950
Anal. Chem. 1907, 59, 950-954
(8) Malinowski, E. R. Anal. Chim. Acta 1962, 734, 129. (9) Malinowskl, E. R.; Cox, R. A.; Haldna, U. L. Anal. Chem. 1984, 56, 778. (10) Ozeki, T.; Kihara, H.; Hikime, S.BunsekiKagaku 1986, 3 5 , 885. (11) Murata. K.; Ikeda, S . Anal. Chim. Acta 1963, 757,29. (12) Murata, K.; Ikeda, S . Po/yhedron 1983, 2, 1005. (13) Murata, K.; Ikeda, S . Spectrochim. Acta, Part A 1983, 39A, 787. (14) Sasaki, Y.: Lindqvist, I.; SillBn, L. G. J . Inorg. Nucl. Chem. 1959, 9 , 93. (15) Sasaki, Y.; SillBn, L. G. Acta Chem. Scand. 1964, 78,1014. (16) Sasaki, Y.; SillBn, L. G. Ark. Kemi 1967, 29, 253. (17) Freedman, M. L. J . Inorg. Nucl. Chem. 1963, 25, 575. (18) Aveston, J.; Anacker, E. W.: Johnson, J. S. Inorg. Chem. 1984, 3 , 735.
(19) Griffith, W. P.; Lesniak, P. J. B. J . Chem. Soc. A 1969, 1966. ( 2 0 ) Tytko, K. H.; Schonfeld, B. 2.Naturforsch., B 1975, 308, 471. (21) Tytko, K. H.; Baethe, G.; Hirschfeld, E. R.; Mehmke, K.; Stellhorn, D. Z . Anorg. Allg. Chem. 1983, 503, 43. (22) Malinowski, E. R.; Howery, D. G. Factor Analysis in Chemistry; Wiiey: New York, 1980. (23) Malinowski, E. I?.,private communication, 1985. (24) Lindqvist. I . Ark. Keml 1950, 2, 349. (25) Lindqvist, 1. Ark. Kemi 1950, 2 , 325. (26) Shimao, E. Bull. Chem. SOC.Jpn. 1967, 4 0 , 1609.
RECEIVED for review August 4,1986. Accepted November 24, 1986.
Doubly Stopped Flow: A New Alternative to Simultaneous Kinetic Multideterminations in Unsegmented Flow Systems Fernando Ldzaro, M. D. Luque de Castro, and Miguel ValcBrcel* Department of Analytical Chemistry, Faculty of Sciences, University of Cbrdoba, Cbrdoba, Spain
We have deslgned a new assembly for simultaneous multldetermlnatlons by the stopped-flow technique. I t involves both the kinetk and the nonlclnetlc modes commonty used in flow Injection analysts. The device Includes a dual Injection valve that Inserts the sample Into two channels: bolus, Is retalned at the detector as the flow Is halted throughout the system for the flrst t h e and Is used to monHor the development of the reaction of Interest; meanwhlle, bolus, Is kept In a reactor, where a step prior to the Indicator reaction takes place. The start of the pump allows the second bolus to reach the detector after merging with the reagent, the slgnal corresponding to the evolution of the reaction of this second analyte being measured durlng the second halting of the flow. The device has been successfully used in the determination of free and bound SO2 in wines (bound SO2 requlres a prlor hydrolytic release).
