Automated On-Line Sample Pretreatment System for the

Division of Occupational Medicine, Institute of Occupational Safety and Health, Council of Labor Affairs Executive Yuan,. Min Sheng E., RD, Taipei, Ta...
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Anal. Chem. 1997, 69, 3930-3939

Automated On-Line Sample Pretreatment System for the Determination of Trace Metals in Biological Samples by Inductively Coupled Plasma Mass Spectrometry Chuan-Chieh Huang and Mo-Hsiung Yang*

Department of Nuclear Science, National Tsing Hua University, 30043 Hsinchu, Taiwan Tung-Sheng Shih

Division of Occupational Medicine, Institute of Occupational Safety and Health, Council of Labor Affairs Executive Yuan, Min Sheng E., RD, Taipei, Taiwan

A fully automated on-line sample pretreatment system combining microwave digestion with sample preconcentration/matrix separation for the determination of trace metals (Fe, Ni, Cu, Zn, and Pb) in blood and serum samples by inductively coupled plasma mass spectrometry (ICPMS) was designed and evaluated. The samples were mixed with an appropriate reagent and digested in a flow-through, focused microwave-heated oven. After digestion, the sample solution was transferred on-line to a column packed with iminodiacetate-based resin for separation of matrix elements like Na, K, Ca, Mg, Cl, P, and S that might interfere with the measurement by ICPMS. The analytes chelated on the resin were subsequently eluted and led to ICPMS for multielement determination. The whole sample pretreatment process was automatically controlled by a self-designed expert system. The analytical reliability of data from this on-line system was confirmed to be good with the analysis of SRM samples (Seronorm Whole Blood and NIST SRM 1598 Bovine Serum), and the limits of detection (3σ) for Fe, Ni, Cu, Zn, and Pb were 68, 0.34, 3.5, 13.4, and 0.22 µg/L, respectively. With this fully automated on-line system, the determination of analytes in biological fluid samples down to micrograms-per-liter levels has been proven to be feasible, and the sample throughput can achieve up to 6 samples/h.

like Na, Mg, Ca, and K, may cause a signal suppression; furthermore, for quadrupole MS, interferences may occur due to polyatomic ions and doubly charged ions.12,13 To overcome these problems, some researchers simply diluted the samples with dilute nitric acid solution1,3,10 or (NH4)2H2(EDTA) and Triton X-100 solution14 prior to the ICPMS determination; others preferred to use preanalysis microwave digestion followed by either direct instrumental determination or further preconcentration prior to determination.6,15 The latter method can offer many advantages, such as a wide application to various materials, a high preconcentration factor, and matrix element removal. Haswell and Barclay16 designed an on-line microwave digestion system for slurry samples followed by dilution of the digested solution and subsequent determination of Ca, Fe, Mg, and Zn with FAAS. Tsalev17,18 et al. developed an on-line system combining focused microwave digestion with cold vapor and hydride generation AAS (CVAAS and HGAAS) for the determination of Hg, As, Bi, Pb, and Sb in urine and environmental water samples. Guo and Baasner19 also designed an on-line microwave digestion system to determine Hg in blood samples by FI-CVAAS. More-

* To whom correspondence should be addressed. Tel.: +886-3-5727309. Fax: +886-3-5723883. (1) Vanhoe, H.; Dams, R.; Versieck, J. J. Anal. At. Spectrom. 1994, 9, 23-31. (2) Ward, N. I.; Abou-Shakra, F. R.; Durrant, S. F. Biol. Trace Element Res. 1990, 26 (7), 177-187. (3) Vandecasteele, C.; Vanhoe, H.; Dams, R. J. Anal. At. Spectrom. 1993, 8, 781-786.

(4) Lam, J. W. H.; McLaren, J. W.; Methven, B. A. J. J. Anal. At. Spectrom. 1995, 10, 551-554. (5) Taylor, A.; Briggs, R. J. J. Anal. At. Spectrom. 1995, 10, 1033-1037. (6) Ebdon, L.; Fisher, A. S.; Worsfold, P. J.; Crews, H.; Baxter, M. J. Anal. At. Spectrom. 1993, 8, 691-695. (7) Campbell, M. J.; Demesmay, C.; Olle, M. J. Anal. At. Spectrom. 1994, 9, 1379-1384. (8) Gelinas, Y.; Youla, M.; Beliveau, R.; Schmit, J.-P. Anal. Chim. Acta 1992, 269, 115-122. (9) Jarvis, K. E.; Gray, A. L.; Williams, J. G.; Jarvis, I. Plasma Source Mass Spectrometry; The Royal Society of Chemistry: Cambridge, UK, 1990. (10) Mulligan, K. J.; Davidson, T. M.; Caruso, J. A. J. Anal. At. Spectrom. 1990, 5, 301-306. (11) Jarvis, K. E.; Gray, A. L.; Houk, R. S. Handbook of Inductively Coupled Plasma Mass Spectrometry; Chapman and Hall: New York, 1992. (12) Evan, E. H.; Giglio, J. J. J. Anal. At. Spectrom. 1993, 8, 1-18. (13) Vanhoe, H.; Goossens, J.; Moens, L.; Dams, R. J. Anal. At. Spectrom. 1994, 9, 177-185. (14) Delves, H. T.; Campbell, M. J. J. Anal. At. Spectrom. 1988, 3, 343-348. (15) De Pena, Y. P.; Gallego, M.; Valcarcel, M. J. Anal. At. Spectrom. 1994, 9, 691-696. (16) Haswell, S. J.; Barclay, D. Analyst 1992, 117, 117-120. (17) Tsalev, D. L.; Sperling, M.; Welz, B. Analyst 1992, 117, 1729-1733. (18) Tsalev, D. L.; Sperling, M.; Welz, B. Analyst 1992, 117, 1735-1741. (19) Guo, T.; Baasner, J. Talanta 1993, 40 (12), 1927-1936.

