Anal. Chem. 1087, 5 9 , 1859-1863
of data interpretation must be explored to increase investigators’ comprehension of these complex data. Broader use of chemometrics could add much to their ability to describe complex mixtures of contaminants and to quantitate their biological and ecological impacts.
Registry No. Aroclor 1242,53469-21-9;Aroclor 1248, 1267229-6; Aroclor 1254, 11097-69-1; Aroclor 1260, 11096-82-5. LITERATURE CITED Stalling, D. L.; Schwartz, T. R.; Dunn W. J., 111; Petty, J. D. in Envlronmental Applications of Chemometrics; Breen, J. J., P. E. Roblnson, Eds.; ACS Symposium Serles, 292; American Chemical Society, Washlngton, DC, 1985; pp 1-15. Kowalski, 6.;Chem. Ind. (London) 1978, 18, 882-884. Dunn. W. J.. 111: Stalllna. D. L.: Schwartz. T. R.: Hoaan. J. W.: Pettv. ,. J. D. Anal. Chern. 1984, 56, 1308-1313. Wold, S. I n ChemonmMcs-Mathematlcs and Statistics in Chemistry; Kowaiski, 8. R., Ed.; Reldel: Boston, MA, 1984; pp 17-95. Wold, S.; Albano, C.; Dunn, W. J., 111; Ebensen, E.; Helberg, S.; Johansson, E.; Sjostrom, M. I n Multivariate Data In Food Research and Data Ana/ysls; Martens, H., Russwurm, H., Jr., Eds.; Applied Science: New York, 1983; pp 147-188.
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(6) Wold, S. Technometrlcs 1978, 20, 397-406. (7) Onuska, F. J.; Mudroch, A.; Davies, S. HRC CC J . H/gh Resolut. Chromatogr. Chromatogr. Commun. 1985, 8, 748-754. (8) Schwartz, T. R.; Stalling, D. L. Rice, C. L. Environ. Sci. Techno/. 1987, 21, 72. (9) Schwark T. R.; Campbell, R. D.; Stalling, D. L.; Little, R . L.; Petty, J. D.; Hogan, J. W.; Kaiser, W. M. Anal. Chem. 1984, 56, 1303-1308. (10) Shannon, C. E.; Weaver, W. I n The Mathematical Theory of Comrnunication; University of Illinois Press: Urbana, IL, 1947. 111) Cieij, P.; Dijkstra, A.; Fresenius’ Z.Anal. Chem. 1979, 298,97-109. i12j Marlen, G.; Dijkstra, A. Anal. Chem. 1976, 48, 595-598. (13) Scott, D. R. Anal. Chem. 1986, 58, 881-890. (14) Alford, A. L.; Bellar, T. A.; Eichelberger, J. W.; Budde, W. L. Anal. Chem. 1988, 58, 2014-2022. (15) Capei, P. D.; Rapaport, R. A.; Eisenreich, S. J.; Looney, B. 6.Chemosphere 1985, 14, 439-450.
RECEIVED for review June 24, 1986. Resubmitted December 17,1986. Accepted March 17,1987. Reference to trade names, commercial products, or manufacturer does not imply or government endorsement Or recommendation for use.
Individual and Simultaneous Determination of Ethanol and Acetaldehyde in Wines by Flow Injection Analysis and Immobilized Enzymes Fernando Ldzaro, M. D. Luque de Castro, and Miguel Valclrcel*
Department of Analytical Chemistry, Faculty of Sciences, University of CGrdoba, Cdrdoba, Spain
Several methods for the individual and simultaneous photometric determinatbnof ethanol and acetaldehyde In wines by use of hnmoblllzed alcohol and acetaldehyde dehydrogenases that improve on those available are proposed in this paper. The Individual determinations are carried out on a straighlforward flow injection analysis (FIA) manifold that includes a suitable enzyme reactor. The features of the methods are as follows: linear range of the calibration curves, (2-14) X IO-‘% (v/v) of ethanol (0.5-11.0 pg/mL of acetaldehyde); correlation coefficient, 0.996 (0.999) relative standard deviation *0.6% (*0.5%) for ethanol (acetaldehyde), with a sampling frequency of 40 h-’ in both cases. The simuttaneous determination uses a dual injection valve, which inserts the sample into channels of different length, each of which indudes an enzyme reactor. One peak per analyte is obtained. The appkatlon of the methods to white, red, and sweet wlnes provlded excellent results. I n spite of the different concentrations of these analytes in wines, the analysis is performed with a single dilution of the samples.