The high cost and sophistication of the instrumentation used in the application of the conventional stopped-flow technique limits its scope of application to fast reactions (half-lives less than 10 s). Flow injection analysis (FIA) (I) is a novel way to implement this technique involving the use of a timer synchronizing the start and stop of the peristaltic pump with the injection and the stopping of the reacting bolus either in the reactor or in the flow cell according to a nonkinetic mode, that makes possible the use of slow reactions in FIA and provides increased sensitivity of the method in all cases, or a kinetic mode. The FJA/stopped-flow methodology can be applied to reactions with half-lives in the range 5 s to 10 min. The methods described so far deal with the determination of individual species and, occasionally, of two analytes (I). The design presented here is aimed to the resolution of complex mixtures in which the determination of one of the analytes requires a step prior to the indicator reaction and involves a simultaneous dual injection with parallel valves and stopping the flow twice sequentially. The first halting coincides with the passage of one of the reacting boluses (bolusl) through the detector and the monitoring of its evolution. The other bolus (bolusz) is kept in a reactor, where reaction preceding that of the interest takes place. In the second halting, bolus2 is kept in the detector, where the development of the
corresponding indicator reaction is monitored. One of the salient applications of this assembly is the determination of free and bound SOz in wines. Sulfur dioxide is added to wine during its elaboration to avoid undesirable oxidation processes along the different steps involved. It is usually found in wine either bound to carbonyl or unsaturated compounds and/or phenol derivatives or free, as HS03- and SOz, this last being the sole species with antiseptic properties. The interest in the determination of SO2 arises from (a) the differences in the maximum allowed levels established by legislation in each country, (b) the monitoring of the disappearance of SO2from wine during aging (diffusion, oxidation, and binding) to determine the amount of SOz to be added, and (c) the need to avoid the disagreeable aroma and taste that SOz gives wine (2). The analysis of free and total SO2is conventionally carried out directly or with the prior separation of the analyte. The direct determination is carried out titrimetrically with iodine (3-5) and calls for the use of one aliquot to determine free SOz,another for total SO2 (after a prior alkaline hydrolysis), and a third one to measure the blank after addition of a chelating agent for SOz. The prior separation step (dragging vacuum (8), or distillation (9))requires the by N2 (61, air (7), use of two aliquots. Measurements are either titrimetric (6, 10)or gravimetric (these last are made after oxidizing SO2 to H2S04with HzOz (9)). The FIA methods proposed so far for the determination of this analyte in different samples and with different types of detection (11-22) show the potential of FIA for analysis of SO2. The interferents in the determination of this analyte in wines are eliminated by using a gas-diffusion cell (15,22) or the stopped-flow technique (21).However, only free SO2 (15,21)or free and total SOz individually (22)can be determined in this way. We have developed a method for the simultaneous determination of free and total SOz by use of the new assembly and a doubly stopped flow mode based on the formation of a colored compound (ArnB 578 nm) between the analyte, formaldehyde, and p-rosaniline (23).Simultaneous determinations carried out with the proposed assembly (Figure 1)involve the following: Simultaneous injection of the sample, which merges at point b in bolusl with the p-rosanilinelformaldehyde mixture, and halting of the stream containing this bolus as it reaches the detector, where the indicator reaction is mon-
0003-2700/87/0359-0950$01.50/00 1987 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 59, NO. 7, APRIL 1, 1987
951
Table I. Optimization of Variables
optimization methoda MSM MSM MSM MSM MSM MSM
uv
MSM MSM NO
MSM NO NO MSM
uv uv
optimum
variable
value
PHsample
2.0-10.0
PHcarrier VI
3.5-10.0 0.2 0.432 1.0 4.0 20-40
[NaOH],M [HzS0419 M [p-rosaniline],g/L [formaldehyde],g/L temperature, "C flow rate, q,, q4, mL/min flow rate q2, q3, mL/min R,, R4,cm (i.d. 0.5 mm) RBIcm (i.d. 0.7 mm) R3, cm (i.d. 0.7 mm) R5,cm (i.d. 0.5 mm) VI, vz, ILL
delay and stop times for VI, s delay and stop times for Vz, s
0.92 0.46 6.0
90 60 41 70 12 and 25 24 and 25
Key: MSM, modified simplex method; UV, univariate method; NO, nonoptimized.
