Anal. Chem. 1987, 5 9 , 1112-1119
1112
similar to that of Au than for those elements like Cu.
ACKNOWLEDGMENT The authors acknowledge the useful comments provided by T. Rettberg and G. Christopher. Registry No. Cu, 7440-50-8;Au, 7440-57-5;graphite, 7782-42-5. LITERATURE CITED (1) L'vov. B. V. Atomic Absorption Spectrochemical Analysis; Elsevier: New York, 1970; p 116. (2) Sturgeon, R. E.; Chakrabarti, C. L.; Langford, C. H. Anal. Chem. 1976. 4 8 . 1792-1807. (3) Srnets. B,'; Spectrochim. Acta, Part B 1980, 356, 33-42. (4) Zhou, N. G.; Frech, W.; DeGalan, L. Spectrochim. Acta, Part B 1984, 3 9 8 , 225-235. (5) Akrnan, S.; Genc, 0.; Osdural. A. R.; Balkis, T. Spectrochim. Acta, Part B 1980, 3 5 8 , 373-378. (6) Frech, W.; Zhou, N. G.; Lundberg, E. Spectrochim. Acta, Part 6 1982, 376, 691-702. (7) Guerrieri, A.; Lampugnani, L.; Tessari, G. Spectrochim. Acfa, Part B 1984, 3 9 8 , 193-203. (8)Torsi, G.; Tessari, G. Anal. Chem. 1975, 4 7 , 839-842. (9) Torsi, G.; Tessari, G. Anal. Chem. 1976, 4 8 , 1318-1324. (10) Paveri-Fontana. S. L.; Tessari, G.; Torsi, G. Anal. Chem. 1974, 4 6 , 1032-1038. (11) Holcornbe, J. A.; Rayson, G. D. Prog. Anal. A t . Spectrosc. 1963, 6. 225-251. (12) Musil, J.; Rubeska, I. Analyst (London) 1982, 707, 588-590. (13) Black, S. S.; Riddle, M. R.; Holcornbe, J. A. Appl. Spectrosc. 1986, 40, 925-933. (14) L'vov, B. V.; Bayunov, P. A. Zh. Anal. Khim. 1985. 4 0 , 614-624. (15) Langmuir, I.Phys. Rev. 1918, 72, 368-370. (16) Langmuir, I. Phys. Rev. 1912, 34, 401-421. (17) Styris, D. L. Fresenius' Z.Anal. Chem. l 9 8 & 323, 710-715. (18) Holcombe, J. A,; Rayson, G. D.; Akerlind, N., Jr. Spectrochim. Acta, Part B 1982, 378, 319-330. (19) McCarroil, 6.J. Chem. Phys. 1987, 4 6 , 863-869. (20) McCarroll, B. J. Appl. Phys. 1969, 4 0 , 1-9. (21) Redhead, P. A . Vacuum 1982, 72.203-211.
(22) Arthur, J. R.; Cho. A. Y. Surf. Sci. 1973, 3 6 , 641-660. (23) Menzel, D. Interactions on Metal Surfaces ; Springer-Verlag: New York, 1975, Chapter 4. (24) Hennig, G. R. Chemistry and Physics of Carbon; Marcel Dekker: New York, 1966; Vol. 2, Chapter 1. (25) Baird, T.; Fryer, J. R.; Arbuthnott, A. R.; McAneney, B.; Riddell, E. V.; Walker, D. Carbon 1974, 72, 381-390. (26) Hidy, G. M.; Brock, J. R . The Dynamics of Aerocolloidal Systems; Pergamon: New York, 1970; Voi. 1, pp 14-151. (27) Seaver, A. E. Aerosol Sci. Techno/. 1984, 3 , 177-185. (28) Dean, J. A.; Rains, T. C. Flame Emission and Atomic Absorption Spectrometry; Marcel Dekker: New York, 1971: Vol. 2, p 334. (29) L'vov. B. V. Atomic Absomtion SDectrochemical Analysis ; Elsevier: New York, 1970; p 204. (30) Niemczyk, T. M.; Yin, I.H. Appl. Spectrosc. 1985, 3 9 , 882-883. (31) Rayson. G. D. PhD. Dissertation, University of Texas at Austin, Austin, TX 1983 ... (32) Hoicombe, J. A.; Rayson, G. D.; Bass, D. A. "Mass Spectral Monitoring of Graphite Furnace Atomization Processes"; Colloquim Spectroscopiurn Internationale XXII, Amsterdam, 1983. (33) Shabushnig, J. G.; Hieftje, G. M. Anal. Chim. Acta 1983, 748, 181-192. (34) Smith, D. D.; Browner, R. F. Anal. Chem. 1984, 5 6 , 2702-2708. (35) Kahn, H. L.; Conley, M. K.; Sotera, J. J. Am. Lab. (fairfield, Conn.) 1980, 72(8), 72-79. (36) Holcornbe, J. A,; Koirtyohann, S. R. Spectrochim. Acta, Part B 1984, 398 243-248. (37) Huitgren, R.; Desai, P. D.; Hawkins, D. T.; Gleiser, M.; Kelley, K. K.; Wagman, D. D. Selected Values of the Thermodynamic Properties of the Elements; American Society for Metals: Metals Park, OH, 1973; pp 47-53. (38) Barin, I.; Knacke, 0.; Kubaschewski, 0 ThermochemicalProperties of Inorganic Substances, Supplement: Springer-Verlag: New York, 1970; p 53-54 I
RECEIVED for review September 23,1986. Accepted December 24, 1986. This work was supported, in part, by National Science Foundation Grant CHE-8409819and Robert A. Welch Foundation Grant F-787.
Instrumentation for Zeeman Electrothermal Atomizer Laser Excited Atomic Fluorescence Spectrometry Joseph P. Dougherty, Francis R. Preli, Jr., John T. McCaffrey, Michael D. Seltzer, and Robert G . Michel* Department of Chemistry, University of Connecticut, Storrs, Connecticut 06268
A dye laser pumped by an exclmer laser was used wlth a graphlte tube electrothermal atomizer and an ac electromagnet to demonstrate Zeeman background correctlon for laser excited atomic fluorescence spectrometry (ZETA LEAFS). Laser radlatlon was propagated parallel to the magnetic fleld through the pole pieces of the electromagnet In order to observe the longltudlnal Zeeman effect. A description of the Instrumentationfor ZETA LEAFS is presented together with prelimlnary information concerning the determlnation of cobalt in aqueous solutions. LEAFS detection limits for cobalt, with and without Zeeman background correctlon, were 0.7 pg (with a relatlve standard devlatlon at 100 pg of 30 % ) and 0.3 pg (with a relatlve standard devlatlon at 100 pg of 13%), respectlvely. The linear dynamlc range for cobalt extended for 5 orders of magnitude above the detection limit whether or not Zeeman background correction was applied.