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S0003-2700(97)00284-9 CCC: $14.00

Because of its extreme importance for the human organism and easy accessibility, human blood, plasma, or serum has been selected by many clinical and analytical scientists for the determination of trace and ultratrace elements.1 Recently, inductively coupled plasma mass spectrometry (ICPMS) has been successfully applied to the multielement analysis of a wide range of biological materials and fluids.1-11 However, it has a number of disadvantages: the major elements present in biological matrices,

© 1997 American Chemical Society

over, Stewart and Barnes20 evaluated different heating coil designs and developed a flow-through, microwave-heated digestion chamber for automated sample preparation. The methods so far reported for the on-line microwave digestion coupled with spectroscopic determination are limited to the measurement of hydride-forming elements (As, Se, Hg, Pb, Bi, and Sb) and some minor elements (Ca, Mg, Zn, and Fe) in biological and environmental samples. To further explore the feasibility of determining trace and ultratrace metals, aside from the above-mentioned cold vapor and hydride-forming elements, in biological materials, a preconcentration step prior to instrumental determination becomes a necessity. The on-line procedure, when used in combination with flow injection techniques, offers the advantage of reducing sample preparation time, sample volume required for analysis, and the risk of contamination from airborne. Ebdon et al.,6 as well as Heithmar et al.,21 used an iminodiacetate-based resin column (Chelex-100) for on-line removal of interferences from predigested biological samples followed by ICPMS determination of Ti, V, Mn, Fe, Cu, Co, Ni, Zn, Cd, and Pb. Beauchemin and Berman22 successfully applied silica-immobilized 8-hydroxyquinoline (I-8HOQ) for on-line preconcentration of metals in water samples followed by determination with ICPMS. The on-line systems so far reported consisted mainly of a preconcentration of trace metals from water samples or predigested biological samples followed by instrumental determination. No fully automated analytical system coupling digestion of biological samples with column preconcentration followed by instrumental determination of ultratrace metals has been reported. Development of an on-line analytical system capable of ultratrace metal determinations in biological samples is of interest to the analytical community. Automation is of key importance to an on-line sequential process since it can reduce the risk of operational errors, increase analytical repeatability, and accelerate sample throughput. The purpose of this study is to develop an on-line, fully automated system for the determination of trace and ultratrace metals in biological samples by coupling the sample digestion with preconcentration/separation followed by an automated introduction into the ICPMS. Elements of significant biological importance, such as Cu, Ni, Zn, Fe, and Pb, were selected to test the applicability of the proposed system. EXPERIMENTAL SECTION Instrumentation. The inductively coupled plasma mass spectrometer used was the Perkin-Elmer Sciex Elan Model 5000 (Perkin-Elmer, Uberlingen, Germany). The instrumental operating conditions used for determining the analytes after the pretreatment process are summarized in Table 1. The on-line sample pretreatment system was composed of two individual parts, digestion and preconcentration, as shown in Figure 1. A Maxidigest MX 350 microwave station with a TX 31 Maxidigest programmer (Prolabo, Paris, France), two 205U/BA peristaltic pumps (Watson-Marlow, Wilmington, MA) with Viton pump tubing (Cole-Parmer, Niles, IL), two two-position valves, and a stream selection valve were incorporated in the digestion part of the system. Two high-pressure, metal-free HPLC pumps (Altech, Model 325, Deerfield, IL), an iminodiacetate (IDA) resin (20) Stewart, L. J. M.; Barnes, R. M. Analyst 1994, 119, 1003-1010. (21) Heithmar, E. M.; Hinners, T. A.; Rowan, J. T.; Riviello, J. M. Anal. Chem. 1990, 62, 857-864. (22) Beauchemin, D.; Berman, S. S. Anal. Chem. 1989, 61, 1857-1862.

Table 1. ICPMS Operating Conditions

torch rf power plasma gas flow auxiliary gas flow nebulizer gas flow

Plasma Condition normal “short” type 1.0 kW 15 L/min 0.850 L/min 0.800 L/min

Mass Spectrometer Settings Bessel box plate lens -63.26 V Bessel box lens 10.95 V Einzel lenses 4.98 V photon stop lens -9.05 V sampler orifice diameter pt, 1.14 mm skimmer orifice diameter pt, 0.89 mm interface pressure 2 Torr mass spectrometer pressure 1.5 × 10-5 Torr peaks monitored m/z 54, 56, 57, 60, 61, 63, 64, 65, 67, 207, and 208 Isotope Ratio Determination resolution high dwell time 20 ms sweeps per reading 250 reading per replicate 1 replicate 4

column, two two-position valves, and an on-line filter (Altech) were used in the preconcentration part of the system. A column of 5 mm i.d. × 100 mm long, with an adjustable endpiece (Omnifit, Cambridge, England), and containing approximately 1.5 g of resin, was used. The two-position valves and stream selection valve used in this system were Omnifit Model 11110 10-way PTFE valves and a Model 11109 8-way PTFE valve, respectively. The manifold tubing used was 1.0 mm i.d. × 1.6 mm o.d. PTFE tubing (Altech). All manifold channels were connected using high-pressure gripper fittings (Omnifit). The manifold components and flow rates used are listed in Table 2. Digestion Device Design. (A) Knitted Coiled Teflon Tubing. Ten meters of Teflon tubing (1.0 mm i.d. × 1.6 mm o.d.) was knitted23-25 and then coiled around a PTFE backbone rod (6.0 mm o.d.). The device was placed inside the Prolabo MX 350 microwave digestion cavity, as shown in Figure 2a. Sample and reagent were passed through the coil and heated with microwave energy. (B) Chamber Arrangement. The quartz chamber digestion device, as shown in Figure 2b, was modified from Stewart’s design.20 Instead of using glass tubing as the transfer line, two pieces of Teflon tubing were used in our system. The chamber device had a 52.6 mL capacity. The sample, reagent, and buffer were pumped into the chamber through the respective Teflon tubings. Upon completion of the digestion procedure, the digests were transferred to the next step through the other Teflon tubing for the subsequent preconcentration process. Automation. In order to set up a fully automatic system, some modifications of the control function of the commercial instruments were made, including the ICPMS and microwave digestion system. An electromagnetic switch controllable by personal computer was installed on the computer keyboard of the ICPMS. In the eluting step, this switch can press the “READ” function key to trigger the data acquisition procedure of the ICPMS software. The door safety switch of the microwave station was (23) Selavka, C. M.; Jiao, K. S.; Krull, I. S. Anal. Chem. 1987, 59, 2221-2224. (24) Fang, Z. L.; Welz, B. J. Anal. At. Spectrom. 1989, 4, 543-546. (25) Fang, Z. L.; Guo, T. Z.; Welz, B. Talanta 1991, 38, 613-619.

