Anal. Chem. 1907, 5 9 , 2005-2010
ACKNOWLEDGMENT The authors thank Quinis Long and Eileen S. Morgan for typing this manuscript. We also thank Thomas Bernert, Omar Henderson, and Wayman Turner for performing the lipid analyses and James Pirkle for technical support. We also thank Brenda Lewis and Carolyn Newman for providing the materials and logistics for encoding, handling, and sample receiving. Registry No. TCDD, 1746-01-6;dibenzo-p-dioxin, 262-12-4. LITERATURE CITED Karasek, F. W.; Hutzinger, 0. Anal. Chem. 1986, 58, 633A-642A. Ballschmlter, K.: Buchert, H.; Nlemczyk, R.; Munder, A,; Swerev, M. Chemosphere, in press. Henck, I. M.; New, M. A.; Kociba, R. J.; Rao, K. S . Toxicoi. Appi. Pharmacol. 1981, 59, 405. Tuchmann-Duplessls in AccMental Exposure to Dioxins , Human Health Aspects; Coulston, F., Ed.; Academic: New York, 1983; p 201. Wassom, J. S.; Huff, J. E.; Loprleno, N. Mufat. Res. 1977/1978, 4 7 , 141. Poland, A.; Knutson, J. Annu. Rev. Pharmacol. Toxicol. 1982, 22, 517. Hoffman, R. E.; Stehr-Oreen, P. A,; Webb, K. A,; et al. JAMA, J . Am. Med. Assoc. 1986, 2 5 5 , 2031-2038. Stehr, P. A.; G. Stein; H. Falk; et al. Arch. Environ. HeaRh 1986, 4 1 , 16-22. Rappe, C.; Nygren, M.; Gustafson, G. Chbrlnated Dloxins and Furans in the Total Environment; Choudhary, G.; Keith, L., Rappe, C., Eds.; Butterworth: MA; Stoneham, 1983; pp 355-365. Heath, R. G.; H a r k s , R. L.; Gross, M. L.; et al. Anal. Chem. 1986, 58, 463-468. Rappe, C.; Bergovist, P. A.; Hansson, M.; Kjeller, L. 0.; Llndstrom, 0.; Marklund, S.; Nygren, M. Banbury Report 18: Biological Mechanisms of Dioxin Action 1984, pp 17-25. Schecter, A.; Ryan, J. J.; Gliltz, 0. I n Chlorinated Dioxins and Dibenzofurans in Perspective; Choudhary, G.; Kelth, L. H., Rappe, C., Eds.; John Lewis: Chelsea, MI, 1986; Chapter 4 pp 51-65. Gross, M. L.; Lay, J. O., Jr.; Lyon, P. A,; et al. Environ. Res. 1984, 33, 261-268. Patterson, D. G., Jr.; Hoffman, R. E.; Needham, L. L.; et al. JAMA, J . A m . Med. Assoc. 1988, 2 5 6 , 2683-2686. Patterson, D. G., Jr.; Holler, J. S.; Smith, S. J.; et al. Chemosphere 1986, 15, 2055-2060. Aibro, P. W.; Crummett, W. B.: Dupuy, A. E. Jr.; et al. Anal. Chem. 1985, 57, 2717-2725.
