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Determination of nitric oxide saturated (stock) solution by chronoamperometry on a porphyrine microelectrode
ANALVICA CHIMICA ACTA
ELSEVIER
Analytica Chimica Acta 339 (1997) 265-270
Determination of nitric oxide saturated (stock) solution by chronoamperometry on a porphyrine microelectrode $. Mes&ogay*,
S. Grunfeldb, A. Mesh-oEov&“, D. Bustina, T. Malinskib
aDepat?ment ofAnalytical Chemistry Slovak Technical University, Radlinskt!ho 9, SK-812 37, Bratislava, Slovakia b Department of Chemistry Oakland University, Rochestec MI 48309, USA
Received 30 July 1996; revised 16 September 1996; accepted 23 September 1996
Abstract A porphyrinic-based microelectrode was applied for the determination of nitric oxide (NO) saturated solution by absolute chronoamperometry. This method is based upon the separation of the current component limited by linear diffusion from that limited by the cylindrical contribution to the total flux of the determined substance to the cylindrical electrode. Precision and accuracy were tested and compared with the UVNIS Griess method. The concentration of saturated NO solution was 1.74~tO.03 mmol-‘. Keywords: Amperometry; Nitric oxide; Porphyrin
1. Introduction Nitric oxide (NO) plays a role in vasodilatory responses which maintains normal blood pressure and it also mediates the antitumor effects of cytotoxic activated macrophages [l-3]. Recently, NO release has been detected in the central nervous and it may possibly function as a retrograde messenger for long term memory [4,5]. Quantitative measurement of NO and its decay product in biological systems is important in the understanding of their physiological role. NO can be measured by chemiluminescence, bioassay, and UV spectroscopy [6]. However, these methods are less suitable for real time monitoring with high spatial
*Corresponding author. Tel.: +421 7 326 021 (313); fax: +421 7 393 198; e-mail: [email protected]. 0003-2670/97/$17.00 Copyright PII SOOO3-2670(96)00466-7
0
resolution. In 1992, Malinski’s group reported that a carbon fiber microelectrode coated with an electrodeposited p-type semiconducting porphyrinic film [7] could be used to detect NO by its oxidation [8]. Electrodes based on this concept have been used to detect NO in several biological environments including in vivo [9] and single cells [8,10,11]. However, all methods for the direct determination of NO requires calibration. Saturated solutions of NO in phosphate buffer, prepared from NO in a gas cylinder or from acid decomposition of nitrite has been used. Unfortunately, the concentration of the prepared solution is usually unknown. Therefore, we describe a very simple and short-time procedure for the determination of NO concentration of the saturated solution. The method is based on a cylindrical microelectrode made from carbon fiber coated by porphyrine [S] for “absolute” chronoamperometric determination of NO of standards.
1997 Elsevier Science B.V. All rights reserved.
Z?.MescimS et nl./Analytica
266
In previous papers [ 12,131 we proposed that the use of the chronoamperometric method of electroactive species determination would avoid the necessity of standardization. The current-time dependence which could be evaluated so that the current component caused by linear diffusion is separated from that caused by the spherical contribution to the total current. The concentration of the analyzed electroactive species was calculated from these components. Results of the analysis were accurate in the concentration range 10d3 to 10v4 mol 1-i and are independent of the temperature of the analyzed solution. An approximate expression for chronoamperometric curves at stationary microcylindrical electrodes was described by Aoki and co-workers [14], in the following form: -
+ 0.422 - 0.0675 log 19
where 19= Dt/?, c the bulk concentration, D the diffusion coefficient, and the upper sign holds for log 821.47 and the lower sign log 8~1.47; I, current; F, Faraday constant; A, area of the electrode and r, is the radius of the electrode. This equation was in good agreement with the theoretically predicted curves measured at times ranging from 40 ms to 8 s [ 141. In a previous paper [12] we presented an accurate simplified approximation of Eq. (1) without logarithmic terms I = zFcDA(&+
y).