SOIUS,
soI"51 dalrr ~ o l l e c l 8 O n P r i o r reOCI,Qn
Flgure 1. (a) Schematic dngram of the device for doubly stopped flow: P, pump; q, flow rate; DIV, dual injection valve; V, Injecting volume; R, reactor; a, b, confluence points; W, waste. (b) Time sequence of
events (for details, see text). itored (determination of free SO2). Bolus2 is stopped in R2, where its alkaline hydrolysis takes place. The start of P2 flushes bolus, out of the system and drives bolusz to the detector after merging at point a with the reacting mixture. The second halting is effected as bohs2 reaches the detector and is followed by the monitoring of the change in absorbance, which is now made up of the contribution of the both free and bound SO2. The new start of pump P1sends bolus2 to waste. The study of this chemical system by means of the proposed assembly confirms its great potential.
EXPERIMENTAL SECTION Reagents. The solutions utilized (Figure la) were as follows: 0.2 M NaOH, 1.0 g/L p-rosaniline hydrochloride+ 0.432 M H$04 + 20% (v/v) ethanok and 4.0 g/L formaldehyde+ 0.432 M HzS04. All reagents used were pro-analysi grade Merck. Apparatus. Two peristaltic pumps (an Ismatec mini-S 840 (PI)and a Gilson minipuls-2 (P2),a variable-volume dual-injection valve (DIV), a SP6-500 Pye Unicam photometer furnished with a 178.12QS Hellma flow cell, and a REC 80 Radiometer recorder made up the FIA manifold (Figure la). A Hewlett-Packard HP 85 microprocessor collected the data from the photometer through an HP 3478A multimeter and an HP-IB 82937A interface and calculated the absorbance increments and the tangent of the straight line absorbance-time obtained from the plot corresponding to the two haltings of the flow. Then, the calibration curves run with standard solutions were used to calculate the free and total SOzcontent in the injected wine sample. A homemade timepiece controlled the start and stop of the peristaltic pumps (PIand RZ)and the operation of the dual-injection valve. The timepiece consisted of six timers programming as many cycles. The homemade injection system included two variable-volume Rheodyne 5041 injection valves connected to an electric motor controlled by the timer; this made possible the automatic filling of the loops and the insertion of their contents into their respective carriers at the preselected times. Manifold. A general scheme of the proposed assembly is shown in Figure la, while a diagram of the go and stop intervals ( T )of the pumps appears in Figure lb. The instant at which each
interval begins is determined by t. Pump Pl: The interval T1 corresponds to the loop-filling positions, at the end of which P1 is stopped until the start of the new cycle and a fresh sample is injected (emptying position) at tl. Pump P2: The carrier and reagent streams circulate through the system during T,, merging at tl with the injected samples, bolus, and bolus2, which are transported to the flow cell and R2, respectively, during T zand stopped at these points at t2 During the halting corresponding to T3,the detector monitors the change in absorbance due to free SOz and sends the data to the microprocessor, while bolusz undergoes the hydrolytic reaction in Rz,subsequently being driven after to the detector during T4 and monitored in it during T5, which a new cycle is started.