2005
Patterson, D. 0.. Jr.; Holler, J. S.;Lapeza, C. R. , Jr.; et al. Anal. Chem. 1986, 5 8 , 705-713. Lapeza, C. R., Jr.; Patterson, D. G., Jr.; Llddle. J. A. Anal. Chem. 1988, 58, 713-716. Halogenated Biphenyls, Terphenyls , Naphthalenes, Dibenzodioxins and Related products ; Kimbrough, R. D., Ed.; Elsevier/North-Holland Bbmedlcal Press: New York, 1980. National Research Council Prudent Practices for Handling Hazardous Chemicals in Laboratories; National Academy Press: washington, DC, 1981. EPA Method 613 Fed. Regist. 1079, 44(233), 69326-69330. H a r k s , R. L.; Oswaid, E. 0.; Wilkinson, M. K.; Dupuy, A. E.; McDaniel, D. D.; Tai, Han Anal. Chem. 1980, 5 2 , 1239-1245. Alexander, L. R.; Patterson, D. G.; Myers, G. L.; Holler, J. S. Environ. sci. Techno/. 1986, 20, 725-730. Safe Handling of Chemical Carcinogens, Mutagens, Teratogens and Highly Toxic Substances; Walters, D. B., Ed.; Ann Arbor Science Publishers: Ann Arbor, MI, 1980. Patterson, D. G.. Jr.; Alexander, L. R.;Gelbaum, L. T.; et al. Chemo sphere 1986, 75, 1601-1604. DuPont Aufomatic Clinical Analyzer (ACA ) Chemistry Manual; DuPont: Wllmlngton, DE, 1983. Beveridge, J.; Johnson, S. Can. J . Res., Sect. B . 1949, 2 7 , 159-163. Folch, J.; Lees, M.; Sloan-Stanley, G. H. J. Bioi. Chem. 1957, 226, 497-509. Cooper, G. R.;Duncan, P. H.; Haziehurst, J. S.; et al. Selected Methods for the Small Ciinicai Chemistry Laboratory;American Association Clinlcai Chemist, 1981. Cheek, C. S.; Wease D. F. Clin. Chem. (Winston-Salem, NC.) 1969, 75, 102-107. Graham, M.; Hlleman F. D.; Wendling J.; et al. Chlorocarbons in Adipose Tissue Samples. Presented at the 5th International Symposium on chlorinated Dioxins and Related Compounds, Bayreuth, West Germany, Sept. 17, 1985. Ryan J. J.; Wililams D. J., Abstracts of Papers, 186th National Meetlng of the American Chemical Society, Washington DC; American Chemistry Society: Washington DC, 1983; Environmental Chemistry Section, Abstract 55, p 157. Graham M.; Hileman F. 0.; Wendling J.; et al. Background Human Exposure to 2,3.7,8-TCDD. Presented at the 4th International Symposium on Chlorlnated Dioxins and Related Compounds, Ottawa, Canada, October 16-18, 1984.
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RECEIVED for review January 5,1987. Accepted April 27,1987. Use of trade names is for identification only and does not constitute endorsement by the Public Health Service or the U.S. Department of Health and Human Services.
Spatiotemporal Description of the Diffusion Layer with a Microelectrode Probe Royce C. Engstrom,* Trevor Meaney, and R a y Tople Department of Chemistry, University of South Dakota, Vermillion, South Dakota 57069
R. Mark Wightman Department of Chemistry, Indiana University, Bloomington, Indiana 47405 Carbon fiber microelectrodes were used to measure the mlcroscoplcally local concentratlon of chemkal specles wlthln the dmusion layer of another electrode. concentration profWes of terrlcyanlde were obtalned at a platlnum electrode durlng a double potentlal step experlment. On the forward step, reliable concentratlon profiles could be obtalned at times as short as 20 ms after the potentlal step, when the dtffuslon layer thlckness Is only 4 pm. On the reverse step, reWable proflles were obtalned at times greater than 100 ms. The technlque was used to study the electrochemlstry of epC nephrlne, In which the oxldatlon prdduct undergoes chemical transformation wlthln the dlffuslon layer. Concentration profiles of both the Immediate electrochemlcal product and the chemical reactlon product were obtalned. Dlfferentlatlon between two possible mechanlsms was accompllshed by comparing experimentally determined concentratlon profiles wlth proflles generated by dlgltal slmulatlon. 0003-2700/87/0359-2005$01.50/0
Under typical electroanalytical conditions, concentrations of chemical species are perturbed from their bulk solution values at distances away from the electrode surface measured in tens of micrometers or less. While theories exist that describe interfacial concentrations as a function of distance and time for a number of electroanalytical situations, the experimental observation of interfacial concentrations has not been possible due to the lack of techniques with the necessary spatial and temporal resolution. To provide direct observation of concentration profiles relevant to electroanalytical chemistry, the probe must provide spatial resolution in the micrometer range and temporal resolution approaching the millisecond range. In addition, the ideal probe would not perturb the interfacial concentrations from their normal profiles. Two approaches to interfacial chemical analysis meeting those requirements have been reported. Jan et al. (1,2) have 0 1987 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 15, AUGUST 1, 1987