Various atom cells have been used for laser excited atomic fluorescence spectrometry (LEAFS) including flames (I-I6), electrothermal atomizers (ETAs) (17-B), and plasmas (24-33). Weeks et al. ( 4 ) reported detection limits in an air/acetylene 0003-2700/87/0359-1112$01.50/0
flame that were comparable to the best detection limits reported for flame atomic absorption spectrometry (AAS) (34). The flame LEAFS detection limits reported by Weeks et al. have since been improved by as much as 2 orders of magnitude for lead (8), iron ( I ) , and calcium ( 3 ) . For these elements, detection limits for flame LEAFS have been shown to be comparable to those for AAS with electrothermal atomization. Further improvement in detection limits for LEAFS has been achieved with electrothermal atomization. Bolshov et al. (24) have taken advantage of the higher atom density provided by ETA in comparison to flame techniques and achieved subpicogram detection limits for the determination of seven elements. Tilch et al. (26)have reported similar success with ETA LEAFS for the determination of six elements. The detection limits for ETA LEAFS are up to 3 orders of magnitude better than those for ETA AAS (35). Although very low limits of detection have been achieved for LEAFS in flames and in ETAs, the full analytical utility of LEAFS has yet to be demonstrated. In particular, methods of background correction have not been studied in detail for flames and no method has been used for ETAS that simultaneously corrects for background during a single furnace atomization. The primary sources of background in LEAFS are scattered laser radiation and ETA blackbody emission. C' 1987 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 59, NO. 8, APRIL 15, 1987
Atomic and molecular fluorescence from concomitant species may also be important. Bolshov et al. (24) estimated the background for ETA LEAFS by averaging several furnace atomizations with the dye laser detuned 0.2-0.3 nm away from the analytical wavelength. For flame LEAFS, several methods have been used to correct for background. The advantages and disadvantages associated with these background correction techniques are discussed here: Intermodulated fluorescence (5,6)and harmonic saturation spectroscopy (7) rely on the nonlinear response of fluorescence, under conditions of optical saturation, to discriminate between fluorescence and scatter. Both techniques require saturation of the atomic transition, a condition that may be difficult to obtain for some elements with the tunable laser systems currently available. Optical polarizers have been used to discriminate against scattered laser radiation for LEAFS (22). Discrimination by polarization, however, results in losses of fluorescence (36)and does not discriminate against any scattered radiation that may have been depolarized in the atom cell. Two-channel boxcar detection of LEAFS has been used with pulsed laser systems to correct for continuous background (25). One channel was gated during the laser pulse and the other was gated temporally before, or after, the laser pulse. This scheme does not discriminate against any laser-induced background and is therefore only effective against thermally induced atomic emission and ETA blackbody emission. Time-resolved fluorescence has been used for background correction by measuring the fluorescence signal a t some time after laser excitation ( 2 ) . This technique requires the use of a laser system with picosecond pulses to temporally resolve short-lived ( 4 0 ns) fluorescence signals from scatter. Variation of the excitation spectral profile has been investigated for LEAFS (37). Laser radiation was periodically passed through or around a vapor cell (containing a high concentration of the analyte of interest) before it reached the atom cell. The laser excitation profile, resulting from passage through the vapor cell, was used to measure background because the center of the laser line was totally absorbed. This system requires two atom cells, which makes it difficult to implement, and is only suitable for laser systems with spectral bandwidths greater than the atom line width. Such lasers increase the amount of scattered light, that needs to be corrected for, compared to narrow bandwidth lasers. Wavelength modulation has been used to correct for background in flame LEAFS using a continuous-wave laser (10). An etalon and an electroscan tuning device were used inside the dye laser cavity to shift the wavelength. Such devices are expensive (especially if several etalons are required to cope with all elements) and can result in losses in laser power. These losses in power would degrade detection limits for LEAFS for transitions that are not saturated or may prevent achievement of saturation in some cases. Wavelength modulation measures background slightly away from the analyte line and may be ineffective if the spectral background is structured within the wavelength modulation interval. An alternative approach to background correction for LEAFS is Zeeman background correction. Zeeman background correction for AAS (ZAAS) has been studied extensively, and detailed reviews of the subject are available in the literature (38-40). ZAAS has been shown to be a very accurate method because the background is measured ut the wavelength of interest. Zeeman background correction has been applied to atomic fluorescence spectrometry (AFS) with conventional light sources (41). It offers easy optical alignment and a relatively high modulation frequency. For the studies reported here, Zeeman background correction was applied to LEAFS using an ac magnetic field around an electrothermal
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atomizer (ETA). Since we are applying Zeeman background correction to an ETA for LEAFS, we call the technique ZETA LEAFS. The longitudinal Zeeman effect was used, whereby laser radiation was propagated parallel to the magnetic field. Flame LEAFS has been successfully applied to the analysis of analytes in matrices by detecting nonresonance fluorescence transitions ( I , 23,15). In those investigations, no extensive background correction technique was used to discriminate against laser-induced background. Bolshov et al. (24, 27) determined elements in complicated matrices by ETA LEAFS. They reported matrix interferences that suppressed LEAFS signals, particularly in the determination of cobalt in agricultural samples. They attributed the suppression of LEAFS to the occurrence of gas-phase reactions and quenching in the region above the cup atomizer where LEAFS was measured. They were only able to reduce these interferences by atomizing under vacuum conditions (27). Vapor-phase interferences have been rigorously investigated in ETA AAS. Rapidly heated graphite tube atomizers, equipped with L’vov platforms, have reduced many vapor-phase interferences in ETA AAS by enabling the analyte to be vaporized at the optimized temperature under approximately isothermal conditions (42). Tube furnaces, similar to those used in AAS, have been shown to be feasible atom cells for LEAFS (19, 28). Dittrich and Stark (28) compared LEAFS detection limits for lead in a graphite tube furnace with a carbon rod atomizer. They reported better detection limits