RESULTS AND DISCUSSION Variables Influencing the Indicator Reaction. Variables were optimized by the modified simplex method, MSM (24),and univariate method (Table I). The optimum H2S04 and NaOH concentrations found by the MSM were 0.432 and 0.2 M, respectively. As checked later, this NaOH concentration is sufficient to accomplish the fast dissociation of bound SOz, the HzS04 concentration being the best to neutralize the NaOH stream and provide a very acidic medium for the optimum development of the indicator reaction. Because of the high concentration of NaOH and H2S04the pH of the carrier of bolusl and the sample pH had no influence the analytical signal in the ranges studied (3.5-10.0 and 2.0-10.0, respectively). The signal intensity increased with the p-rosaniline concentration up to 1.0 g/L, above which it decreased as the base-line absorbance increased dramatically. The formaldehyde concentration had a similar influence, though the signal remained constant after attaining a maximum a t 4.0 g/L. The study of the effect of temperature revealed an increase in the signal between 10 and 25 "C, and an interval (25-40 O C ) over which the signal remained constant; nevertheless, the increase in the signal was unappreciable above 20 "C, so that room temperature (20-25 "C) was chosen. The joint study of the influence of the flow rate (changed simultaneously in all channels), loop volumes, and length of R2 and R5 by the MSM yielded the values shown in Table I. Reactor R2 was not very long since the dissociation of bound SO2 was completed in the first halting of the flow, while R5 was very short to ensure as small a dispersion of the sample as possible at the detector. The length of reactors R1, R3,and R4 was not optimized, as their influence was well-known: R1 and R4 were made as short as possible because their only mission was to hold bolus, and the reagents for the indicator reaction. The length of R3 was the minimum reactor length
952
* ANALYTICAL CHEMISTRY, VOL. 59, NO. 7,APRIL
1, 1987
Table 11. Features of the Calibration Curves parameter
free SO2 Aabsorbance method
tangent method
determination range (pg/mL) intercept slope corr coeff re1 std dev, % sampling freq, h-l
1.0-16.0
o.oooa i 0.0003 0.00155 f 0.00003 0.998 3.1 55
tangent method
1.0-16.0 0.020 f 0.006 o . o m i 0.0006 0.996 2.8 35
total SO, Aabsorbance method
1.0-16.0 0.0011 f 0.0001 0.00104 f 0.00001 0.999 2.9 55
1.0-16.0 0.019 f 0.003 0.0130 f 0.0003 0.998 2.1 35
Table 111. Recovery of Total SO2 in Wine tangent method % recovery of the following added amts amt found, type of wine" white, white, ros6
sweet amontillado
% recovery of the following added amts
ccg/mL
2.0 wg/mL
4.0 rg/mL
pg/mL
amt found, %/mL
3.9 2.3 1.3 7.0 4.7
102.3 94.6 105.9 92.0 94.5
98.8 97.8 98.6 96.5 97.7
97.7 100.5 98.9 102.8 98.8
3.9 2.2 1.4 7.1 4.8
6.0
mean recovery 98.5% re1 std dev 3.5% a
Aabsorbance method
2.0
&mL 106.0 103.0 105.0 92.6 94.0
4.0 wg/mL
Fg/mL
6.0
98.0 97.5 97.7 98.3 98.5
96.5 99.7 96.8 100.8 100.2
mean recovery 99% re1 std dev 3.7%
Each tvDe is representative of a set of ten different samples.
needed to achieve a reasonable degree of homogenization of two merging streams (q2 = q3) a t the working flow rate. A sample volume of 70 pL was chosen as a compromise between sensitivity and sample consumption. Variables Involved in the Application of the Method to the Analysis of SO2 in Wines. Once the variables influencing the indicator reaction had been optimized, some aspects of the specific application of the method to the enological analysis were considered. I t was found that ethanol content in the samples between 0 and 20% did not affect the signal appreciably. On the other hand, when SOz solutions were prepared in media containing 12% (v/v) ethanol, 2.0 g/L tartaric acid, and 0.02 M Na2S04and the p H was adjusted to 3.6 to operate with samples containing the various ingredients of wine a t known concentrations, the signal remained unalterated with respect to that obtained from an aqueous solution of SOz, although the shape of the