used a spectroelectrochemical approach, in which a column of light from a laser source was made to pass in a direction parallel to the electrode-solution interface and a t a position that included the interface boundary. After traversing the interfacial region, the light was magnified and imaged onto a diode array detector. Absorption by chromophores in the interfacial region was monitored as a function of time and distance away from the electrode. Concentration profiles were obtained at times as short as 50 ms after a potential step, when the diffusion layer had a thickness of only 8 pm. We have reported on another approach to interfacial chemical analysis, which uses a microelectrode to measure chemical species produced at another electrode (3). In that experiment, the microelectrode tip was positioned near the surface of another electrode, the specimen electrode, and the microelectrode amperometricdy detected the product of the specimen electrode reaction. With the use of carbon fiber microelectrodes ( 4 , 5 ) , it was possible to spatially resolve concentrations in the diffusion layer in planes both parallel and perpendicular to the interface. The microelectrode probe experiment was not the f i s t example of two working electrodes spaced only microscopic distances apart. "Twin-electrode" arrangements (6-8),interdigitated arrays (9, I O ) , and paired-band electrodes (11,12)have been reported, each involving electrodes held at independent potentials and separated by only micrometers, but none were applicable to making spatially resolved measurements. Recently, a report has appeared on the use of microelectrodes for the in situ measurement of electrode surface topography with submicrometer resolution in an adaptation of scanning tunneling microscopy (13). In the present paper, we report on the further development and characterization of the microelectrode probe for the determination of concentration profiles within the diffusion layer. Improvements in microelectrode design, microelectrode positioning, and data acquisition have led to improved spatial and temporal resolution over that reported earlier (3). High-resolution concentration profiles are presented for a diffusion-controlled system and for a system involving homogeneous chemical reaction of an electrogenerated species.
EXPERIMENTAL SECTION Apparatus. A laboratory-built bipotentiostat of conventional design (14) was used to control the potentials of the specimen electrode and the microelectrode independently. The potentials were fed to the bipotentiostat from an IBM PC (Boca Raton, FL) via a Tecmar Labmaster interface (Scientific Solutions, Solon, OH). The specimen electrode potential could be pulsed between two preselected values with a minimum pulse duration of approximately 25 ms. The microelectrode potential was held at a constant value during the pulsing of the specimen electrode. Current through the microelectrode was amplified with a Kiethley Model 427 current amplifier (Cleveland, OH), usually operated at a gain of lo9 VIA and at a rise time of 3 ms. The output of the current amplifier was fed to the analog-to-digital converter of the interface for storage and processing by the computer. Microelectrode positioning was accomplished with a Kliiger Model CC1.2 controller (Richmond Hill, NY) and stepper-motor driven positioners capable of 1-pm increments. The specimen electrode used for the work reported here was made from a 0.5-mm-diameterplatinum wire sealed in glass and polished to a final finish with 0.05-~malumina. The carbon fiber microelectrodes were prepared as described in the literature ( 4 , 5) from Thornell P-55 fibers (Union Carbide Corp., Danbury, CT) of 10-pm diameter. Before use, the microelectrode tips were beveled at an angle of approximately45O on a commercial beveling apparatus (WP Instruments, Inc., New Haven, CT) as previously described (15). If beveling was not carried out, reproducibility of the microelectrode response suffered due to the nonreproducibility of the interelectrode geometry. The microelectrode was positioned over the specimen electrode in a holder designed t o reproduce the angle and rotational orientation of the microe-
"
I
07 06 05 0 4 0 3 0 2 01 0 0 - 0 1 Microelectrode Potential, V Figure 1. Current-tlme response of microelectrode to a potential step
at the specimen electrode. lectrode. A microscope (Bausch and Lomb Stereozoom,Rochester, NY) was used to aid in positioning the microelectrode. A saturated sodium calomel electrode (SSCE) and a platinum wire served as reference and auxiliary electrodes, respectively. The cell, microscope, and micropositioning stage were all mounted on a vibrationally damped platform, which was in turn placed inside a Faraday cage. Digital simulations were carried out on the IBM PC equipped with a math coprocessor. In all simulations,100 volume elements and 500 time iterations were used. Reagents. All inorganic chemicals were reagent grade and used without further purification. (-)-Epinephrine was purchased from Sigma Chemical Co. (St. Louis, MO) and used as received. In the epinephrine experiments, buffers were prepared from potassium dihydrogen phosphate adjusted to the appropriate pH. Doubly distilled water was used throughout.
