ANALYTICAL CHEMISTRY, VOL. 50, NO. 7, JUNE 1978
LITERATURE CITED
(7) B. N. Bhaduri and A. Fowler, R o c . R . (1934). (8) H, G,Howell, proc, D C n r I n n A n n
(1) K. Fuwa and B. L. Vailee, Anal. Chem., 41, 188 (1969). (2) H. Haraguchi and K. Fuwa, Anal. Chem., 48, 784 (1976). (3) K. Tsunoda. K. Fujiwara, and K. Fuwa, Anal. Chem.. 49, 2035 (1977). (4) E. Yoshimura, Y . Tanaka, K . Tsunoda, S.Toda, and K . Fuwa, Bunsekl Kaaaku. 26. 647 (1977). (5) R. %'. B,. Pearce and A: G. Gaydon, "The Identification of Molecular Spectra , 3rd ed., Chapman & Hall, London, 1976. (6) K. Tsunoda, et al., 38th Annual Symposium of Japan Society for Analytical Chemistry, June 3, 1977.
865
SOC.London, Ser. A , 145, 321 Car
A
'IAR
COC 110'2Kl
RECEIVED for review January 5 , 1978. Accepted March 6, 1978. This work is supported by the Government Grant-in Aid No. 203015, and No. 211204, and u. s. Cooperative Project No. 6R023.
Determination of Ultra Trace Cadmium by Laser-Induced Photoacoustic Absorption Spectromery Shohei Oda," Tsuguo Sawada, and Hitoshi Kamada Department of Industrial Chemistry, Faculty of Engineering, The University
Trace determination of cadmium was carried out by laserinduced photoacoustic absorption spectrometry. The heavy metal-tolerant fungus, Penicilium ochro-chloron, was decomposed and cadmium was measured after extraction into chloroform. The results obtained were in good agreement with those of atomic absorption spectrometry. The detection limit for cadmium was 0.02 ng mL-' Cd (14 ppt). This value was approximately two orders of magnitude lower than that for colorimetric and conventional flame atomic absorption spectrometry.
Absorption spectrometry utilizing the photoacoustic effect has become of major interest. With the photoacoustic absorption technique, it is possible to measure absorption spectra of opaque solids and liquids which previously had been extremely difficult using conventional transmission or reflectance spectrometry. Rosencwaig ( I ) and Adams et al. ( 2 )have been actively investigating possible applications of the technique. Lasers, used extensively as light sources in many areas of analytical spectrometry, have provided sensitive detection when employed in photoacoustic absorption spectrometry. Ultra-low gas concentration has been determined by this approach, while Kreuzer ( 3 )has discussed photoacoustic gas analysis in detail. Lahmann et al. ( 4 ) were the first workers to apply the laser-induced photoacoustic absorption technique t o analysis of liquids. They determined trace (3-carotene in chloroform with a n argon ion laser, and a detection limit of 9 X 1O'O molecules per cm3 (12 ppt) was achieved. The latter authors ( 5 ) also determined Mn0,- in aqueous solution with the same method. T h e detection limit obtained was about two orders of magnitude lower than that of the standard colorimetric method. In the present paper, the determination of cadmium in the heavy metal-tolerant fungus, Penicilium ochro-chloron, was carried out with this new technique. The fungus, important in the biochemical and medical fields, is used for studies concerning the mechanism of resistance t o heavy metals in vivo. Satisfactory results were obtained, compared with those of flame atomic absorption spectrometry.
