simple and rapid wet ashing procedure described was preferred for the atomic absorption analysis of chromium with the graphite atomizer. This procedure had the further advantage that it was generally applicable to the determination of chromium in biological materials other than plasma and urine. It has been used as described with equal effectiveness to the determination of chromium in whole blood, red blood cells, and to 50-mg samples of other tissues such as human hair and finger nails. It is concluded that atomic absorption spectrometry with the graphite tube atomizer provides the basis for a simple and very rapid method for the quantitative measurement of chromium in biological samples. The inherent specificity and sensitivity of this technique satisfies the requirements for the determination of chromium in very small biological
samples at concentrations of a few parts per billion. Pretreatment of the sample by wet ashing is recommended because : a) chemical interferences due to sample composition are avoided, b) there is general applicability to a variety of biological materials, and c) considerable reduction of instrument analysis time with wet ashed samples occurs. For simplicity, sensitivity, reliability, and speed of analysis, the method as proposed has distinct advantages over other current procedures which require extensive sample preparation and analytical time.
RECEIVED for review March 27, 1972. Accepted May 25, 1972. This work was supported by National Institutes of Health Grant No. AM13322.
Ultrasonic Nebulization in a Low-Emission Flame for Atomic Fluorescence Spectrometry M. B. Denton’ and H. V. Malmstadt Deparfmenf of Chemistry, School of Chemical Sciences, University of Illinois at Urbana-Champaign, Urbana, Ill. 61801 The use of an ultrasonic nebulizer in conjunction with a low turbulence, argon-hydrogen-entrained air flame provides improved performance of almost two orders of magnitude as compared with a conventional pneumatic total consumption burner. Increased sensitivity results from more efficient nebulization, reduced light scattering, and lower flame background emission. An automated sample changing system is described which provides the required freedom from sample cross-contamination. THEHIGH SENSITIVITY in trace analysis offered by atomic fluorescence spectrometry A F ( I ) , has been demonstrated by many workers (2-4). Various different flame-burner-nebulizer combinations have been investigated for use in generating the required atomic vapor (3). Desirable atomizer characteristics include efficient conversion of sample matrix into analyte atoms suitable for excitation, low radiational background, a low concentration of quenchers, a long residence time of analyte atoms in the optical path, as well as low cost and simplicity of operation ( 4 ) . An ideal atomizer should also produce a minimum of scattering centers. This is particularly important when using high intensity sources in resonance line fluorescence (5) where any remaining droplets or salt crystals can scatter the exciting radiation into the entrance optics of the detection system.
Present address, Department of Chemistry, University of Arizona, Tucson, Ariz. 85721. (1) J. D. Winefordner and T. J. Vickers, ANAL.CHEM.,36, 161 (1964). (2) Richard Smith, “Spectrochemical Methods of Analysis,” J. D. Winefordner, Ed., Wiley-Interscience, New York, N.Y., 1971, Chapter 4. (3) J. D. Winefordner and T, J. Vickers, ANAL.CHEW,42, 206 R (1 970). (4) J. D. Winefordner and R. C. Elser, ibid.,43 (4),24 A (1971). (5) M. B. Denton and H. V . Malmstadt, Appl. Phys. Lett., 18, 485 (1971).
This requirement would tend to indicate the desirability of an extremely hot flame or plasma to ensure complete desolvation and conversion into atomic vapor; however, this increased temperature is often accompanied by an undesirable increase in background emission. For many applications, better results can be achieved in a low-temperature flame through generating an aerosol composed of very fine droplets of sample solution which can be rapidly and efficiently desolvated. The salt crystals resulting from desolvation of smaller droplets will, consequently, be smaller and, therefore, more readily converted to atomic vapor. Bratzel, Dagnall, and Winefordner ( 6 ) compared premixed and turbulent air-hydrogen and argon-air-hydrogen flames. They noted that turbulent flames suffered from several problems: having less than 100% atomization efficiency (particularly in the lower regions); being difficult to illuminate and collect light from the larger high regions ; and having very high rise velocities, shortening the time allowed for solvent and solute evaporation. However, the better results they achieved with turbulent flames were attributed to the higher total volume of solution reaching the flame. Considerable effort has been expended by many workers to characterize various types of nebulizers. Dean and Carnes (7) studied the drop-size distribution of the Beckman integral aspirator, total consumption burner and found that with water, 31 of the drops were over 20 pm in diameter. Stupar and Dawson (8) have contrasted pneumatic and ultrasonic methods for the production of aerosols. They have observed that ultrasonic devices generate more homogeneous aerosols. At higher ultrasonic frequencies, the resulting droplet size decreases (8, 9). The proper ultrasonic nebulizer offers a solution to the problem of producing very small droplets with (6) M. P. Bratzel, Jr., R. M. Dagnall, and J. D. Winefordner, ANAL. CHEM., 41.713 (1969). (7) John A. Dean and William J. Carnes, ibid.,34,192 (1962). (8) J. Stupar and J. B. Dawson, Appl. Opt., 7, 1351 (1968). (9) J. Spitz and G.Uny, ibid.,p 1345.