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Figure 1. Schematic diagram of the proposed on-line sample pretreatment system. P1 and P2, HPLC pumps; P3 and P4, peristaltic pumps; V1-V4, two-position valve; V5, stream selection valve; L1, sample loop; L2, reagent loop; L3, collection loop; MWD, microwave digestion device; A, air; B, buffer solution; C, carrier solution (H2O); D, drain; E, eluent; F, on-line filter; R, oxidation reagent; S, sample; 3488A, switch/ control unit; and PC, personal computer (for details, see text). Table 2. Optimized Manifold Components and Flow Rates

sample loop reagent loop cooling coil collection loop sample loading reagent loading carrier and buffer (to MWD) collector preconcentration elution

Sample Loops, Cooling Coil, and Collection Loop PTFE, 1.0 mm i.d.; 0.5 mL (L1) PTFE, 1.0 mm i.d.; 0.4 mL for serum (L2), 0.8 mL for blood PTFE, 3.5 m × 1.0 mm i.d. PTFE, 1.0 mm i.d.; 6.0 mL (L3) Pump Tubing and Flow Rates 2.4 mL/min (Viton, 1.42 mm i.d., 40 rpm, P3) 3.0 mL/min (Viton, 1.52 mm i.d., 40 rpm, P3) 5.3 mL/min (Viton, 2.05 mm i.d., 40 rpm, P3) 8.0 mL/min (Viton, 2.05 mm i.d., 60 rpm, P4) 1.5 mL/min (P2) 1.5 mL/min (P1)

also modified into a microwave on/off switch. To control the flow rate of the HPLC pumps and peristaltic pumps, four home-made adjustable dc power supplies (0-15 V) were used. The dc power supply, based on the regular electric circuit design, was made of transformers, variable resistors, and other standard electric devices. A switch/control unit (model 3488A, Hewlett Packard, Palo Alto, CA) equipped with relay modules (Model 44471A, Hewlett Packard) was used to control the rotation direction of the valves and the start/stop of the pumps and microwave station. Finally, a personal computer was used to operate a self-designed software (based on Microsoft QuickBASIC language) for controlling the prescribed function of the HP 3488A unit. If the controlled system functions properly, the on-line analytical system can achieve the full automation of sample digestion, matrix separation, analyte preconcentration, and signal measurement. Operation of the System. The stepwise sample pretreatment process in this on-line microwave digestion and preconcentration is depicted in Figure 3. The detailed operation of the manifold in each step of this automated system is also shown in Table 3. A total of 12 steps was designed to complete the whole process of the sample pretreatment. In the on-line microwave digestion process, the sample and reagent were first transferred to the injection loops (L1 and L2) in step 1 with a peristaltic pump (P3). With the aid of water and air as the carrier (V5), the sample and reagent were then delivered to the digestion chamber (MWD) in step 2. After 2 min of digestion (heated with 300 W microwave 3932 Analytical Chemistry, Vol. 69, No. 19, October 1, 1997

energy in steps 3 and 4), the buffer was added to neutralize the digests in step 5 to a pH of 5.0-5.5. The neutralized solution was then pumped out in step 6 with a peristaltic pump (P4). On leaving the digestion chamber, the heated solution was cooled in a water bath and then collected on a collection loop (L3) in step 6 for the subsequent preconcentration process. In the preconcentration process, a HPLC pump (P2) was used to transfer the digests in steps 7-11 to the iminodiacetate resin column (H2O as the carrier). The analytes that chelated on the resin were subsequently eluted by a nitric acid solution in step 12 with a HPLC pump (P1) and led to the ICPMS for multielement determination. To shorten the average time needed to complete the whole cycle for sample analysis, a time-sharing process was devised as follows. When the digested sample goes to the next step for preconcentration, the digestion chamber can be treated with two washings of acid and water mixture in steps 7-12. As the sample continues on to the eluting step, the system is now ready to accept the next sample for digestion. With the proper arrangement of the treatment sequence, as shown in Table 3, the average time required for completing the whole procedure for one sample is about 10 min. Reagents. All reagents used were of analytical reagent grade, unless otherwise stated. High-purity water, which was purified by demineralization, two-stage quartz distillation, and subsequent subboiling distillation, was used throughout. Nitric acid (E.

Figure 2. (a) Knitted coiled Teflon tubing microwave digestion device design: A, 200; B, 30; and C, 35 mm. (b) Quartz chamber microwave digestion device design: A, 35; B, 90; C, 340; D, 410; and E, 10 mm.

Merck, Darmstadt, Germany; Tracepure grade, further treated by in-house subboiling), sulfuric acid (Fisher Scientific, Pittsburgh, PA; Tracemetal grade), hydrogen peroxide (E. Merck), and perchloric acid (E. Merck) were used for digestion of the samples. Standard solutions of the analytes in 0.14 mol/L nitric acid were freshly prepared by the dilution of aqueous 1000 mg/L stock solutions (E. Merck). A 0.56 mol/L nitric acid solution was prepared and used as an eluent. The enriched stable isotope solutions of Fe-54, Cu-65, and Zn-67, with concentrations of 10.0 µg/mL each (Teknolab, Drobak, Norway) were used for isotope dilution analysis. The IDA chelation exchange resin used was obtained from Sigma (Poole, Dorset, UK; sodium form, 50-100 mesh, 400 mequiv/L). The preparation and purification of the column followed the procedures described in the literature.6 A buffer solution of ammonium acetate was prepared by mixing ammonia (25%, E. Merck) and acetic acid (E. Merck) at a ratio of 6:1 (v/v). Buffer, 1.8 and 0.9 mL, respectively, was added to blood and serum to adjust them to a pH of 5.0-5.5. To prepare the synthetic matrix solution, sodium chloride (E. Merck), potassium chloride (E. Merck), 1000 mg/L stock solutions of calcium, magnesium, and phosphate (E. Merck), and diammonium sulfate (E. Merck) were used. The solution prepared was adjusted with an ammonium acetate buffer to a pH of 5.5 and contained 200 mg/L of sodium, 200 mg/L of potassium, 10 mg/L of calcium, 10 mg/L of magnesium, 40 mg/L of phosphorus, 400 mg/L of chloride, 16 000 mg/L of sulfate, and

Figure 3. Flow chart of sample pretreatment of on-line microwave digestion and on-line preconcentration/matrix separation.