with the tube furnace than with the carbon rod. The aim of the work reported here was t o incorporate Zeeman background correction and facets of AAS tube furnace technology into instrumentation for LEAFS. The instrumentation for ZETA LEAFS is described here along with some preliminary data for the determination of cobalt. Our on-going work is aimed a t evaluating the potential of ZETA LEAFS for ultratrace elemental analysis in complicated matrices. Zeeman Effect for Background Correction in ETA LEAFS. Yasuda et al. (39) have reviewed the various instrumental configurations possible for Zeeman background correction in MS. The configurations include the use of either an ac or dc magnetic field, which can be placed around the excitation source (direct Zeeman effect), or the atom cell (inverse Zeeman effect). The field may be oriented perpendicular (transverse) or parallel (longitudinal) to the excitation beam. Many of these concepts are directly applicable to AFS. Three instrumental configurations that use the inverse Zeeman effect are possible for AFS. For graphite tube ETA LEAFS, the longitudinal Zeeman effect, using an ac magnetic field, is the most promising configuration. The longitudinal Zeeman effect is depicted in Figure 1. An ac electromagnet is used to alternately “switch” the magnetic field on and off. When the field is off, atomic absorption of the laser radiation is possible. The “field off‘ signal is comprised of the analyte atomic fluorescence + background. When the field is on, absorption of the laser radiation by the analyte atoms is no longer possible because the u components are displaced away from the excitation wavelength, and the T transition is forbidden for longitudinal excitation. The “field on” signal is therefore comprised of background only. The background corrected fluorescence signal results from a subtraction of the two signals (“field off - field on”). In the ZETA LEAFS instrument, the collimated laser beam is passed through the pole pieces of the magnet to provide excitation parallel to the magnetic field. The fluorescence signal can be collected at a right angle with respect to the excitation beam without obstruction from the magnet core. A polarizer is not required (because the K transition is forbidden for this configuration), and therefore all of the laser
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Table I. Instrumentation description/model no.
manufacturer
excimer laser/SOOXR dye laser/DL-lSP frequency doubler/5-12 boxcar averager/165/162 wide-band preamplifier/VVlOOBTB monochromator (f/3.5, 0.1 m focal length, 8 nm/mm linear dispersion)/H-10 photomultiplier tube/9893QB/350 PMT gating board/GB1001B furnace power supply/HGA-2000 PDP 11/03 minicomputer/Hll-A electromagnet (44) magnet power supply (45) triggering circuitry furnace electrode assembly commter interface
Tachisto, Needham, MA Molectron, Santa Clara, CA Inrad, Northvale, NJ Princeton Applied Research, Princeton, NJ LeCroy, Spring Valley, NY Instruments SA, Metuchen, NJ Thorn-EMI, Fairfield, NJ Thorn-EMI, Fairfield, NJ Perkin-Elmer, Norwalk, CT Heath Corp., Benton Harbor, MI laboratory constructed laboratory constructed laboratory constructed laboratory constructed laboratorv constructed
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Figure 1. Longitudinal Zeeman effect. Atomic fluorescence follows laser excitation in the absence of a magnetic field. Application of a magnetic field splits the atomic transition into two CT components and atomic fluorescence can no longer occur. energy can be used for excitation.
Detection Limits for ZETA LEAFS. The detection limits for ZETA LEAFS, in comparison to ETA LEAFS, are primarily dependent upon two factors. First, the noises in each of the two boxcar channels are added during the twochannel subtraction process. The second consideration is the ability of the magnetic field to split the a components away from the laser emission profile during the measurement of the background. For the parallel ac field configuration, the degradation of detection limit that results from incomplete splitting can be reduced by a n increase in magnetic field strength. An alternative approach would be t o narrow the laser line width using an intracavity etalon t o reduce t h e a overlap without requiring an increase in the magnetic field strength.
EXPERIMENTAL S E C T I O N Procedures. Standard solutions of cobalt were made by serial dilution of a 1000 pg/mL stock solution. The detection limits for ETA LEAFS and ZETA LEAFS were based on three standard deviations of 16 measurements of the blank and were obtained by use of a photomultiplier tube (PMT) voltage of 1.9 kV and a slit width of 1.0 mm. The linear dynamic ranges for ETA LEAFS and ZETA LEAFS were obtained by use of two PMT voltages. Two calibrated neutral density filters (1.0% and 10% transmittance, Ealing Corp., South Natick, MA) were used individually or together to attenuate the fluorescence signal by a factor of 10, 100, or 1000 to ensure a linear response from the PMT. Instrumentation. A block diagram of the ZETA LEAFS instrument is shown in Figure 2 and the components and their manufacturers are listed in Table I. Some of the instrumentation used in this work was previously described, by us, for LEAFS work in a flame (16) and in a direct current plasma (43). The atom cell used here, for ZETA LEAFS, is a laboratory-constructed
Figure 2. Block diagram of ZETA LEAFS instrumentation. MAGNET POLE PIECES
2 FURNACE f l ’I / ’/
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Figure 3. Graphite tube furnace positioned between the magnet pole pieces. The laser beam passes through the tube furnace via the magnet pole pieces and fluorescence is collected at a right angle. graphite tube ETA (Figure 3). It is held by two graphite electrodes and positioned between the pole pieces of an ac elecromagnet. Two holes in the side of the tube allow passage of the laser beam through the furnace while fluorescence is collected through the bore of the tube. Laser radiation reaches the furnace tube through 4-mm-diameter holes in the magnet pole pieces. The laser beam is propagated parallel to the direction of the magnetic field and atomic fluorescence is detected a t a right angle to the incident laser beam. The ac electromagnet operates at the line frequency of 60 Hz while the laser system is externally triggered a t 80 Hz. Proper synchronization of the laser triggering results in laser pulses which alternately coincide with magnetic field maxima (“field on”) and minima (“field off‘) (see later discussion and Figure 4). The measurements made a t “field on” and “field off” are processed by two separate boxcar integrator channels. The boxcar channels are alternately triggered so that each channel separately processes a signal corresponding to either “field on” (background) or “field off‘! (fluorescence + background). A subtraction of the two channels by the boxcar results in the background-corrected atomic fluorescence signal. Laser System. Our pulsed laser system consisted of an excimer laser operating with xenon chloride (308 nm) which pumped a tunable dye laser. The dye laser grating was tuned with a stepper
ANALYTICAL CHEMISTRY, VOL. 59, NO. 8, APRIL 15, 1987 60 Hz mag. field ref.