Chimica Acta 339 (1997) 265-270
kis(3-methoxy-4-hydroxyphenyl) porphyrin (TMHPP), with nickel(I1) as the central atom [7]. We deposited poly-TMHPP-Ni from a solution of 0.1 M NaOH containing monomeric porphyrine by continuous scan cyclic voltammetry in the potential -0.2-1.2 V vs. SCE with a scan rate range 100 mV s-‘. Sensor fabrication is accomplished by dipping the electrode in a Nafion solution (1% w/w in alcohol) for 15 s. The completed sensor is left to dry (5 min) and is stored in phosphate buffer at pH 7.4. 2.2. Nitric oxide saturated solution The phosphate buffer (pH 7.4) was transferred to a sample bottle with a silicon rubber stopper. The solution was then boiled for about 5 min followed by a cooling period. Finally the sample bottle was submerged in an ice bath and connected to the nitric oxide generation apparatus, which was in an inert atmosphere due to nitrogen gas bubbling. Next, 125 ml of 6 M sulfuric acid and approximately 50 g of sodium nitrite were added in the nitric oxide generation apparatus. The nitric oxide generation apparatus was degassed by purging with nitrogen for about 10 min followed by turning on the three-way switch to let sulfuric acid slowly drop into sodium nitrite. Consequently, the reaction generated nitric oxide. Nitric oxide was continuously purged into the degassed buffer until the buffer became saturated approximately 20-25 min from the onset of the reaction. Nitric oxide saturated in the aqueous phase had an approximate concentration of 2 mM at 0°C. This saturated solution is stable for 48 h at 0°C. 2.3. Chronoamperometric
The difference in current calculated from Aoki equation and the simplified form does not exceed 3% in the interval 0.16)&<2.5. For typical values of D and r from the microelectrode range, this corresponds approximately to the time interval O.O4
measurement
Chronoamperometric curves were measured using the EG&G PAR Potentiostat/Galvanostat M273A (Princeton, NJ) with custom data acquisition electrochemical software made available by the manufacturer. A three electrode arrangement was used with an NO-sensor working electrode, saturated calomel reference electrode (SCE) and a platinum wire as a counter electrode.
microsensor 2.4. UV-VIS
Polymeric porphyrine was deposited on carbon fiber with a diameter of 70 urn (BASE USA) [8]. The polymeric film was obtained from monomeric tetra-
measurement
The Griess reaction as used in UVNIS spectrophotometry involves a two-step assay based on the
3. Mescid
et al./Analytica
Chimica Acta 339 (1997) 265-270
261
observation that the adduct of nitric oxide and sulfanilic acid (1 .O%) interacts with N-( 1-naphty1)ethylenediamine (0. l%), to generate an azo derivative product that is readily monitored by spectrophotometry. The spectrum of this product shows an absorption band with a maximum at 548 nm. All absorbance measurements were performed on a Milton Roy Spectronic 3000 Diode Array UVNIS Sectrophotometer with 1 cm cells.
The radius is important for the calculation of concentration and is microscopically measured before experiments. An additional important parameter is the area of the electrode which is calculated from measured length and diameter of the electrode. The optimum radius of electrode for this experiment is 46f2 urn [12]. As we previously described [ 121 the chronoamperometric experiment and its evaluation were carried out with the following sequence of operations:
3. Results and discussion
1. the acquisition of l-t curve of the analyzed NO; using 3000 acquisition points per second; 2. the acquisition of l-t curve of the background (phosphate buffer without NO) at the same working potential as the indication electrode; of the 3. the correction of the chronoamperogram analyzed component by subtracting the Z-t background curve; 4. the transformation of the corrected Z-t curve to the form (l/,/?/A) vs. Ji; of U and S 5. a linear regression-calculation (including standard deviations); of concentration (including standard 6. calculation deviation).
The usage of chronoamperograms for the determination of the concentration of an electroactive species is based on the equation of the limiting current-time function for cylindrical electrodes Eq. (1). As mentioned in Section 1 we used a simplified equation for the evaluation Eq. (2), without loss of precision and accuracy [ 121. The chronoamperograms were obtained at constant potential with a porphyrine microsensor. The best potential of each electrode was determined from a differential pulse voltammetry (DPV) experiment. We used approximately 25 urn concentration of NO in a phosphate buffer (pH 7.4). The potentials obtained from the DPV experiments were in the range 0.68-0.75 V vs. SCE. The chronoamperometrical data were used to construct the graph (ItiA) vs. 4. This graph should be linear with the slope S and the intercept U. S=
0.422zFDc
)
r
u=-.
zFD’/=c 7$/=
(3)
U and S/fi are the components of the chronoamperometric constant caused by linear and cylindrical contribution to the diffusion current, respectively. Values U and S determined from a single chronoamperometric experiment were used for calculating the concentration of the electroactive substance according to the equation c = U2n0.422 zFSr
’
(4)
where z is the charge of NO+, the electrochemical oxidation product of NO NO-e--+NO+.