RESULTS AND DISCUSSION Current-Time Response in a Diffusion-Controlled System. To characterize the performance of the microelectrode probe experiment, the electrodes were bathed in a solution containing approximately 2 mM potassium ferrocyanide and 1.0 M potassium chloride. The specimen electrode potential was stepped from a value of 0.0 to 0.6 V vs. SSCE, the latter being in the mass-transfer limited region of ferrocyanide oxidation a t platinum. With the microelectrode positioned at the distance of closest approach (see below), its current was recorded during a 2-s pulse of the specimen electrode potential. As shown in Figure 1,when the microelectrode potential was a t 0.2 V or less, its current increased rapidly from zero to a steady positive value, indicating reduction at the microelectrode of ferricyanide produced at the specimen electrode. Upon return of the specimen electrode back to 0.0 V, production of ferricyanide ceased and the microelectrode current immediately dropped back to zero. When the microelectrode was held at poentials positive of 0.4 V, its current before and after the potential pulse was a t a steady negative value indicating direct oxidation of ferrocyanide at the microelectrode. During the potential pulse, the microelectrode current went nearly to zero as ferrocyanide near its tip was consumed by the specimen electrode. In other words, the specimen electrode very efficiently shielded the microelectrode from the electroactive species in a manner similar to that at a ring-disk electrode when the disk and ring are a t the same potential. Current-time curves a t varying interelectrode distances were recorded during 2-s potential pulses of the specimen electrode from 0.0 to 0.6 V with the microelectrode potential set a t 0.0 V. Solution conditions were the same as for the previous experiment. Figure 2 shows the results at 5-pm intervals, with the first one taken at the distance of closest approach. As the interelectrode distance increased, the response time on both the forward and reverse step increased due to the greater time needed for ferricyanide to diffuse across the gap. In addition, the response amplitude decreased with increasing interelectrode distance, reflecting the decrease in ferricyanide concentration as it diffuses away from its source, the specimen electrode. In practice, the position of closest approach was determined by moving the microelectrode in fairly close to the specimen electrode with the aid of the microscope. Final positioning was accomplished by pulsing the specimen electrode potential and monitoring the microelectrode response amplitude and
ANALYTICAL CHEMISTRY, VOL. 59, NO. 15, AUGUST 1, 1987
P
5t
,
~
2007
.. 1
”_,_....-..f \ e, -, i ., -,,-. I’ ~
40
Distonce,pm
0
2 Time, s
4
Flgure 2. Current-time response of mlcroelectrode at varlous Interelectrode dlstances.