ANALYTICAL CHEMISTRY, VOL. 44, NO. 1 1 , SEPTEMBER 1972
1813
Aerosol t o Burner
Figure 1. Jebulizer assembly with automatic sample removal and cleaning The ultrasonic transducer is a Macrosonics HFS801 assembly
120 VAC from t r i m
are the result of this improved nebulization efficiency. An automatic sequencer system is described that greatly facilitates sample changing by automatically removing an old solution and washing the nebulizer chamber. EXPERIMENTAL Cld
4
Figure 2. Details of the shutter valve (VI) used in the automated nebulizer Internal valve dimensions should be sufficient so as not to restrict the aerosol flow to the burner a small size distribution which can be efficiently desolvated even in very low-temperature flames. It is the purpose of this study to demonstrate that improved detection limits are possible through the combination of an ultrasonic nebulizer and a low-emission flame. The small droplets generated by a high-frequency ultrasonic nebulizer are shown to be efficiently desolvated even in a very lowtemperature flame. An increase in the concentration of atomic vapor and a substantial reduction in light scattering 1814
Measurement System. The measurement system is basically that previously described (IO) with the addition of a n Osram lamp and associated power supply in the A F source compartment. A 10-mm focal-length, quartz lens was used to focus the excitation energy on the flame. In order to increase the read-out time constant, 0.01-pF capacitors were placed across the operational feedback resistors for the 5 X A and 2 X 1O-Io A scales on the Heath log-linear module. This increased the respective full scale time constants to 10 sec and 25 sec. Burners. The Beckman 4020 total consumption burner was compared to a previously described, fabricated burner (Ref. IO, Figure 4). Zinc and cadmium Osram lamps (Edmund Scientific Company, Barrington, N.J.) were operated from a Heath prototype power supply. Temperature Measurements. Flame temperature measurements were made using the sodium line reversal technique (11). A G E 30AT24/13 ribbon filament, ultraviolet spectrum lamp was calibrated using a Pyrometer Instruments Company (Bergenfield, N.J.) model M-6028 optical pyrometer. An Electro-Nuclear Laboratories (Menlo Park, Calif.) model LS-32 He-Ne laser was used as the source for the laser scattering photographs. Nebulizer. Through the addition of solenoid valves and sequencing circuitry to the nebulizer system of Reference IO, sample changing has been facilitated. The automated nebulizer is shown in Figure 1. Skinner (New Britain, Conn.) V52DA2022 normally-closed, two-way, stainless steel, solenoid-operated valves are used to control both the vacuum waste removal and wash water lines. These devices have sufficient orifices to eliminate any possible restriction at this point. A Skinner V56DA2 three-way, stainless steel valve directs support gas flow around the nebulizer. The burner is isolated from the nebulizer by a speciallyconstructed shutter valve shown in Figure 2. During the wash cycle, this valve blocks the main aerosol line while (10) M. B. Denton and H. V. Malmstadt, ANAL.CHEM., 44, 247
(1972). (11) H. M. Strong and F. P. Bundy,J. Appl. Phys.. 25, 1521 (1954).