Steps 1 to 3 are obtained by the EG&G PAR M273A electrochemical software and points 4 to 6 by our simple program written in Quick-Basic. 3.1. Elimination species
of influence from other oxidisable
The porphyrinic-based microsensor was coated by Nafion (see Section 2) to eliminate the diffusion and adhesion of anionic species easily oxidisable at the surface of the electrode. As can be seen from Fig. 1 (curve b) the addition of nitrite at a concentration ten times greater than NO did affect neither the height of the signal, nor the potential of the peak (Fig. 1, curve a) (0.63 V vs. SCE). A porphyrinic sensor lacking a coating of Nafion, or just carbon fiber without porphyrine, shifted the potential to a more positive value (0.77 V vs. SCE), and the current of the peak is larger than the identical concentration of NO detected by a Nafion coated electrode (Fig. 2, curve a). This may be due to air oxidation of the NO solution to nitrite and the oxidation of both species on the porphyrinic elec-
268
s. MesriroS et al. /Analytica 1.6,
-0.21 0.2
I 0.4
I 0.6
I 0.6
, 1.0
, 1.2
ElVvs.SCE
Fig. 1. Differential pulse voltamrnograms of a Nafion coated NO biosensor; scan rate: 10 mV SC’; pulse height: 25 mV, pulse width: 50 ms; medium: phosphate buffer pH 7.4 (a) NO standard 2.5x10-s mall-’ (solid line); (b) NO standard 2.5x10-s mol I-‘+2.5x 10m4 nitrite (solid circle).
trode. As can be seen from Fig. 2, curve b after the addition of a nitrite solution ten times more concentrated than NO, the current of the oxidation peak has increased. From these experiments we conclude that Nafion is not affected by nitrite and the peak current is exclusively due to NO. 3.2. Determination
of NO saturated solution
Before the sensor can be used for the determination of NO, it is necessary to obtain the exact value of the potential. This can be determined by differential
a)
-1
0 0.2
0.4
0.6
0.6
1.0
1
1.2
ElVvs.SCE
Fig. 2. Differential pulse voltammograms with an uncoated Nafion NO biosensor; scan rate: 10 mV s-‘; pulse height: 25 mV, pulse width: 50 ms; medium: phosphate buffer pH 7.4 (a) NO standard 2.5x 10m5 mol 1-l (solid line); (b) NO standard 2.5x 10-s mol 1-l + 2.5 x 10e4 nitrite (solid circle).
Chimica Acta 339 (1997) 265-270
pulse voltammetry (DPV). The value of the constant potential is a couple of millivolts larger than the peak potential. The applied electrochemical reaction was the one-electron oxidation of NO. Also, it was necessary to determine optimum duration of the chronoamperometric experiment to avoid the influence of convection caused by the density change in the diffusion layer as well as by the instability of the apparatus. This influence demonstrates itself by the bending of the otherwise straight line (Id/A) vs. 4 dependence toward higher l/d/A values. NO was determined in phosphate buffer. The precision and accuracy of absolute chronoamperometric analysis was tested within the concentration range of 10e4 to 10e3 mol 1-l. The results are summarized in Table 1 and compared with the Griess UV-VIS method. The chronoamperometric experiment gives a concentration of the saturated solution of 1.74f0.03 mm01 1-l in comparison to the experimental value obtained by UVNIS of 2.27f0.20 mmol 1-l. The higher value obtained from the UV/VIS experiment results from the presence of nitrite. The Griess reaction is not specific to NO, it will also react with nitrite. The theory of absolute chronoamperometric analysis predicts that the sensor for NO measurement should be independent of the temperature of the analyte. This theory was experimentally investigated within the range of 5°C to 40°C. The results are summarized in Table 2. A statistical test of the arithmetic means showed that none of the average different concentrations, c(experimenta1) at temperatures are significantly different. None of the c(experimenta1) values can be considered as outlier with respect to their arithmetic mean (1.72~tO.02 mmol 1-l). From this it can be concluded that the temperature change does not significantly influence the results of chronoamperometric analysis. During the analysis, however, the analyte temperature must be kept constant and homogeneous, but due to our short response time (about 1 s), this was not a problem. The effect of electroinactive substances present in the analyte at high concentrations was also investigated. These substances can significantly alter the density and viscosity of the analyte. To test the effect of dextran (MW 70000), on the NO determination. The viscosity of the prepared solution of dextran has
