1882
Anal. Chem. 1983. 55, 1682-1684
with matrix matching, the slope is somewhat non-Nernstian.
Comparison of Results with Different Analytical Finishes. In Table V, we present results for assessing distillates for cyanide by both the photometric and cyanideelectrode potentiometric procedures and thereby confirm that free cyanide is being determined. ACKNOWLEDGMENT We thank R. Luthy of Carnegie-Mellon University for helpful discussions and C. C. Keiser of the J. T. Baker Chemical Company for Contributions and Experimental Assistance.
Registry No. Cadmium carbonate,513-780; cadmium nitrate, 10325-94-7;cyanide, 57-12-5; nitrite, 14797-65-0;sulfamic acid, 5329-14-6; water, 7732-18-5. LITERATURE CITED "Methods for Chemical Analysis of Water and Wastes", EPA-600/479-020, STORET NO 00720; Environmental Protectlon Agency: Cincinnati, OH 1979; 7 pp. "Standard Methods for the Examinatlon of Water and Wastewaters" 15th ed.; American Public Health Association: Washington, DC; 1980; pp 312-328. "Annual Book of ASTM Standards"; American Soclety for the Testing of Materials: Philadelphia, PA, Method D2036-75. "Standard Methods for the Examination of Water and Wastewaters", American Public Health Assoclatlon: Washington, DC, 1975; p 427. Ludzack, F. J.; Moore, W. A.; Ruchhoft, C. C. Anal. Chem. 1954, 26, 1784- 1792. Ruchhoft, C. C.; Moore, W. P.; Terhoeven, G. E.; Middieton, F. M.; Krleger, H. L. Bulletin 355; Robert A. Taft Sanitary Engineerlng Center: Cinclnnati, OH, 1949; pp 10-18 (PB-215059).
(7) Serfass, E. J.; Freeman, R. B.; Dodge, 8. F.; Zabban, W. Platlng (East Orange, N . J . ) 1952, 39,267-273. (8) Serfass, E. J.; Muraca, R. F. Platlng (East Orange, N . J . ) 1956, 43, 1027-1030. (9) Kruse, J. M.; Mellon, M. G. Anal. Chem. 1953, 25, 446-450. (10) Kruse, J. M.; Melion, M. G. Sewage Ind. Wastes 1951, 23, 1402-1407. (11) Komatsu, M.; Kaklgama, H. Bunseki Kagaku 1973, 21, 315-321; through Anal. Abstr. 1973, 30, 1330. (12) Ammon, F. Metalloberflaeche 1956, 10,161-164. (13) Frant, M. S.; Ross, J. W.: Riseman. J. H. Anal. Chem. 1972. 44. 2227-2230. (14) Harzdorf, C.; Dorn, L. Z.Anal. Chem. 1977, 284, 189-192. (15) Schleuter, A. EPA Report 600/4-76.020, June 1976, 20 pp (PB2558521. ingersiil, D.; Harrls, W. E.; Bomberger, D. C. Anal. Chem. 1981, 53, 2254-2258. Hofton, M. Environ. Sci. Techno/. 1976, IO,277-280. Owerbach, D. J.-Water Pollut. Control Fed. 1980, 52, 2647-2654. Cussbert, P. J. Anal. Chim. Acta 1978, 87,429-435. Barton, P. J.; Hammer, C. A,; Kennedy, D. C. J.-Water Pollut. Control Fed.1978, 5 0 , 234-239. Luthy, R. G.; Bruce, S. G.; Waiters, R. W.; Nakles, D. V. J.-Water Pollut. Control Fed. 1979, 51,2267-2282. Kunz, R. G.; Casey, J. P.; Huff, J. E. Hydrocarbon Process. 1978, 5 , 98-106. Williams, H. F. "Cyanogen Compounds"; Edward Arnold: London, 1948; p 258. Rohm, T. J.; Davidson, R. Anal. Lett. 1978, A l l , 1023-1037. 1982 draft revision of ref 1, and Luthy, R., Carnegle-Meilon University, personal cohmunicatlon, July 1982. Schlesinger, H. I.; Brown, H. C.; Finholt, A. E.; Giibreath, J. R.; Hoerkstra, H. R.; Hyde, E. K. J . A m . Chem. SOC. 1953, 75, 215-2 19. Pratt, J. M.; Swinden, G. J . Chem. Soc., Chem. Commun. 1969, 1321-1322.
