capac microwave induced plasma

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Anal. Chem. 1994,66, 31-535

Capacitively Coupled Microwave Plasma Atomic EmissionSpectrometer for the Determination of Lead in Whole BloodMlchael W. Wenslng, Benjamln W. Smlth, and James D. Wlnefordner'Department of Chemistrv, University Of Florida, Gainesville, Florida 326 1

The determination of lead in whole blood by atomic em issionspectrometry using a capacitively coupled microwave plasmaand a tungsten filament electrode is presented. When theplasma-supportingelectrode is a lso used a s the sample holder,transfer of the sample to the plasma is 100%.Microwaves areused to dry the sample and, at higher powers, ignite a heliumplasma which results in the atom ization and excitation of Pb.Using this methodology, a de tection limit of 3 pg of Pb wasobtained using ~ - M Lqueous samples. The precision was 9%.Whole blood samples were subjected to a drying stage similarto that of the aqueous samples. Following this drying stage,a low-power (3 0 W) helium plasma was ignited and used toash the blood sample. Higher power plasmas (>150 W) wereused to atomize and excit e the Pb. Recovery of Pb from theblood samples was 88%,when compared to aqueous standards.

It has become apparent that lead is toxic to the humanbody at lower concentrations than previously thought. As aresult, in 1991 he Centers for Disease Con trol (CD C) loweredthe level of concern for blood lead to 10 pg/d L.' Th e mostpopular method previously used to screen blood lead, anindirect measurement involving the fluorescence of zincprotoporphyrin, cannot determine lead below 20 pg/dL.2Therefore, it is important to develop new methods ormethodologies which are sensitive enough to determine leadat these levels, yet affordable enough t o be used as a screeningdevice.

There are several methods which are sufficiently sensi-tive to determin e lead at these levels in blood: anodic strip-ping voltammetry (ASV), po tentiometric stripping analysis(PSA ), and graphite furnace atomic absorption spectrometry(GF AAS ). However, all of these methods require dilution ofthe blood sample, and the add ition of chemical reagents, whichincreases the likelihood of samp le contam ination. ASV andPSA require the ad dition of acids to lyse the red blood cellswhere lead is boun d, a buffer solution, and a surfa ctant whichhelps stabil ize the s~ lu ti o n .~ccura te and precise results forthe rapid determination of lead in whole blood by GFAASrequire dilution of the blood sample with Triton X-100 (asurfactant) and the use of a matrix modifier containingphosphate such as ammonium phosphate, diammoniumhydrogen phosphate, or phosphoric acida4 The Triton X-100

lyses the red blood cells, while the ph osphate-containing saltsallow higher ashing temperatures, ensuring the completeremoval of carbonaceous material. Using simple dilutionprocedures, i.e.,dilutionof the blood sam plewithTriton X-100and water, lower ashing temperatures are used, resulting incarbonaceous deposits which build up inside the furnace. Thesedeposits retain lead and are not completely removed duringthe atomization step or the cleaning step. This results in acontin ual decrease in instrum ental sensitivity for subsequentana lyse^.^ Using GFAA S, th e end result is at least a 5-folddilution of the sample.6 In our laborato ry, we ar e developinga technique which requires no sample pretreatm ent for theanalysis of lead in whole blood. This paper describes theoptimization of a capacitively coupled microwave plasma(CM P) for the direct determination of lead in blood.Since the CMP is less well known to analytical atomicspectroscopists than t he m icrowave induced plasma (MIP ),it will be briefly compared to the M IP . These plasmas differby the way the energy is transferred to the plasma. In anMI P, microwaves are transferred from th e microwave gen-erator usually via a coaxial cable to an external resonatorcavity. The cavity supports a standing microwave and sustainsa gas discharge by interaction of the discharge gas with themicrowave magnetic field. Th e discharg e gas is contained bya quartz tube.7 MIPShave been operated a t a variety of powers(30-1600 W) and, in general, can handle greater sampleloading at higher powers. MIP S operated below 120 W havebeen used primarily in the analysis of G C eluates, wh ere plasmaloading is minimal. At these low powers, pressure gradientsupercritical fluid chromatography (SF C) was not successfulas the analyte signal was depressed as the pressure of the SF Cmobile phase was increased.8 For these reasons,500-WMIPShave been developed and, due to their higher power, havebeen used sucessfully n handling SF C eluent. Limited successhas been achieved in solid sampling into an MI P, where 1-mgquantities of finely ground coal hav e been directly introducedinto a 500-WM IP and analyzed for carbon. The precisionwas limited to 20%.9CM Ps require an electrode to sustain the plasma. Mi-crowaves are transferred via a waveguide to the electrode,which is supported in a widened, central channel of an ICPtorch. The electrode couples to the microw ave field, and whenthe microwave power is strong enough, a plasma ignites a t the