s. MesimS
et al./Analytica
Chimica Acta 339 (1997) 265-270
269
Table 1 Precision and accuracy of the chronoamperometric determination of the concentration of NO saturated solutions c(experimental), in phosphate buffer (pH 7.4) at different concentrations of NO solution (estimated concentration from thermodynamic tables for saturated solution) c(est). Current sampling interval 0.04-l s after starting the electrolysis at the potential E=0.7 V, prior to electrolysis the potential is E=O.O V, all vs. SCE. Working electrode-porphyrine biosensor; ~35 urn and 1=4 mm; T=310 K; volume of phosphate buffer V=4 ml; number of analysis n=lO Volume of standard (ml)
c(est.) x lo4 (mall-‘)
0.25 0.50 1.00 1.50
Z(experimental)x a (mol 1-l)
1.17 2.22 4.00 5.45
lo4
E(experimental)x b (mol 1-l)
lo4
*
(11
*
**
1.04 1.91 3.48 4.73
17.68 17.19 17.40 17.35
1.31 2.69 4.62 5.77
22.27 24.17 23.12 21.18
a Chronoamperometric experiment. t UVNIS experiment. concentration obtained from experiment. ** concentration of NO saturated solution. s, - standard deviation of chronoamperometric sa - standard deviation of UVNIS experiment
** s, (mol 1-l)
** % (mol I-‘)
0.5 1 0.32 0.25 0.23
3.23 2.58 2.24 2.17
experiment (calculated for saturated NO solution). (calculated for standard NO solution).
Table 2 Influence of analyte temperature and viscosity on the precision and accuracy of NO determination by absolute chronoamperometric analysis. Concentration of standard (mean from Table 1)=1.74x 10m3 mol 1-l. Current sampling interval 0.04-l s after starting the electrolysis at the potential E=0.7 V, prior to electrolysis the potential is E=O.O V, all vs. SCE. Working electrode - prophyrine biosensor; r=35 pm and 1=4 mm; volume of phosphate buffer V=4 ml; number of analysis n=lO Temperature
278 288 298 310 313 310 310 310 310 310
(K)
Viscosity
(lo3 N s m-r)
1.53 1.15 0.90 0.76 0.67 1.00 1.50 2.00 3.00 3.50
* concentration obtained from experiment. ** concentration of NO saturated solution. s - standard deviation (calculated to saturated
f(experimental)x
lo4 (mol 1-l)
s** (mol 1-t)
*
**
3.41 3.43 3.46 3.48 3.46 3.47 3.45 3.48 3.50 3.46
17.05 17.15 17.30 17.40 17.30 17.35 17.25 17.40 17.50 17.30
0.34 0.27 0.31 0.25 0.24 0.26 0.28 0.32 0.34 0.31
NO solution).
a similar viscosity as blood (2.8x lop3 N s mm*). The results of the analysis are summarized in Table 2. As can be seen from Table 2, the determined values of NO concentration are independent of the viscosity (concentration of dextran) with respect to their arithmetic mean (1.74f0.01 mm01 1-l). The described and discussed results demonstrate that the chronoamperomeric analysis with porphyr-
ine-based microsensor can be regarded as an absolute method for the determination of NO saturated solutions, within the concentration range lop4 to lop3 mol 1-i. In the described method, to obtain the concentration of the NO saturated solution is easy, fast and it eliminates the influence of nitrite, which is also present in standard stock solutions. The proposed method enables also to determine values of diffusion
270
3. Mesa’&
et al. /Analytica
coefficient of NO. The accuracy and precision comparable with that given for concentration.
Chimica Acta 339 (1997) 265-270
is
References [l] S. Moncada, Acta Physiol. Stand., 145 (1992) 201. [2] M.S. Mulligan, S. Moncada and PA. Ward, Br. J. Pharmacol., 107 (1992) 1159. [3] C.R. Lyons, G.J. Grloff and J.M. Cunningham, J. Biol. Chem., 267 (1992) 6370. [4] C. Nathan, FASEB J., 6 (1992) 3051. [5] Y. Izumi, D.M. Clifford and C.F. Zorumski, Science, 257 (1992) 1273. [6] S.J. Chung and H.L. Fung, Anal. Lett., 25 (1992) 2021.