Since the early 1980s the use of immobilized enzymes in flow injection analysis (FIA) has increased considerable, as reflected in the publication of over 50 papers on this subject (most of them in the last 3 years (I)). This is a result of the advantages lying in enzyme immobilization, which helps to overcome the two most serious shortcomings involved in the use of these catalysts, namely enzyme instability and high cost per analysis. In addition, immobilization endows determinations with convenience and simplicity. These are features of great relevance to areas of chemical analysis in which a large number of samples have to be processed with minimum costs 0003-2700/67/0359-1859$0 1.50/0
and in as short a time as possible (e.g. clinical, environmental, and food analysis). Thus, the association of FIA with immobilized enzymes offers a promising future to food analysis as far as the determination of enological parameters are concerned on account of the fact that most of the methods commonly used in this field are slow and scarcely selective and entail a prior separation step (2). This problem has been satisfactorily solved by the FIA immobilized enzyme association in joining the rate, sensitivity, and precision of the FIA technique with the selectivity, economy, and convenience of immobilized enzymes. Enological-enzymatic analyses by FIA has been infrequently applied up to now; in fact, only eight papers (3) have been published on this topic. Among them, two use dissolved enzymes for the photometric determination of ethanol by conventional FIA ( 4 ) and for ethanol and acetaldehyde by the merging-zones mode (5), while another describes the enthalpy determination of glucose with immobilized enzymes ( 6 ) . Thus, the methods proposed herein for the individual and simultaneous determination of ethanol and acetaldehyde in wines by the use of immobilized enzymes constitutes an important contribution to the modernization of enological analysis. These methods are based on the oxidation of ethanol by oxidized @-nicotinamideadenine dinucleotide (NAD+), catalyzed by alcohol dehydrogenase (ADH) at an alkaline pH (7), with semicarbazide being used as a trapping agent for the acetaldehyde formed in the reaction. Acetaldehyde is determined by oxidation with NAD+ in the presence of aldehyde dehydrogenase (AlDH) in a basic medium (8). NADH is monitored photometrically a t 340 nm in both cases.
EXPERIMENTAL SECTION Apparatus. A four-channelGilson minipuls-2 and a n Ismatec mini-S-840peristaltic pump, a variable-volumeTecator L100-1 @ 1987 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 14, JULY 15, 1987
A)
S
Pump (C’)
c
[B’)B
91
4
1‘
‘2
‘3
Photometer I
W
Recorder
Pump
C’
B‘
Figure 1. Configuration used for the individual (A) and simultaneous (B) determination of ethanol and acetaldehyde: S,C, and B denote sample, carrier, and buffer, respectively: w stands for waste: r 2 is the enzyme reactor for the individual determinations and for ethanol in the simultaneous determination; r2’ is the enzyme reactor for acetaldehyde in the simultaneous determination. The meaning and value of each variable (q q 2 , r o , etc.) is shown in Table I
,,