time course. The response would continue to become larger and sharper until contact between the two electrodes was achieved. At that point, the electrodes would either short out or the response would simply no longer increase. In either case, the microelectrode was moved away by a single 1-pm increment, and that position was assigned a distance of zero. Additional uncertainty due to nonparallel orientation between the two electrodes is estimated to be no more than 2 pm, giving a total absolute uncertainty of 3 pm to the interelectrode distance. The reproducibility of positioning the microekrode at the distance of closest approach was asseased by mounting and positioning a microelectrode three different times, and recording the current-time curve for each mounting. The relative standard deviation of the response amplitude was 1.2%. With unbeveled microelectrodes, that same operation produced a relative standard deviation of 30%. Concentration Profiles i n a Diffusion-Controlled System. Concentration profiles were obtained by storing a set of current-time curves such as those in Figure 2 in the computer and retrieving the currents at a given time as a function of distance. The responses at that time were normalized to the response at the distance of closest approach to facilitate comparison with theory. Plots of normalized response on the forward step of ferrocyanide oxidation are shown in Figure 3, where response is on the y axis, distance is on the x axis, and time is on the z axis. The current was sampled at 10-ms intervals and the raw current-time data were subjected to smoothing by a three-point running average prior to construction of the concentration profiles. The solid lines in Figure 3 represent theoretical concentration profiles (normalized to the bulk concentration of reactant) calculated for the product of a diffusion-limited reaction under potential step conditions (ref 14,p 180) by using a diffusion coefficient of 7.63 X lo4 cm2/s (16). Figure 3a shows that at times greater than 30 ms, the normalized response profile is in excellent agreement with the theoretical concentration profile, indicating that the microelectrode current is a reliable probe of the microscopically local ferricyanide concentration within the diffusion layer. At 20 ms, significant deviation is apparent a t low responses, and that time represents the lower limit accessible in the present experimental configuration. At that time, the width of the diffusion layer measured at a normalized concentration of 0.5 is only 4 pm. At times between 50 and 500 ms (Figure 3b), agreement between theory and experiment is excellent, with no evidence of systematic deviation. At times greater than 1s (Figure 3c) significant deviation is present and becomes more noticeable as time increases. Several factors can be identified that would contribute to deviation at long times. First, recycling of electroactive material between the two electrodes would be expected when the interelectrode distance is smaller than the microelectrode diameter and when the time of measurement exceeds the time needed for diffusion over a distance ap-
Dietonce,pm C
I 0)
E
3 a 0 I50 ’
0 Distance, p
Flgure 3. Experimental (dots) and theoretical (lines) concentration profiles for the forward step of ferrocyanlde oxidation.
proximately 3 times the interelectrode distance. (The material would need to diffuse to the microelectrode, back to the specimen electrode, and again to the microelectrode to be deteded more than once.) The current under those conditions would be greater than that in the absence of recycling; however when the data are normalized to the response at the distance of closest approach, the result would be a negative deviation of experiment from theory, as is evident in Figure 3c. Another source of error is blocking of the specimen electrode from incoming electroactive material by the microelectrode tip, which would also be most noticeable at small distances and long times. The result would be decreased production of product at the specimen electrode and therefore a decreased current at the microelectrode, which would result in positive deviations when plotted in the normalized fashion. A third source of long-time deviation would be convection due to imperfect vibration isolation. Although the effects of shielding and recycling could be explored with two-dimensional digital simulation, we have not done so yet due to the good agreement that does exist at times less than 1s. Further improvements in the microelectrode probe technique will be directed toward making measurements at shorter times, where the above processes are less important. Results of the reverse potential step, from 0.6 back to 0.0 V, are shown in Figure 4. Theoretically, the concentration of ferricyanide should immediately be driven to zero at the electrode surface, but the 50-ms profile in Figure 4a shows that the microelectrode current does not respond immediately. However, by 100 ms the microelectrode current is a reasonably accurate representation of the concentration on the reverse
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 15, AUGUST 1, 1987
oxidized anodically to its quinone (EQ), which proceeds via deprotonation and cyclization to leucoadrenochrome (L)
a
OH
EO
E
Time, m \S
500
..e..
... ...
0
2H+
40
-
'
O
m
o
HO
Disiance,lm
H
+
2H+ ( 1 )
I CH3
b L
L is an easily oxidizable species and can undergo oxidation at the electrode in a classical ECE mechanism to form adrenochrome (A)
Distance,prn
Table I. Potentials Involved in the Epinephrine Experiments solution
cathodic wave
1.0 M HzS04
I I
0.59
pH 4.4 buffer
-0.08
pH 6.1 buffer
I1 I1
Epeska 0.20 -0.14
CH3
CH,
Flgure 4. Experimental (dots) and theoretical (lines) concentration profiles for the reverse step of ferrocyanide oxldatlon after a 1-s potential pulse.