[7] T. Malinski, A. Ciszewski, J. Bennet, J.R. Fish and L. Czuchajowski, J. Electrochem. Sot., 138 (1991) 2008. [S] T. Malinski and Z. Taha, Nature, 358 (1992) 676. 191 T. Malinski, F. Bailey, Z.G. Zhang and M. Chopp, J. Cerebral Blood Flow Metab., 13 (1993) 355. UOI T. Malinski, Z. Taha, S. Grunfeld, A. Burewicz, P. Tombouhan and F. Kiechle, Anal. Chim. Acta, 279 (1993) 135. Hll T. Malinski, Z. Taha, S. Grunfeld, S. Patton, M. Kapturczak and P. Tomboulian, Biochem. Biophys. Res. Comm., 193 (1993) 1076. [W S. Me&o& M. Rievaj and D. Bustin, Collect. Czech. Chem. Commun., 58 (1993) 281. S. Me&o& M. Rievaj and P. Tomrik, [131 D. Bustin, Electroanalysis, 7 (1995) 329. J. [141 K. Aoki, K. Honda, K. Tokuda and H. Matsuda, Electroanal. Chem., 186 (1985) 79.
ELSEVIER
Analytica Chimica Acta 339 (1997) 265-270
Determination of nitric oxide saturated (stock) solution by chronoamperometry on a porphyrine microelectrode $. Mes&ogay*,
S. Grunfeldb, A. Mesh-oEov&“, D. Bustina, T. Malinskib
aDepat?ment ofAnalytical Chemistry Slovak Technical University, Radlinskt!ho 9, SK-812 37, Bratislava, Slovakia b Department of Chemistry Oakland University, Rochestec MI 48309, USA
Received 30 July 1996; revised 16 September 1996; accepted 23 September 1996
Abstract A porphyrinic-based microelectrode was applied for the determination of nitric oxide (NO) saturated solution by absolute chronoamperometry. This method is based upon the separation of the current component limited by linear diffusion from that limited by the cylindrical contribution to the total flux of the determined substance to the cylindrical electrode. Precision and accuracy were tested and compared with the UVNIS Griess method. The concentration of saturated NO solution was 1.74~tO.03 mmol-‘. Keywords: Amperometry; Nitric oxide; Porphyrin
1. Introduction Nitric oxide (NO) plays a role in vasodilatory responses which maintains normal blood pressure and it also mediates the antitumor effects of cytotoxic activated macrophages [l-3]. Recently, NO release has been detected in the central nervous and it may possibly function as a retrograde messenger for long term memory [4,5]. Quantitative measurement of NO and its decay product in biological systems is important in the understanding of their physiological role. NO can be measured by chemiluminescence, bioassay, and UV spectroscopy [6]. However, these methods are less suitable for real time monitoring with high spatial
*Corresponding author. Tel.: +421 7 326 021 (313); fax: +421 7 393 198; e-mail: [email protected]. 0003-2670/97/$17.00 Copyright PII SOOO3-2670(96)00466-7
0
resolution. In 1992, Malinski’s group reported that a carbon fiber microelectrode coated with an electrodeposited p-type semiconducting porphyrinic film [7] could be used to detect NO by its oxidation [8]. Electrodes based on this concept have been used to detect NO in several biological environments including in vivo [9] and single cells [8,10,11]. However, all methods for the direct determination of NO requires calibration. Saturated solutions of NO in phosphate buffer, prepared from NO in a gas cylinder or from acid decomposition of nitrite has been used. Unfortunately, the concentration of the prepared solution is usually unknown. Therefore, we describe a very simple and short-time procedure for the determination of NO concentration of the saturated solution. The method is based on a cylindrical microelectrode made from carbon fiber coated by porphyrine [S] for “absolute” chronoamperometric determination of NO of standards.
1997 Elsevier Science B.V. All rights reserved.
Z?.MescimS et nl./Analytica
266
In previous papers [ 12,131 we proposed that the use of the chronoamperometric method of electroactive species determination would avoid the necessity of standardization. The current-time dependence which could be evaluated so that the current component caused by linear diffusion is separated from that caused by the spherical contribution to the total current. The concentration of the analyzed electroactive species was calculated from these components. Results of the analysis were accurate in the concentration range 10d3 to 10v4 mol 1-i and are independent of the temperature of the analyzed solution. An approximate expression for chronoamperometric curves at stationary microcylindrical electrodes was described by Aoki and co-workers [14], in the following form: -
+ 0.422 - 0.0675 log 19
where 19= Dt/?, c the bulk concentration, D the diffusion coefficient, and the upper sign holds for log 821.47 and the lower sign log 8~1.47; I, current; F, Faraday constant; A, area of the electrode and r, is the radius of the electrode. This equation was in good agreement with the theoretically predicted curves measured at times ranging from 40 ms to 8 s [ 141. In a previous paper [12] we presented an accurate simplified approximation of Eq. (1) without logarithmic terms I = zFcDA(&+
y).