FIA peak was modified as a result of increase absorbances or slight increases in the time parameters. The recovery of bound SOz was studied by using variable amounts of acetaldehyde; it was complete (100%) up to an acetaldehyde-sulfur dioxide ratio of 20:l and was 96% for a 40:l ratio. Finally, the influence of the first halting on the dissociation of bound SOz was also studied. The reaction was found develop to completion within very short times ( 5 s). CalibrationCurves. Although the FIA stopped-flow mode corresponds to the conventional kinetic method involving the measurement of the initial tangent of the kinetic curve, the increment in the signal obtained a t a fixed time was taken as the analytical signal. A comparative study of the results obtained from the two analytical signals recorded simultaneously by means of a simple computer program is made below. To study the influence of the stop time on the calibration curves, stop times of 5, 10,20, and 30 s were run. From the results obtained it follows that as the stop time increases (a) the correlation coefficient diminishes, (b) the absorbance increment increases, (c) the intercept and slope of the calibration curves run from the absorbance increment increases and that obtained from the tangent of the kinetic curve decreases, and (d) the linear range width remains constant. Consequently,
times of 5 and 20 s for measurements of the tangent and absorbance increment, respectively, were considered as optimum. The delay time for bolus, and bolusz was 12 and 24 s, respectively, and the flow was stopped immediately after the peak maximum, at which the signal was more reproducible than in the zone preceding it. The linear ranges of the calibration curves corresponding to free and total SO, listed in Table I1 were obtained under these conditions. As can be seen, such ranges are quite wide (1.0-16.0 pg/mL) and feature good correlation coefficients (better than 0.996 in all cases). The relative standard deviation obtained for 11 samples of 7.4 pg/mL of SO, is also shown in Table 11. In both cases (free and total SO2) the precision was better when the absorbance increment was taken as analytical signal. The lesser sensitivity of the determination of total SOz and its higher precision are due to the higher dispersion undergone by bolus,; this compensates for the possible irregularities in the control of the start and stop times of Pz. The sampling rate achieved is 55 and 35 samples per hour (initial tangent and absorbance-increment methods, respectively). Recovery of the Total SO,. As the recovery of free SO2 in wines could not be studied owing to the fact that some species present would bind added SOz partially, only that corresponding to total SO2was performed. In Table I11 appears the percent recovery obtained for SO, after adding 2.0, 4.0, and 6.0 g / m L to five types of different wines and diluting to 1:lO. The values obtained were acceptable in all cases, the average recovery being 98.5% and 99.0% and the relative standard deviation being 3.5% and 3.7% when the initial tangent or absorbance increment, respectively, was taken as the analytical signal. Free SOz recovery was studied on synthetic samples prepared as above (12% (v/v) ethanol, 2.0 g/L tartaric acid, 0.02 M Na2S04and 100 pg/mL acetaldehyde for total SOz) a t pH 3.2. T o five of these samples were added different amounts of SO, (2.0, 4.0, 6.0, 8.0, and 10.0 pg/mL) and then 2.0, 4.0, and 6.0 Mg/mL. Table IV shows the results obtained. The average recovery was excellent and the relative standard deviation quite acceptable, the results being slightly better when the absorbance increment was taken as analytical signal. As the total SOz recovery from synthetic samples and wines was
ANALYTICAL CHEMISTRY, VOL. 59, NO. 7, APRIL 1, 1987
% recovery of added total SOz
% recovery of added free SO2
tangent method concn,
2.0
4.0
pg/mL
pg/mL
a/mL
2.0 4.0 6.0 8.0 10.0
92.8 96.4 99.3 102.1 100.1
92.5 97.0 95.9 92.7 95.7
Aabsorbance method 6.0
953
tangent method
Aabsorbance method
2.0
4.0