(1) US. enters for DiseaseControl (CD C) PreuentingLeadPosioningin YoungChildren. A Statement by the Center for Disease Control in October, 1991.U S . Department of Health and Human Services/hblic Health Service/tip of the electrode. Powers ranging from 100 to 2000 W ar eused' CMP plasmas are very robust and have been

Centers for Disease Control: Atlanta, G A, 1991.

Chem. 1988, 34 , 563 .397.

(2) Noble, D. Anal. Chem. 1993,65, 267A.(3) Roda, S.M.; Greenland, R. D.; Bornschein, R. L.; Hammond, P. B. Clin.(4 ) Benzo, Z.; Fraik, R Carrion, N.; Loreto, D. J . Anal. At . Spectrom. 1989,4,

( 5 ) Subramanian, K. S . Prog. Anal. Spectrosc. 1986, 9, 237.(6) Jacobson, B. E.; Lockitch, G.; Quigley, G. Clin.Chem. 1991, 37, 515 .(7) Zander, A. T.; Hieftje, G. M. Appl. Spectrosc. 1980, 35, 357.(8) Wu, M.; Carnahan, J. W. J . A n d . At. Spectrom. 1992, 7 , 1249.(9 ) Gehlhausen, J. M .; C a r n a h a n , J. W . A na l . C he m . 1991, 63, 2430.0 0 0 3 - 2 7 0 0 /9 4 /0 3 6 6 - 0 5 3 1 $ 0 4 .5 0 /00 1994 Amerlcan Chemical Society Analytical Chemistry, Vol. 66, o. 4, February 15, 1994 531


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successfully with aqueous aerosols, and such matrices a s toma toleaf residue and coal fly ash residue.IO In addition, CMPatomic emission spectrometry (CMP-AES) is very sensitive.Ali and Winefordner" obtained CMP -AE S detection limitsin the 1-100-pgrangefor Ag, Ba, Cd, Cu, Ga, Ge, In, Li, Mg,an d Zn by vaporizing samples directly off a small- (1-3 mm )diameter tungsten loop electrode.

Discrete sample introduction is simplified in a C M P versusan M IP or IC P because microliter sa mple volumes can bedeposited directly on he plasma-supporting electrode, ensuringcomplete transfer of the sample to the plasma with minimaldilution. At low microwave powers, the samp le is dried bydirect absorption of microwaves and also indirectly throughresistive heating of the electrode. At higher m icrowave powers,a plasma ignites on he electrode, around thesam ple, atomizingand exciting the sample. Th e emission signal is detected asa transient signal. Ali and Winefordne rl0Jl used this approachwith the tungsten filament loop and with a graphite cupelectrode. This approach is advantage ous over other sampleintroduction methods, including electrothermal v aporization,where the sample is diluted by the ca rrie r gas and some of thesample is retained on the tubing walls leading from thevaporization device to the plasma.12 Direct insertion of thesample into an A r-IC P plasma was first performed by Salinand Horlick,13 who inserted previously dried samples carriedon a grap hite electrode directly into the IC P; they introducedthe electrode axially through the injector tub e of an I C P orch,which also ignited the IC P upon insertion. Oth ers haveintroduced th e graph ite electrode directly into a continuouslyrunning ICP.l"-l7 Abdullah, Fuwa, and Haraguchi18 raisedthe samp le-containing graph ite electrode in stages, first usingindirect heating from the IC P to ash the sample, before it wasintroduced into the continuously running IC P. For the CM P,we report the use of a plasma ashing step, where after firstcha rring the blood with microwaves, a very low power (30 W )microwave plasma is ignited on the electrode and is allowedto ash the sample.