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1115
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Flgure 7. Furnace electrode assembly. The tube ETA is held between two graphtte electrodes under spring tension. The assembly is designed to fit between the pole pieces of the ac electromagnet.
W Figure 5. Zeeman ac electromagnet. The core of the magnet is placed at a 45' angb to ailow a large collection angle for fluorescence.
motor with a resolution of approximately 0.001 nm per step. The dye laser output was focused into an autotracking frequency doubler with a 300 mm focal length lens to enhance the conversion efficiency. The excimer laser was operated with a pulse repetition rate of 80 Hz and was synchronized with the modulation of the Zeeman magnet. An optical data link was used to transmit trigger pulses to the excimer laser. This helped to minimize radio frequency (rf) interference by isolating the excimer laser from the rest of the instrument. Zeeman Electromagnet. The construction of the ac electromagnet was based on the design of de Loos-Vollebregt and de Galan (44). The magnet had an iron core and aluminum foil windings. The pole faces were 12 X 12 mm with a 12-mm air gap between them. A 4-mm-diameter hole was drilled through the center of the pole pieces to allow passage of the laser light. The magnet windings were each made by wrapping 70 m of 0.13 mm X 46 mm metalloxyd anodized aluminum foil (International Foil, Alliance, OH) around a coil former fabricated from a Kevlar 285/epoxy composite (Hexcel F-1616, Dublin, CA). The core was made from transformer E-plates which were machined, assembled into the two coil formers, and bonded together with epoxy resin (Miller Stephenson, Danbury, CT). An aluminum base was constructed to support the magnet and furnace assemblies as shown in Figure 5. The base supported the magnet at a 45' angle to allow efficient light collection perpendicular to, but in the same plane as, the laser beam. The design of the magnet power supply was based upon that of van Uffelen et al. (45). The power supply was modified to
operate at 60 Hz (instead of 50 Hz)by changing the RC timing circuit. An ammeter and a Hall effect probe (Walker Scientific, Inc., Worcester, MA) were used to measure the magnetic field strength as a function of the current drawn from the 240-V single-phase line for the two-magnet coils which were connected either in parallel or in series (Figure 6). The parallel configuration was adopted for all ZETA LEAFS work because it provided the strongest field. A 566-rF bank of capacitors was connected in parallel with the magnet to minimize the amount of current drawn from the line by forming a resonant circuit. The resonant circuit allows the magnet field strength to be increased by increasing the applied voltage without drawing additional current from the mains. In Figure 6, the maximum field strength of 12.5 kG was obtained at 25 A with the capacitor bank, as opposed to 44 A without the capacitors. Design and Fabrication of Graphite Tube Furnaces. Furnace tubes were machined from high-purity, solid graphite rods (Ringsdorff RW-0, Sigri Corp., Somerville,NJ). The tubes were 8 mm in length with a 10-mm 0.d. and a wall thickness of 1.25 mm. Two 4-mm holes were drilled through the walls of the furnace for passage of laser radiation. Furnace electrodes were fabricated from 4.6-mm-diameter graphite rods (Ringsdorff RW-0, Sigri Corp., Somerville, NJ). The ends of the electrodes were contoured to match the outer diameter of the furnace tubes. The furnace tubes and electrodes were pyrolytically coated at 2000 OC for 2 min by adding 5% methane to an argon atmosphere. A furnace electrode assembly (Figure 7 ) was constructed to fit between the pole pieces of the electromagnet and to allow easy access to furnace tubes. A spring plunger located on the top of the assembly provided positive pressure on the upper electrode
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 8, APRIL 15, 1987
I
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u Flgure 8. Simplified block diagram of ZETA LEAFS trigger circuitry. ZETA LEAFS trigger circuitry produces the outputs represented in Figure 4. to ensure electrical contact between the furnace tube and the electrodes. The furnace electrode assembly was mounted on a three-dimensional translational stage to allow alignment of the furnace tube between the magnet pole pieces. A Perkin-Elmer HGA-2000 power supply was used to heat the furnace. An aluminum and Plexiglas enclosure was constructed around the furnace assembly and was purged with argon to minimize oxidation of the furnace tube. A removable aluminum door a t the front of the enclosure allowed access to the furnace. A set of three optical baffles, located in the front door of the furnace enclosure, was designed to reduce furnace emission while allowing a large solid angle for collection of fluorescence. Samples were introduced into the graphite furnace by removing the front door and depositing the 10-pL sample directly on the wall of the tube with a micropipet. Triggering Circuitry. Electronic circuitry was designed to synchronize the magnetic field of the ac electromagnet, the laser pulses, and the alternate channel triggering of the boxcar (Figure 4a,f,g). The laser system was triggered alternately during “field onn and “field off” of the ac magnetic field. The ac field was modulated by the 60-Hz line frequency. (Le., two field maxima and two field minima occurred during each cycle of the 60-Hz mains.) The maximum possible repetition rate of the excimer laser used here was 100 Hz. Therefore, the highest laser repetition rate that could be synchronized for alternate laser firing to occur at “field onn and “field off“ was 80 Hz (Figure 4). The block diagram in Figure 8 shows how the required boxcar steering and laser triggering were obtained from the ac line that was used to drive the magnet. A sine-wave phase shifter is used to shift the magnetic field reference signal so that it is exactly in phase with the magnetic field. A zero-crossing detector is then used to produce a 120-Hz pulse train from the 60-Hz sine wave. The positions of these pulses are illustrated in Figure 4b. A digital phase shifter then shifts the pulses to the points corresponding to the 60-Hz magnetic field maxima (Figure 4c). The 120-Hz zero-crossing pulses and the 120-Hz phase-shifted pulses are combined into a 240-Hz pulse train using an OR gate. A pulse thus occurs at each “field on” and “field