78 000 mg/L of ammonium. This solution was estimated as having a similar inorganic matrix composition to that of the solution which was obtained from digestion of 0.5 mL of blood sample with 0.4 mL of nitric acid and 0.4 mL of sulfuric acid and further adding 1.8 mL of ammonium acetate buffer solution. Samples and Reference Materials. Pool blood and serum samples from the Hsinchu Blood Transfusion Center (Hsinchu, Taiwan) were used for the manifold optimization studies. Lyophilized human reference whole blood sample (Seronorm Trace Elements Whole Blood, Levels I, II, and III) and bovine serum sample (NIST SRM 1598 Bovine Serum) were used to check the accuracy of the proposed method. Isotope Ratio Determination and Isotope Dilution Analysis. To determine the isotope ratio, the samples were first treated with the automated sample pretreatment system (off-line from ICPMS), and the analytes eluted from the column were then collected in a 15 mL test tube (approximately 4 mL was collected). The isotope ratios of Fe (54:57), Ni (60:61), Cu (63:65), and Zn (64:67) in the treated samples were determined by ICPMS in a steady-state signal mode with the conditions shown in Table 1. The measured isotope ratios have to be further corrected for mass bias of ICPMS. For this purpose, mass discrimination correction factors of the respective elements were determined by comparing the calculated isotope ratios26 and the experimental ratios by introducing 100 µg/L of standard solution of respective ions to the ICPMS. (26) Friedlander, G.; Kennedy, J. W.; Miller, J. M. Nuclear and Radiochemistry; John Wiley & Sons: Singapore, 1981.

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Table 3. Operation of Manifolds Involved in Each Step of the Automated Systema step

time, s

V1

V2

V3

V4

V5

P1

P2

P3

P4

MWD

read

1 2 3 4 5 6 7 8 9 10 11 12

25 36 19 101 16 54 30 60 100 36 60 60

injection injection injection injection injection injection load load load load load injection

load load load load load load injection injection injection injection injection injection

load injection load load load load load load load load load load

load injection load load load load injection load load injection load load

H2O air buffer H2O H2O H2O H2O H2O air air air air

on on on on on on on on on on on on

on on on on on on on on on on on on

on on on off on off on on off on off off

off off off off off on off off on off off on

off off on on off off off on off off on off

x

a P1 and P2, HPLC pump; P3 and P4, peristaltic pump; V1-V4, two-position valve; V5, stream selection valve; MWD, microwave digestion device.

In conducting the isotope dilution analysis, the amounts of sample and enriched isotopes (Fe-54, Cu-65, and Zn-67) were carefully weighted to achieve a final isotope ratio of approximately 1 for respective isotope sets. The isotope ratios of analyzed sample and spiked sample were determined by a method similar to the one described above. The final concentration of the analytes in the sample was calculated using the following equation:27

(Asp - BspR) atomic mass (BR - A) sample mass

concn (µg/g) ) WC

(1)

where R is the measured isotope ratio, corrected for mass bias, Asp and Bsp are the spike isotope abundances, A and B are the natural isotopic abundance, W is the mass of spike in grams, and C is the concentration of spike in micromoles per gram. RESULTS AND DISCUSSION Optimization of Digestion Condition. The digestion conditions or degree of dissolution in the microwave cavity are generally governed by four variables, namely, digestion device design, sample resident time, microwave power, and power of oxidation reagent. As the manifolds are typically small and most commercial microwave ovens have a rather large irradiated space, the volume ratio of the oven to the reaction area is unfavorably high and the power absorption inefficient. Moreover, the percentage of nonabsorbed power with such small loads was rather high, and its reflected portion could damage the magnetron or, at least, impair its performance. Considering these problems, a focused microwave digestor (FMD) was chosen for this work. The focused microwave digestion system produced by Prolabo has a relatively small irradiated zone (microwave cavity) of approximately 31 mm in height and 48 mm in diameter.17 To be used for on-line purposes, the digestion device must satisfy some criteria. These include capability to operate automatically, freedom from contamination, and operation flexibility toward the change of digestion conditions. The traditional Teflon bottle is no longer suitable for an on-line system because of its need for manual operation to open and close the vessel. According to the literature, two digestion device designs, namely a coiled (27) Beary, E. S.; Paulsen, P. J.; Fassett, J. D. J. Anal. At. Spectrom. 1994, 9, 1363.