El: 0.20
A
L
or it may be chemically oxidized by EQ in a homogeneous reaction, again forming A and regenerating E in an ECC process (20, 21)
E2c"
-0.30 EQ
0.00
-0.40
L
"Volts vs. SSCE. step and remains so until approximately 500 ms. A t longer times (Figure4b) the current is significantly lower than theory. The positive deviation at short times is probably due to radial diffusion to the microelectrode of ferricyanide that has accumulated in the solution adjacent to the microelectrode tip during the potential step. The long time deviations are probably the same ones operating on the forward step, except that recycling would not be operative because the specimen electrode is at a nonoxidizing potential. In summary, the experiments with ferrocyanide show that on the forward step, useful concentration profiles can be obtained at times between 20 and approximately 2000 ms, where the "half width" of the diffusion layer is 4 and 35 pm, respectively. On the reverse step, the useful time frame is 100 to approximately lo00 ms. Although the microelectrode must be considered an "invasive probe", since it consumes the material being measured, disturbance of the diffusion layer is minimal over the time frame specified. Concentration Profiles in a System Involving Homogeneous Reaction. The microelectrode should be capable of detecting a chemical species generated at the specimen electrode as long as that species has a lifetime comparable to or greater than the transit time between the two electrodes. We demonstrated earlier the detection of the free radical intermediate in the reduction of nicotinamide adenine dinucleotide (3),a species considered to have a lifetime in the millisecond time domain (17, 18). In the present work, we have collected concentration profiles that characterize the chemical conversion of the product of epinephrine oxidation. The electrochemistryof epinephrine was studied by Hawley et al. (19). In their proposed mechanism, epinephrine (E) is
PH l CH3
A
E
A
Hawley et al. concluded that the ECC mechanism was operative based on the apparent number of electrons in the initial oxidation step. It was our intent to observe the concentration profiles for the immediate product of oxidation, EQ, as well as the end product of the homogeneous reactions, A, since both are electroreducible, but at distinct potentials. The overall rate of the chemical transformation of EQ to A is pH dependent below pH 6 because of the deprotonation step. In dilute sulfuric acid, the chemical reaction rate is negligibly slow on the time scale of cyclic voltammetry at 100 mV/s, so the reduction of only EQ is observed on the negative-going scan. At pH 6 or higher, the chemical reaction rate is fast, so that the reduction of only A is observed. A t intermediate pH values reduction of both species is observed. This pattern was reproduced in the present study, with the peak potentials for the various reductions given in Table I. In a 2 mM solution of E, the specimen electrode potential was stepped from 0.0 V to a value in the diffusion-limited region of the oxidation G f E (0.70 V at pH 6.1 and 0.80 V at pH 4.4). Concentration profiles were first obtained with the microelectrode potential at El, (see Table I) where only the immediate product of the electrochemical reaction, EQ, was detected. Profiles were then obtained at a potential, E2,,where both EQ and A were detected. The concentration of A alone was obtained by subtracting the responses at El, from those
ANALYTICAL
CHEMISTRY, VOL.
pH 6.1
pH 4.4
c C-
IO
-
C
E Or-
59, NO. 15, AUGUST 1, 1987
2009
2,"
AAA4 Microelectrode Potential, V
Flgure 8. Current-time responses in the epinephrine system at various microelectrode potentlals. U.3
Q I
\
C
n 01
a 0 I
-0
n
20
20
0
Distance, prn
Flgure 5. Experimental concentrations profiles for the epinephrine system. The z axis is time, ranging from 50 to 500 ms in 50-ms increments.