ANALYTICAL CHEMISTRY, VOL. 44, NO. 11, SEPTEMBER 1972
WASH J E T
ADD DTM
h k
TO TRlAC DRIVERS
I
I COUNT
-
io
NEBULIZER
LIGHT DRIVERS
M4 PRESET
WASH
CYCLES bypaar
Figure 3. Schematic diagram of sequencing logic built using the Heath 801 Analog Digital Designer (ADD) and standard ADD logic cards
I
-t,
-
n n n
Timlnq Pulse from ADD
Wash Jet On- Period
A-
LI )
preset at four warhinqs
!+ ts -+I
Figure 4. Timing diagram for automatic sequencer (illustrated for four wash cycles)
Nebulizer Oar Eyparr
venting the nebulizer chamber. In the open position, it must provide a reasonably unobstructed flow of the aerosol. To eliminate the need for an excessively long travel by the actuating solenoid, the gas flow is formed into a long, thin area. The internal orifice is 2.22 cm by 0.476 cm, or an area of 1.06 square cm comparable to the 0.79 square cm inside area of the tube. This valve is operated by a Guardian (Santurce, Puerto Rico) 1Al-INT-115 AC solenoid. Sequencer. The required sequencing circuitry is shown in Figure 3. This was built on an EU-801A Heath Analog Digital Designer (Heath Company, Benton Harbor, Mich.). To perform an analysis, a sample of approximately 30 ml is injected through the sample septum and the determination is made. The automatic sequencer eliminates the need for manually changing the valves when removing a n old sample and washing the nebulization chamber. A “start” pulse causes solenoid valves VI, Vy, and V4 to change state, directing gas flow around the nebulizer and starting the removal of the previous sample. A counter causes V3 to open for a set interval for any preset number of washing cycles between one and fifteen. A delay allows the last washing solution to be removed; then V, is closed, followed by the opening of Vi and Va to direct the carrier gas flow again through the nebulizer
chamber. Injecting the new sample through the septum completes sample changing. Sequencing Logic. The timing can best be understood by referring to both the schematic diagram (Figure 3) and the timing sequence (Figure 4). The leading edge of a bounceless start pulse causes the output of NAND gate 2 to go to zero, triggering monostable Ms, and allows the preset number (N) of wash cycles to be read into the preset binary downcounter ,‘madeup of flip-flops A through D, while holding the output of NAND gate 1 at one. The Q output from M2, which is set at 100 microseconds, resets the downcounter flip-flops. Since the start pulse is long compared to the output of M,, gates 3 through 6 are still held open, and the counter is set to the proper number. Gate 7 determines when the counter is at zero. As soon as the counter is set, gate 7 goes to the one state, causing gate 8 to turn on gate 9. This turns on the vacuum valve and causes gate 10 to turn gate 11 on. Gate 11 provides the signal to actuate the nebulizer bypass valves. The next positive-going spike output from the 801 Digital Timing Module, which is set at approximately 0.2 Hz, turns off gate 1 , triggering monostable MI for a time set to t2. This is the period the wash water jet (valve V,) is turned on. The one-to-zero transition triggers flip-flop D. Pulses are al-
ANALYTICAL CHEMISTRY, VOL. 44, NO. 11, SEPTEMBER 1972
e
1815
A
B
Figure 5. ( A ) Background spectra of the Ar-H2-entrained air flame in a total consumption, turbulent burner, and ( B ) in a low-entrainment rate, “laminar type” burner
Table I. Relative Detection Limits Obtained with the Beckman 4020 Burner Employing Oxy-Hydrogen and Argon-Hydrogen-Entrained Air Flames and Limits Obtained with the Described Ultrasonic Nebulizer, Low Turbulence, Argon-Hydrogen-Entrained Air Flame Beckman Low Ar-H2turbulence entrained Ar-H2air burner entrained air (pneumatic burner Beckman nebuliza(ultrasonic Element Line H2-02 tion) nebulization) (Detection limit in ppm) 2139 A 0.1 0.002 0 .oooO25 Zn Cd 2288 A 0.05 0.001 O.oooO13
lowed through gate 1 until the counter reaches zero as indicated by gate 7’s going to zero. This locks gate 1 in the high state, stopping the washing jet bursts. Gate 7’s dropping to zero causes monostable M3 to be triggered. M3 takes over from gate 8 and continues to hold gate 9 a t one for the length of time, t 4 , required to remove the last wash water. This results in the vacuum’s being applied t 5 from the beginning of the first wash jet to a set time, r4, following the last wash jet. When the vacuum signal turns off, monostable M4 is triggered, taking over from gate 10, and continues to hold gate 11 at one for a time, f 6 , resulting in the nebulizer bypass being actuated for a total time, r7, from the start of the first wash jet to a set time, t 6 , following removal of vacuum. The 0- and 5-volt TTL logic signals from the A D D unit are converted to the 120-volt alternating current required by the solenoid valves through the use of a triac driver (12) which has proved to be free from transient noise spikes. Solutions. Stock solutions of 1000 ppm were made directly from the metals. All other solutions were prepared by successive dilutions from these stock solutions using high purity water. The stability of the trace level solutions varied. Fresh dilutions were prepared daily.