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tube made of Teflon16-19,28-30 or glass20 and a glass chamber,20 have been used for on-line digestion. Though both of them can be appropriately used for the digestion of biological samples, the superior choice of mode for use in the automated sample preparation system needs to be further evaluated. (A) Knitted Coiled Teflon Tubing. The apparent advantages of sample digestion in coiled tubing are a low risk of contamination, high sample throughput, simplicity, and the capability of automation. To evaluate the performance of sample digestion in coiled tubing, 10 m of Teflon knitted coiled tubing, as shown in Figure 2a, was used. Knitted coiled tubing is used because the sample dispersion in the axial direction can be much less than that in a straight tube of the same length and inner diameter.23-25 An investigation was conducted in this study by loading 0.5 mL of blood and 0.5 mL of nitric acid into the knitted coiled tubing with water as carrier to lead the flow through a microwave heating zone. The performance of sample digestion was studied in terms of two important parameters: microwave power applied and sample flow rate. The observation of bubble formation and flow disturbances through the coil were pronouncedly increased with the increasing of microwave power, even when the power setting was only in slight excess of 60 W. However, the bubble formation was found to be markedly decreased with an increasing of the rate of sample flowing through the microwave heating zone. As the flow rate increased to about 4.0 mL/min, the bubble formation could no longer be observed, even when the microwave power setting was at 300 W (the maximum power available with this FMD). Since copious bubble formation may result in irregular flow, incomplete digestion, and impaired precision, proper measures to overcome this bubble-forming has become the major problematic factor in the digestion with a tubing design. On the basis of the above observation, it seems that the problem of bubble formation might be overcome by lowering the microwave power heating and/or increasing the flow rate of sample. However, this is not necessarily the best solution. Digestion of sample with low microwave power may result in incomplete decomposition of the sample, especially for complicated samples such as blood; increasing the sample flow rate will (28) Arruda, M. A. Z.; Gallego, M.; Valcarcel, M. J. Anal. At. Spectrom. 1995, 10, 501-504. (29) Sturgeon, R.; Willie, S. N.; Methven, B. A.; Lam, J. W. H.; Matusiewicz, H. J. Anal. At. Spectrom. 1995, 10, 981-986. (30) Arruda, M. A. Z.; Gallego, M.; Valcarcel, M. J. Anal. At. Spectrom. 1996, 11, 169-173.

shorten the resident time of sample in the microwave cavity and, consequently, result in incomplete digestion as well. To overcome the problem of bubble formation, one possible alternative, using a back-pressure regulator installed downstream, was also reported.16,31 We found that a back-pressure of up to about 100 psi was effective in increasing the efficiency of the digestion and also in reducing the bubble formation. However, high back-pressure and high flow rate operation conditions may sometimes cause the Teflon tubing to break. Although the Teflon tubing can be operated up to 1000 psi, the high temperature and knitted coiled condition might reduce its strength. Besides the easy susceptibility to system instability possibly caused by copious bubble formation, the coiled tubing digestion also exhibits a lack of flexibility toward a change of digestion conditions. Some typical limitations of this tubing design are that sulfuric acid, a high boiling point acid known as an effective digestion agent for blood sample, cannot be used with Teflon tubing coil and, also, that the digestion conditions cannot be flexibly adjusted by changing the volume and composition of reagents in accordance with different kinds of sample. In view of these unfavorable features, the method of microwave-heated digestion with Teflon tubing design was thus abandoned. (B) Chamber Arrangement. A flow-through quartz digestion chamber similar to that of Stewart and Barnes20 was modified in this research. This system, as shown in Figure 2b, possesses many desirable characteristics. First, the sample and reagent are sequentially introduced by the peristaltic pump, which enables the volume and composition of reagent to be easily changed for different kinds of samples. Second, since the sample and reagent stay in the quartz chamber, the microwave energy and heating time can be easily controlled. Third, the product gas can escape from the open end of the chamber, while the reagent vapors condense in the condenser and drain back to the reflux chamber. This reduces the bubble formation and pressure buildup. Fourth, concentrated sulfuric acid, which is very effective for organic sample decomposition, can be used because of the quartz chamber’s ability to tolerate its high boiling point. The use of sulfuric acid can increase the digestion efficiency and thus decrease the digestion time needed, as well as decrease the volume of reagent used. Fifth, digestion of solid sample is possible. Sixth, although it is not a real on-line system, automation is possible for this flow-through device by suitable arrangement of the system. Since sample digestion in this chamber design is operated in an open system, the probability of contamination from ambient air and apparatus, cross contamination between samples, and loss of analytes is expected to increase. To solve these problems, a quartz-made chamber and condenser are used in this study to minimize the risk of contamination and the loss of analytes adsorbed on the equipment surfaces. Two washing steps of the chamber by acid and water mixture in sequence between samples is used to avoid cross contamination. For on-line purposes, the shortest possible time for microwave digestion is desirable. From a practical point of view, a 2 min heating at 300 W of microwave power is considered acceptable and is used in the subsequent studies. In the preliminary study, a combination of nitric acid and perchloric acid was used because this acid mixture was known to be effective for digestion of biological samples in a closed system.2,4,6,8,31 However, we found (31) Welz, B.; He, Y.; Sperling, M. Talanta 1993, 40 (12), 1917-1926.

that it did not work in our system because a large amount of precipitate was still observed in the digested sample, even if the heating time was prolonged to 5 min. Although adding hydrogen peroxide could further assist digestion of the sample into a colorless solution, it needs additional treatment by heating and bubbling, thus making the digestion process impractical. The mixture of sulfuric and nitric acids was eventually found to be the best choice for blood digestion because it can effectively result in the digestion of sample into a clear solution with only a slightly tinted solution. Finally, for the digestion of 0.5 mL blood samples, 0.8 mL of the sulfuric acid and nitric acid mixture at the ratio of 1:1, and for serum samples, 0.4 mL of the same acid mixture at the ratio of 1:1 was used in this on-line system. Optimization of Preconcentration Conditions. After microwave digestion, the chemical components in the digested biological sample consist mainly of ions such as Na+, K+, Ca2+, Mg2+, Fe3+, PO43-, and Cl- and others from the digesting and buffering agents like NH4+, NO3-, and SO42-. A significant interference effect may generally not be observed for the determination of elements in minor or trace levels in the presence of these chemical components; however, as the analyte concentrations further decrease to levels, the detrimental effect of matrix for their determination by the matrix substances would be no longer negligible. For the ICPMS measurement, the interference comes mainly from spectral overlap and signal intensity change. This might induce the spectral overlap and signal intensity change for the ICPMS measurement. To prevent the occurrence of interference, a preconcentration procedure separating the analytes from interferences and increasing the analyte concentration becomes the method of choice. The commercially available IDA copolymer has long been recognized as an important chelating resin for preconcentration purposes.21,32-34 Although this resin is very effective in separating a number of trace elements from anions, alkalis, and alkaline earth metals, its routine use suffers from the disadvantage of pronounced changes in volume upon changing ionic form and may, consequently, result in impeding the flow of sample.21 However, this problem can be relatively easily overcome with the use of a constant flow HPLC pump in the on-line preconcentration system. The characteristic properties of IDA resin for adsorption of metal ions has been extensively reviewed.21,32-34 Most transition metal ions can be retained on the chelating resin with a pH between 5.0 and 5.8, while alkali and alkaline earth metal ions can only be retained at pH g 8. Aside from pH, the ionic strength of the sample solution is another important factor influencing the preconcentration of metal ions on the resin. Some reports revealed that lowering the ionic strength by diluting the buffer solution would result in a decreasing of the adsorption efficiency of metal ions.21 This indicates the apparent effect of ionic strength on the affinity of ions on the resin. It is, therefore, of interest to investigate the adsorption behavior of metal ions on the resin at a substantially high ionic concentration under the fixed pH of 5.5, as this will later be encountered in our preconcentration system. In order to facilitate this on-line analytical system which can achieve rapid separation and minimum contamination, the sample and reagents added must be restricted to the smallest possible (32) Pai, S.-C. Anal. Chim. Acta 1988, 211, 257-270. (33) Pai, S.-C. Anal. Chim. Acta 1988, 211, 271-280. (34) Raje, N.; Kayasth, S.; Asari, T. P. S.; Gangadharan, S. Anal. Chim. Acta 1994, 290, 371-377.