at E& The profiles are shown in Figure 5, where the left-hand column was taken at pH 4.4 and the right-hand column was taken at pH 6.1. The responses at a given pH and a given time were all normalized to the response at Ezc. Qualitatively, in the time frame shown (50-500 ms) the profiles at Ezc show diffusion-controlled behavior as expected for the undifferentiated detection of two species with similar diffusion coefficients. The profiles at E,, show that the concentration of the immediate electrochemical product, EQ, is relatively low at pH 6.1 where chemical reaction proceeds rapidly and relatively high at pH 4.4 where the reaction is slower. The difference profiles, Ezc - Elc, show the concentration of the chemical reaction product, A, already at a high value at pH 6.1, but at a low value and slowly increasing at pH 4.4. Differentiation between the two possible mechanisms was accomplished by examining the shapes of the concentration profiles. In the ECE mechanism, the overall chemical reaction is first order with respect to the concentration of EQ, characterized by a first-order rate constant, k1 kl
EQ-L
(4)
The behavior of EQ and E would therefore be described by the relationships
(5)
In the ECC mechanism, the overall chemical reaction of EQ would be second order, with a second-order rate constant, k2
2EQ
k2
A
+E
(7)
The behavior of EQ and E would be described by
We attempted to differentiate between these two possible mechanisms by comparing experimentally obtained concen-
Distance, urn
Figure 7. Concentration profiles of adrenalinequinone: (0)experisecond-order simulation. mental; (- - -) flrst-order simulation; (-)
tration profiles of EQ with profiles produced by digital simulation. Experimentally, profiles were obtained in a 1.76 mM epinephrine solution, buffered at pH 5.7. Since it was crucial that the concentration of EQ alone was measured, it was important to set the microelectrode at the correct potential. Current-time curves were first taken with the microelectrode at the distance of closest approach and at various microelectrode potentials. In all cases, the specimen electrode potential was stepped from 0.0 to 0.8 V for 2 s. The responses are shown in Figure 6. At 4.1 V or less, both EQ and A were detected, and from 0.0 to 0.2 V, only EQ was detected. The decrease in current with time in the potential region where EQ is detected reflects its consumption by chemical reaction, whereas when both EQ and A are detected, the response approaches a steady value. A potential of 0.05 V was selected to monitor the concentration of EQ alone. The concentration profile of EQ taken 200 ms after the potential step is shown in Figure 7 (dots). By use of finite difference simulation (ZZ),the first-order disappearance of EQ (ECE mechanism) was modeled by using the finite-difference forms of eq 5 and 6 and the second-order process (ECC) was modeled by using eq 8 and 9. As can be seen in Figure 7, the second-order disappearance of EQ follows the experimental results more closely; thus it can be concluded that the ECC mechanism dominates, in agreement with Hawley et al(19). A third fit was attempted by allowing for both first- and second-order disappearance of EQ, since it is reasonable to expect that both chemical oxidation of L (ECC mechanism) and electrochemical oxidation (ECE) might occw. The behavior of EQ would then follow equation
-WQI - - DEQat
- k,[EQ] - k2[EQI2 ax2
(10)
and E would still be regenerated according to eq 9. A series of simulations was carried out by using the simplex optimization method (23,24)to find the combination of kl and k2 that best fits the data. However, the fit was not significantly better than that obtained for the second-order process alone.
CONCLUSIONS The microelectrode probe technique permits the visualization of concentrations within the microscopic domain of the diffusion layer at a combined spatial and temporal resolution
2010
Anal. Chem. 1987, 59, 2010-2012
equaled only by the spectroelectrochemical approach of Jan et al. (1, 2). Selectivity is available through control of the microelectrodepotential, making it possible to study the fate of different chemical species in the diffusion layer. Previous workers have determined the mechanism of homogeneous reactions by probing the diffusion layer spectroscopically(25, 26), but this appears to be the first example of studying mechanisms by direct observation of concentration profiles. It should be possible with the present system configuration to obtain concentration profiles for chemical species that are so reactive they exist no more than 5-10 km away from the electrode where they were generated, implying first-order rate constants up to approximately 200 s-l.
LITERATURE CITED (1) Jan, C.C.; McCreery, R. L. Anal. Chem. 1986, 58, 2771. (2) Jan, C.-C.; McCreery, R. L.: Gamble, F. T. Anal. Chem. 1985, 57, 1763. (3) Engstrom, R. C.; Weber, M.; Wunder, D. J.; Burgess, R.; Wlnqulst, S. Anal. Chem. 1986, 5 4 , 844. (4) Dayton, M. A.; Brown, J. C.; Stutts, K. J.; Wightman, R . M. Anal. Chem. 1980, 82,848. ( 5 ) Ewlng, A. 0.;Dayton, M. A.; Wightman, R. M. Anal. Chem. IS81, 5 3 , 1842
(6) &Duffle, B.; Anderson, L. B.; Reilley, C. N. Anal. Chem. 1986, 38, 883. (7) Anderson, L. B.; Rellley, C. N. J. Hectroanal. Chem. 1985, IO, 295. (8) Hubbard, A. T.; Anson, F. C. Anal. Chem. 1964, 36, 723.
