Anal. Chem. 1982, 5 4 , 429-434 (25) Gullbault, G. G.; Durst, R. A.; Frant, M. S.; Frelser, H.; Hansen, E. H.; Light, T. S.; Pungor, E.; Rechnltz, G.; Rice, N. M.; Rohm, T. J.; Simon, W.; Thomas, J. D. R. Pure Appl. Chem. 1976, 48, 127-132. (26) Meier, Peter C.: Ammann, Daniel: Morf, Werner E.: Slmon, Wllhelm I n Medical and Biological Applications of Electrochemical Devices"; KOM a , J., Ed.; Wiiey: Chiichester, New York, Brisbane, Toronto, 1980; pp 13-91. (27) Schulthess, P.; Shijo, Y.; Pham, H. v.; Pretsch, E.; Ammann, D.; simon, W. Anal. Chlm. Acta 1961, 131, 111-116.
429
(28) Niedrach, Leonard W. United States Patent 3 898 147, 1975.
RECEIVED for review June 12, 1981. Accepted October 28, 1981. This work was partly supported by the Swiss National Science Foundation and Orion Research. p. s.thank corning Ltd. for a grant.
Series Dual-Electrode Detector for Liquid Chromatography/Electrochemistry Daryl A. Roston and IPeter T. Kissinger" Department of Chemistty, Purdue Universiv, West La fayette, Indiana 47907
By utilization of a duai-electrode detector wlth the electrodes orlented In series with respect to the flow axis, oxldatlon products from the upstream detector are detected by reductlon at the downstream detector. Exploiting the oxidativereductive mode allows Improvements In three aspects of liquid chromatography/electsochemistry: seiectlvlly, peak identification, and high gain detection at extreme potentials. The above Improvements are demonstrated in the context of two pertlnent analytical problems: the determlnatlon of phenolic constituents In commercilal beverages and the determlnatlon of the metabolites of tho analgeslc acetaminophen in urine. Also included Is a discussion concerning the factors that arc important in determining the magnitude of the fraction of upstream products that are converted at the downstream detector (the so-called collection efficiency).
Liquid chromatography combined with electrochemistry (LCEC) has become a well-established technique for traceorganic determinations (I). The technique couples the advantages of high selectivity, low detection limits, and low cost. An innovative approach to electrochemical detection involves the use of dual-electrode transducers. Several studies have reported the use of dual-electrode detection for liquid chromatography (2-6). In each case, the orientation of the elec trodes with respect to the flow axis was one of the three configurations shown in Figure 1,which we have termed the parallel-adjacent, series, (andparallel-opposed configurations. Fenn e t al. explored the possibility of parallel-opposed dual-electrode detection to improve detection of catecholamines in blood plasma @). Blank employed a series configuration detector for dual-potential monitoring to improve specificity (3). Both electrodes were used in an oxidative mode. Roston and Kissiinger performed dual-potential monitoring with a parallel-adjacent detector to aid in the identification of phenolic constituents in commercial beverages (4). The design and use aC a series coulometric-amperometric detector was reported by Schieffer (5). Removal of easily oxidized components by the ''upstream* coulometric cell was shown to increase selectivity at the downstream detector. A potentially useful detection scheme for the series dualelectrode detector involves the detection of products from the upstream detector at the downstream detector. When used in this manner, the series detector is analogous to the wellstudied ring-disk electrolde in which products from the disk 0003-2700/82/0354-0429$01.25/0
are detected a t the ring. This study characterizes the analytical utility that results when oxidation products from the upstream detector are monitored in a reductive mode at the downstream detector (oxidative-reductive detection). This detection mode is analogous to the scheme described in a study by MacCrehan and Durst which appeared shortly before the submission of the present work (6). These workers employed series dual-electrode detection consisting of the downstreamoxidative detection of reduction products from an upstream mercury amalgam electrode (reductive-oxidative detection). Emphasis was on the elimination of oxygen interferences during reductive mode LCEC and improvements in detection limits for compounds that reduce at very negative potentials. The use of series dual-electrode detection for elimination of oxygen interferences in reductive LCEC has also been demonstrated by Bratin and Kissinger (7). The results reported here demonstrate that oxidative-reductive series detection using glassy carbon electrodes can be used to improve three aspects of liquid chromatography/ electrochemistry: selectivity, peak identity confirmation in complex samples, and high gain detection a t extreme potentials. These points are demonstrated in the context of two analytical problems: the determination of phenolic constituents in food materials and the determination of acetaminophen metabolites in urine.