6.0
2.0
4.0
6.0
rg/mL
6.0 wg/mL
pg/mL
rg/mL
wg/mL
a/mL
pg/mL
wg/mL
97.0 98.4 93.3 92.7 94.9
96.1 96.8 102.8 100.3 97.9
101.0 95.1 101.1 104.0 97.8
102.0 102.7 99.4 95.1 100.1
100.1 95.4 105.4 96.0 95.4
105.0 100.8 100.9 104.3 94.8
103.4 105.6 96.9 98.3 96.4
101.0 101.0 102.8 96.3 98.9
2.0
4.0
&mL
pg/mL
92.4 97.4 103.2 99.0 105.2
106.5 96.8 99.6 102.1 98.0
"The samples contained: 129'0 (v/v) ethanol, 2.0 g/L tartaric acid; 0.02 M NaZSO4(100 pg/mL acetaldehyde for total SOz), and 2.0, 4.0, 6.0, 8.0, or 10.0 pg/mL SOz, pH 3.2. Table V. Comparison between the Proposed and the Recommended Method total so2
free SO2
found (pg/mL)
found
% error
found
% error
recommended method found (pg/mL)
19.5 16.7 33.1 14.8 12.8 23.3 42.4
18.0 17.2 34.5 15.0 12.7 22.5 40.5
-7.7 3.0 4.2 1.4 -0.8 -3.4 -4.5
19.1 17.3 34.9 14.5 12.7 23.6 40.9
-2.1 3.6 5.5 -2.0 -0.8 1.3 -3.5
61.6 56.8 48.0 59.2 79.3 63.2 85.9
recommended
wine sample 1 2 3 4
5 6 7
method
mean error:
tangent method
method
3.5
good, the presence of interferents can be safely ruled out. Although not demonstrated, a similar behavior can be assumed for the determination of free SOz, because the basic reaction is the same and the recovery from synthetic samples is also quite good. In any case, the following study confirmed this assumption. Simultaneous Determination of Free and Total SOz in Wines. Different types of wines (dry, sweet, amontillado, ros8, etc.) were chosen to demonstrate the selectivity of the proposed method with respect to the color and matrix of the samples. To test the accuracy of the method, it was contrasted with the standard method recommended by the EEC (25), which involves the direct titration with iodine for the determination of free SOz and with a prior alkaline hydrolysis for that of total SOz. As shown in Table V, the results obtained by both methods are quite consistent. The average errors were 3.5% and 2.8% for free SOz and 3.3% and 3.0% for total SOz, as obtained by taking the initial tangent or absorbance increment, respectively, as analytical signal. The latter method slightly excells the former. (The relative standard deviation obtained by the EEC method oscillates between 5.1 and 14.1% depending on the type of sample (26).)
CONCLUSION The assembly proposed here for implementation of a doubly stopped flow mode is one more proof of the versatility of FIA. It widens the scope of multideterminations with this technique in combining the two possibilities of stopped flow used so far in FIA: the kinetic mode, involving the halting of the reacting mixture at the detector (where signal-time measurements are made), and the nonkinetic mode, with stopping of the reacting mixture at the reactor, generally intended to increase sensitivity of slow reactions. The proposed method is particularly useful (a) when the analyte is in two different forms in the sample and one of them has to be converted to the other prior to determining the overall concentration (speciation studies) and (b) in the determination of two analytes, one of which requires a prior chemical step to bring about the indicator reaction. The
tangent method found % error 62.8
55.7 43.6 58.6 82.0 62.9 81.4
2.0 -1.9 -9.2
-1.0 3.4 -0.5 -5.2
2.8
3.3
Aabsorbance method found % error 60.8 54.6 46.1 56.9 77.8 61.5 83.1
-1.3 -3.9 -4.0 -3.9 -1.9 -2.7 -3.2 3.0
potential of this method is further increased by the use of a configuration with two flow cells in a dual-beam spectrophotometer or aligned with the optical path of a single-beam photometer instead of that involving the confluence of the sample channels. This last manifold allows the use of different reagents for each analyte, which reacts with them a t very different rates via a variety of reaction sequences. The dual-injection system used can be substituted by a single one including splitting point after the injection port. Other possibilities of the proposed assembly are currently being investigated.
ACKNOWLEDGMENT We gratefully acknowledge Gonzglez Byass for the wine samples supplied. Registry No. SOz, 7446-09-5.