RECEIVED for review February 18, 1983. Accepted June 8, 1983.
Determination of Formaldehyde with an Enzyme-Coated Piezoelectric Crystal Detector George G. Guilbault'
Laboratoire de Biologie et Technologie des membranes du CNRS, Universite Claude Bernard, Villeurbanne, France
Formaldehyde dehydrogenase was placed onto a plezoelectric quartz crystal, together wlth the cofactors reduced glutathlone and nicotlnamlde adenine dlnucleotide; a reverslble reaction wlth formaldehyde occurred in the gas phase, the frequency change of which was directly proportlonai to the concentration of the aldehyde. Excellent selectivity resulted, wlth llttie response to other aldehydes or alcohols (>1000:1 seiectlvlty ratio at 1 and 10 ppm formaldehyde).
In recent years, coated piezoelectric crystal detectors have become of increasing interest for detection of traces of toxic atmosphere pollutants, not only as highly sensitive and selective detectors (1) but also as simple, inexpensive, and portable devices, which are even small enough to be carried in a worker's pocket (2). King (3) developed a sensitive piezoelectric crystal detector for monitoring hydrocarbons in the atmosphere. Frechette and Fashing ( 4 ) have proposed their use in a static system for the detection of sulfur dioxide. Karasek applied them as detectors for gas chromatography 'On sabbatical leave from Orleans, LA 70148.
the
University of New Orleans, New
(5-7). Guilbault et al. (8-15) developed sensitive and selective detectors for organophosphorus pesticides, sulfur dioxide, ammonia, nitrogen dioxide, hydrogen chloride, hydrogen sulfide, and explosives in the atmosphere. The principle of the detector is that the frequency of vibration of an oscillating crystal is decreased by the adsorption of a foreign material on its surface, A gaseous pollutant is selectively adsorbed by a coating on the crystal surface which is specific for that substance, thereby increasing the mass on the crystal and decreasing the frequency. The decrease in frequency is proportional to the increase in mass owing to the presence of gas adsorbed on the coating, according to the Sauerbrey equation (16, 17)
AF = K-AC
(1)
where AE'is the frequency change in Hz,K is a constant which refers to the basic frequency of the quartz plate (MHz), area coated (cm2),arid a factor to convert the mass adsorbed into concentration (ppm) of sample gas (AC). The theoretical limit of detection is about g (18) and the mass sensitivity is about 400 Hz/pg for a 9-MHz crystal and 2600 Hz/pg for a 15-MHz crystal. By coating the surface of crystal with a substance which will selectively adsorb a particular gas, we can determine the
0003-2700/83/0355-1682$01.50/0 0 1983 Amerlcan Chemical Soclety
ANALYTICAL CHEMISTRY, VOL. 55, NO. 11, SEPTEMBER 1983
I
2
rlw
3
5
4
J
COATED
ivr
.%+
P I E Z O E L E C T R i C CRYSTAL
/f
SAMPLE
FLOW M E T E R
Figure 1. ExperimiBntal apparatus: (1) recorder, (2) digital to analog converter, (3) frequency counter, (4) oscillator, (5) power supply.
concentration of that gas quantitatively. Mandenius, Guilbault e t al. (19) have studied the use of protein coatings on piezoelectric detector for assay of alcohols and formaldehyde. This investigation was directed t o a preliminary study of the detection of substrates with the enzymes and cofactors placed as a coating on a crystal and demonstrates the utilization of enzymes for assay of inhibitors directly in the g,as phase, entirely analogous t o their use in solution. Various methods for the determination of formaldehyde in air with solid iiorbent sampling tubes have been described (20-24), but because of a lack of sensitivity, long sampling times (15 xnin) are required. Rietz et al. (25)have proposed a method for monitoring formaldehyde a t the parts-per-billion level-this procedure involves collection of formaldehyde by chemisorption on a coated solid sorbent and desorption with concentrated sulfuric acid followed by a colorimetric or fluormetric determination of the reaction product. No direct, sensitive and selective assay of formaldehyde exists. This paper represents one of the first published papers on the use of enzyme for assay of substrates directly in the gas phase.