EXPERIMENTAL Apparatus. A block diagram of the photoacoustic spectrometer is shown in Figure 1. The output beam of argon ion 0003-2700/78/0350-0865$01 .OO/O
of Tokyo, Hongo, Bunkyo-ku, Tokyo, Japan
laser (Spectra Physics Model 164-03), operating in a single line mode of 514.5 nm, was modulated at a given frequency by a light chopper and was directed into the sample cell through a collecting lens (f = 20 cm). The pressure fluctuation induced in the sample solution by absorbed radiation was detected by a piezoelectric ceramic (NPM, N-6 supplied by Tohoku Kinzoku Co. Ltd.). A lock-in amplifier/preamplifier (NF Co. Ltd., Model LI-574) was used to amplify the modulated output signal. The piezoelectric ceramic acts simultaneously as a sample cell and a pressure sensor. The middle part of the cell was of cylindrical piezoelectric ceramic (length 50 mm and inner diameter 24 mm) which was sealed on both sides by Pyrex tubes incorporating a stopcock and a quartz window. The cell was placed inside an airtight chamber which was secured to a vibration-free stand to prevent pressure fluctuations caused by vibrations from external sources. An atomic absorption spectrophotometer (Dainiseikosha Co. Ltd., Model SAS 7 2 5 ) using the air-acetylene flame was used for atomic absorption measurements. Reagents and Procedure. All reagents were of ultrapure grade or spectral grade and were used without further purification. Water was prepared by distilling (twice) deionized water. Cadmium was extracted into chloroform as cadmium dithizonate according to Saltzman's procedure ( 6 ) ,to separate cadmium from interfering metals. A stock solution of cadmium dithizonate (0.5 pg/mL Cd) was prepared by diluting cadmium solution (10 mL, 1 Fg/mL Cd) to 20 mL with chloroform. The calibration graph was then obtained for standard cadmium solutions which were prepared by appropriate dilution of the stock cadmium solution (0.5 Fg/mL Cd). Peniczlzurn ocho-chloron fungus, 0.3 g (dry weight), was decomposed by nitric acid (4 mL) and sulfuric acid (1 mL). The solution was adjusted to 25 mL with distilled water. The extracted solution was diluted with chloroform to provide a solution whose cadmium concentration was within the range of the standard solutions. The procedure was carried out for the blank solution. The photoacoustic signal intensity of the blank solution was subtracted from that of the cadmium solutions to obtain the cadmium concentration.
RESULTS AND DISCUSSION Kohanzadeh e t al. (7) reported that the laser-induced pressure fluctuations in a sample cell depend upon several physical constants of the solute and solvent. In very dilute solution, however, the photoacoustic signal intensity is considered to be proportional to the incident laser power, the absorptivity of the sample a t different wavelengths, and the concentration of the solute. In a given solvent, the photoacoustic signal intensity is proportional to the concentration C 1978 American Chemical Society
866
ANALYTICAL CHEMISTRY, VOL. 50, NO. 7, JUNE 1978 Sample Cell and Detector
T a b l e I. C o m p a r i s o n of C a d m i u m D e t e r m i n a t i o n s in Penicilium ochro-chloron by T h i s M e t h o d a n d A t o m i c Absorption Spectrometry
Cd concentration,
fig/mL
II I
I Preamp. I
Sample
Lock-in Amp.
1 2 3
No.
4 5
This method
0.23
0.28 11.7 0.55 1.36
A.A.S.
0.25 0.28 9.0 0.35 0.98
Figure 1. Block diagram of the laser-induced photoacoustic spectrometer
\
of 2:l. This value was about two orders of magnitude lower than that for colorimetric analysis (3 ng/mL) (6) and for conventional flame atomic absorption measurement (10 ng/mL) (8). As mentioned previously, the laser-induced pressure fluctuations in a sample cell depend upon several physical constants of the solvent and the absorptivity of the solute in very dilute solution, if the concentration of the solute, the laser power, and other experimental conditions are constant. A detection limit of MnO, in aqueous solution was achieved to be 3 ng/mL Mn ( 5 ) . The molar absorptivity of Mn0,- in aqueous solution is 1800 (L mol-' cm-') for 514.5 nm. On the other hand, cadmium dithizonate has a molar absorptivity of -79000 (L mol-' ern-') at 514.5 nm. The ratio of the pressure fluctuation of chloroform in comparison with water is 16:l according to Kohanzadeh et al. (7). From the above results, a detection limit of cadmium dithizonate was estimated to be 0.008 ng/mL Cd. Our experimental results agree roughly. The results for determinations of cadmium in Penicilium ochro-chloron utilizing the laser-induced photoacoustic absorption method and atomic absorption are shown in Table I. The numerical values in Table I represent concentrations of cadmium in 1 mL of the acid-decomposed solution. Preliminary analysis of the fungus sample (0.3 g dry weight) also indicated the presence of a few pg of copper, iron, and zinc. Saltzman has reported that the presence of 5 10 mg of such metals did not interfere with determinations of