off‘ (Figure 4d). Dividing this 240-Hz pulse train by 3 and then reshaping the pulses results in the required 80-Hz pulses (Figure 4e). These pulses are stretched to a 10-ms pulse width to accommodate the external triggering requirements of the excimer laser (Figure 4f). The two gated integrators that were used to detect the ZETA LEAFS signal require a 50% duty cycle and alternate triggering at half the laser repetition rate. This was obtained with a divide-by-two counter (Figure 8). The resultant 40-Hz waveform (Figure 4g) was used to trigger the two gated integrators of the boxcar, which were set at opposite logic levels. This allowed the “field on” and “field off“ signals to be processed separately. More complete circuit diagrams for the ZETA LEAFS triggering system are available from the authors. Detection System. Parts of the detection system have been described in a previous publication (16)and are briefly summarized here. A monochromator with an f/3.5 aperature was used. A gated photomultiplier tube was operated with a laboratoryconstructed dynode chain specifically designed for high current, pulsed signals. The PMT was surrounded with a magnetic shield
(Perfection Mica Co., Bensenville, IL) that fitted inside the PMT housing. A wide-band (200 MHz) preamplifier with a gain of 10 and an input impedance of 1kQ was used between the PMT and the boxcar (Figure 2) to boost the signals above the level of noise originating in the boxcar. The boxcar averager was operated in a two-channel mode with alternate channel triggering. A 5 0 4 input impedance was used a t the boxcar integrator modules to satisfy the output load requirements of the wide-band preamplifier. Exponential signal averaging with a 0.5-w~integrator time constant and a 10-ns boxcar gate width were used for all measurements. The time constant used for the boxcar mainframe output was 1 ms. The boxcar was triggered by the excimer laser pulse as detected by a fiber-optic photomultiplier system which has been described in a previous publication (16). Two 50 mm focal length, biconvex lenses were used to image the fluorescence onto the horizontal entrance slit of the monochromator. The optical baffle system inside the door of the furnace enclosure allowed a sharp ring of furnace emission to be focused around the slit while fluorescence from the center of the furnace was focused through the slit. Alignment of the lenses was critical to minimize the amount of furnace emission reaching the detector. The two lenses provided physical separation between the ac magnet and the PMT to reduce interference caused by the magnetic field. Flame LEAFS was used to optimize the wavelength of the dye laser prior to furnace fluorescence measurements. Flame fluorescence was imaged with a lens and a removable mirror onto the entrance slit of the same monochromator used for the ZETA LEAFS measurements. Computer Control and Data Acquisition. A DEC PDP 11/03 minicomputer and a laboratory-constructed analog-to-digital (A/D) and digital-to-analog (D/A) interface (46) were used for data acquisition and instrumental control. The interface provided 12-bit A/D and D/A resolution with selectable acquisition rates of 8 to 8000 points/s. Twelve lines of a 16-line parallel interface module (PIM) on the PDP 11/03 bus were used to transfer data between the computer and the interface. The other four lines of the PIM were used by software routines to control the ZETA LEAFS instrument. A user-written program in RT-11 Macro Assembly language (Digital Equipment Corp.) determined the control sequence of the ZETA LEAFS measurement and provided fast data acquisition. A user-written FORTRAN program was used for data manipulation. The sequence for a ZETA LEAFS measurement was as follows: After a sample was placed in the tube furnace, the heating program of the HGA-2000 power supply was manually initiated. The heating sequence consisted of drying and atomization. (Charring was omitted for aqueous standards.) After the sample was dry, the closing of a relay in the furnace power supply was used to trigger a bounceless switch located in the computer interface. The changing logic level of the bounceless switch initiated a timing sequence within the macro program. Three seconds prior to atomization, the laser and the ac electromagnet were activated. Computerized data collection from the boxcar was initiated 2 s prior to atomization, allowing time for the laser output and magnetic field to stabilize. Data collection continued until the atomization step was complete. Data were stored directly in computer memory by the macro routine at the rate selected for the A/D interface. (A data acquisition rate of 512 points/s was typically used.) After atomization, the laser and magnet were disabled and the ZETA LEAFS signal was sent through the D/A interface to a chart recorder a t a rate which allowed for the slow response time of that device. The data were then stored on disk and computer control was transferred to the FORTRAN routine. The FORTRAN program used a two-point “moving gate” smoothing routine on the raw data. The average value of the data collected in the 2 s prior to atomization was subtracted from the rest of the data to establish a base line and compensate for electronic drift in the boxcar output. The peak height and peak area of the ZETA LEAFS signal were calculated and the output was sent to the CRT and a printer. Computer control was then automatically transferred back to the macro routine which waited until the next heating cycle of the HGA-2000 furnace power supply was initiated. RESULTS AND DISCUSSION Preliminary data are reported here for cobalt which dem-
ANALYTICAL CHEMISTRY, VOL. 59, NO. 8, APRIL 15, 1987
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51 field off
x
11.5
12
c
field on
field off corrected signal
4
Flgure 9. ZETA LEAFS blank signals. Chart recorder traces of signal vs. time. Correction for a blank signal caused primarily by furnace emission is shown using a magnetic field strength of 12 kG with excitation at 304 nm and detection at 341 nm. I
