted for quantification. A FIAlab-3000 sequential injection (SI) system (FIAlab Instruments, Bellevue, WA), equipped with two 24 000step syringe pumps, SP1 and SP2 (Cavro, Sunnyvale, CA) with capacities of 2.5 mL, and a six-port selection valve was employed for sample processing. Both syringes were connected to the selection valve via a mixing tee (T) (see Figure 1). A single computer was employed to control both the spectrometer and the SI system to synchronize the sample processing in the lab-onvalve and the ETAAS determination. Surface charge (ζ-potential) of the C18 immobilized surface was measured by using a microelectrophoretic system (WD-9408D, Beijing Liu-Yi Instruments). The lab-on-valve microconduit was fabricated by PVC (50-mm diameter, 10-mm thickness), and was mounted atop the 6-port selection valve, as illustrated in the flow manifold (see Figure 1). The six microchannels can individually communicate with the central channel via the orifice of the selector. A small piece of PEEK tubing (i.d./o.d. ) 125 µm/1.6 mm, Upchurch Scientific) was inserted into the central channel, and the one was connected to port 4, to serve as microcolumn cavities. The channel was (15) Wang, J.-H.; Hansen, E. H.; Miro, M. Anal. Chim. Acta 2003, 499, 139147. (16) Wang, J.-H.; Hansen, E. H. TrAC, Trends Anal. Chem. 2003, 22, 225-231. (17) Long, X. B.; Chomchoei, R.; Gala, P.; Hansen, E. H. Anal. Chim. Acta 2004, 523, 279-286. (18) Hartwell, S. K.; Christian, G. D.; Grudpan, K. Trends Anal. Chem. 2004, 23, 619-623.

Figure 1. Schematic diagram of the sequential/bead injection labon-valve system for cadmium separation/preconcentration via precipitation with a renewable microcolumn packed with C18-bound silica microbeads. SP1, SP2: syringe pumps. HC1, HC2: holding coils. C1, C2: renewable microcolumn positions. T: T-connector.

designed such that the space between the channel wall and the PEEK rod allowed fluid to flow freely while the sorbent beads were prevented from escaping with the fluid flow and were caught inside the cavity to form a microcolumn. All the external channels were 1.0-mm-i.d. PTFE tubing connected to the lab-on-valve unit with PEEK nuts/ferrules (Upchurch Scientific), except for the eluate-delivering line from C2 to ETAAS, which was made from 0.5-mm-i.d. PTFE tubing (1.2mm o.d.). The lengths of the two holding coils were 235 cm, with capacities of ∼2.4 mL. Chemicals. The three media tested for cadmium hydroxide precipitate collection included C18 immobilized silica microbeads (amorphous; nominal bead size, 30 µm; Tianjin Di-er Chemicals Co., Tianjin, China), C18-immobilized poly(styrenedivinylbenzene) coploymer microbeads (spherical; nominal bead size, 40 µm; Polysorb MP-1, Transgenomic, San Jose, CA), silica microbeads (amorphous; nominal bead size, 65 µm; Aldrich). Microbead suspensions, 1:10-1:20 (w/v), were prepared in 50% ethanol to facilitate bead injection. All reagents used were at least of analytical reagent grade, and deionized water (18 MΩ cm-1) was used throughout. Cadmium working standard solutions were obtained by stepwise dilution of a 1000 mg L-1 stock solution. Other chemicals used were NaOH (G. R., Beijing Chemicals), HNO3 (Suprapur, Tianjin Kemiou Chemicals, Tianjin, China), HClO4 (G. R., 70%, Tianjin Dongfang Chemicals, Tianjin, China), HF (40%, G. R., Xinhe Chemicals, Shenyang, China), and ethanol (99%, Beijing Chemicals). Deionized water was used as carrier stream. Samples and Sample Pretreatment. Community Bureau of Reference CRM 320 (river sediment) and CRM 279 (sea lettuce) were pretreated with a previous procedure after minor modification:19 3.0 mL nitric acid (65%) and 3.0 mL hydrofluoric acid (40%) were added to 0.2 g CRM 279 or 0.5 g CRM 320, respectively, in a quartz beaker. The mixtures were soaked for 1 h and then heated gently on a sand bath until fumes appeared and the solution (19) Wang, J.-H.; Hansen E, H. J. Anal. At. Spectrom. 2002, 17, 1278-1283.