A disadvantange of the C M P electrode is that it erodeswith time and appears in the emission spectra, and so it isnecessary to choose an electrode material which will notproduce interfering spectral emission lines. Th e electrode mustbe conductive and have a higher m elting point than th e plasmatem per atur e. Several materials have been successfully usedas electrodes in our laboratory, including tungsten, ta ntal um ,and graphite.19.20

This paper characterizes the C M P for the atomic emissionspectrometric determination of Pb in blood using a tungstenfilament electrode and the discrete sampling techniquementioned above. Th e optimization procedu re is describedfor aqueous samples, and the technique is applied to blood

~~~ ~ ~~

(IO) Ali, A. H.; Ng , K. C.; Winefordner,J. D. J . Anal. At. Spectrom. 1991,6,21 .(11) Ali, A. H.; incfordner, J. D. Anal . Chim. Acta 1992, 264, 327.(12) Alvarado, J.;Cavalli, P.; Omenctto, N.; Rossi, G. nal. Lett . 1989, 22 , 2975.(13) Salin, E. D.; Horlick, G . Anal. Chem. 1979. 51, 2284.(14) Sommer, D.; Ohls, K. Fresenius 2. nal. Chem. 1980, 304, 97.( I S ) Kirkbright,G. F.; Walton, S. A ~ I y s t 982, 107, 276.(16) Kirkbright,G. F.; Zhang, L.-X. Analysf 1982, 107, 617.(17 ) Zhang, L .-X.; Kirkbright,G. .; Cope, M . J.; Walton, J. M . Appl. Specfrosc.(18) Abdullah, M.; Fuwa, K.; Haraguchi, H. Specfrochim. Acta 1984,398,1129.(19) Masamba, W. R. L.;Sm ith,B. W.; Winefordner, J . D. Appl. Spectrosc. 1992,(20) atel, B. M.; Heithmar, E. ; Winefordner,J. D. Anal. Chem. 1987,59,2374

1983, 37, 250.

46 , 1741.

voltageglass chlmney

Flgurs 1. Capacltively coupled microwave plasma setup.Table 1. CMP Equlpmenldc high-voltage power supply; 803-330 Hipotron ics,magnetron OM75A Samsungaluminum waveguide lab construc tedtwo concentric tube lab constructedquartz torches0.5 m spectrometer; 1870 Spex, Edison, NJ120 0 grooves/",300-nm blaze wavelengthintensified photodiode TN-6122A Tracor Northern,

array Middleton,WI

item model manufacturerBrewster, NYax output 1.2kW,voltage regulation 15%

analysis. Helium is used as the plasma gas due to its excellentexcitation efficiency and low background characteristics.EXPER I MENTAL SECTIONApparatus. The experimental apparatus is shown in Figure1; the instrumental components ar e described in Ta ble 1. Thesetu p is typical of CM P- AE S systems, with the exception thata high-voltage switch is used to gat e power to the mag netronand water cooling of the electrode is unnecessary. Theelectrode is constructed from 0.25-mm-diameter high-purity(99.98%) tungsten wire (Aesar, Ward Hill, MA). Theelectrode loop is formed by tying a knot 3 mm in diameter inthe wire (sam ples deposited on he loop are held by adhesion)and then bending the ends of the wire so that they areperpendicular to the loop. Th e ends extend 63 mm downwardfrom the loop. This length was found to be optimum forefficient coupling to the waveguide. Th e electrode is held inthe 1-mm-diameter central channel of a modified ICP torchby compression of the wire against the central channel w alls.The bottom of the central channel is sealed.

Procedure. Aqueous samples of 5-pL volume are depositedon the electrode with a pipet and dried a t 75 W for 90 s. Afterthe drying step, the high-voltage switch is opened, and thevoltage is increased. Th e helium plasma gas is set to 10L/min,and when the switch is closed, the plasma autoignites. Closingthe switch also triggers the detector, which integrates signalsover 0.1-s eriods for a tota l of 4 . A total of 40 time-resolvedspectra are obtained. The results are stored and analyzedwith a computer. For all determinations, the monochromatorwas operated with a slit width of 120 pm .For blood samples, 2-1L aliquots are deposited on th eelectrode. Th e sample is dried first at 75 W for 30 s. Theplasma g as is then ignited a t low power (30 W ) and allowedto ash the sample for 3 min. The switch is opened, and the

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1.0

250 1