onstrate the analytical and diagnostic capabilities of the ZETA LEAFS instrument. All studies reported here were performed by using laser excitation a t 304 nm and nonresonance detection of the fluorescence a t 341 nm. The detection limit and linear dynamic range for cobalt were determined for both ETA LEAFS and ZETA LEAFS. The effect of field strength on the “field on“, “field off“, and background corrected signals was studied. Atomic fluorescence spectral profiles of the “field on”, ”field ofF, and background corrected channels, as a function of excitation wavelength, were obtained and were used to compare ETA LEAFS with and without Zeeman background correction. Precision. The precision of the ZETA LEAFS measurement was determined to be 30% relative standard deviation (RSD) for 10 successive atomizations of 100 pg of cobalt. The precision of ETA LEAFS measurements made under similar conditions was 13% RSD. For ZETA LEAFS, the laser was operated with a pulse repetition rate of 80 Hz, of which 40 Hz was for the signal measurement and 40 Hz was for the background measurement. For ETA LEAFS, the full 80 Hz was used for the signal measurement. An increase in noise, for ZETA LEAFS, in comparison to ETA LEAFS, was expected because of the subtraction process, but a detailed study of noise sources in ZETA LEAFS has not yet been undertaken. A higher laser pulse repetition rate would probably improve the ZETA LAFS precision because the transient signal would be sampled more frequently. Detection Limit for Cobalt. The ZETA LEAFS detection limit for cobalt was found to be 0.7 pg and the ETA LEAFS detection limit was 0.3 pg. The ZETA LEAFS detection limit was 4 times better than the ETA AAS detection limit for cobalt (3 pg) (35),while the ETA LEAFS detection limit was 10 times better. The ETA LEAFS detection limit was 5 times worse than the graphite cup ETA LEAFS detection limit reported by Bolshov et al. (24),obtained under saturation conditions. It was determined that the maximum laser power available for this work (10 pJ/pulse) did not saturate the 304-nm cobalt transition. An increase in laser power, to saturate the atomic transition, would probably improve the detection limit reported here. Calibration Curve for Cobalt. The ZETA LEAFS calibration curve for cobalt was linear (within l o % ) , with a relative slope of 1,for 5 orders of magnitude from the detection limit, and started to curve a t 0.1 pg (10 pg/mL). The ZETA LEAFS linear dynamic range was the same as the linear dynamic range that we obtained for ETA LEAFS, but there was more curvature for the ZETA LEAFS case between 0.1 pg (10 pg/mL) and 10 pg (1 mg/mL). This does not degrade the utility of ZETA LEAFS, in comparison to ETA LEAFS, because the curved portion of the calibration curve is not normally used to make analytical measurements. Background Correction. ZETA LEAFS was shown to correct for furnace emission, which was the primary source of background for the determination of cobalt. Figure 9 shows typical blank signals obtained a t a field strength of 12 kG. Continuum emission is not affected by the presence of a magnetic field, and therefore the “field onn and “field off”
85
9
95
10
10.5
11
FIELD STRENGTH ( k G 1
Flgure 10. Fluorescence signals vs. magnetic field strength (for 0.1 ng of cobalt at 304 nm excitation and 341 nm detection). The “field off” signal is unaffected by field strength. The “field on” signal decreases as the u components are split away from the laser emission profile. The corrected signal (Le. “field off - field on”) consequently increases with increasing field strength.
signals were the same. The RMS noise on the corrected signal (“field off - field on”) was about a factor of 2 or 3 greater than the noise present in either of the two boxcar input channels. The integrated area of the corrected signal was very close to zero. Scatter was a negligible component of the blank signals because a nonresonance fluorescence transition was used. Additional experiments have demonstrated the ability of ZETA LEAFS to correct for scatter, using resonance fluorescence transitions, and these will be detailed in a future publication (47). Field Strength Dependence of ZETA LEAFS Signals. ZETA LEAFS signals (“field on”, “field off“, and corrected signal) were studied, as a function of field strength, for cobalt. The sensitivity of the ZETA LEAFS measurement was dependent upon the ability of the magnet system to split the Zeeman u components away from the laser excitation profile. The experimentally observed behavior of ZETA LEAFS signals, as a function of field strength, is shown in Figure 10. The “field onn data were normalized to account for a small difference in sensitivity between the two input channels of the boxcar averager (approximately 10%). The background emission component of the “field on” and “field off“ signals was manually subtracted from the data. This was done automatically by the ZETA LEAFS instrument for the corrected signal data. As expected, the “field off“ signal was constant regardless of field strength. The magnitude of the “field on” signal was dependent upon a-component overlap with the laser emission profile. The data indicate that the overlap was substantial at a field strength of 8.5 kG where the “field on” and “field off“ signals were equal. A t this field strength, virtually no Zeeman splitting had occurred with respect to the laser line width (0.008 nm), and an equivalent fluorescence signal was measured in both the “field on” and “field off” channels. Hence, subtraction of the two signals produced a ZETA LEAFS sensitivity of zero a t 8.5 kG. As the field strength was increased, the u-component overlap with the laser emission profile was reduced and the background channel signal (“field on”) approached zero. The corrected signal increased in magnitude with increasing field strength. At a field strength of 12 kG, the separation of the u components from the laser emission profile was almost complete, as indicated by the small difference in signal size between the “field off“ signal and the corrected signal. A t this field strength, the magnitude of the fluorescence signal was approximately equal for both ETA LEAFS and ZETA LEAFS. Cobalt fluorescence peaks obtained a t a field strength of 12 kG are depicted in Figure 11. The “field off‘ signal was comprised of the analyte fluorescence and furnace background
1118
ANALYTICAL CHEMISTRY, VOL. 59, NO. 8, APRIL 15, 1987
’*1
/?,
a field on
field o f f
field o f f
(a)
fieid on 12 k G
(b)
c7
corrected signal
Flgure 11. Typical chart recorder traces of ZETA LEAFS signals for 0.1 ng of cobalt (at 12 kG magnetic field strength, 304 nm excitation and 341 nm detection). The “field on” signal is comprised of furnace emission and u-component overbp. The “field off” signal is comprised of atomic fluorescence furnace emission. The corrected signal (Le., “field off - field on”) is atomic fluorescence only.