injection valve, a Tecator type I11 chemifold, a Hellma 178.12 QS flow cell with an inner volume of 18 pL, and a Perkin-Elmer A - 1 spectrophotometer connected to a Perkin-Elmer RlOO recorder were employed to set up the individual determinations. A home-made variable-volume dual injection valve was used instead of the single one to carry out the simultaneous determination. PVC pump tubing of different diameter suited to the required flow rate and Teflon connecting tubing, 0.5-mm i.d., were also used. Reagents. Standard absolute ethanol and distilled acetaldehyde (assayed by iodometric back titration (9))were used. Alcohol dehydrogenase (EC 1.1.1.1,ca. 400 units/mg of protein), aldehyde dehydrogenase (EC 1.2.1.5, ca. 26.5 units/mg of protein) and oxidized p-nicotinamide adenine dinucleotide (NAD’) were supplied by Boehringer Mannheim. Controlled-pore glass (CPH 02000, 80/ 120 mesh, 202.5-nm mean pore diameter) purchased from Electro-Nucleonics, Inc., (3-aminopropy1)triethoxysilane(Sigma),protected from moisture and stored at 4 OC, and 25% glutaraldehyde (Merck) were employed to immobilize ADH and AlDH. The following buffer solutions were used: For the individual determination of ethanol (buffer B), a mixture of 33.0 g/L Na4Pz07,1.6 g/L glycine, 8.8 g/L NaCl, 2.0 g/L NAD’, and 8.0 g/L semicarbazide hydrochloride adjusted to pH 8.2 with sodium hydroxide; for the individual determination of acetaldehyde and simultaneous determination of ethanol and acetaldehyde (buffer B’), a mixture of 33.0 g/L Na4P,07, 5.0 g/L KC1, and 1.0 g/L NAD+, adjusted to pH 8.2 with hydrochloric acid. Carrier solutions (C and C’) and samples (S)contained 40% of buffer (B or
B’) to ensure a similar refractive index and the samples were adjusted to an alkaline pH suitable for release of the bound acetaldehyde prior to injection. Configurations. The individual determinations were performed with the manifold shown in Figure 1A. Both are based on the insertion of a sample (S) volume (VI) into a carrier stream C (or C’), which then merges with the buffer solution B (or B’). As the sample plug passes through r2 the immobilized enzyme catalyzes the above described reactions, which give rise to an FIA peak on passage of the reacting plug through the detector. The simultaneous determination is founded on the simultaneous insertion of two sample volumes (VI, V J into carrier streams (C’), which later merge with the buffer solution (B’). The plugs circulate through reactors r1 and rl’, of different length (rl >> r:), react in r2 (ethanol) and r2) (acetaldehyde),and reach the detector at different times, yielding two peaks. Immobilization and Reactor Packings. The glass, after cleaning, was alkylaminated with (3-aminopropy1)triethoxysilane and the cross-linking agent (glutaraldehyde)coupled as described by Masoom and Townshend (IO). Immobilization was performed by the following procedure: Alcohol dehydrogenase, 4000 units (or aldehyde dehydrogenase, 250 units), was dissolved in 2.0 mL of cold (4 “C), deoxygenated phosphate buffer (0.1 M, pH 6.0) and added to 500 mg of activated glass. This solution was kept at 4 OC overnight in an argon atmosphere, then the glass was packed in a PVC reactor (1.14 mm i.d. and the required length) and was washed with cold phosphate buffer to remove unlinked enzyme. Each reactor can be used for about 500 sample injections if stored at 4 “C and in a phosphate buffer after usage. In this
ANALYTICAL CHEMISTRY, VOL. 59, NO. 14, JULY 15, 1987
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Table I. Optimum Values of the Variables determination
variables
both analytesb in ethanola acetaldehyde" mixture 1.0 33.0
1.0 33.0
2.0 33.0 8.8 1.6 8.0 8.2 7.0-10.3 25-40 0.5 0.7
8.0 11.0 5.0
5.0
-
.-
8.2 7.0-10.3 35-40 0.5 0.7 8.0 621 11.0
5.0
20.0
50.0
50.0
30.0
200.0
20.0 6.0 50.0 30.0 200.0
0.8Q,
U
0.6-
n L
0 ul
0.L0.2 -
A
.-c>
0
8.2 7.0-10.3 35-40 0.5 0.7 8.0 11.0
1.0-
Q
LO
U
Manifold A. Fieure 1. Manifold B. Finure 1.
fi
-
e
5.0
$
>.