LITERATURE CITED Valdrcel, M.; Luque de Castro, M. D. Flow Injection Analysis:
Principles and Appllcatlons , Ellis Horwood: Chichester, in press. Amerlne, M. A.; Ough, C. S. An.#sis de vinos y mostos; Acribia: Zaragoza, Spain, 1976; p 109. Ripper, M. J . Prakt. Chem. 1892, 4 5 , 428; 1892, 4 6 , 428. Jaulmes, P. VI1 Congr6s International de la Vigne et du Vin, Rome, 1953. Tanner, H.; Rentschler, H. Mitt. Geb. Lebensmiftelunters. Hyg. 1951, 42, 514. Deibner, L. Chim. Anal. (Paris) 1966, 4 8 , 66 and 143. Paul, F. MItteilungen 1858, 8 , 21. Burroughs, L. F.; Sparks, A. H. Analyst (London) 1964, 8 9 , 1054. Monk-Williams, G. W. Rep. Public Health Med. 1927, 4 3 , 1. Deibner, L.; BBnard, P. Ind. Agric. Aliment. 1955, 7 2 , 565 and 673. Ramasamy, S. M.; Monola, H. A. Anal. Chem. 1982, 5 4 , 283. Rios, A.; Luque de Castro, M. D.; ValcBrcel, M.; Monola, H. A. Anal. Chem. lS87, 5 9 , 666. Alexander, P. W.; Haddad, P. R.;Trojanowicz, M. Anal. Chem. 1984, 56, 2417. Marshall, G. B.; Mldgley, D. Analyst (London) 1983, 108, 701. Granados, M.; Maspoch, S.; Blanco, M. Anal. Chim. Acta 1986, 179, 445. Wang. J.; Dewald, H. D. Anal. Chim. Acta 1983, 153, 325. Masoom, M.; Townshend, A. Anal. Chim. Acta 1986, 179, 399. Yamada, M.; Nakada, T.; Suzuki, S. Anal. Chim. Acta 1983, 147, 401. Burguera, J. L.; Burguera, M. Anal. Chim. Acta 1984, 157, 177. Williams, T. R.; McElvany, S. W.; Ighodalo, E. C. Anal. Chim. Acta 1981, 123. 351.
Anal. Chem. 1987, 59,954-958 Hansen, E. H.; Ruzicka, J. Anal. Chim. Acta 1980, 774, 19. Moiler, J.; Winter, B. Fresenius' Z . Anal. Chem. 1985, 320, 451. West, P. W.; Gaeke, G. C. Anal. Chem. 1958, 28, 1816. Deming. S. N.; Parker, L. R., Jr. CRC Crit. Rev. Anal. Chem. 1978.
187. Journal Officiei des Communautirs Europeennes, L. 133, 14 Mai 1982: D 55.
(26) Buechsenstein, J. W.; Ough, C. S. Am. J . Enol. Viric. 1978,29, 161.
RECEIVED for review July 18, 1986. Accepted November 25, 1986. The CAICyT is thanked for financial support (Grant NO. 2012-83).
Tetrathiafulvalene Tetracyanoquinodimethane Xanthine Oxidase Amperometric Electrode for the Determination of Biological Purines Kevin McKenna and Anna Brajter-Toth*
Department of Chemistry, University of Florida, Gainesuille, Florida 3261 1
An amperometrk xanthlne oxldase electrode constructed wlth the conducting salt tetrathlafulvalene tetracyanoqulnodlmethane, TTF+TCNO-, as the electrode materlal Is evaluated for the determlnatlon of purine, hypoxanthlne, and xanthlne. Under unsaturated enzyme condltlons, substrate-dependent current response linear in the concentration range to 6 X lo4 M. Substrate dmuslon through the membrane Is the rate-lknnlng step. Appkation to the determlnatlon of purlnes in blood plasma Is described.