Chimica Acta 339 (1997) 265-270
kis(3-methoxy-4-hydroxyphenyl) porphyrin (TMHPP), with nickel(I1) as the central atom [7]. We deposited poly-TMHPP-Ni from a solution of 0.1 M NaOH containing monomeric porphyrine by continuous scan cyclic voltammetry in the potential -0.2-1.2 V vs. SCE with a scan rate range 100 mV s-‘. Sensor fabrication is accomplished by dipping the electrode in a Nafion solution (1% w/w in alcohol) for 15 s. The completed sensor is left to dry (5 min) and is stored in phosphate buffer at pH 7.4. 2.2. Nitric oxide saturated solution The phosphate buffer (pH 7.4) was transferred to a sample bottle with a silicon rubber stopper. The solution was then boiled for about 5 min followed by a cooling period. Finally the sample bottle was submerged in an ice bath and connected to the nitric oxide generation apparatus, which was in an inert atmosphere due to nitrogen gas bubbling. Next, 125 ml of 6 M sulfuric acid and approximately 50 g of sodium nitrite were added in the nitric oxide generation apparatus. The nitric oxide generation apparatus was degassed by purging with nitrogen for about 10 min followed by turning on the three-way switch to let sulfuric acid slowly drop into sodium nitrite. Consequently, the reaction generated nitric oxide. Nitric oxide was continuously purged into the degassed buffer until the buffer became saturated approximately 20-25 min from the onset of the reaction. Nitric oxide saturated in the aqueous phase had an approximate concentration of 2 mM at 0°C. This saturated solution is stable for 48 h at 0°C. 2.3. Chronoamperometric
The difference in current calculated from Aoki equation and the simplified form does not exceed 3% in the interval 0.16)&<2.5. For typical values of D and r from the microelectrode range, this corresponds approximately to the time interval O.O4
measurement
Chronoamperometric curves were measured using the EG&G PAR Potentiostat/Galvanostat M273A (Princeton, NJ) with custom data acquisition electrochemical software made available by the manufacturer. A three electrode arrangement was used with an NO-sensor working electrode, saturated calomel reference electrode (SCE) and a platinum wire as a counter electrode.
microsensor 2.4. UV-VIS
Polymeric porphyrine was deposited on carbon fiber with a diameter of 70 urn (BASE USA) [8]. The polymeric film was obtained from monomeric tetra-
measurement
The Griess reaction as used in UVNIS spectrophotometry involves a two-step assay based on the
3. Mescid
et al./Analytica
Chimica Acta 339 (1997) 265-270
261
observation that the adduct of nitric oxide and sulfanilic acid (1 .O%) interacts with N-( 1-naphty1)ethylenediamine (0. l%), to generate an azo derivative product that is readily monitored by spectrophotometry. The spectrum of this product shows an absorption band with a maximum at 548 nm. All absorbance measurements were performed on a Milton Roy Spectronic 3000 Diode Array UVNIS Sectrophotometer with 1 cm cells.
The radius is important for the calculation of concentration and is microscopically measured before experiments. An additional important parameter is the area of the electrode which is calculated from measured length and diameter of the electrode. The optimum radius of electrode for this experiment is 46f2 urn [12]. As we previously described [ 121 the chronoamperometric experiment and its evaluation were carried out with the following sequence of operations:
3. Results and discussion
1. the acquisition of l-t curve of the analyzed NO; using 3000 acquisition points per second; 2. the acquisition of l-t curve of the background (phosphate buffer without NO) at the same working potential as the indication electrode; of the 3. the correction of the chronoamperogram analyzed component by subtracting the Z-t background curve; 4. the transformation of the corrected Z-t curve to the form (l/,/?/A) vs. Ji; of U and S 5. a linear regression-calculation (including standard deviations); of concentration (including standard 6. calculation deviation).