60
r
r-
10 11 pH Flgure 2. Influence of pH on the analytical signal for the systems: (a) ethanol/NAD+/alcohol dehydrogenase: (b) acetaldehyde/NAD+/ aldehyde dehydrogenase. way, 0.5 unit of ADH and 0.1 unit of AlDH per sample are consumed.
RESULTS AND DISCUSSION Determination of Ethanol. The optimization of the chemical and FIA variables affecting the system was performed by univariate method; the values taken as optimum are listed in Table I. The study of the pH of the pyrophosphate buffer shows an increase in the analytical signal with this variable up to p H 10.3, above which the signal decreases (Figure 2). The rising trend is a result of the chemical reaction being favored by the alkalinity of the medium, while the falling trend is due to the loss of enzymatic activity at very basic pHs. Figure 3 shows the loss of activity with the pH after 10 min of reactor use. The rate of loss decreases with increasing time, so that, for a p H of 10.3, the decrease in the absorbance of the FIA peak after 10 min is 25%, while after 30 min it is 52%; therefore a p H of 8.2 was chosen for further experiments. The pH of
0
20-
ul 0
-J
0-
6
7
8
9
10
11 p H
Flgure 3. Influence of pH on the stability of the immobilized enzyme expressed as the loss of activity after 10 min of reactor use: (a) alcohol dehydrogenase; (b) aldehyde dehydrogenase.
the sample (containing 40% pyrophosphate buffer) can range between 7.0 to 10.3 without appreciable changes in the signal arising from its mixing with the buffer-carrier before arriving a t the reactor. Neither the glycine, pyrophosphate, nor sodium chloride concentration affect the analytical signal over a wide concentration range; conversely, the semicarbazide concentration exerts a significant influence through its trapping effect, increasing the peak height at concentrations up to 8.0 g/L, above which the signal remains constant. The influence of NAD+ is similar, its optimum concentration being 4.0 g/L. Temperature positively influences the reaction development up to 25 "C, having no further effect on the peak between 25 and 40 "C. The signal increases as the flow rate decreases (longer reaction time) and the sample volume increases (higher analyte concentration); thus, a flow rate of 1.2 mL/min and a sample volume of 200 KLwere chosen. The peak height increases with the reactor length up to 18.0 cm, from which the signal remains constant. The lengths of the rest of the reactors are as small as possible, because their sole purpose is to interconnect the different units of the system. Under these working conditions, a calibration curve was linear between 2.0 X lo-' and 14.0 X lo4% (v/v) ethanol, with a small intercept (0.013 f 0.006 absorbance units) and good slope (108.3 f 0.7% (v/v)-l) and correlation coefficient (r2= 0.996), the relative standard deviation (RSD) being &0.6% (for 11determinations of 8.0 X lo4% ethanol). Nevertheless, 1:20000 dilution of the wine sample is necessary, which, in addition to introducing significant error, is not suitable for the simultaneous determination tackled later. For these reasons, and in order to widen the determination range in the upper part, the following steps were taken: The sample volume was decreased from 200 to 30 gL, as was the NAD+ concentration (from 4.0 to 2.0 g/L) and the enzyme reactor length (from 18.0 to 5.0 cm). Under these conditions, the calibration curve showed a logarithmic shape between 2.5 X and 50.0 X (v/v) of ethanol: log (absorbance) = 0.48 (f0.04) + 0.64 (f0.02) log [ethanol]; correlation coefficient = 0.996. The RSD for 11determinations of 2.5 X loT3%(v/v) analyte was f0.3% and the sampling frequency was 40 h-l. The study of the recovery was performed by adding two different amounts (0.01% and 0.02%, v/v) of analyte to different types of wine (white, red, sweet) and beer (pilsen, lager), obtaining a mean recovery of 100.5%, and an average
ANALYTICAL CHEMISTRY, VOL. 59, NO. 14, JULY 15, 1987
1862
Table 11. Recovery for the Determination of Ethanol in Winen and Beerb Samples sample
concn found, % (v/v)
white wine, white wine, white wines white wine, sweet winel red wine, pilsen beer, pilsen beer, lager, lager,
17.5 15.6 12.9 17.2 16.1 12.4 4.6 4.4 4.8 4.0
Dilution, 1:2600.