EXPERIMENTAL SECTION Materials. Formaldehyde dehydrogenase (E.C.1.2.1.1), from Pseudomonas putida, 1U/mg glutathione (reduced form), and nicotinamide adenine dinucleotide (NAD+)were all obtained from Sigma (St. Louis, MO). Formaldehyde, acetaldehyde, propionaldehyde, methanol, and benzaldehyde were all spectrophotometric quality (Baker, Phillipsburg, NJ). All solutions were made with distilled water. Piezoelectric crystals (9 MHz), AT cut quartz with gold electrodes, were used in a piezoelectric crystal monitoring device PZ 101 (Universal Sensors, New Orleans, LA). Apparatus. A typical experimental setup with the piezoelectric quartz crystal detiector is shown schematically in Figure 1. The cell design is the most sensitive one for use in a flow system. The piezoelectric crystals used are 9-MHz quartz crystals with silver-plated metal electrodes on both sides. The crystals are AT-cut mounted in HC 25/U holders (JAN Crystals Mfg. Co.). The instrumentation consisted of a low-frequency OX transistor oscillator (International Crystal Mfg. Co., Oklahoma City, OK) powered by a regulated power supply (Heath Kit, 1P-28). The applied voltage was kept constant at 9 V dc. The frequency output from the oscillator was measured by a frequency counter (Health-Scblumbesrger,Model SM-4100),which was modified by a digital-to-analog converter, so that the frequency could be recorded. The frequency (peak maximum) could be read on either the frequency counter or the recorder. Portable Monitoring Device. A portable detector, which is 20 X 14.7 X 9 cm in dimension and less than 3 lbs in weight, was developed for field use. The detector included a piezoelectric crystal monitor, a miniature pump, a sampling valve, and batteries. The piezoelectric crystal monitor was build by Universal Sensors, New Orleans, LA, and included reference and sensor oscillators, a frequency mixer, and solid-state display of the readout. The readout is the frequency differencebetween a sensor crystal, which is coated with the enzyme/cofactor, and a reference uncoated crystal. A miniature pump (Sipin personal pump) was used to
1683
Table I. Response of a Piezoelectric Crystal Coated with Ternary Mixture for 4 Days day
AHZ~
1 2 a
480 550
day
AHza
3 4
500 200
Response to 10 ppm of formaldehyde at setting of 6 .
sample contaminated air into the cell. The reversibility of the detector was achieved by turning a sample valve to let clean air, which was purified by activated carbon and silica gel, pass through the detector cell, thus desorbing the sample off the surface of the coating. The detector operates on NiCd batteries which can be recharged after 8 h of operation. The detector chamber has a 10-mL volume, with two inlets placed symetrically with the inlets and one outlet connected to the pump. The samples were introduced by the syringe injection method, in which definite volumes of gas are introduced by syringe injection into a controlled air flow through walls of PVC tubing. Alternatively the tubing was placed into the flask for continuous sampling. The concentration of formaldehyde was calculated by collecting a definite volume of air sample using the SKC formaldehyde sample tubes (26) followed by fluorometric analysis (25). The SKC tubes have a stated validity over the range tested in this study. The crystal was a circular quartz plate, 15 mm in diameter and 1 mm thick. On each side was attached a gold electrode, 5 mm in diameter, connected to the oscillator circuit. Onto the electrode was placed a thin layer of coating by dropping 3-5 pL of an aqueous solution of the enzyme and cofactors dissolved in distilled water onto each side. The concentrations of formaldehyde dehydrogenase, reduced glutathione and NAD were each l mg/mL. An accurate calculation of the quantity adsorbed can be realized from the frequency change, since 100 Hz corresponds to 1 Wg. The return to base line after adsorption is easily followed by the frequency change, and a stable value was always obtained prior to the next measurement.