(9) Sanderson, D. E.; Anderson, L. B. Anal. Chem. 1985, 57, 2388.
(IO) Chklsey, C. E.; FeMman, B. J.; Lundgren, C.; Murray, R. W. Anal.
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RECEIVED for review January 20, 1987. Accepted April 23, 1987. This work was supported in part by the National Science Foundation, Grant No. CHE-8411000 and CHE8500529, and was presented in part at the National Meeting of the American Chemical Society, New York, 1986.
Radioimmunologic Determination of Human C-Peptide and Proinsulin in Plasma with Anti-C-Peptide Serum after Prepurification by High-Performance Liquid Chromatography Martin Komjati,*JPeter Nowotny, and Werner Waldhausl
I. Medizinische Uniuersitatsklinik, Division of Clinical Endocrinology and Diabetology, Wien, Austria
A method Is descrlbed for analytical separatlon and subsequent determination of human GpepUde and proln8uln In plasma. To this end detennlnatlons of C-peptide were performed after Its separatlon from prolnsulln by hlgh-performance Uqukl chromatography (HPLC). Chromatographic p r a pur'flcatlonwas peformed wRh methanol and Sep-PAK CIS extractlon of plasma, foilowed by HPLC elutlon wlth acetonitrile In an ammonlum acetate/acetk acld buffer (pH 4.2). Thereby C-peptlde, lnsulln, and prolnsuln were separated skndtaneotrsiy In 35 % acetonitr#ewlthln 10 mln. Thereafter, Gpeptlde contsbllng fractkns were pooled and deealted, and Cpeptkle was measwed by radldmnwloaeray uslng antLCpeptlde antibodies as blndlng reagent. Subsequently, prolnsulln concentratlon was calculated as the dmerence between Its apparent molar concentratlon In plasma minus absolute plasma C-peptlde concentratlon, the lower llmlt for the detection of the latter belng 50 pmoVL. Thls approach for the determlnatlon of plasma C-peptlde and prdnsulln Is of value whenever crogs-reactlansbetween the two hormone mdeHes are to be absolutely avolded.
The availability of pure intact human proinsulin through recombinant DNA technology (I) raised interest in studies dealing with its physiological and therapeutical potential (2, Dedicated t o Prof. Werner Waldhausl, h i s 50th birthday.
M.D., on t h e occasion of
3). Since proinsulin is antigenically related to insulin and C-peptide, severe difficulties arise when one of the two latter compounds has to be estimated in plasma enriched by exogenously administered proinsulin. In this case proinsulin can only be removed either by physicochemical methods, e.g. high-performance liquid chromatography (HPLC), or by immunoprecipitation. The latter method requires highly specific antibodies directed against human proinsulin with negligible cross-reactivity vs. insulin and C-peptide. The generation of such antibodies and their application for human proinsulin radioimmunoassay have been described recently (4-6). Furthermore, to avoid the use of specific anti-human proinsulin antisera, separation by anti-insulin antiserum of insulin and human proinsulin has been used prior to estimation of C-peptide immunoreactivity in serum (7, 8). A number of papers have been published in the recent years on successful separation and analysis of pancreatic polypeptide hormones by reversed-phase HPLC (9-13). However, only one of these studies describes a method, in which pancreatic peptides were separated from biological fluids in order to determine the immunological activities of insulin, C-peptide, and glucagon by radioimmunoassay (13). To overcome the uncertainities of immunological crossreactivity, this paper describes a method for separation by HPLC of human proinsulin from endogenous C-peptide with subsequent measurement of chromatographed C-peptide by radioimmunoassay. Such a procedure is of considerable importance whenever analytical separation of proinsulin and C-peptide is required, particularily so during exogenous ad-
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