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was nearly dried by controlling the temperature, not to exceed 140 °C. After the solution had cooled, 1.0 mL of perchloric acid was added, and the contents were further heated to dryness. After cooling, the residue was dissolved with 2 mL of nitric acid (v/v ) 1:1) and afterward was diluted to 100 mL with water. Certified Reference Material GBW09140 (frozen cattle blood) was pretreated by applying a minor modification to the reported procedure:20 4.0 mL of nitric acid and 0.4 mL of perchloric acid were successively transferred into a quartz beaker containing 300 µL of the whole blood sample. The mixture was afterward heated gently on a sand bath at a temperature not to exceed 200 °C until fumes appeared and the contents nearly dried. After cooling, the residue in the beaker was soaked with 1% (v/v) nitric acid and diluted to 10 mL. Operating Procedure. Before starting the operation, an appropriate amount of the bead suspension was aspirated into a 1.0-mL plastic syringe, which was mounted vertically on port 6 of the valve. The bead suspension facilitated bead injection and column packing/renewal by slowly aspirating desired amount of beads into the lab-on-valve. As illustrated in the flow manifold in Figure 1, a complete operating cycle runs through the following steps: System Preconditioning. A 500-µL portion of carrier and 500 µL of HNO3 (1%, v/v) were aspirated successively by SP1 from the carrier reservoir (as illustrated in Figure 1) and port 1, respectively; the two zones were afterward dispensed through port 4 to clean up the microcolumn. Bead Loading/Column Packing/Precipitate Collection. A 30-µL portion of bead suspension was aspirated from port 6 into column position C1 and then transferred into C2 to pack a microcolumn. Thereafter, SP1 was set to successively aspirate 200 µL of carrier, 600 µL of NaOH solution, and 50 µL of air into HC1, while SP2 aspirated 200 µL of carrier, 600 µL of sample solution, and 50 µL of air into HC2. The aspirated air was used to evacuate the tubing between the central port and the T-connection. The solutions were afterward dispensed through the T while mixing and flowing through the microcolumn. Precipitate was subsequently collected on the surface of the beads. Post washing of the column surface was facilitated by dispending the previously stored carrier in the syringes. Precipitate Dissolution. A 1300-µL portion of air was aspirated via SP1 into HC1, 1200 µL of which was dispensed through port 4 to evacuate the microcolumn. Then 20 µL of nitric acid (1%, v/v) was aspirated and immediately directed through the microcolumn to dissolve the precipitate. The eluate was thence transferred into the graphite tube of the ETAAS via the 100 µL of air left in HC1. Renewal of Column Packing. Whenever necessary, that is, after undergoing ∼100 cycles of precipitation/dissolution, the used beads were aspirated back to the column position C1 along with a small amount of carrier. The beads were afterward directed to waste via port 3, which was regularly employed for aspirating NaOH solution. Thereafter, another aliquot of bead suspension could be aspirated to pack a new microcolumn for the ensuing processes. (20) Wang, J.-H.; Yu, Y. L.; Du, Z.; Fang, Z.-L. J. Anal. At. Spectrom. 2004, 19, 1559-1563.