RESULTS AND DISCUSSION The enzyme formaldehyde dehydrogenase catalyzes the oxidation of formaldehyde to formic acid, in the presence of NAD and reduced glutathione as cofactors. Hence a ternary mixture of these three substances was placed onto the crystal in approximately equal amounts. Exposure to formaldehyde vapor using the syringe dilution method with lab air (Le., about 50% relative humidity) was utilized. A linear response from 10 ppb t o 10 ppm was obtained, with frequency changes of from 2 to 500 Hz or about 200 Hz per decade of concentration. Both cofactors were found to be necessary for good results, much lower sensitivivy being observed with crystals coated with enzyme alone or without one of the two cofactors present. Thus, the reaction is belived due to an enzymatic reaction occurring on the crystal electrode surface. E cofactors formaldehyde [complex] products
+
+
-
However, if the reaction time is kept short (less than 1 min), only the complex forms reversibly, and not products. This is important if the crystal coating is to be used continuously for several days and many assays. Table I shows that the results obtained with coatings of soluble enzyme and soluble cofactors indicate consistent analysis can be performed for 3 days or 100 analyses. A drop in response is obtained on day 4. This decrease is due t o loss of enzyme activity-this was shown by testing the coating in solution. If chemically bound enzyme (glutaraldehyde method) is placed onto the crystal with soluble cofactors, the same crystal can be used for about 10 days, but since it is impossible to remove the enzyme coating, the crystal must be discarded. Thus, it is recommended that soluble enzyme be used. After 3 days the ex-
1664
ANALYTICAL CHEMISTRY, VOL.
55, NO. 11,
SEPTEMBER 1983
Table IV. The coating is fairly specific for formaldehyde, little response being obtained for other aldehydes and alcohols a t concentrations of 1 ppm and 10 ppm (at least a 1OOO:l selectivity ratio is observed). In a study of the reproducibility of the procedure, a sample of 1 ppm was determined 10 times by using the PZ 101 detector. Values of 300 f 6 Hz were obtained, with a u of 1.5 and a coefficient of variation of 1.5%. Thus, it is believed that an enzyme coating can be successfully used on a piezoelectric crystal for the direct analysis of substrates in air, analogous to the determination of such substrates using the enzyme in an aqueous solution. This should open up exciting new areas of research possibilities.
Table 11. Effect of Electronic Gate Setting on PZ 101 on Sensitivity of Response setting 5 6 a
AHZ~ 25 500
setting 7 8
AHZ~ 800 300
ResDonse to 1 0 m m of formaldehvde.
Table 111. Effect of Weight of Coating on Sensitivity of Response wt of coating,
a
wt of coating,
!Jk2
dHza
!Jg
AHza
10 17 32 68
100 200 480 550
95 160 170 200
500 2055 1850 450
ACKNOWLEDGMENT Carl-Frederick Mandenius (University of Lund, Sweden) is thanked for his collaborative support in this project. Registry No. Formaldehyde dehydrogenase,9028-84-6; glutathione, 70-18-8; nicotinamide adenine dinucleotide, 53-84-9; quartz, 14808-60-7;formaldehyde, 50-00-0.
Response to 1 0 ppm of formaldehyde.
Table IV. Effect of Other Substrates on the Assay of Formaldehyde compound formaldehyde MeOH EtOH acetaldehyde propionaldehyde benzaldehyde
AHZ~ 500 0 2 10 5 0
LITERATURE CITED
AH^ 300 0 0 1 1 0
a At a concentration of 1 0 ppm of formaldehyde. a concentration of 1ppm of formaldehyde.