'70 recovery +0.01% (v/v) +0.02% (v/v) 105.0 102.2 97.8 103.0 101.4 98.0 101.6 103.4 101.5 98.3
100.3 96.6 96.0 102.8 99.8 97.0 102.8 101.3 100.2 100.3
Dilution 1:666.
Table 111. Recovery for the Determination of Acetaldehyde in Winesn wine
concn found, #g, mL
whitel white, whites white, white5 whites sweet, sweet, red, red,
173.2 220.2 160.9 204.5 493.3 204.0 245.3 190.5 200.5 210.0
R recovery +3.0 pg/mL +6.0 Mg/mL 100.9 100.3 102.4 101.0 100.2 101.6 100.3 103.2 98.2 103.2
99.2 101.7 99.2 101.7 97.2 100.2 99.6 101.7 97.5 102.4
Dilution. 1:150.
deviation of the recovery with respect to 100% of *2.1% (see Table 11). These results show the excellent selectivity of the proposed method, attributable to the use of enzymes and to the great dilution necessary (1:2600 for wines and 1:666 for beers), this
last being the cause of the negligible blank signal. Determination of Acetaldehyde. The influence of the pH of the pyrophosphate buffer on the analytical signal is similar to that exerted on the determination of ethanol (Figure 2). The loss of activity over time, Figure 3, is more marked than for alcohol dehydrogenase; thus, for the same reason, a pH of 8.2 was chosen. The sample pH and the pyrophosphate concentration have no effect on the reaction. The presence of a high concentration (above 5.0 g/L) of potassium ion, as KC1, increases the enzymatic activity of AlDH (8)and stabilizes the signal. An increase in the NAD+ concentration between 0 and 0.5 g/L results in a considerably increased signal, which remains constant above 1.0 g/L. The temperature favors the reaction up to 35 "C; the reaction rate stays constant between 35 and 40 O C . The influence of the FIA variables on this system is analogous to that described above for ethanol; thus, a flow rate of 1.2 mL/min, a sample volume of 200 wL, and an enzymatic reactor length of 20.0 cm were selected as the most suitable. The calibration curve was linear between 0.5 and 11.0 pg/mL, with an almost nil intercept (0.006 0.005 absorbance units), a slope of 0.0535 f 0.0009 (pg/mL)-l, and an excellent correlation coefficient (r2= 0.999). The RSD was f0.5% for 11determinations of 5.0 wg/mL. The sampling frequency was 40 h-l. To test the validity of the method the recovery of acetaldehyde (3.0 and 6.0 pg/mL) in several types of wines (white, red, and sweet) of different origin was assayed, obtaining the results shown in Table 111. The mean recovery was 100.6% and the deviation from 100% was very good (f1.5%). The determination of acetaldehyde in beer was not attempted because of the low concentration level of this analyte.