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RESULTS AND DISCUSSION Cadmium Hydroxide Precipitate Retention on C18 Surface. Inorganic coprecipitation reaction, for example, the magnesium hydroxide coprecipitation system, is frequently used for the isolation of heavy metals. In the present study, this was initially adopted to separate cadmium, but the experiments showed that it does not seem to work for cadmium. It indicated that within a cadmium concentration range identical to that cited in the present study, adsorption of cadmium hydroxide was not observed using either hydrophobic or hydrophilic surfaces as collecting media. Further investigations revealed that in the absence of magnesium hydroxide, cadmium hydroxide itself as a precipitate could be effectively collected on an octadecyl immobilized surface. The employment of a renewable microcolumn for precipitate collection via bead injection in a lab-on-valve system facilitated reproducible precipitation/dissolution by on-site renewal of the packed microcolumn, as further illustrated in the section titled The Renewal of the Microcolumn. Surface adsorption of a specific species depends strongly on surface characteristics, namely, surface hydrophobicity/hydrophilicity and surface charge.21 The adsorption of nonpolar (neutrally charged) hydrophobic metal complexes on the octadecyl surface is obviously dominated by the compatibility of their hydrophobic natures. The mechanism for retention of cadmium hydroxide precipitate on the C18 surface could be quite different and might be explained by the electrostatic interaction attributed to the surface charge22,23 and the charged bulk of the fresh precipitate. In an alkaline medium, hydrated cadmium ions [Cd(H2O)n]2+, through an olation process, form the bulk of the cadmium hydroxide precipitate. The so-called olation process denotes the condensation of the hydrated ions via a hydroxyl bridge: a metal-oxygen-metal bond is formed, accompanied by the release of a water molecule. The olation process of hydrated cadmium ions includes the following: First, a hydroxide ion coordinates into a hydrated cadmium ion and becomes a hydroxyl group, and hydroxyl groups in various cadmium ions [Cd(OH)(H2O)n-1]+ condense each other by forming a divalent ion [Cd(H2O)n-1-O-Cd(H2O)n-1]2+. In the subsequent olation process, positively charged and highly anisotropic cadmium hydroxide clusters are formed.24 The clusters assemble to the bulk of the precipitate and leave a positive charge on the surface of the fresh precipitate. The electrostatic interaction between the negatively charged C18 surface and positively charged fresh cadmium hydroxide precipitate thus facilitates the precipitate retention. The above mechanism was further proved by the measured surface charge of the octadecyl surface. The experiments indicated that in the range of pH 5-11 (higher pH value was not measured), the C18-immobilized surface is negatively charged, and the amount of charge density (expressed as ζ potential) is -15.5 mv. The experimental results indicated that the process of precipitate adsorption by the C18 surface follows a different mechanism, as in the case of chromatography, in which C18 beads are (21) Cessner, A.; Lieske, A.; Paulke, B. R.; Muller, R. H. Eur. J. Pharm. Biopharm. 2002, 54, 165-170. (22) Knox, J. H.; Hartwick, R. A. J. Chromatogr. 1981, 204, 3-21. (23) Kitagawa, S.; Nishitama, H.; Kametani, F. Biochim. Biophys. Acta 1984, 775, 197-202. (24) Ichinose, I.; Kurashima, K.; Kunitake, T. J. Am. Chem. Soc. 2004, 126, 71627163.

Table 1. ETAAS Parameters Selected for the Determination of Cadmium

Figure 2. The recorded ETAAS signals by employing C18 immobilized silica beads (A), C18 immobilized copolymeric beads (B), and silica beads without C18 immobilization (C). Sample volume, 600 µL; sample/reagent volume ratio, 1:1; volume of dissolution solution (1% HNO3), 20 µL; pyrolysis temperature, 400 °C; holding time, 20 s; atomization temperature, 1700 °C; integration time, 4 s.

Figure 3. The effect of pyrolysis temperature on the integrated absorbance recorded at the following conditions: sample volume, 600 µL; sample/reagent volume ratio, 1:1; 0.1 µg L-1 Cd; volume of dissolution solution (1% HNO3), 20 µL; holding time, 20 s; atomization temperature; 1700 °C, integration time, 4 s.

frequently used for withholding neutrally charged complexes/ chelates, which are ruled by the compatibility of their hydrophobic nature. To make sure whether the residual silanol groups on the surface of the C18 immobilized silica beads contribute to the adsorption of cadmium hydroxide precipitate, C18-immobilized silica and copolymeric microbeads as well as pure silica beads without immobilization of C18 groups were investigated for cadmium hydroxide precipitate collection. The experimental results revealed that residual silanol groups contribute virtually nothing to the adsorption of the precipitate. Figure 2 shows that same amount of C18 microbeads immobilized on silica and copolymeric matrixes gave identical signals, but a significantly lower readout was recorded using pure silica beads. For the ensuing investigations, C18-immobilized silica microbeads were used. The ETAAS Parameters. As key parameters in the atomization process, the effects of pyrolysis and atomization temperature were investigated, with sensitivity and reproducibility as the figures of merit. Figure 3 indicates that optimal pyrolysis temperature was in the range of 400-450 °C. Thereafter, the integrated absorbance decreased with the elevation of temperature