EXPERIMENTAL SECTION Apparatus. The liquid chromatographic system was a Bioanalytical Systems LC-154 (Bioanalytical Systems Inc., West Lafayette, IN). A Biophase CIS column (25 cm X 4.6 mm) was employed. Dual-electrode LCEC experiments were carried out by use of two modified BAS LC-4A controllers. Technical details of this apparatus will be reported elsewhere (8). The dual-electrode detector was of the thin-layer amperometric design used in previous studies (9). Two circular glassy carbon electrodes, each 3 mm in diameter, were positioned in series in the thin-layer channel. A distance of less than 0.1 mm separated the electrodes. The rectangular cross-sectionof the thin-layer channel was 0.013 cm X 0.5 cm. For adequate potential control at both detectors, it is necessary to minimize ohmic losses by positioning the auxiliary electrode across the thin-layer channel from the working electrodes (detectors). The merits of this cell geometry have been described elsewhere for single-electrode transducers (10). The Ag/AgCl reference electrode was positioned downstream in the conventional manner. Reagents. Phenolic compounds were purchased from the following sources: gallic acid, vanillic acid, vanillin, ferulic acid, sinapic acid, 3,4-dihydroxyphenylaceticacid, 4-methylcatechol, Aldrich Chemical Co.; gentistic acid, caffeic acid, 4-hydroxy0 1982 American Chemlcal Soclety
430
ANALYTICAL CHEMISTRY, VOL. 54, NO. 3, MARCH 1982 P a r a l l e l -Adlacent
Series
Parallel-Opposed
Figure 1. Detector conflguratlons for thin-layer dual-electrode detection.
1 1 511 n A
iC,
0
12
8
4
MINUTES
I
6
0
8
4
12
16
MINUTES
Figure 3. Series dual-electrode chromatograms for caffeic acid. Conditions: (A) W1 = 4-1.0 V, W2 = +1.0 V; (B) W1 = +1.0 V, W2 = 0.0V; 25 cm Biophase C,Bcolumn; flow rate, 1 mL/min; 0.09 nmol of caffeic acid.
Figure 2. Cyclic voltammogram of caffeic acid. Conditions: 2.7 mM caffeic acld; medii, mobile phase; scan rate, 100 mV/s; glassy carbon electrode.
phenylacetic acid, Sigma Chemical Co.; 2-hydroxy-5-methoxybenzoic acid, p-coumaric acid, Eastman Kodak Co.; 4-hydroxybenzoic acid, Matheson, Coleman and Bell; hydroquinone, J. T. Baker Chemical Co. Sinapic and p-coumaric acids were recrystallized once from warm water. The other standards were used without purification. With the exception of methanol, reagent grade organic solvents were used without purification. Methanol was distilled once prior to use. Procedures. Extractions. A commercial beer sample was acidified to pH 2 and extracted with ethyl acetate as previously described ( 4 ) . A urine sample was obtained 4 h after a normal acetaminophen dosage (two Tylenol tablets) from an apparently healthy adult female. The urine sample was diluted 1:50 prior to injection onto the LCEC system. Liquid Chromatography. Pertinent experimental parameters were as follows: ambient temperature;flow rate, 1mL/min, unless otherwise specified; injection loop volume, 20 pL. For all of the liquid chromatograms except the urine and gentisic acid chromatograms, the mobile phase compositionwas as follows: 2.18% 1-propanol,1.98% acetic acid, 8.71% methanol, 87.13% deionized distilled water, 0.036 M ammonium acetate. For the urine and gentisic acid chromatograms,the mobile phase was 4.0% methanol, 96% 0.1 M acetate buffer, pH 4.