cadmium. Consequently, the above metals were estimated to affect within the range of experimental errors. In fact, the results obtained by the laser-induced photoacoustic method were in good agreement with those of atomic absorption. From the above results, it is clear that the photoacoustic absorption method is more sensitive than conventional flame atomic absorption spectrometry for the determination of ultra trace amounts of cadmium. The need for ultra trace determinations of cadmium in heavy metal-tolerant fungi will undoubtedly increase, as studies concerning the role played by cadmium in the biochemical field progress. The ultra trace determinations of cadmium in waste water and foods as well as bio-samples have also become an important problem. The photoacoustic absorption method can be applied to such problems. Saltzman's method is most suitable for determinations within the concentration range of 1-10 gg/mL. In our experiments, concentrations of cadmium extracted into chloroform satisfied this concentration range. However, for the case where the laser-induced photoacoustic absorption method is applied to ultra trace determinations of cadmium in real samples in the concentration range of ng/mL to pg/mL, more detailed experiments are required. For example, some of the excess dithizone is known to be extracted into the organic solvent as well as the metal dithizonate. However, the absorption of free dithizone can be corrected by measuring the photoacoustic signal intensity at two wavelengths (9). For the case where the argon ion laser is used, two wavelengths
-
200
LOO
600
800
Chopping Frequency ( Hz ) Figure 2. Relation between photoacoustic signal and chopping frequency. Concentration of cadmium dithizonate, 5 ng/mL Cd. Laser power, 500 mW
of the solute, if the laser power is constant. The dependence of the photoacoustic signal intensity upon chopping frequency was investigated in order to obtain the optimum frequency for measurement. The results are shown in Figure 2 . The photoacoustic signal intensity decreased beyond 200 Hz. Adams et al. (2) observed a similar relationship in the measurement of solid samples and pointed out that it is due to the l / f thermal lag. However, this explanation seems hard to accept for liquid samples. At the present stage, this relationship in liquid samples cannot be clarified. From the above results, subsequent experiments were carried out at the frequency of 185 Hz. At 500 mW of laser power, the relationship between the photoacoustic signal intensity and the concentration of cadmium in chloroform was linear over a range of three orders of magnitude, that is, from 0.05 ng/mL Cd to 50 ng/mL Cd. The coefficient of variation was 1.3% (8 determinations) for 0.5 ng/mL Cd. The intensity exhibited a little saturation beyond 50 ng/mL Cd, and the saturation was considered to be caused by an increase in the temperature of solution. In fact, a t laser power less than 500 mW, the linear range was extended to concentrations greater than 50 ng/mL Cd. In comparison to colorimetric and conventional atomic absorption spectrometry, the linear concentration range offered by the laser-induced photoacoustic method is at least one order of magnitude greater. Moreover, appropriate selection of the laser power may extend the linear concentration range. The detection limit was calculated to be 0.02 ng/mL Cd (14 ppt), based on a limiting photoacoustic signal to noise ratio
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ANALYTICAL CHEMISTRY, VOL. 50, NO. 7, JUNE 1978
of 488.1 nm and 514.5 nm are available. Moreover, as the existences of small amounts of foreign metals might cause large errors, a complete separation will become an important requirement. T h e laser-induced photoacoustic absorption method is applicable to a wide range of liquid samples and application of lasers operating in the UV, visible, and IR is recommended. Further development in surface pre-treatment of piezoelectric ceramic is needed in order to measure materials in concentrated acidic and basic media. Then, ultra trace determinations of various kinds of metals beside cadmium and organic compounds would become possible utilizing the laser-induced photoacoustic absorption method.
867
heavy metal-tolerant fungus as samples, and are also grateful to M. Suzuki for her help in atomic absorption measurements.
LITERATURE CITED A. Rosencwaig, Anal. Chem., 47, 592A (1975). M. J. Adams, A. A. King, and G. F. Kirkbright, Ana/yst(London),101, 7 3 (1976). L. B. Kreuzer, Anal. Chem., 48, 239A (1974). W. Lahmann, H. J. Ludewig, and H. Welling, Anal. Chem.. 49, 549 (1977). S. Oda, T. Sawada, and H. Kamada, BunsekiKagaku, 27, No. 5, (1978). B. E. Saltzman. Anal. Chem.. 25, 493 (1953). Y. Kohanzadeh, J. P. Whinnew, and M. M. Corroll, J . Acoust. Soc. Am.. 5 7 , 67 (1975).
W. Slavin, "Atomic Absorption Spectroscopy", Interscience, New York, N Y 1968 E. B.'Sandell,"ColorimetricDeterminathn of Trace of Metals", Interscience, New York, N.Y., 1959.