step

temp °C

ramp s

hold s

argon flow rate mL min-1

drying pyrolysis atomization cleaning

100 400 1700 1900

5 10 0 2

20 20 4 3

800 800 0 800

due to the loss of cadmium in the absence of a chemical modifier.17,25,26 Considering that the precipitate dissolution solution was relatively free of matrixes, it is thus not mandatory to incorporate chemical modifiers and employ long ashing times before atomization. For the ensuing investigations, a pyrolysis temperature of 400 °C along with a holding time of 20 s was employed. The atomization temperature was optimized likewise. Experiments showed that the peak shape tended to broaden with a decreased absorbance and a deteriorated precision at an insufficient atomization temperature. On the other hand, too high a temperature led to a negative tailing in the recorded peak and resulted in deterioration of the analytical performance. An atomization temperature of 1700°C gave rise to well-shaped peaks with the highest integrated absorbance. Deuterium background correction was used throughout. The temperature program of the ETAAS determination is listed in Table 1. The Concentration of Precipitating Reagent. Sodium hydroxide concentration, the only parameter affecting cadmium hydroxide precipitation and its collection efficiency on the C18 surface, was investigated. The studied cadmium concentration was defined by the linear range of this procedure, that is, 0.01-0.2 µg L-1. This was achieved by varying their concentrations while, for the sake of convenience, keeping the flow rates of sample and NaOH solutions constant. Figure 4 illustrates the recorded results, which were acquired by fixing identical sample and NaOH solution flow rates of 10 µL s-1 and employing a cadmium concentration of 0.1 µg L-1. It is obvious that when the concentration of precipitating reagent was insufficient, that is, less than 0.05 mol L-1, the precipitation/collection efficiency increased with the increase of NaOH concentration. Beyond that, the curve leveled off, and further increasing the NaOH concentration resulted in only a minor increase of the blank signal along with a slight deterioration of the precision. A NaOH concentration of 0.05 mol L-1 was thus selected for further experiments. It has also been verified that this concentration was sufficient for the precipitation (25) Wang, J.-H.; Hansen, E. H. Anal. Chim. Acta 2002, 456, 283-292. (26) Miro, M.; Jonczyk, S.; Wang, J.-H.; Hansen, E. H. J. Anal. At. Spectrom. 2003, 18, 89-98. (27) Anthemidis, A. N.; Zachariadis, G. A.; Stratis, J. A. J. Anal. At. Spectrom. 2003, 18, 1400-1403. (28) Alonso, E. I. V.; Gil, L. P.; Cordero, M. T. S.; de Torres, A. C.; Pavon, J. M. C. J. Anal. At. Spectrom. 2001, 16, 293-295. (29) Hosten, E.; Welz, B. Anal. Chim. Acta 1999, 392, 55-65. (30) Silva, M. M.; Arruda, M. A. Z.; Krug, F. J.; Oliveira, P. V.; Queiroz, Z. F.; Gallego, F.; Valcarcel. M. Anal. Chim. Acta 1998, 368, 255-263. (31) Benkhedda, K.; Infante, H. G.; Ivanova, E.; Adams, F. C. J. Anal. At. Spectrom. 2000, 15, 1349-1356. (32) Heithmar, E. M.; Hinners, T. A.; Rowan, J. T.; Riviello, J. M. Anal. Chem. 1990, 62, 857-864.

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Figure 4. Effect of sodium hydroxide concentration on the integrated absorbance (9) and the blank (b) under the conditions described in Figure 2.

Figure 5. Optical microscopy photographs of the C18-immobilized silica beads (nominally 30 µm in diameter) (A) before use (as purchased) and (B) after ∼150 operation cycles by processing CRM 320 digest.

of cadmium within the linear concentration range of the present procedure. The Concentration and Volume of Dissolution Fluids. Experiments indicated that among a few inorganic acids, nitric acid was the most appropriate for dissolving the retained cadmium hydroxide precipitate on the C18 surface. In the present case, the effects of nitric acid concentration in the range of 0.1-2% (v/v) and the corresponding volume for complete dissolution of the precipitate were investigated. The results showed that within the studied cadmium concentration range, that is, 0.01-0.2 µg L-1, 20 µL of 1% (v/v) nitric acid sufficed for quantitative dissolution of the retained precipitate. Further increase of the concentration and volume of dissolution solution did not improve the performance. Thus, 20 µL of 1% (v/v) nitric acid was employed throughout. Under these conditions, no significant carryover was observed. The Effects of Flow Rates During Precipitation/Dissolution. The effect of the flow rates of sample and precipitating reagent solutions were investigated by fixing their flow rate ratio at 1:1. To avoid the buildup of flow resistance at faster flow rates, the precipitation of cadmium hydroxide on the C18 microcolumn in the lab-on-valve system was conducted by employing relatively low flow rates. The experimental results indicated that no flow impedance was observed within flow rates of 6-14 µL s-1, and the retention efficiency of the precipitate remained virtually unchanged at the level of ∼93%. Beyond this range, that is, at flow rates exceeding ∼25 µL s-1, leakage from the connecting port in the lab-on-valve unit was observed due to the creation of flow impedance during the precipitation process. As a compromise, a flow rate of 10 µL s-1 for both sample and NaOH solutions was selected. For the same reason, the flow rate for precipitate dissolution was also carefully optimized. A dissolution flow rate of more than 20 µL s-1 gave rise to incomplete dissolution of the precipitate. In addition, the creation of flow resistance further deteriorated the analytical performance of the whole procedure. It is indicated that quantitative dissolution of the precipitate could be achieved at flow rates within 8-14 µL s-1, while no problems resulting from the flow resistance were encountered. For the ensuing experiments, a dissolution flow rate of 10 µL s-1 was employed. The Renewal of the Microcolumn. The experiments revealed that the C18 microcolumn surface is quite durable for dealing with the precipitation/dissolution of cadmium hydroxide precipitate;