-04.0 404.5 405.0 405.5 406.0 406.5 407.0 407.5 408.0Wovelength (nm)Flguro 2. Aqueous emission spectrum near 405 .8 nm.voltage is increased. Closing the switch results in thevaporization, atomization, and excitation of lead. Detectionis accomplished in the same way as in the case of aqueoussamples.RESULTS AND D ISCU SSIONAqueous Background. The electrode and aqueous back-ground around the 405.8-nm Pb line is shown in Figure 2.On e can identify several tungsten lines, all adequately resolvedfrom the Pb line. In addition, one can identify a very weakmolecular peak near the P b emission line. This peak is mostlikely due to t he second positive system of nitrogen a nd c anbe corrected for by careful subtrac tion of the blank. Th enitrogen originates from air that is entrained into theatmospheric pressure plasma.

Radial, Axial, and Power Optimizations. It was importantto determin e spatial variation of the Pb signal/backgroundratio since the whole plasma was not imaged into thespectrometer. Two microliters of 1 ppm Pb was used. ThePb signal-to-background ratio was measured by integratingthe Pb signal over time . For high concentrations of Pb (>100ppb), the full width at half-maximum (fwhm) of the transientPb emission signal was determined to be 0.4 s. Therefore, alllead signals were integrated over this time period. Th ebackground was also added over this same period of time. Th ePb signal-to-background ratio is optimum near th e electrodeand decays rapidly in both the axial and rad ial directions. Theresults are shown in Figure 3A,B.For the power optimization, 2 pL of 10 ppm P b was used.Th e integrated Pb signal was measured over the fwhm of thetrans ient Pb signal, which was different a t different powers.The Pb signal profiles as a function of time are plotted inFigure 4 and show tha t the signal profile becomes shar per andmore intense as the power is increased. However, above 172W, there is no gain in the integrated signal-to-backgroundratio. At 172 W, the electrodecould sustain 100 firings, whileat powers greater than this, the electrode lasted for only 5firings. Th e filament loop slowly thinned a t the edges of theloop until it broke. Therefore, a power of 17 2 W was usedin furth er studies. Th e detector was triggered immediatelyafter the high-voltage switch was closed, however, it took afew tenths of a second for the plasma to ignite. This producesthe app arent delay preceding the ap pearance of the P b signal.Aqueous Calibration Curve. A calibration curve wasobtained for Pb trsing 5-pL aliquots and the optimum

0PB

0 1O 10 .5 I I I I I I I I I-0.5 0.0 0 . 5 1.0 1 .5 2 .0 2 .5 3.0 3.5Radial Distance from Electrode (mm)

0

--0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5Distance above Electrode (mm)

Flguro 5. Radial (A, top) and axial (B, bottom) emission profiles of Pb.

5000ea 4000eee 30005E

e

2000Ps- 1000C0

-10000.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 7.8 2.0Tlme ( 8 )Figure4. Temporal emission pro filesof Pb as a function of microwavepower.

atomization power of 17 2 W and viewing the center of theplasma less than 1mm above the electrode. The P b solutionswere in the form of the nitrate salt an d were made by serialdilution. Th e calibration curve was linear over three decadesAnalytical Chemistry, Vol. 66,No. 4, February 15, 1994 533


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0 1 2Time (s)

Figure 5. Temporal emission profile of 5 ng of Pb.

-50

from thedetectionlimit (0.7 ppb) to 1 ppm. Thema ssdetectionlimit corresponded to 3 pg of Pb. Th e limit of detection wasbased on three st and ard deviations of the background obtainedby averaging 10 diodes on either side of the Pb peak. Th eCM P-A ES detection limit in aqueous samples is better tha nthat necessary for the CD C (10 ppb) and rivals that of GF AA S(5.5 pg, 0.3 ppb) while being better than for ICP-AES (14ppb).21 Figure 5 shows the background-corrected, time-resolved emission profile of 5 ng of Pb. The Pb emissionsignal decays to the baseline quickly as the majority of thesignal is captur ed in 1 s. It actually takes 3 s for the P b signalto return