RESULTS AND DISCUSSION A comparison between the argon-hydrogen-entrained air flame’s background emission observed for the total consump(12) E. S. Iracki, M. B. Denton, and H. V. Malmstadt, ANAL. CHEM., 44, 1924 (1972). 1816
tion burner and the low turbulence (“laminar flow”) burner is shown in Figure 5 . The lower background observed for the low turbulence burner is the result of decreased temperature due t o the lower air-entrainment rate. For a direct current readout, this lower background improves signal-to-noise and allows use of a larger slit width, improving the monochromator throughput and, therefore, sensitivity. Table I compares the relative detection limit for zinc and cadmium using the total consumption, pneumatic nebulizer Beckman burner and the described ultrasonic nebulizerburner system. The detection limit is defined as a signal that is twice the peak-to-peak backgromd noise. In each case, the slit width, gas flow rates, and flame areas viewed were adjusted for optimum performance. Photomultiplier voltage was held constant a t 750 volts. An improvement of almost two orders of magnitude is evident for the ultrasonic nebulizer-burner system. In order t o gain an insight into what factors contribute t o the observed increase in sensitivity, a comparison study was undertaken. The results are shown in Table 11. The scattering signal Is,is defined as Is
= IT,
-
re
(1)
where IT,is thr: total readout current with water aspirating and I , is the current resulting from the flame background emission. The observed fluorescence current IF is defined as I F = IT,
-
(IB
+
IS)
(2)
where IT, is the total readout current with a 10-ppb cadmium solution being nebulized. The 2288 A region used for cadmium was observed with a 600-pm slit. To determine the contribution of the ultrasonic nebulizer to this sensitivity, one additional configuration was included in this study. A small spray chamber was fabricated so as to utilize the Beckman total consumption burner as a pneumatic nebulizer for the premixed burner. The resulting aerosol dispersion is routed to the premixed burner in place of that generated by the ultrasonic nebulizer. Table I1 shows the comparatively high background emission, IB, and scattering signals, Is, that are observed with the Beckman total consumption, turbulent-flow burner. Background is reduced by almost two orders of magnitude in the premixed, low turbulence flame because of its decreased temperature, which is a result of the relatively slow rate of atmospheric oxygen entrainment. This decrease in background offers improved signal-to-noise for a given fluorescence
ANALYTICAL CHEMISTRY, VOL. 44, NO. 11, SEPTEMBER 1972
Table 11. Comparison of Background Emission, Light Scattering, and Relative Fluorescence Efficiency for Cadmium at 2288 A, Holding a Constant Slit Width at 600 pm Flame background A Fluorescence signal (IF)for Burner-nebulizer with 600 pm slit ( 4 ) Scattering signal, A (IS) 10 ppb Cd A. Beckman 4020, 18 x 10-lo A 1 . 5 x 10-lOA 1 . 7 x 10-lOA integral pneumatic nebulizer total consumption burner, turbulent Ar-Hz-entrained air flame B. 0.7-cm round multihole premixed below 2 x lo-” A 3.5 x 10-1O A below 3 X 10-11 A burner (Ref. IO), spray chamber pneumatic nebulization, low turbulence Ar-Ha-entrained air flame C. 0.7-cm round multihole premixed below 2 X 10-11 A below 2 X 10-11 A 4 x 10-lo A burner (Ref. IO), ultrasonic nebulization, low turbulence Ar-HI-entrained air flame
signal. However, when this cooler flame is used with a conventional pneumatic nebulizer (second example in Table 11), the relatively large drops are not efficiently desolvated ; and the total observed signal for a 10-ppb cadmium solution is almost entirely the result of light scattering from solution droplets. The third example results from the use of the low-emission flame with a n ultrasonic nebulizer. At this slit width, almost n o scattering is observed in the upper regions of the flame (11 cm). An increase in absolute fluorescence signal exceeding twofold is also observed. The absence of scattering centers and increased fluorescence signal indicate improved atomization efficiency (Le., the conversion of solution t o atomic vapor) over the hotter turbulent-flow flame resulting from the ease with which the small droplets from the ultrasonic nebulizer can be vaporized. Laser Scattering Study. Figures 6 A and B illustrate the scattering problem. In these photographs, the 1.5-milliwatt output beam of a n He-Ne laser is directed through the upper portion of the burner flame into which distilled water is nebulized. Presence of scattering centers causes the laser beam t o be visible. Figure 6 A shows the high degree of scattering from within the flame of the total consumption burner. Figure 6B shows the lack of scattering centers when using ultrasonic nebulization in conjunction with the described low turbulence flame. With water aspirating, n o trace of the laser beam is visible, indicating a relative freedom from scattering centers. The lower background emission of this flame is also clearly evident, making it almost invisible. Flame Temperature Measurement. The sodium line reversal technique gives only a n average temperature across the flame. In a study using the optical pyrometer to determine the temperature of a platinum wire, it was observed that for lower regions a n average temperature was misleading since combusticn takes place only a t the outside edges; however, near the top of the flame where analyses were carried out, the temperature is reasonably constant. Flame temperature a t the height (11.5 cm from the burner) and flow rates (3.2 l./min argon and 4.8 l./min hydrogen) used for analysis was determined t o be 1505 “C. However, this could be somewhat high in view of work by de Galan and Winefordner (13). A more direct temperature profile should be possible ~-
(13) L. de Galan and J. D. Winefordner, J. Quam. Spect. Radiar. Tratisfer, 7,703 (1967).
