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T h e analysis results for each storm group were averaged to obtain a single datum per storm (11). Samples which contained snowfall from two or more storms were rejected from the data set. The resulting data set was subdivided in seeded and nonseeded subsets of 14 and 43 averaged measurements, respectively. Seeded samples were those collected when substantial seeding occurred in conjunction with favorable winds for transport of seeding material to the sampling area. Evaluation of each data subset via the Half-Normal Method described by Daniel (12) indicated that both fit a log-normal (Geometric) rather than a normal distribution. Evaluation for the presence of outliers resulted in the rejection of 1 and 5 inordinately high values from the seeded and nonseeded subsets, respectively, a t the 95% confidence level. The remaining data in these subsets of 13 and 38 average measurements were also shown to fit a log-normal rather than a normal distribution. This is consistent with the stochastic behavior of trace substances in natural systems as pointed out by Rustagi (13) and Ahrens (14). The geometric mean and standard deviation of the seeded subset were determined to be 1.7 X 10 f 0.3 X 10 pg Ag/mL; the values fur the nonseeded subset were 7.8 X & 2.4 X l o 4 Ag/mL. The difference between these means was determined t o be significant a t the 95Y~confidence level. This effectively indicates that seedling resulted in a mean increase in the silver content of the snowfall by approximately a factor of 2 as previously noted by Warburton (3). In this context, the results suggest the occurrence of extended area seeding effects at distances approximating 100 km or more when meteorological conditions are favorable. This is supported by the analysis of the silver data and the associated meteorology presented by Mulveq and Sheaffer (11). T h e accurate determination of Ag a t these concentration levels is more than a trivial problem. Results obtained in the
present study have indicated that precautions must be taken to avoid contamination. The presence of appreciable amounts of Ag in many reagents and the difficulty of purifying these further argues for the use of a minimal reagent approach such as that adopted herein. The accumulation of the majority of the Ag and the acid preservative in the portion of an aqueous sample which does not freeze at -20 "C emphasizes the necessity of completely thawing such samples in order to obtain a representative sample of the original for analysis. Finally, the analysis of rainfall for the present purpose must be based on the utilization of a sample collection system which remains closed until precipitation starts and closes soon after it ends. Otherwise, contamination by fugitive dust and insects will render the data highly suspect.
LITERATURE CITED (1) L. 0 . Grant and A. M Kahan, "Weather Modification for Augmenting Orographic Precipitation": J. Sinrpson and A. S. Dennis, "Cumulus Clouds and their Modification", in "Weather and Climate Modification", W. N. Hess, Ed., John Wiley and Sons, New York, N.Y., 1974. (2) E. Bollay Associates, Final Report, Bureau of Reclamation Contract No. 14-06-D-5573, 20 pp, 1965. (3) J. A. Warburton, Proceedings of International Conference on Weather Modification, Canberra, Australia, 1971, p 185. (4) R. Woodriff, R. B. Culver, D. Schrader, and A . B. Super, Anal. Chem., 45, 230 (1973). (5) J. A. Warburton, and L. G. Young, Anal. Chem., 44, 2043 (1972). (6) D. A. Segar, Department of Commerce, NOAA Laboratories, Miami, Fh., personal communication, 1976. (7) A. W. Strumpler, Anal. Chem., 45, 2251 (1973). (8) A. W. Strumpler, Armos. Environ., 10, 33 (1975). (9) J. Wisnuski, Proceedings of the Fourth Conference on Weather Wlication, 516 (1974). (10) J. E. Dye, C. Knight, and T. W. Cannon, Proceedings of the Fourth Conference on Weather Modification, Fort Lauderdale, Fla., 1974. (1 1) G. Mulvey and J. D. Sheaffer, submitted to J . Appl. Mefeorol. (12) Cuthbert Daniel, Technometrics, 1, 311 (1959). (13) J. S.Rustagi, Arcb. Environ. Hea/fh, 8, 68 (1964). (14) L. A. Ahrens, Geochim. Cosmochim. Acta, I , 49 (1954).
RECETCTD for review March 30,1978. Accepted May 12,1978.
Chemiluminescence Fiber Optic Probe for Hydrogen Peroxide Based on the Luminol Reaction Thompson M. Freeman and W. Rudolf Seitz" Department of Chemistry, University of New Hamsphire, Durham, New Hampshire 03824
cence (CL) fiber optic probe. This approach is applicable to any enzyme-catalyzed process that leads to the production of light. The enzyme catalyst is immobilized on the surface of a fiber optic. When the fiber optic is immersed in a solution of substrate, light will be generated as substrate diffuses into the immobilized enzyme phase. This light is transmitted through the fiber optic to a photomultiplier tube. The light level will reach a steady state when the rate at which substrate diffuses into the enzyme phase equals the rate at which substrate reacts in the enzyme phase. If the light-producing reaction is first order in substrate, then steady-state CL will be proportional to substrate concentration. CL fiber optic probes offer several potential advantages for chemical analysis. By immobilizing the enzyme catalyst, it is possible to reuse the enzyme many times. Often immobilization leads to an increase in enzyme stability. The advantages of immobilization have been well documented and have led to considerable work to develop analytical methods utilizing immobilized enzymes (1-3). The main advantages of CL methods are low detection limits and wide linear dynamic ranges with simple instrumentation ( 4 , 5 ) . It should
A chemliuminescence (CL) fiber optic probe for hydrogen peroxide based on the iumlnol reaction has been constructed by immobilizing peroxidase in a polyacrylamide gel on the end of a fiber optlc. When the probe is immersed in a solution of peroxide in the presence of excess lumlnol, CL is generated as peroxide diffuses into the peroxidase phase. The fiber optic transmks CL to a detector. The theory of CL fiber optic probes has been developed assuming a first-order reaction In the enzyme phase. However, the peroxide probe shows response which is second order in peroxide. The detection limit is close to lo-' M peroxide. The time required to reach steady state M H,02, steady varies with peroxide concentration. At state is reached in about 4 s. Under the conditions usually employed, the CL intensity is limited by the rate of mass transfer from solution to the surface of the enzyme phase rather than by the activity of the peroxidase.