ACKNOWLEDGMENT We are greatly indebted to S. Toda and Y. Dokiya of the Faculty of Agriculture, The University of Tokyo, for providing
RECEIVED for review January 13, 1978. Accepted February 27, 1978.
Fluorometric Determination of Manganese(I1) via Catalyzed Enzymatic Oxidation of 2,3-Diketogulonate Verne L. Biddle and E. L. Wehry" Department of Chemistry, University of Tennessee, Knoxville, Tennessee 379 16
A fluorometric technique for the determination of Mn(I1) is reported. The specific requirement for Mn(I1) in the peroxidase-catalyzed Oxidation of 2,3-diketogulonate is utilized to produce H,Oz, and the signal observed in the concurrent fluorometric Hz02"fixed time" assay procedure is related to the Mn( 11) concentration using a modified standard addition method. The utility of this procedure is demonstrated at pH 8.0 and 25 O C in a synthetic seawater matrix wherein the detection limit Is 8 pM Mn( 11); accurate determinations can be performed up to ca. 50 pM Mn(11). Analysis time is less than 30 min.
Fluorescence and chemiluminescence methods of analysis for trace metals have achieved popularity due to their inherently high sensitivity. The required instrumentation is relatively inexpensive and discrimination against interferences can often be achieved in fluorescence techniques by changing the excitation or emission wavelength. Interest in the use of enzymes for analytical purposes has also been widespread (1-3);however, apart from their use in ion-selective electrodes, little use has been found for them in the area of metal-ion assay. T h e present paper describes a new method for fluorometric determination of Mn(I1) in aqueous media, based upon acceleration by Mn(I1) of the rate of enzymatic oxidation of 2,3-diketogulonate (hereafter abbreviated "DGA"; IUPAC name of acid form: threo-2,3-hexodiulosonicacid, formula 0
7
)
.
Only three fluorometric methods for Mn(I1) appear to have been described. Pal and Ryan (4)found that the oxidation reaction of MnO,- with 8-hydroxyquinoline-5-sulfonic acid, which produces a highly fluorescent species, could be used to detect 2.5 ppb-2.5 ppm Mn(I1) following oxidation to Mn04via the Ag(1)-catalyzed persulfate reaction. Though few ions were found whose interference was not eliminated by boiling the sample with persulfate, partially oxidized organic matter with absorption or fluorescence spectra in the region of 375
nm or 485-490 nm, respectively, would interfere in the determination. Guilbault, Brignac, and Zimmer ( 5 ) reported a procedure in which Mn(I1) was assayed by its inhibition of the fluorometric determination of H202with the enzyme peroxidase. From 0.3-12 ,ug/mL Mn(I1) could be determined, but the method was not specific for Mn(I1). The only other published fluorometric method for Mn(I1) involved the measurement of the fluorescence intensity of extracted ionic associates, formed between cationic complexes of Mn(I1) with neutral nitrogen-containing ligands (e.g., 1,lO-phenanthroline, 2,2'-bipyridine) and anions of hydroxyxanthene dyes (6). Severe interference in Mn(I1) determinations by this procedure was observed in the presence of other metal ions (e.g., Co(II), Cu(II), Pb(II)), since their complexes had very similar spectra to that of the Mn(I1) species. When interfering ions were removed from the sample, 1-10 ,ug Mn(I1) could be detected by this method. Chemiluminescence methods for the determination of trace amounts of manganese have also been developed (7,8). These procedures suffer, however, from a lack of specificity for Mn(I1); a number of other metal ions (e.g., Cu(II), Co(II), Cr(II1)) also catalyze the chemiluminescence reactions of luminol ( 7 ) or lucigenin (8) in the presence of HzOz. An alternative chemiluminescence method for Mn(I1) is based upon the observation t h a t aqueous suspensions of siloxene luminesce in the presence of MnO,- in highly acidic solutions (9). Mn(I1) was first oxidized to Mn04- via the Ag(1)-catalyzed persulfate reaction; the Mn04- was then assayed by the luminescence it produced. The relative standard deviation was on the order of f 1 5 % . The procedure described herein is based on a specific requirement for Mn(I1) in the photo-oxidation of DGA, as first noted by Habermann, Gaffron, and Homann (10-12). Oxidation of DGA has also been observed in several nonphotochemical reactions, with the presence of Mn(I1) ions being a specific requirement (13). One of these reactions involved the presence of the enzyme horseradish peroxidase (HRP),
0003-2700/78/0350-0867$01,00/0 C 1978 American Chemical Society