RESULTS AND DISCUSSION The series dual-electrode detection approach can be understood by considering the cyclic voltammogram of the compound caffeic acid shown in Figure 2. Well-defined oxidation and reduction currents are evident because the redox couple is chemically reversible. Figure 3A depicts the series chromatogram for caffeic acid when both electrodes are poised at +1.0 V. Oxidative responses are observed at the upstream (Wl) and downstream (W2) electrode. The downstream response is smaller because of the depletion of caffeic acid at the upstream detector. Figure 3B shows the chromatogram resulting when the downstream detector potential was changed to 0.0 V. An oxidative response was observed at the upstream detector and a reductive response was recorded at the downstream detector. The cathodic downstream current corresponds to the reduction of the oxidized form of caffeic acid produced a t the upstream detector. The fraction of upstream products that are converted at the downstream detector, termed collection efficiency, is important for analytical applications of the downstream signal. Detection limits utilizing the downstream signal will be defined by the magnitude of the collection efficiency. An additional requirement
is that the collection remain constant over the range of quantities evaluated to ensure linearity in the downstream response. The maximum achievable collection efficiency for a dualelectrode hydrodynamic system is termed No (11). The collection efficiency, No,represents the ratio of the current at the indicator electrode (downstream detector) to the current a t the generator electrode (upstream detector) when the product of the generator electrode is stable and the potential of the downstream detector is sufficiently positive or negative that the magnitude of the downstream current is limited solely by mass transport. In addition, it is assumed that the number of electrons transferred for the forward and reverse reactions are the same. For the series dual-electrode detector, collection efficiency is defined by the expression
No =
i(downstream) i (upstream)
(1)
Collection Efficiency. The collection efficiency, No,for dual-electrode hydrodynamic systems is defined by the cell dimensions. Complex expressions for No with dual-electrode systems in rectangular channels have been derived. Although the expressions have not been thoroughly experimentally validated, they clarify which detector dimensions are important in determining collection values. Matsuda put forth an expression showing that the determinant detector dimension for No is the ratio of the distance between the electrodes to the length of electrodes along the flow axis (12). Both electrodes were assumed to have the same length. As the ratio of the distance between the electrodes to the electrode length increases, No decreases. Braun derived an expression for collection at a dual-electrode series cell that is similar to the expression derived by Albery and Brukenstein for No at rotating ring-disk electrodes (13). The expression derived by Braun contains parameters based on two cell dimensions: the ratio of the distance between the electrodes to the length of the upstream electrode along the flow axis and the ratio of the lengths of the two electrodes along the flow axis. Limited experimental data on the effect of detector dimensions on collection with series dual-electrode systems are available. Using a cell consisting of two rectangular gold electrodes of equal size in rectangular channel, Gerischer et al. observed that the log of No was a linear function of the distance between the electrodes (14).The ferricyanide couple was used. The range of electrode spacing examined encompassed 10 to 110 pm. Collection efficiencies varied from -17% to -38% with the values increasing as electrode
- -
ANALYTICAL CHEMISTRY, VOL. 54,
0.368 0.367
4-methyl-1,2-dihydroxybenzene
0.348 ? 0.5
(4-methylcatechol) 3,4-dihydroxycinnannicacid (caffeic acid) 3-methoxy-4-hydroxybenzoic acid (vanillic acid) 3-methoxy-4-hydroxybenzaldehyde (vanillin) 3-methoxy-4-hydroxycinnamicacid (ferulic acid) 2-hydroxy-5-methoxybenzoicacid 4-hydroxycinnamic acid (p-coumaric acid) 3,5-dimethoxy-4-hyclr.oxycinnamic acid (sinapic acid) 4-hydroxybenzoic acid 3,4,5-trihydroxybenzaic acid (gallic acid) 4-hydroxyphenyl acetic acid
0.340
f f
f
0.5 0.5
0.8
~-
0.213 f 1.0 0.187
f
3.4
0.174 rt 1.7 0.141 i 3.7 0.132 t 4.1 0.109 0.044
i
0.1
?:
3.7
-
spacing decreased. Thus, the findings of Gerischer and coworkers (14) are consistent with the expressions derived by Matsuda (12) and Braun (13). No for the series dual-electrode detector utilized in this study is 0.37. This value was determined with hydroquinone, a compound that has bleen used to determine No values for rotating ring-disk electrodes (11). The value was determined as hydroquinone eluteld from the liquid chromatography column. The upstream detector potential was +1.0 V and the downstream detector was at -0.1 V. An No value of 0.37 is consistent with the collection values reported by Gerischer et al. (14) and MacCrehan et al. (6) in the studies mentioned above, which employed series dual-electrode hydrodynamic systems similar to the one used in this study. It is important to note that in the present work disk electrodes were used and therefore the gap between the electrodes varies across the channel. Effective