Simultaneous Determination of Ethanol and Acetaldehyde. The manifold in Figure 1B has been used for this determination; this is a combination of the manifolds used for the individual determinations. One shortcoming of this method is the different concentration level of both analytes in wines, which calls for very different dilutions (1:2600 and
Table IV. Recovery for the Simultaneous Determination of Ethanol and Acetaldehyde in Wines ethanol
acetaldehyde
Yo recovery
70 recovery wine
concn found, % (v/v)
+0.1%
+0.2%
concn found, Mg/mL
+3.5 pg/mL
+7.0 pg/mL
white, white, whites white, sweet, sweet, redl red2
17.4 15.5 13.0 17.2 16.3 14.9 12.5 12.9
102.3 101.6 102.9 101.8 103.2 106.2 98.0 102.4
96.2 95.5 97.7 99.4 99.6 101.3 98.9 98.9
175.0 218.2 160.5 204.0 248.0 188.0 201.0 212.0
101.0 101.7 103.5 100.3 102.2 99.4 101.5 100.4
96.3 98.1 96.9 100.5 99.8 99.7 100.1 99.7
Table V. Comparative Study of the Use of Dissolved and Immobilized Enzymes
parameter RSD, % linear range ratioC determination limitd enzyme consumption per analysis, U mean recovery, % mean deviation of recovery from 100% sampling frequency, h-'
determination of ethanol S" Ib 0.7 8 0.002 8.0 55
0.3 20 0.0025 0.5 100.5 f2.1 40
determination of acetaldehyde S I 0.5 8 1.0 0.7 102 f2.0 50
0.5 22 0.5 0.1 100.6 f1.5 40
simultaneous determination ethanol acetaldehyde S I S I 0.3 10 0.02 8.0 101.4 f4.7 25
0.3 32 0.025 0.5 100.4 f2.3 23
0.5 7 0.3 0.7 98.8 f3.3 25
0.4 16 0.9 0.1 100.1 11.3 23
Use of dissolved enzymes. bUse of immobilized enzymes. Ratio of the upper to the lower limit of the calibration curve. dConcentrations are expressed in 70 (v/v) for ethanol and fig/mL for acetaldehyde.
1863
Anal. Chem. 1987, 59, 1863-1867
1:150 for ethanol and acetaldehyde, respectively). In a previous paper (5) this problem was solved by using a diode array spectrophotometer, allowing for application of dilution and amplification methods (11). In this paper the problem was solved by using the optimum working conditions for determination of acetaldehyde and by circulating the plug for determination of ethanol through a very long and wide reactor (rJ. This reactor (621 cm long, 0.7 mm i.d.1 exerts two recognizable effects: (a) It provides a longer residence time for the ethanol plug, whose peak appears after that of acetaldehyde has attained the base line. (b) It dramatically dilutes ethanol in plug VI, making possible the use of sample dilution suitable for acetaldehyde (1:150) in the simultaneous determination. With this manifold, a calibration curve for acetaldehyde was run between 0.9 and 15.0 pg/mL: absorbance = 0.012 (f0.008) 0.0272 (fO.0001) [acetaldehyde]; r2 = 0.996; the RSD being *0.4% (11 determinations of 7.0 pg/mL). For ethanol, the experimental points were ajusted to a logarithmic curve in the range 0.025%-0.800% (v/v): log absorbance = 0.825 (f0.005) 0.509 (fO.009) log [ethanol]. The sampling frequency was 46 h-l. The recovery of both species in different types of wines was assayed by adding 0.1% of ethanol + 3.5 pg/mL of acetaldehyde and 0.2% ethanol + 7.0 pg/mL of acetaldehyde to the samples. From the experimental data were calculated a mean recovery of 100.4% and 100.1%, and an average deviation from 100% of f2.370 and f1.370 for ethanol and acetaldehyde, respectively (see Table IV).
+
+
CONCLUSIONS A comparative study between methods involving dissolved ( 5 ) and immobilized enzymes (see Table V) leads to the following conclusions: (a) Precision, expressed as RSD, for the use of immobilized enzymes is equal to of higher than with dissolved ones. (b) The use of immobilized enzymes dramatically widens the linear range of the calibration curve, increasing the ratio between the lower and upper limit at least 2-3-fold with respect to that achieved with dissolved enzymes. The determination limit for ethanol is similar in both methods; yet, while in the individual determination of acetaldehyde the lower determination limit corresponds to methods with im-
mobilized enzymes, in the simultaneous determination the lower determination limit is achieved in the method with dissolved enzymes. This apparent contradiction can be clarified by considering that the use in the last case of a diode array detector, which allows application of a method for amplification of the analytical signal, results in increased sensitivity and a lower determination limit (11). (c) Enzyme consumption per analysis is considerably lower (between 7and 16-fold) for immobilized enzymes. (d) The recovery is better when enzymes are immobilized and so is the stability and convenience. (e) The sampling frequency is only slightly higher for dissolved enzymes. All these arguments testify to the suitability of the proposed methods for use in enological control laboratories.