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Table 4. Isotope Ratio of Analytes before and after Matrix Separationa element

reference

before treated

after treated

Fe (54:57) Ni (60:61) Cu (63:65) Zn (64:67)

2.658 ( 0.025 22.04 ( 0.025 2.235 ( 0.025 11.90 ( 0.025

2.517 ( 0.025 22.93 ( 0.19 0.3949 ( 0.0028 85.47 ( 0.85

2.661 ( 0.013 22.13 ( 0.18 2.241 ( 0.009 11.75 ( 0.06

a

Figure 4. Effect of dilution factor from the original sample matrix on the preconcentration recovery of iron (0), nickel ([), copper (9), zinc (b), and lead (O). Concentration of respective analytes, 100 µg/ L. Sample, 4 mL. Resin weight, 0.8 g. Preconcentration flow rate, 2.0 mL/min. Original matrix concentrations: Na+, 200 mg/L; K+, 200 mg/L; Ca2+, 10 mg/L; Mg2+, 10 mg/L; P, 40 mg/L; Cl-, 400 mg/L, SO42-, 16 000 mg/L; and NH4+, 78 000 mg/L.

volume. This is because a 2-fold increase of sample volume will require a double expenditure of column separation time, and increased amount of reagents will naturally cause an increase of the blank. To meet the above criteria, proper measures must be taken to adjust the digested sample solution to a condition suitable for on-line preconcentration purpose. There are two alternative possibilities: one is to decrease the matrix composition simply by diluting the digested sample solution, and the other is to add buffer solution to adjust the sample solution to a proper pH without much increase in the sample volume. Although large dilution of the digested solution is a favorite choice to reduce the matrix effect, it will inevitably prolong the column separation time, thus making this on-line analytical system impractical. To the contrary, if the sample volume is intentionally limited to a minimum by adding only buffer solution to the digested sample, this may result in the problem of low separation efficiency due to a high ionic concentration of the analytical solution. In an attempt to determine the optimum preconcentration condition suitable for this on-line analytical system, a test sample solution is prepared by simulating the digestion of 0.5 mL of blood sample with concentrated HNO3 (0.4 mL) and H2SO4 (0.4 mL), followed by the addition of an ammonium acetate buffer to adjust to a final volume of 4 mL at pH 5.5. The simulated sample matrix solution so obtained, consisting of a high ionic concentration (200 mg/L Na+, 200 mg/L K+, 10 mg/L Ca2+, 10 mg/L Mg2+, 40 mg/L PO43-, 400 mg/L Cl-, 78 000 mg/L NH4+, and 16 000 mg/L SO42-), was applied to an IDA resin column to test its effect on the recovery of analytes. Figure 4 shows the effect of matrix concentration of the simulated sample (expressed as the dilution factor from the original solution) on the preconcentration recovery of analytes. In this study, 4 mL of test sample solution, with varying dilution from the original sample solution, containing 100 µg/L of each analyte was applied to the preconcentration system and subjected to separation with the established condition as described in Table 2. The result reveals that the analyte recovery of the tested analytes lies at about 60-80% for the original sample (dilution factor of 1), whereas the recovery of most analytes, except Fe, notably increases to nearly quantitative as the dilution factor increases to 10. Iron, being an element which behaves 3936 Analytical Chemistry, Vol. 69, No. 19, October 1, 1997

Results are means of three measurements ( standard deviation.

somewhat differently, can achieve quantitative recovery only after being diluted to about 50 times. According to the above results, it is obvious that, in order to ensure quantitative recovery of the analytes by this on-line preconcentration system, at least a 10-fold dilution of the digested sample solution is needed. This implies that a 10-fold longer separation time is required, making this analytical system obviously impractical. In order to achieve simple and efficient operation of the system, the alternative possibility of adding buffer to the digested sample solution, without further dilution, followed by on-line preconcentration, seems to be the method of choice. This method will inevitably encounter a high concentration of matrix components and, consequently, result in incomplete recovery of the analytes. Under these circumstances, an additional calibration technique is required to match the matrices of samples and standards. For eluting the adsorbed analytes from the column, various acid mixtures of HNO3 and HCl can be employed.21-22,34 In considering possible interferences caused by the polyatomic ions from chloride ions, HNO3 was selected as the eluent in the desorption process. An acid concentration of 0.56 mol/L at a flow rate of 1.5 mL/min was found to be favorable in this on-line separation system. Evaluation of Preconcentration Efficiency by ICPMS. For the determination of trace and/or ultratrace amounts of transition metals and metalloids (As and Se) by ICPMS, the potential interference elements are mainly Cl, P, S, Na, Mg, and Ca in the biological samples. From the literature reports,3,6,9-11,13,21 the spectral interferences of 64Zn by 31P16O21H, 32S16O2, and 32S2, 63Cu by 40Ar23Na, 65Cu by 33S16O2 and 32S33S, 56Fe by 40Ar16O, 58Ni by 58Fe, and 60Ni by 44Ca16O and 43Ca16O1H are the cases most probably encountered. For evaluating whether the presence and extent of the matrix substances can cause interferences for analyte determination, an investigation of the measured isotope ratio in the sample in comparison with the reference one of that element can provide useful information. Basically, if an obvious deviation of the measured isotope ratio from the reference ratio is observed, it may indicate the existence of spectral interferences in this analyte measurement. Table 4 shows the reference isotope ratios for Fe, Ni, Cu, and Zn and the ratios for the measured ones for the same elements in the blood sample prior to the preconcentration process. Since mass spectrometers may exhibit mass discrimination, resulting from a deviation of the experimentally measured isotopic ratio from the true ratio, the mass bias correction factor of ICPMS used in this work has to be determined and applied for data correction.35 Clearly from the table, this exhibited a notable deviation of the measured isotope ratios from (35) Beary, E. S.; Paulsen, P. J.; Fassett, J. D. J. Anal. At. Spectrom. 1994, 9, 1363.