i.e., its analytical performance remained virtually constant in terms of retention efficiency, precision, and flow resistance within ∼100 cycles of successive sample processing. At this point, it is reasonable to employ the C18 microcolumn as a semipermanent component of the system, and it is renewed only when flow impedance has been encountered or the performance of the system has deteriorated. As illustrated in Figure 5, after ∼150 precipitate/dissolution cycles, a substantial change on the morphology of the C18-immobilized silica microbeads was observed. In this case, not only was flow resistance created in the microcolumn, but also a substantial decline in the retention capability of the surface was recorded. The renewal of the microcolumn can easily be achieved, as described in the Operating Procedure. It is worth mentioning that polymeric beads employed in lab-on-valve applications are perfectly spherical and therefore are easy to manipulate. Although the silica particles used in this work have an amorphous morphology, making them somewhat difficult to handle, they did not cause any trouble during this study, and the column renewal was carried out by circulating the beads suspension with continuous stirring, as described previously.15 Interferences. The potential interfering effects of some foreign species, which are frequently encountered in biological and environmental samples, were tested with the present procedure by gradually increasing the amount of foreign ions. At a cadmium concentration level of 0.1 µg L-1 and within a (5% error range, 100 mg L-1 of alkali and alkaline earth metal ions does not interfere with the determination (no tests for higher concentration levels). The common heavy metal ions, Co2+, Ni2+, Pb2+, and Cu2+, can be tolerated up to 10 mg L-1. At this concentration level, however, the most important heavy metal ion in biological samples, Fe3+, suppressed the signal by more than 5%. For common biological and environmental samples, including some of the most problematic matrixes, for example, seawater, the contents of the above metal ions in sample digests or after appropriate dilution will not exceed the tolerance concentration levels; therefore, the present procedure can be directly employed, and no further treatment or masking reagents are needed. Performance and Validation of the Procedure. Under the optimized experimental conditions, the peak shapes were recorded for the preparation of calibration graph within the concentration range of 0.01-0.2 µg L-1 and are shown in Figure 6. The performance data obtained for the sequential/bead injection labon-valve on-line precipitation procedure for cadmium separation/

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Table 3. Determination of Cadmium in CRM 279, CRM 320, and GBW09140 Certified Reference Materials values (µg g-1)a samples

certified

found

CRM 320 CRM 279 GBW09140

0.533 ( 0.026 0.274 ( 0.022 6.7 ( 1.0

0.519 ( 0.009 0.296 ( 0.023 6.9 ( 1.1

a Results are expressed as the mean of three replicates ( confidence interval at the 0.05 significance level.

Figure 6. Peak shapes recorded for the preparation of calibration graph within the range of 0.01-0.2 µg L-1 under the conditions described in Figure 2. Table 2. Analytical Performance of the Present Protocol via Collecting Cd(OH)2 Precipitate on a C18 Surface, along with Some On-Line Preconcentration Methods with Detection by ETAAS or ICPMS sample volume 600 µL

1200 µL

0.01-0.2 µg L-1 AA ) 2.5874CCd + 0.0370 correlation coefficient r2 ) 0.998 sampling frequency 13 h-1 detection limit (3σ, n ) 11) 1.7 ng L-1 RSD (0.05 µg L-1, n ) 7) 2.1% bead consumption