Collection Efficiency. As described above, No represents the maximum collection efficiency that can be achieved with the dual-electrode hydrodynamic system. We have termed the collection efficiency observed under given experimentalconditions as the “effective”collection efficiency, Nefp For most compounds N,ff will be less than No. This situation will be observed when the downstream detection potential is not sufficiently positive or negative to render the downstream detector current mass-transport limited. Under these conditions, Neffcan be increased by increasing the downstream detector potential. When the downstream detector potential is such that the downstream current is mass transport limited and NeIfremains less than No,either or both of two additional factors may be responsible. Frequently,the chemical instability of the upstream product affects collection. (For simplicity, the following discussion assumes the number of ellectrons transferred at each electrode and the diffusion coefficients of all species are the same.) If the initial product (B) of the upstream detector ( W l ) reacts to form a secondary prolduct (C) that is electroactive at the downstream detector (W2), Neff is not changed by the homogeneous reaction. When the product of the upstream -
L
B
L
A
I* C
L
D
0.3 0.5
0.8 1.3
2.5 4.2
1.0
1.6 2.7 0.3 0.5
4-hydroxybenzoic acid
7.1 0.8
1.6
1.3 2.5 4.2
2.7
7.1
1.0
0.37 0.37 0.37 0.37 0.39 0.09
0.09 0.10 0.11
0.14 a Mean value from three chromatograms at each flow rate. detector reacts to form an electroinactive compound (C’), Ne, can be diminished. The extent of the decrease will be deA
L
B
w
e
A
1
0.032 f 5.2
-~
~~
hydroquinone
0.219 ? 2.2
Mean a w1 = t 1.0 V, W2 = -0.1 V, flow = 1 mL/min. from five chromatograms i relative standard deviation,
A
431
% collectionb
hydroquinone 2,5-dihydroxybenzoic acid (gentisic acid)
~
MARCH 1982
Table 11. Effect of Effluent Flow Rate on Collection
Table1.- Neffsof Several Phenolic Compoundsa compound
NO. 3,
C’
pendent on the reaction rate; only extremely short-lived species will not be detected at the downstream detector. A t a flow rate of 2 mL/min, the time required to traverse from the center of the upstream detector to the center of the downstream detector is -50 ms. This value can easily be decreased by decreasing the gasket thickness for the thin-layer cell, increasing linear velocity. It is also possible that an electroinactive product (0’) is subject to a subsequent reaction which yields an electroactive compound (E). The extent that collection decreases is dependent on the rates of the coupled chemical reactions. In the latter example, collection might go through a minimum as a function of flow rate depending on the relative values of kl and kp, the electroreactivity being temporarily “stored” in the reactive intermediate C”. Exw2
A - B - A
1 C”
/Xe E - F
pressions for collection efficiency when the upstream detector product is subject to chemical reactions have also been derived by Braun (13). The expressions incorporate the rate constant of the post electron transfer chemical reaction. The effect of chemical reactions on N,ffis reflected in the range of values summarized in Table I. Nef$observed for 2,5- and 3,4-dihydroxysubstituted phenolic acids were close or identical with No. The oxidized form of such compounds are stable. Decreased Nefawere observed for the hydroxymethoxy and monohydroxy substituted phenolic acids which participate in various post-electron transfer chemical reactions. When Ne, is diminished by the chemical instability of the upstream electrode product, N,ff can also be shown to be dependent on two additional factors, flow rate and the upstream detector potential. If the upstream detector potential is incrementally increased in the potential range where the magnitude of the upstream current is potential dependent, the concentration gradient produced at the upstream detector increases with detector potential. Therefore, the reaction rate can be dependent on the upstream detector potential for reactions which are second order. As the potential increases, the reaction rate increases and diminishes Neff.The occur-
ANALYTICAL CHEMISTRY, VOL. 54, NO. 3, MARCH 1982
432
trically similar dual-electrode hydrodynamic system, When a flow-rate range of -30 to -100 cm/s was examined, it was found that collection was not dependent on flow rate below velocities of -70 cm/s (14). Although it is possible to change collection with changes in flow rate when the upstream product is subject to chemical reactions, the small range of flow rates used for liquid chromatography limits the usefulness of such an approach for significantly decreasing detection limits. An additional situation which can render Neff< No when the downstream detector current is mass transport limited is when the reaction at each electrode involves a different number of electrons. For example, it is possible to irreversibly reduce an aromatic nitro group to the corresponding hydroxylamine, a 4e- process, and oxidize the latter compound to a nitroso compound, a 2e- process:
WI
PhNOz
’
/I
i
4e-
PhNHOH
PhNHOH
5
PhNO
Ne, will therefore be half of the value observed if the steps Series dual-electrode chromatograms for an ethyl acetate extract of a commercial beer sample. Conditions: W1 = +0.95 V, W2 = +0.25 V; 25 cm Biophase C,, column; flow rate, 1 mL/min. Figure 4.