z
i f i e i d o f f -field on
+
emission and was equivalent in nature to ETA LEAFS signals. With the “field on“, the signal was comprised of furnace emission and atomic fluorescence arising from overlap of the u components with the laser excitation wavelength. The furnace blackbody emission signal was determined to be only a small fraction of the “field on” and “field o f f signals. Therefore, the “field on” signal was primarily due to u-component fluorescence. The area of the “field on” signal was approximately 10% of the “field off” signal, and therefore noise considerations, not u overlap, were responsible for the degradation of the detection limit for ZETA LEAFS, in comparison to ETA LEAFS. In the figure, the background corrected peak shows the result of the subtraction of the “field on” signal from the “field off’ signal. ZETA LEAFS Fluorescence Spectral Profiles. The relationship between fluorescence and excitation wavelength was studied for the “field o f f , “field on”, and background corrected (“field off - field on”) conditions. The excitation wavelength of the laser was incremented over small intervals between successive atomizations of a 1 ng cobalt standard. Figure 12a shows the data collected from the “field off“ channel. Each point on the figure represents the signal obtained from a single furnace atomization cycle. The normal analytical excitation wavelength (304 nm) is the zero point on the figure. A smooth curve was drawn through the data points to represent the fluorescence profile. The profile is a convolution of the atomic fluorescence line width with the laser line width. The laser line width was measured to be 0.008 nm fwhm, using an interferometer. The convoluted full width a t half maximum of the fluorescence profile and the laser profile was measured to be 0.012 nm. Figure 12b shows the wavelength scan for the “field on’‘ channel a t a field strength of 12 kG. Even with the relatively broad laser emission profile, the two Zeeman u components can be seen to have been split away from the analytical wavelength. These data complement the fluorescence vs. field strength data, which were discussed above and in Figure 10. In Figure 10, at a field strength of 12 kG, the ZETA LEAFS signal approached the magnitude of the ETA LEAFS signal. Figure 12b verifies that the u components were split away from the laser wavelength, and therefore no appreciable atomic fluorescence could occur in the background (“field on”) channel. We have also studied the wavelength dependence of the u components at a field strength of 8.5 kG. At this field strength no u splitting could be detected, and the profiles obtained, from both the signal and background channels of the boxcar, resembled that shown in Figure 12a. Finally, the data obtained from the background-corrected channel of the boxcar (“field off - field on”), as a function of wavelength, are shown in Figure 12c, for a field strength of
(C)
-8”
+ooz
’
’
”
”
”
4012 o -mi2 -a024 RELATIVE WAVELENGTH ( n m )
Figure 12. Wavelength scans for cobalt (at 304 nm excitation and 341 nm detection). The “field off” profile (a) is a convolution of the laser emission profile with the atomic fluorescence profile. The “field on” profile (b) shows the u profiles split away from the analytical wavelength. The ”field off - field on” profile (c) shows the subtraction of the u components from the normal fluorescence profile.
12 kG. The u components are inverted because of the subtraction process. In summary, ZETA LEAFS was shown to correct for background furnace emission under the experimental conditions used to determine cobalt. Studies are currently under way to evaluate the effectiveness of ZETA LEAFS in correcting for background in real sample matrices.
LITERATURE CITED (1) Epstein, M. S.;Bayer, S.; Bradshaw, J.; Voightman, E.; Winefordner, J. D. Spectrochim. Acta, Part 8 1980, 358, 233-237. (2) Russo, R . E.; Hieftje, G. M. Anal. Chlm. Acta 1982, 134, 13-19. (3) Horvath, J. J.; Bradshaw, J. D.; Bower, J. N.; Epstein, M. S.; Winefordner, J. D. Anal. Chem. 1981, 53,6-9. (4) Weeks, S. J.; Haraguchi, H.; Winefordner, J. D. Anal. Chem. 1978, 50,360-368. (5) Omenetto, N.; Hart, L. P.; Winefordner, J. 0.Appl. Spectrosc. 1984, 38. .., 619-624. . . .- . (6) Hart, L. P.;Alkemade, C. Th. J.; Omenetto, N.; Wlnefordner, J. D. Appl. Spectrosc. 1985, 39, 677-688. (7) Fruehoiz, R. P.;Geibwachs, J. A. Appl. Opt. 1980, 19,2735-2741. (8) Human, H. G. C.; Omenetto, N.; Cavaili, P.; Rossi, G. Spectrochim. Acta, Part B 1984, 398, 1345-1363. (9) Omenetto, N.; Hatch, N. N.; Fraser, L. M.; Winefordner, J. D. Spectrochim. Acta, Part 8 1973, 266,65-78. (10) Goff, D. A.; Yeung, E. S. Anal. Chem. 1978, 50, 625-627. (11) Kachin, S. V.; Smith, B. W.; Winefordner, J. D. Appl. Spectrosc. 1985, 39. 587-590. (12) Fruehoiz, R. P.; Gelbwachs, J. A. Spectrochim. Acta, Part 6 1984, 398,807-812. (13) Omenetto, N.; Human, H. G. C.; Cavalii, P.; Rossi, G. Anakst (London) 1984, 109, 1067-1070. (14) Omenetto, N.; Hatch, N. N.; Fraser, L. M.; Winefordner, J. D. Anal. Chem. 1973, 45, 195-197. (15) Epstein, M. S.;Bradshaw, J.; Bayer, S.; Bower, J.; Voightman, E.; Winefordner, J. D. Appl. Spectrosc. 1980, 34,372-376. (16) Seltzer, M. D.; Hendrick, M. S.; Michel, R. G. Anal. Chem. 1985, 57, 1096-1 100. (17) Omenetto, N.; Human, H. G. C. Spectrochim. Acta, Part 6 1984, 396, 1333-1343. (18) Hohimer, J. P.; Hargis, P. J., Jr. Anal. Chim. Acta 1978. 97,43-49. (19) Mizlolek, A. W.; Wiiiis, R. J. Opt. Led. 1981, 6, 528-530. (20) Hohimer, J. P.; Hargis, P. J., Jr. Appl. Phys. Lett. 1977, 30,344-346. (21) Wittman, P. K.; Winefordner, J. D. Can. d . Spectrosc. 1984. 29, 75-78. (22) Neumann, S.;Kriese, M. Spectrochim. Acta. Part B 1974, 298, 127-137. (23) Bolshov, M. A.; Zybin, A. V.; Koioshnikov, V. G.; Vasnetsov, M. V. Spectrochim. Acta, Part 6 1981, 368, 345-350. (24) Boishov, M. A,; Zybin, A. V.; Smirenkina, I . I . Spectrochim. Acta, PartB 1981, 368, 1143-1152.