Figure 5. Signal profiles of the respective matrix ions in the preconcentration and elution step. Sample, 4 mL. Resin weight, 1.5 g.

the reference ones, especially for Cu and Zn, was observed, indicating the existence of serious interferences for the determination of these elements in blood sample by direct ICPMS measurement. The need for preconcentration of trace analytes from biological matrix can be, to a certain extent, justified from the above measurement of isotope ratio. In order to examine the effectiveness of the preconcentration method established in this on-line system, the full time scale elution chromatogram from sample loading to desorption of analytes was investigated. A test solution was used which was comprised of the estimated concentration of major matrix elements (Na, K, Ca, Mg, P, Cl, and S) and buffering components (NH4+ and OAc-) in the digested sample solution. Prior to the testing, the flow pattern of the established on-line system was modified to allow the effluents from the preconcentration column, from both the initially introduced sample solution and the desorption solution, to be directed to ICPMS for signal monitoring. Figure 5 shows the chromatograms for Na, Mg, P, S, Cl, K, and Ca so obtained. It can be seen from this figure that, during the sample uptake and washing steps, which takes about 350 s, huge peaks of respective matrix elements appear in the chromatograms, thereby confirmin nonadsorptivity of these ions on the IDA resin at this specific pH (5.5). In the subsequent desorption step with 0.56 mol/L HNO3 as eluent, shown in the time scale from 350 to 800 s, only small peaks of Ca, Mg, Na, and K are observed. The concentration of the respective cations observed in the elution step in comparison with that which was

Table 5. Absolute Blanks, Detection Limits, and Recovery for This On-Line Microwave Digestion and Preconcentration System

b

element

blank,a ng

detection limits,a µg/L

recovery,b %

Fe Ni Cu Zn Pb

162 ( 11 0.71 ( 0.06 12.9 ( 0.58 36.7 ( 2.2 0.81 ( 0.04

68 0.34 3.5 13.4 0.22

75.9 ( 3.0 92.7 ( 8.8 91.3 ( 4.3 77.1 ( 3.5 82.1 ( 4.0

a Results are means of seven measurements ( standard deviation. Results are means of three measurements ( standard deviation.

originally injected is estimated to be as follows: Na, 0.7%; K, 0.5%; Mg, 7%; and Ca, 5%. Though these retained ions can be easily washed out from the column with ammonium acetate as an eluting solution,21 this would inevitably increase operational complexity and the sample throughput rate of the on-line system. To investigate if the presence of these ions (at the estimated levels of milligrams per milliliter) can exert any perceivable effect on the determination of trace analytes, the isotope ratios of the respective analytes in the blood sample were measured again after the on-line preconcentration procedure. The results shown in Table 4 reveal clearly that an agreement between the reference and measured isotope ratios can now be obtained, indicating that the presence of matrix substances to such an extent would not exert any harmful effect on the analyte determination. Based on Analytical Chemistry, Vol. 69, No. 19, October 1, 1997

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Table 6. Results Obtained for the Analysis of Standard Reference Materialsa element sampleb

methodc

blood I

certified method 1 method 2 certified method 1 method 2 certified method 1 method 2 certifiedd method 1

blood II blood III serum

Fe, mg/L

Ni, µg/L 3 2.82 ( 0.25

466 ( 22 475 ( 3

7 7.05 ( 0.32

495 ( 28 501 ( 38 577 ( 35 566 ( 6 2.62 ( 0.10 2.55 ( 0.17

11 10.94 ( 0.55 (0.72) 1.05 ( 0.38

Cu, mg/L

Zn, mg/L

1.25 ( 0.08 1.32 ( 0.01

12.15 ( 0.33 12.23 ( 0.48

0.893 ( 0.035 0.874 ( 0.049

9.85 ( 0.48 10.10 ( 0.04

0.803 ( 0.050 0.812 ( 0.004 0.74 ( 0.04 0.70 ( 0.06

10.43 ( 0.62 10.17 ( 0.37 0.92 ( 0.0.06 0.91 ( 0.04

Pb, µg/L 32-40 36 ( 3 359-409 380 ( 12 630-725 673 ( 27 (0.6) 1.2 ( 0.4

a Results are means of seven measurements ( standard deviation. b Blood I, II, III: Seronorm trace element in whole blood, levels I, II, and III, batch nos. 205052, 205056, and 205053. Serum: NIST SRM 1598 bovine serum. c Method 1: on-line method of this work. Method 2: on-line method of this work with isotope dilution technique. d Values in parentheses are not certified and are given for information only.