Amperometric immobilized-enzyme electrodes offer the possibility of enhancing the selectivity of electroanalytical methods while providing the desired sensitivity. This is possible because of the electrode design in which the immobilized enzyme reacts selectively with analytes in a catalytic reaction (1, 2). In the original design, analysis with amperometric enzyme electrodes relied on the detection of the end product of the enzymatic reaction (1,2). For continuous operation of electrodes with immobilized flavoenzymes, an acceptor was required which regenerated the enzyme following enzyme-analyte reaction. In a newer design, the analytical signal was produced when the acceptor was regenerated electrochemically, acting as a mediator between the electrode and the enzyme (3, 4 ) . The success of the mediated reaction relied on the choice of the mediator, which had to react rapidly with the enzyme and the electrode a t moderate potentials. In the simplest design, the analytical signal would be obtained as a result of a direct regeneration of the enzyme a t the electrode surface following reaction with analyte. It has recently been reported that the reduced flavin redox center (FADHJ of glucose oxidase can be electrooxidized a t electrodes made of organic conducting salts such as tetrathiafulvalinium tetracyanoquinodimethanide (TTF+TCNQ-) (5-7). In this work, we describe the response of a xanthine oxidase (a flavoenzyme) immobilized-enzyme electrode constructed by using the conducting salt TTF'TCNQ- as the electrode material. The analytical response of this electrode to the biological purines hypoxanthine, xanthine, and purine was evaluated, and the electrode response was analyzed based on the theory recently developed by Bartlett and Albery (8).
EXPERIMENTAL SECTION Reagents. Xanthine oxidase from buttermilk (EC 1.1.3.2.2) was obtained from Sigma (grade 111,activity 1.1 units mg-'). The TTF was purchased from Aldrich as was the TCNQ. All other
chemicals were reagent grade and were used as received. All solutions were prepared by using doubly distilled water and all experiments were performed in phosphate buffer, ionic strength 0.5 M. Solutions were deaerated by bubbling N2 through the solutions for at least 10 min. Electrode Preparation. The TTF+TCNQ- complex was synthesized using the procedure described by Jaeger and Bard (9). The dried complex was placed in a darkened vacuum desiccator and refrigerated until used. The electrode consisted of a 3.5-mm-diameterplatinum disk fitted into a Teflon holder with a recessed depth of 1 mm (7). Contact was made by connecting a copper wire to one side of the disk using conducting epoxy (Tra-con, Medford, MA). The cavity was filled with a slurry of the TTF'TCNQ- and PVC. The electrode was allowed to cure for 12 h and the excess conducting salt was trimmed off. The electrodes were stored in the dark at room temperature. The membranes were prepared from dialysis tubing (Spectrapor no. 1,Spectrum Medical Industries, Inc., Los Angeles, CA) which had been pretreated prior to use (7). The enzyme electrodes were prepared by trapping a drop (0.050 cm3) of the 90 NM enzyme solution between the electrode and the membrane. The membrane was fixed in place by an O-ring and Teflon tape. Excess enzyme solution was removed by copious washing. Apparatus. Dc cyclic voltammetry and constant potential experiments were performed by using a Bioanalytical Systems Electrochemical Analyzer, BAS-100, and a Houston Instruments DMP-40 series digital plotter. In addition to the TTF+TCNQworking electrode, the cell contained a 1-cm2platinum foil counter electrode and a saturated calomel electrode (SCE) as reference. All current measurements were made at a constant potential of +225 mV with respect to the SCE following a 30-5 incubation at open circuit in solution of analyte. All measurements were made at 24.5 h 0.5 "C.
RESULTS AND DISCUSSION Electrode Design and Analytical Response. Schematic design of the sensor that was used in this study is shown in Figure 1. In this design, the electrode is a conducting organic salt (TTF+TCNQ-) and xanthine oxidase is the immobilized enzyme. The thin layer of enzyme solution is immobilized at the electrode with a porous dialysis membrane. With purine as analyte two reactions determine the response of the electrode: purine xanthine oxidase/FAD uric acid + xanthine oxidase/FADH, (1)
+
-
-
xanthine oxidase/FADH, xanthine oxidase/FAD
+ 2e- + 2H+ (2)
where reaction 2 occurs a t the electrode surface. Figure 2 shows the response of purine at a bare TTF+TCNQ- electrode
0 1987 American Chemical Society 0003-2700/87/0359-0954$01.50/0