completely to the baseline. A plot of the log of theintegrated Pb signal versus the log of the lead concentrationhad a slope of 0.99. Th e precision, as estimated by th e relativestandard deviation, was 15%. This precision was limited bythe reproducibility of the power supply settings, which variedby 34%. A magn etron is a nonlinear device and has the sam evoltage-current charac teristic as a diode. Therefore, in orderto keep the power constant it is essential to control the cur ren t,not the voltage. However, our power supply was incapableof this. Whe n the data set was limited so that the powervaried only by 5%, the precision improved to 9%.

Measurement of Pb in Blood. Volumes of 2 1 L were usedto minimize the amount of sample that needed to be ashed.The CMP handles plasma loading better than the MIP;however, ashing is still necessary to m inimize the a mo unt ofloading. In addition, a neighboring potassium peak completelyobscured the lead peak unless some of the potassium wasremoved. It was determined tha t the best way to ash thesample was to use the plasma ashing step described above.

In order to assess the effectiveness of the ashing step,aqueous samples containing either potassium chloride, leadchloride, or lead phosphate were dried for 90s, ashed a t variousplasma currents for a period of 3 min, and then atomized at172 W. In order to prepare the lead phosphate, 5 p L f 0.2%phosporic acid was added to 5 p L of lead chloride.(21) V a r i a n Guide to I CP/AAS analyt ica l va lues (PN 8 5 101009 00).

A-I 1

+No Ashing 15 mA 20 mA 25 mAAshing Current (mA)

Figure 8. Relative amounts of KCI, PbCI2,and Pba(P01)2 remaining asa function of ashing current.

1' 1 ' I ' I ' I ' I ' I2000 A "1"1500 t I I Pb i

403 404 405 406 407 408 409200 , I

Pb

1005O i n

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Therefore, blood was ashed at an output power of 30 W .This ashing step resulted in th e efficient removal of potassium.The spectrum of 330 ppb (mass/volum e) P b in blood is shownin Figure 7A,B. Figure 7A identifies the 405.8-nm regionaroun d Pb. Figure 7B shows an expanded view of the Pbpeak. The percent recovery of Pb was 88% and was calculatedby comp aring the Pb in whole blood signal to aqueous standa rdswith the same concentratio n of Pb. The precision in bloodwas 7% at 330 ppb. A detection limit of 6 ppb (1 2 pg) wasobtained for Pb in whole blood. This is comp arable to thatachieved by GFAAS (9 pg).22CONCLUSION

Th e use of the tung sten filam ent electrode in combinationwith the CMP has been shown to be a sensitive way ofdeterm ining lead in both blood and aqueous samples. Pico-gram quantities of lead can be detected. Th e plasma ashingstep is a unique and useful way to ash the sample; however,further studies are necessary and a re underway to en sure thatthis step is sufficiently reproducible in a variety of blood(22) Parsons, P. J; Slavin, W. Specfrochim. Acta 1993, 4 8 4 925.

samples. If this is a problem, matrix modifiers such asammo nium pho sphatec an be used to make the lead lessvolatileand the potassium more volatile (Le., Mg(N O&), and theashing time can also be decreased. In addition, other studiesare underway using a current-regulated power supply toimprove the precision of the method. To further reduce thecost of the device, shorter focal length mon ochromators a nd /or interference filter-photomultiplier tube combinations willbe evaluated. Finally, there is no part of this device tha tcannot be automated to a simple push-button device, whichis necessary for routi ne screening of lead in blood by nonskilledoperators.ACKNOWLEDGMENTThis work was supported by a grant from the NationalCenter for Environm ental Health, Cen ters for Disease Controland Prevention, Atlanta, GA (Contract CCR408614-02).Received for revlew August 10, 1993. Accepted November 29,1993."

* Abstract published in Aduonce AC S Absfracfs, January 1 , 1994 .

Analytical Chemistry, Vol. 66, No. 4, February 15, 1994 535

capac microwave induced plasma

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