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Anal. Chem. 1987, 59, 1119-1121 Boishov, M. A.; Zybin. A. V.; Zybina, L. A.; Koloshinkov, V. G.; Majorov, I. A. Spectrochim. Acta, Part B 1878, 318, 493-500. Tiich, J.; PatzoM, J. H.; Falk, H.; Schimdt, K. P. Paper given at Analytiktreffen 82, Neubrandenburg, DDR, Nov 1982. Bolshov, M. A; Zybin, A. V.; Koloshnikov, V. G.; Mayorov, I. A.; Smirenkina, I. I. Specfrochlm. Acta, Part B 1886 418, 487-492. Dittrich, K.; Stark, H. J . Anal. A t . Specfrom. 1888, 1 , 237-241. Pollard, B. D.; Blackburn, M. B.; Nikdel, S.; Massoumi, A.; Winefordner, J. D. Appl. Specfrosc. 1978, 33, 5-8. Huang, X.; Lanauze, J.; Winefordner, J. D. Appl. Specfrosc. 1985, 39, 1042-1047. Omenetto, N.; Human, H. G. C.; Cavaiii, P.; Rossi, G. Spectrochim. Acta, Part B 1984, 398, 115-117. Epstein, M. S.;Nikdel. S.;Bradshaw. J. D.; Kosinski, M. A,; Bower, J. N.; Winefordner, J. D. Anal. Chlm. Acta, 1980, 713, 221-226. Kosinski, M. A.: Uchida, H.; Winefordner, J. D. Talanfa 1983, 30, 339-345. Perkin-Eimer literature, Perkin-Elmer Corp.: Norwaik, CT, 1982. Slavin, W. Graphite Furnace AAS, A Source Book; Perkin-Elmer Corp.: Ridgefieid, CT, 1984. Omenetto, N.; Winefordner, J. D. frog. Anal. At. Spectrosc. 1885, 8 , 371-449. Gonchakov, A. S. Ph.D. Dissertation, Chemical Sciences, Moscow, 1980. Stephens, R. CRC Crit. Rev. Anal. Chem. 1980, 9 , 167-195. Yasuda, K.; Koizumi, H.; Ohishi, K.; Noda, T. frog. Anal. At. Specfrosc. 1980, 3 , 299-368. de Loos-Vollebregt, M. T. C.; de Gaian. L. frog. Anal. At. Specfrosc. 1985, 8 , 47-81. Naranjit, D. A.; Radziuk. B. H.; van Loon, J. C. Spectrochlm. Acta, Part B 1884, 398, 969-977. Siavin, W.; Manning, D. C. Spectrochim. Acta, Part 6 1980, 358, 701-714.
(43) Hendrick, M. S.; Seltzer, M. D.; Michei, R. G. Specfrosc. Lett. 1988, 19, 141-147. (44) de Loos-Voilebregt, M. T. C.; de Galan, L. Spectrochim. Acta, Part B 1880, 358, 495-506. (45) van Uffien, J. W. M.; de Loos-Vollebregt, M. T. C.; de Gaian, L. Spectrochim. Acta, Part B 1982, 378, 527-531. (46) Wu, M. L.; Michel, R. G. Anal. Insfrum. 1984, 13, 117-134. (47) Preii, F. R.; Dougherty, J. P.: Michel, R. G., in preparation.
RECEIVED for review August 14,1986. Accepted January 5, 1987. This work was supported by the National Institutes of Health under Grant No. GM 32002. It was presented in part at the XI1 Annual FACSS Conference, Philadelphia, PA, Oct 2, 1985, as papers 314 and 315, and a t the 3rd Biennial National Atomic Spectroscopy Symposium in Bristol, England, July 23-25, 1986. R.G.M. also presented parts of this material for the Society of Applied Spectroscopy Tour Speaker Program during April of 1986. R.G.M. was supported by a Research Career Development Award from the National Institute of Environmental Health Sciences under Grant No. E S 00130. Some of the equipment used in this research was purchased under grants from the Research Corporation, the University of Conneticut Research Foundation, and the donors of the Petroleum Research Fund, administered by the American Chemical Society.
Peroxidase-Based Colorimetric Determination of L-Ascorbic Acid Robert Q. Thompson Department of Chemistry, Oberlin College, Oberlin, Ohio 44074
An air-segmented contlnuous flow method for the determination of L-ascorblc acid In aqueous samples has been developed. Ascorbate Interferes wlth the enzyme peroxidase, causing a decrease In the rate of oxldatlve coupllng of the chromogenic reagents, 4-aminoantipyrlne and 3,5-dlchioro-2hydroxyphenylsutfonate. The resulting change in absorbance at 510 nm can be related to the concentration of ascorbate. No filtering, dialysis, extraction, or oxidation of samples is necessary, and the dally cost of the analysis is less than 4 dollars. Throughput Is 80 samplesh. The linear range of the determination is 0.4-18 ppm ascorbic acid, and the relative standard deviation is less than 3 % Analysis of muitivltamln samples shows excellent correlation with the nominal values given by the manufacturers and wlth the resuits of lodometric titrations of the same samples. Only ferrous ion is a slgnificant Interference.
.
It has long been known that ascorbic acid interferes with the peroxidase (EC 1.11.1.7)-catalyzed reactions of hydrogen peroxide and hydrogen donor molecules (D), reactions that are often used for visualization of the peroxide produced by oxidase enzymes. Ascorbate has been noted to interfere with solution assays ( I ) as well as clinical test strips (2), reducing the amount of colored product (D-D) formed. The reaction scheme (3) for peroxidase (E) is given below H202 + E compound I
--
+D compound I1 + D
compound I
compound I1
E
+ D'
+ D' + HOH
D'
+ D'
-+
D-D
The mechanism of the interference may be a direct reaction of ascorbate and compound 1/11 ( 4 ) )reaction of ascorbate and oxidized hydrogen-donor molecules (D') ( I ) , or both. In any case, the interaction of ascorbate and the other substrates produces a lag phase in the reaction progress curve, the duration and degree of which depends on the ascorbate concentration. Figure 1 shows some progress curves and the signals that result by measuring each sample in turn at a fixed reaction time. The absorbance change can be related to the concentration of ascorbate. This paper describes an automated procedure that makes use of these facts to determine L-ascorbic acid in aqueous samples. Current methods for ascorbic acid determination include titrations with 2,6-dichloroindophenol (5),iodine, and tetrachlorobenzoquinone (6); differential pulse polarography (7); chemiluminescent detection (8);fluorescence detection after oxidation to dehydroascorbate (9); and methods based on ascorbate oxidase (EC 1.10.3.3) and electrochemical detection (10-12). None of these methods are ideal: titrimetry and chemiluminescence detection are slow and not very specific; the dropping mercury electrode in polarography is cumbersome; the fluorometric method is complicated and may be flawed because aerobic oxidation of ascorbate to dehydroascorbate is nonstoichiometric (13);the ascorbate oxidase based determinations are costly at present because the enzyme must be tediously isolated from cucumber, zucchini, or squash. The proposed determination is simple, rapid, sensitive, and inexpensive. The chromogenic reagents were chosen to be 4-aminoantipyrine and 3,5-dichloro-2-hydroxyphenylsulfonate because of the product's large molar absorptivity and because
0003-2700/87/0359-1119$01.50/00 1987 American Chemical Society