ACKNOWLEDGMENT We gratefully acknowledge “Gonzdez Byass” for providing samples of wine. Registry No. EtOH, 64-17-5; acetaldehyde, 75-07-0; alcohol dehydrogenase, 9031-72-5; aldehyde dehydrogenase, 9028-88-0.
LITERATURE CITED Yao, T. J . Flow Injection Anal. 1985, 2(2), 115. Ribereau-Gayon, J.; Peynaud, E.; Sudrau, P.; Riberau, P. Trait6 d ’ E n ologie. Sciences Techniques do Vin; Tome I. Dunod: paris, 1976. Lizaro, F.; Luque de Castro; M. D., Valclrcel, M. Enologia Enotecnica , in press. Worsfold, P. J.; Ruzicka, J.; Hansen, E. H. Anal. Chim. Acta 1981, 106, 1309. Lizaro, F.; Luque de Castro, M. D.; Valcircel, M. Anal. Chim. Acta 1986, 185, 87. Kiba, N.;Tomiyasu, T.; Furusawa, M. Talanta 1984, 31, 131. Bernt, E.; Gutrnann, I. I n Methods of Enzymatic Analysis. 2nd ed.; Bergmeyer, H. U., Ed.; Verlag Chemie: Weinheim, and Academic: New York, 1974; Vol. 3, pp 1499-1502. Lundquist, F. I n Methods of Enzymatic Analysis, 2nd ed.; Bergmeyer, H. U., Ed.; Veriag Chemie: Winheim, and Academic: New York, 1974; Vol. 3, pp 1509-1513. Official Methods of Analysis, 12th ed.; Association of Official Analytical Chemists: Washington, DC, 1975; p 603. Masoom, M.; Townshend, A. Anal. Chim. Acta 1984, 166, 111. Lizaro, F.; Rios, A,; Luque de Castro, M. D.; Vaicircel, M. Anal. Chim. Acta 1988, 179, 279.
RECEIVED for review December 3, 1986. Accepted March 9, 1987. The “Comisi6n Asesora de InvestigaciBn Cientifica y TBcnica” is thanked for financial support (Grant No. 2012/83).
Electrochemical Pretreatment of Carbon Fibers for in Vivo Electrochemistry: Effects on Sensitivity and Response Time Jian-Xing Feng,’ Michael Brazell, Kenneth Renner, Richard Kasser, and Ralph N. Adams* Department of Chemistry, University of Kansas, Lawrence, Kansas 66045
Oxidative electrochemical pretreatment of carbon flbers greatly improves thelr sensitivity for in vivo electrochemical detection of catecholamine species. I t is shown that the extent of the anodic potentlal excursion In the pretreatment is a major factor in both the sensltivlty and the response time of the resulting flber electrode. The high sensitlvlty for neurotransmitter specles such as dopamlne appears malniy due to adsorptlon on the oxldized carbon fiber surface states. Practlcal protocols for flber electrodes to be used In In vivo brain studies are evaluated. Permanent address: N a n k a i University,
Republic of China.
Two quite diverse applications have spurred studies of the electrochemical pretreatment of carbon fibers in recent years. The fibers are used extensively in resin composites to provide high strength-low weight structural materials, especially for jet aircraft. Oxidative treatments are known to improve the fiber-resin bonding and electrochemical oxidation is a versatile means of pretreatment. Sherwood and co-workers have utilized a variety of spectroscopic techniques to study the surface states of these pretreated fibers (1-5). Although of far less industrial importance, equally intense interest has developed in the use of carbon fiber microelectrodes as in vivo electrochemical detectors to detect biogenic amine neurotransmitters and their metabolites in the extracellular fluid
0003-2700/87/0359-1863$01.50/0 0 1987 American Chemical Society