the results, the washing step after sample uptake can, therefore, be omitted in this on-line analytical system. Establishment of the Automated On-Line Analytical System. Following the optimization of the individual parts of the system, an effort must now be directed toward combining these parts into an automated on-line analytical system. The critical factor underlying the construction of the hyphenated system lies in the interface design for the optimal combination of microwave digestion with a preconcentration system. As indicated previously, sample digestion with a focused microwave digestor was conducted under the normal pressure, while the preconcentration of analytes with IDA resin column was treated under a pressurized condition by a HPLC pump. In order to facilitate transferring the digested sample solution to the pressurized HPLC separation system, a collection loop (L3 as shown in Figure 1) as the interface was devised. With this interface design, the digested sample solution (after adjustment to a proper pH) was first pumped to the collection loop, and from there the solution was then injected into the flow system for further carrying out a series of matrix separation and preconcentration processes. The hyphenated system of this design can be fully operated by an automated control system. The on-line analytical system, as shown in Figure 1, is comprised of three main parts, namely, microwave digestion, matrix separation/preconcentration, and ICPMS measurement. For a sample to be analyzed with this system, as indicated in Figure 3, it must be first introduced together with an acid mixture to the digestion chamber for microwave digestion. After digestion, the digest is adjusted with a buffer to a proper pH and then transferred to the collection loop for further preconcentration. Meanwhile, the digestion chamber is treated with two washings of acid and water mixture. While in the preconcentration step, the sample is transferred with a HPLC pump to an IDA resin column, and subsequently the analytes retained on the column are eluted out and transferred to ICPMS for multielement determination. Throughout this analytical procedure, a sample is estimated to be treated with a total of 12 steps, from injection into the system until the data output from ICPMS. To operate such a complicated analytical system, an automated system with computer control is necessary. Since there is no commercial automation system available for our purposes, a home-made automation control system was developed. In this system, all the equipment or parts involving 3938 Analytical Chemistry, Vol. 69, No. 19, October 1, 1997

the flow rate of the pump are controlled by the magnitude of dc voltage supplied, those involving start/stop of the pump and rotation of the valve are controlled by the logic of TTL signal, and those involving the microwave device and data acquisition of ICPMS are controlled by an on/off switch. The software, containing more than 31 subroutines, 1700 lines, and 50 000 characters, is based on Microsoft QuickBASIC language and is designed by ourselves and run on a PC to control the entire system. The HP 3488A switch/control unit is used in the system as a connection between the PC and the equipment. The final and the most important part for establishing this automated on-line system is the design of an operation sequence for sample digestion and preconcentration. Because the system involves a complicated operation procedure, an effort should be made to devise a time-sharing program in order to increase sample throughput rate. Based on this consideration, the sequence of operation for this on-line microwave digestion and preconcentration was developed as in Figure 3 and Table 3. As revealed in the operation sequence, when the digested sample goes on to the next step for preconcentration, the digestion chamber is treated by two washings with the acid and water mixture. As the sample further goes on to the elution step, the system is now ready to accept the next sample for digestion. Following the operation sequence, a sample can automatically complete on-line digestion, preconcentration, and determination with this automated system in 10 min. The sample throughput rate can achieve up to 6 samples/ with this system. Analysis of Blood and Serum Samples. The analytical performance of this system was evaluated in terms of detection sensitivity, spike recovery, and analytical reliability. The method detection limits as shown in Table 5, defined as the analyte concentration that gives a signal which is three times the standard deviation of the procedure blank (n ) 7), were estimated to be 68, 0.34, 3.5, 13.4, and 0.22 µg/L for Fe, Ni, Cu, Zn, and Pb, respectively. In this study, the evaluation of blank and limits of detection were all based on the analysis of a 0.5 mL blood sample. In the same table, the absolute blanks (in nanograms) for these analytes are also presented. The limits of detection so obtained are low enough to determine the blood and serum levels for the general population.36 Recovery tests were performed by spiking equivalent amounts of Fe, Ni, Cu, Zn, and Pb as those contained in normal blood to the whole blood sample, followed by the operation sequence of

this automated on-line analytical system. The spike recovery was estimated from the ratio of the integrated signal of spiked sample to that obtained from the aqueous standard directly injected into ICPMS through the FIA system. The results of the spike recovery test for these elements are shown in Table 5. As can be seen, the spike recoveries for Fe, Ni, Cu, Zn, and Pb are 75.9, 92.7, 91.3, 77.1, and 82.1%, respectively, all in reasonably good precision (RSD e 10%). The reason why the recovery cannot be quantitative is attributable to the high ionic concentration of the analytical sample used in the on-line system, as previously discussed. To compensate for the incomplete recovery of analytes obtained by this analytical system, the additional calibration method is used for quantitation. For evaluation of data reliability, four certified reference samples, including three Seronorm Trace Element in Whole Blood (Levels I, II, and III, Batch Nos. 205052, 205056, and 205053) and one NIST SRM 1598 Bovine Serum, were used. Among the three whole blood samples cited above, no certified values of Fe, Cu, and Zn were provided. For assessing the accuracy of the data for which no certified values were provided, an alternative verification process using an isotope dilution method was conducted. Table 6 shows the results for the analysis of the reference materials. As can be seen, the results found in this work are all in good agreement with the certified values. Those data for which no reference values were provided are also found to be in agreement with that obtained by the isotope dilution method. (36) Tsalev, D. L. Atomic Absorption Spectrometry in Occurpational and Environmental Health Practice, Volume II: Determination of Individual Elements; CRC: Boca Raton, FL, 1984; Chapters 11, 16, 17, 22, and 34.

CONCLUSION A fully automated on-line system comprised of microwave digestion, preconcentration/matrix separation, and ICPMS measurement was developed. The system is provided with an expert control system which can automatically execute sample uptake, microwave digestion, pH adjustment, matrix separation, preconcentration, measurement, calibration, and data processing. The analytical reliability achievable by this on-line system was confirmed by the analysis of SRM blood and serum samples, and the limits of detection for Fe, Ni, Cu, Zn, and Pb were found to be 68, 0.34, 3.5, 13.4, and 0.22 µg/L, respectively. With this fully automated on-line system, the determination of analytes in biological fluid samples down to the microgram-per-liter level has been proven to be feasible, and the sample throughput can achieve up to 6 samples/h. ACKNOWLEDGMENT The authors gratefully acknowledge the financial support of the National Science Council (NSC 86-2113-M-007-047) of Taiwan. Parts of this work supported by the Council of Labor Affairs of Taiwan are also greatly appreciated. The authors also thank Dr. Jerzy Mierzwa for his critical comments on this paper.

Received for review March 13, 1997. Accepted July 16, 1997.X AC970284E X

Abstract published in Advance ACS Abstracts, September 1, 1997.

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