At
pended coating can be removed, and a fresh one applied. The PZ detector has eight instrument settings, which are electronic gates set from 0.1 s (setting 1) to 32 s (setting 8) sampling time. The higher the gate setting, the higher the sensitivity. However, at high gate times poorer reproducibility can result, especially if the response time (the time required to reach a steady-state value of AF change from base line) is very fast. A setting of 6 was chosen for all studies, since it offers a good compromise between sensitivity and reproducibility (Table 11). In every application of piezoelectric crystal technology,there is an optimum weight of coating substrate which will give an optimum sensitivity of response to the substance to be detected. The results of such an investigation with the enzyme-NAD-reduced glutathione coating are shown in Table 111. Although the coating represented by a frequency change of 16OOO Hz (about 160 pg) is optimum for response to 10 ppm of formaldehyde, the presence of this much coating comes close to the overload value of the crystal (the value at which oscillation ceases). Hence, for best results a coating of only 60 pg was used. Finally, the response of the coated crystal to other aldehydes and alcohols was tested. The results obtained are shown in
Hlavay, J.; Gullbault, G. G. Anal. Chem. 1977, 49, 1890. Schelde, E. P.; Warner, R. B. A m . Ind. Hyg. Assoc. J . 1978, 3 9 , 745. King, W. H., Jr. Envlron. Scl. Technol. 1970, 4 , 1136. Frechette, M. W.; Faschlng, J. L. fnviron. S d . Technol. 1973, 7 , 1135. Karasek, F. W.; Glbblns, K. R. J. Chromatogr. S d . 1971, 9 , 535. Karasek, F. W.; Guy, P.; HIII, H. H.; Tiernay, J. M. J. Chromatogr. 1976, 124, 179. Karasek, F. W.; Tlernay, J. M. J. Chromatogr. 1974, 8 9 , 31. Karmarkar, K. H.; Gullbault, G. G. Anal. Chlm. Acta 1974, 71, 419. Schelde, E. P.; Gullbault, G. 0. Anal. Chem. 1972, 4 4 , 1764. Karmarkar, K. H.; Webber, L. M.; Gullbault, G. G. Envlron. Lett. 1975, 8 , 345. Karmarkar, K. H.; Gullbault, G. G. Anal. Chlm. Acta 1975, 75, 111. Hlavay, J.; Gullbault, G. G. Anal. Chem. 1978, 5 0 , 1044. Hlavay, J.; Guilbault, G. G. Anal. Chem. 1978, 5 0 , 965. Webber, L. M.; Karmarkar, K. H.; Gullbault, G. G. Anal. Chim. Acta 1978, 9 7 , 29. Tomita, Y; Ho, M. H.; Guilbault, G. G. Anal. Chem. 1979, 5 1 , 1475. Sauerbrey, G. 2. Z . Phys. 1959, 755, 206. Sauerbrey, G. 2. Z . Phys. 1964, 178, 457. King, W. H. Anal. Chem. 1964, 3 6 , 1735. Mandenius, C.; Guilbault. G. G.; Danlelsson, 6.; Mosbach, K., manuscript in preparation. Wood, G. 0.; Anderson, R. Amerlcan Industrial Hygiene Conference, Minneapolis. MN, June 1975. DHEW (NIOSH) Publ. ( U . S . ) 1977, No. 77-157, P. & Cam 235-1. Anderson, K.; Anderson, G.; Nilsson, C.;Levln, J. Arbetarskyddsstyrelsen 1979, 2, Stockholm. Beasley, R.; Hoffman, C.; Rueppel, M.; Worley, J. Anal. Chem. 1980, 5 2 , 1110. Kim, W. S.;Geracl, C. L.; Rupel, R. J . Occup. Health Safety, In press. Blsgard, P.; Molhave, L.; Rletz, 6.; Wllhard, P. J. Occup. Health Safety, In press. SKC Formaldehyde Sample Tube, Catalog Number 226-69, SKC, Valley Vlew Road, Elghty Four, PA.
RECEIVED for review December 16, 1982. Accepted May 9, 1983. George G. Guilbault thanks CNRS for the financial support, in the form of a Visiting Director of Research position in the laboratory directed by Daniele Gautheron (LBTMCNRS). Gautheron is also thanked for her assistance in making this position possible.