The usage of chronoamperograms for the determination of the concentration of an electroactive species is based on the equation of the limiting current-time function for cylindrical electrodes Eq. (1). As mentioned in Section 1 we used a simplified equation for the evaluation Eq. (2), without loss of precision and accuracy [ 121. The chronoamperograms were obtained at constant potential with a porphyrine microsensor. The best potential of each electrode was determined from a differential pulse voltammetry (DPV) experiment. We used approximately 25 urn concentration of NO in a phosphate buffer (pH 7.4). The potentials obtained from the DPV experiments were in the range 0.68-0.75 V vs. SCE. The chronoamperometrical data were used to construct the graph (ItiA) vs. 4. This graph should be linear with the slope S and the intercept U. S=
0.422zFDc
)
r
u=-.
zFD’/=c 7$/=
(3)
U and S/fi are the components of the chronoamperometric constant caused by linear and cylindrical contribution to the diffusion current, respectively. Values U and S determined from a single chronoamperometric experiment were used for calculating the concentration of the electroactive substance according to the equation c = U2n0.422 zFSr
’
(4)
where z is the charge of NO+, the electrochemical oxidation product of NO NO-e--+NO+.
Steps 1 to 3 are obtained by the EG&G PAR M273A electrochemical software and points 4 to 6 by our simple program written in Quick-Basic. 3.1. Elimination species
of influence from other oxidisable
The porphyrinic-based microsensor was coated by Nafion (see Section 2) to eliminate the diffusion and adhesion of anionic species easily oxidisable at the surface of the electrode. As can be seen from Fig. 1 (curve b) the addition of nitrite at a concentration ten times greater than NO did affect neither the height of the signal, nor the potential of the peak (Fig. 1, curve a) (0.63 V vs. SCE). A porphyrinic sensor lacking a coating of Nafion, or just carbon fiber without porphyrine, shifted the potential to a more positive value (0.77 V vs. SCE), and the current of the peak is larger than the identical concentration of NO detected by a Nafion coated electrode (Fig. 2, curve a). This may be due to air oxidation of the NO solution to nitrite and the oxidation of both species on the porphyrinic elec-
268
s. MesriroS et al. /Analytica 1.6,
-0.21 0.2
I 0.4
I 0.6
I 0.6
, 1.0
, 1.2
ElVvs.SCE
Fig. 1. Differential pulse voltamrnograms of a Nafion coated NO biosensor; scan rate: 10 mV SC’; pulse height: 25 mV, pulse width: 50 ms; medium: phosphate buffer pH 7.4 (a) NO standard 2.5x10-s mall-’ (solid line); (b) NO standard 2.5x10-s mol I-‘+2.5x 10m4 nitrite (solid circle).
trode. As can be seen from Fig. 2, curve b after the addition of a nitrite solution ten times more concentrated than NO, the current of the oxidation peak has increased. From these experiments we conclude that Nafion is not affected by nitrite and the peak current is exclusively due to NO. 3.2. Determination
of NO saturated solution
Before the sensor can be used for the determination of NO, it is necessary to obtain the exact value of the potential. This can be determined by differential
a)
-1
0 0.2
0.4
0.6
0.6
1.0
1
1.2
ElVvs.SCE
Fig. 2. Differential pulse voltammograms with an uncoated Nafion NO biosensor; scan rate: 10 mV s-‘; pulse height: 25 mV, pulse width: 50 ms; medium: phosphate buffer pH 7.4 (a) NO standard 2.5x 10m5 mol 1-l (solid line); (b) NO standard 2.5x 10-s mol 1-l + 2.5 x 10e4 nitrite (solid circle).