using the thermocouple technique of Smith, Stafford, and Winefordner (14). Solvent Scattering Considerations. It can be argued that scattering from the solvent (water droplets) is a constant factor and can, therefore, be subtracted out. While t o some extent this is true, this procedure opposes one of the basic advantages of A F over atomic absorption (AA) in trace analysis-that A F offers a n inherent advantage over AA because it measures a n absolute fluorescent emission signal and is not limited as with AA by the precision of measuring a small difference between two relatively large signals (15). With very low background emission from the flame and no scattering from centers within the flame, a percentage drift in the source intensity is directly transferred to the fluorescing species. Hence, the observed percentage drift in the fluorescence signal is the same as the percentage of the source drift. With scattering centers present, source drift becomes increasingly difficult t o correct for. Factors determining the concentration and effectiveness of scattering centers can vary (nebulization rate, droplet desolvation efficiency, etc.) resulting in instability and the “constant factor” caused by scattering becomes a variable. Therefore, it is highly desirable t o ensure complete vaporization of all solvent. CONCLUSION AND COMMENTS Through the use of a n ultrasonic nebulizer t o generate very small droplets, light scattering is reduced and efficient atomization is observed in even a low-temperature flame. Overall, increased sensitivity by almost two orders of magnitude for zinc and cadmium is observed as a result of this improvement in atomization efficiency, decreased light scattering, and the extremely low background emission of the low-turbulence, argon-hydrogen-entrained air flame. While this study made use of a low-temperature flame t o demonstrate the improved desolvation and atomization characteristics that were the result of the ultrasonic nebulizer’s capability for generating small droplets, improvements should also be observed in other higher-temperature flames. This should be particularly true in cases where the sample is diffi(14) R. Smith, C. M. Stafford, and J. D. Winefordner, ANAL. CHEM., 41,946 (1969). (15) D. C. Manning and P. Heneage, A I . Absorption Newletr., 6 ,
124 (1967).
ANALYTICAL CHEMISTRY, VOL. 44, NO. 11, SEPTEMBER 1972
1817
A
Figure 6. Photographs contrasting laser light scattering observed when employing total consumption, pneumatic nebulization us. the described optimized ultrasonic nebulizer-burner system (Left) The !Ram from a 1.5-mW He-Ne laser becomes vislble bemuse of scattering fmm centers within the total consumption, turbulent b e r flame (Right) No such Scattering is observed when ultrasonic nebulization is employed. The low emission of the low turbulence, “laminar type”, argonhydrogen-ntrained air Aame is also apparent
cult t o convert to atomic vapor or whL.. a cyLIccLI.Iotion of another species would cause salt crystal light scattering. In these cases, the smaller droplets and the decrease in the size of the resulting individual salt crystals should make their conversion t o atomic vapor easier. While there is no danger of flame flashback with the described low turbulence burner, care must be employed since the flame is almost invisible in a lighted room and is not audible. The lack of flame noise offers an additional advantage over the unpleasantly loud total consumption burner. The authors have been informed that Macrosonics, the manufacturer of the transducer assembly used in these studies, is no longer in business (16). However, initial tests with a (16) Lou Owen, Tomorrow Enterprises, Portsmouth, Ohio, personal communication, 1971.
1818
,...
..--_-”_.
__l___.l_,ufactured by DENCO (Tucson, Ariz.) show even better results. This device provides an increased aerosol generation rate and can be operated at several frequencies. yLy
ACKNOWLEDGMENT
The authors express thanks to DENCO for the use of equipment.
RECE~WD for review January 14, 1972. Accepted May 1, 1972. Work partially supported by National Science FoundationResearch GrantsGP92Wand GP18910.
ANALYTICAL CHEMISTRY, VOL. 44,NO. 11, SEPTEMBER 1972