rence of chemical reactions can also render Ne, flow-rate dependent. As shown in Table 11,Ne@for 4-hydroxybenzoic acid increased from -0.09 to -0.14 when the flow rate was increased from 0.3 mL/min (0.8 cm/s) to 2.5 mL/min (6.5 cm/s). As mentioned above, the oxidation products of monophenols are unstable. At increased flow rates, a lesser portion of the oxidation products become electroinactive before downstream detection and Ne, is increased. NeE does not exhibit flow-rate dependence within the range of flow rates used for liquid chromatography when the oxidized and reduced forms of the compound are stable. When the same flow-rate study was completed on hydroquinone, a compound that is stable in the oxidized and reduced form, no significant change in collection was observed. This is not in disagreement with the study by Gerischer et al. which examined the effect of flow rate on collection with a geome-
involved equal numbers of electrons. Calibration. If the downstream response is to be used for quantitation, the linearity of the upstream and downstream response is important. Hence, it is a requirement that Ne, remain constant for the range of quantities evaluated. The response of several of the phenolic compounds listed in Table I (caffeic acid, 4-methylcatechol, vanillic acid, and 4hydroxybenzoic acid) was evaluated over a 10 to -700 pmol range. Linearity of response (correlation coefficient 20.999) was observed at both detectors for these and all other conipounds examined thus far.
-
Improvements in Electrochemical Detection with the Series Dual-Electrode Detector. Three aspects of electrochemical detection can be improved with the series configuration detector: selectivity, peak identification, and high gain detection at more extreme detector potentials. Selectivity can be improved if the compounds of interest have relatively high N d . If an oxidative chromatographicpeak is comprised of the current responses of two compounds, one with a significantly higher Neffthan the other, the downstream signal
0I 8 O[
0 6-
ic,NORMALIZED
-
04 -
02-
i,,NORMAL .IZED
Figure 5. Normalized oxldative-reductive hydrodynamic voltammograms determined with series dual-electrode detection: (W) 4-hydroxybenzoic acid, (V)vanillic acid, (0)caffeic acld, (0)4-methylcatechol. Cathodic current values are normalized to the value recorded at -0.1 V. Anodic current values are normalized to the value recorded at + 1.1 V.
ANALYTICAL CHEMISTRY, VOL. 54, NO. 3, MARCH 1982
433
A
T
I
5nA
5nA
I
1
----h T I nA
W2
1
0
.