Chimica Acta 339 (1997) 265-270
pulse voltammetry (DPV). The value of the constant potential is a couple of millivolts larger than the peak potential. The applied electrochemical reaction was the one-electron oxidation of NO. Also, it was necessary to determine optimum duration of the chronoamperometric experiment to avoid the influence of convection caused by the density change in the diffusion layer as well as by the instability of the apparatus. This influence demonstrates itself by the bending of the otherwise straight line (Id/A) vs. 4 dependence toward higher l/d/A values. NO was determined in phosphate buffer. The precision and accuracy of absolute chronoamperometric analysis was tested within the concentration range of 10e4 to 10e3 mol 1-l. The results are summarized in Table 1 and compared with the Griess UV-VIS method. The chronoamperometric experiment gives a concentration of the saturated solution of 1.74f0.03 mm01 1-l in comparison to the experimental value obtained by UVNIS of 2.27f0.20 mmol 1-l. The higher value obtained from the UV/VIS experiment results from the presence of nitrite. The Griess reaction is not specific to NO, it will also react with nitrite. The theory of absolute chronoamperometric analysis predicts that the sensor for NO measurement should be independent of the temperature of the analyte. This theory was experimentally investigated within the range of 5°C to 40°C. The results are summarized in Table 2. A statistical test of the arithmetic means showed that none of the average different concentrations, c(experimenta1) at temperatures are significantly different. None of the c(experimenta1) values can be considered as outlier with respect to their arithmetic mean (1.72~tO.02 mmol 1-l). From this it can be concluded that the temperature change does not significantly influence the results of chronoamperometric analysis. During the analysis, however, the analyte temperature must be kept constant and homogeneous, but due to our short response time (about 1 s), this was not a problem. The effect of electroinactive substances present in the analyte at high concentrations was also investigated. These substances can significantly alter the density and viscosity of the analyte. To test the effect of dextran (MW 70000), on the NO determination. The viscosity of the prepared solution of dextran has
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Table 1 Precision and accuracy of the chronoamperometric determination of the concentration of NO saturated solutions c(experimental), in phosphate buffer (pH 7.4) at different concentrations of NO solution (estimated concentration from thermodynamic tables for saturated solution) c(est). Current sampling interval 0.04-l s after starting the electrolysis at the potential E=0.7 V, prior to electrolysis the potential is E=O.O V, all vs. SCE. Working electrode-porphyrine biosensor; ~35 urn and 1=4 mm; T=310 K; volume of phosphate buffer V=4 ml; number of analysis n=lO Volume of standard (ml)
c(est.) x lo4 (mall-‘)
0.25 0.50 1.00 1.50
Z(experimental)x a (mol 1-l)
1.17 2.22 4.00 5.45
lo4
E(experimental)x b (mol 1-l)
lo4
*
(11
*
**
1.04 1.91 3.48 4.73
17.68 17.19 17.40 17.35
1.31 2.69 4.62 5.77
22.27 24.17 23.12 21.18
a Chronoamperometric experiment. t UVNIS experiment. concentration obtained from experiment. ** concentration of NO saturated solution. s, - standard deviation of chronoamperometric sa - standard deviation of UVNIS experiment
** s, (mol 1-l)
** % (mol I-‘)
0.5 1 0.32 0.25 0.23
3.23 2.58 2.24 2.17
experiment (calculated for saturated NO solution). (calculated for standard NO solution).
Table 2 Influence of analyte temperature and viscosity on the precision and accuracy of NO determination by absolute chronoamperometric analysis. Concentration of standard (mean from Table 1)=1.74x 10m3 mol 1-l. Current sampling interval 0.04-l s after starting the electrolysis at the potential E=0.7 V, prior to electrolysis the potential is E=O.O V, all vs. SCE. Working electrode - prophyrine biosensor; r=35 pm and 1=4 mm; volume of phosphate buffer V=4 ml; number of analysis n=lO Temperature
278 288 298 310 313 310 310 310 310 310
(K)
Viscosity
(lo3 N s m-r)
1.53 1.15 0.90 0.76 0.67 1.00 1.50 2.00 3.00 3.50
* concentration obtained from experiment. ** concentration of NO saturated solution. s - standard deviation (calculated to saturated
f(experimental)x
lo4 (mol 1-l)
s** (mol 1-t)
*
**
3.41 3.43 3.46 3.48 3.46 3.47 3.45 3.48 3.50 3.46
17.05 17.15 17.30 17.40 17.30 17.35 17.25 17.40 17.50 17.30
0.34 0.27 0.31 0.25 0.24 0.26 0.28 0.32 0.34 0.31
NO solution).
a similar viscosity as blood (2.8x lop3 N s mm*). The results of the analysis are summarized in Table 2. As can be seen from Table 2, the determined values of NO concentration are independent of the viscosity (concentration of dextran) with respect to their arithmetic mean (1.74f0.01 mm01 1-l). The described and discussed results demonstrate that the chronoamperomeric analysis with porphyr-
ine-based microsensor can be regarded as an absolute method for the determination of NO saturated solutions, within the concentration range lop4 to lop3 mol 1-i. In the described method, to obtain the concentration of the NO saturated solution is easy, fast and it eliminates the influence of nitrite, which is also present in standard stock solutions. The proposed method enables also to determine values of diffusion
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et al. /Analytica
coefficient of NO. The accuracy and precision comparable with that given for concentration.
Chimica Acta 339 (1997) 265-270
is
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