In this paper we report a new approach to using immobilized enzymes for analytical purposes, the chemilumines0003-2700/78/0350-1242$01 OO/O
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ANALYTICAL CHEMISTRY, VOL. 50, NO 9, AUGUST 1978
be pointed out that CL fiber optic probes are only one of several possible analytical configurations involving CL catalyzed by immobilized enzymes. For instance, immobilized enzymes can also be used as sensors in flow systems by having the analyte flow over the immobilized phase which is positioned opposite a light detector. An example of this has been reported by Hornby (6). Also, an ATP assay has been developed by injecting sample into a solution containing firefly luciferase immobilized on a glass rod (7). The CL fiber optic probe is similar in many ways to enzyme electrodes. Both involve a thin layer of enzyme over the surface of a sensor. However, there are several important differences. The enzyme electrode requires not only that substrate diffuse into the enzyme phase but also that product diffuse to the surface of the electrode. The time required t o reach steady state depends on the thickness of the enzyme layer. In the CL fiber optic probe, the reaction of substrate is detected directly via the light generated, and there is no need for product to diffuse to the surface of the fiber optic. As a result, response time is independent of the thickness of the enzyme layer, and it is not necessary to prepare a thin layer of enzyme. I t is, however, necessary to use an immobilized phase which is transparent to the CL emitted. This limits the number of possible enzyme phases suitable for CL fiber optic probes. T h e system chosen for this study was the luminol reaction catalyzed by peroxidase.
&
coo- * coo-
t N2 t 3H20
NY
-
t light Xwx=4x)
nrn
If luminol is in excess, the CL intensity will reflect the amount of peroxide. This system was chosen because peroxidase is inexpensive and available with high specific activity. Also, the authors have previous experience working with luminol and coupling enzymatic processes to the formation of hydrogen peroxide (8, 9). However, it should be emphasized that the advantages of the CL fiber optic probe approach will be more significant for some of the bioluminescence reactions, particularly the firefly reaction used to determine adenosine triphosphate (10) and bacterial bioluminescence used to determine NADH (11). For both these reactions, enzyme cost. availability, and stability are important considerations which have limited analytical usage.
THEORY T h e intensity observed using a CL fiber optic probe is proportional to the number of molecules reacting per unit time in the enzyme phase. Because this differs from other enzyme probes, a brief theoretical treatment relating the observed signal to enzyme activity in the immobilized phase will be presented. Initially, it will be assumed that mass transfer in solution is efficient enough so that the concentration of substrate a t the surface of the enzyme phase equals the substrate concentration in the bulk of the solution. In practice, the solution is stirred so that mass transfer in solution should be considerably more efficient than mass transfer in the enzyme phase, which occurs by diffusion; however, if the activity of the immobilized enzyme is high enough, the substrate may be reacted a t the surface rapidly enough to cause significant concentration depletion a t the surface. It is also assumed t h a t the enzyme-catalyzed reaction follows
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x
0 x-
Figure 1. Model of CL fiber optic probe. The substrate concentration profile assuming mass transfer to the probe surface is fast enough to be neglected is designated by the smooth line. The dotted line shows the profile if the reaction at the surface of the probe is rapid enough to deplete the surface concentration of substrate
Michaelis-Menten kinetics and t h a t the substrate concentration is much smaller than K,, the Michaelis constant. In this case, the rate of the enzyme-catalyzed reaction is proportional to substrate concentration. T h e model of the system is illustrated in Figure 1. At steady state, Le., when the rate of substrate entering the enzyme phase equals the rate a t which substrate reacts, the distribution of substrate within the enzyme phase is given by the following equation (12):
T h e terms in Equation 1 are defined as follows: S(x) = substrate concentration a t position x in the enzyme phase (mol/L); Sbulk = substrate concentration in bulk solution (mol/L); X = membrane thickness (cm); V,, = rate of substrate conversion in the gel under conditions of excess substrate (mol/L-s); K , = Michaelis constant (mol/L); D, = diffusion coefficient for substrate in gel (cm2/s). What we are actually interested in is the number of moles reacting per unit time V. For Michaelis-Menten kinetics with [SI