, 4
,
/
8
I2
‘
16
‘
4
20
0
4
MINUTES
8
12
16
20
MINUTES
Flgure 6. Series dual-electrode chromatograms for four phenolic acids: (1) 0.04 nmol of 4-hydroxybenzolc acid, (2) 0.02 nmol of vanillic acid, (3) 0.07 nmol of caffeic acid, (4) 0.06 nmol of 4-methylcatechoi. Conditions: (A) W1 = +1.10 V, W2 = +0.95; (B) W1 = +1.10 V, W2 = +0.35 V; (C)W1 = 4-1.1 V, W2 = 0.0 V; 25 cm Biophase C,8 column; flow rate, 1 mL/min. will be predominantly comprised of the reduction current of the compound with the higher Nefp Improvements in the selectivity and utility of a chromatogram of a complex sample by series detection are demonstrated in Figure 4, which illustrates chromatograms for an ethylacetate extract of a commercial beer sample. In contrast to the upstream oxidative chromatogram,the dominant peaks of the downstream reductive signal are those of vanillic and ferulic acid, compounds that contribute to the flavor and aroma of beer (15). In ddition, the utility of the early eluting portion of the chromatogram is enhanced with downstream detection. The complexity of the oxidative chromatogram makes it difficult to obtain retention data for many of the early eluting peaks. Retention data are readily discernible from the less complex downstream reductive chromatogram. A surprising aspect of the series dual-electrode system is that discrimination against compounds with chemically unstable oxidation products is not as extensive as was initially anticipated. Because of the short residence time of the chromatographic zone13over the detectors, only extremely short-lived species are not detected at the downstream detector. Consequently, compounds that appear chemically irreversible with “slower” potential scan techniques, such us cyclic voltammetry, often have significant Ne, values. Additional utilization of the series configuration detector concerns peak identification. Comparison of standard and unknown current-potential responses obtained from repeated LCEC experiments coupled with incremental detector potential changes can provide peak identity confirmation. Only oxidative (or reductive) current-potential information can be obtained with single electrode amperometric detectors. Use of the series detector alllows the discernment of oxidative and reductive current-potential information. The more explicit voltammetric data can improve the reliability of the peak identity assignment. Figure 5 depicts the normalized hydrodynamic voltammograms (NHDV) for p-hydroxybenzoic acid, 4-methylcatechol, caffeic acid, and vanillic acid. The NHDVs were determined simultaneously by poising the potential of the upstream detector at +1.1 V and incrementally
0
4
8
12 16 MINUTES
20
24
28
Figure 7. Series dual-electrode chromatograms of a urine sample taken 4 h after normal acetaminophen dosage: (1) acetaminophen. Conditions: urine sample diluted 1:50; W1 = +0.9 V, W2 = 0.0 V; 25 cm Biophase C,, column: flow rate, 1 mL/min. changing the potential of the downstream detector. Initially, the potential of the downstream detector was -0.1 V. Figure 6 shows the W1 and W2 response for the four compounds observed at three different downstream detector potentials. A t the intermediate potential of +0.35 V, both oxidation and
434
ANALYTICAL CHEMISTRY, VOL. 54, NO. 3, MARCH 1982
Table 111. Comparison of Normalized Current Values of an Acetaminophen Standard and Urine Sample Component current
w2 detector potential'"
reductive reductive reductive reductive oxidative oxidative oxidative oxidative
+0.20 t0.25 t0.30 t0.35 t0.55 +0.60 t 0.65 t 0.70
standard
peak 1
1.00b 0.86 0.71 0.54 0.53' 0.86 0.97
1.00b 0.84 0.75 0.58 0.52' 0.88
1.00
0.94 1.00
a W1 = +0.9 V for all experiments. Values normalized to the response recorded at t 0.20 V. ' Values normalized to the response recorded at +0.70 V.
chromatograms were recorded with the upstream detector at +0.9 V while the downstream detector was incrementally changed over a potential range which changed the acetaminophen standard current response from reductive to oxidative. Table 111 summarizes the normalized anodic and cathodic current values for the acetaminophen standard and the urine chromatogram peak. The agreement in the observed values confirms the assignment of the chromatographic peak identity. An additional aspect of LCEC that can be improved with series detection is high gain detection at more extreme potentials. An inherent problem associated with detection at high potentials (>1.0 V) is the significant background current due to the oxidation of water and other mobile phase constituents. When the presence of trace quantities of analyte requires the use of high gains and the magnitude of the base line drift is comparable to the signal, obtaining usable chromatograms becomes a difficult task. In addition, use of the graphite electrode detectors at extreme potentials usually results in rapid decay of performance. If the compound of interest has a significant N e ,downstream detection at lower potentials can be employed to minimize problems associated with base line drift. Figure 8 details the chromatogram of 2 ng of gentisic acid achieved when W1 was at +1.35 V. W2 was maintained at 0.20 V. Because the redox couple of gentisic acid is chemically reversible, the oxidation product can be detected at a lower potential where base line drift is not a problem and the lifetime of the detector is increased. This advantage of series dual-electrode detection has also been demonstrated by MacCrehan and Durst (6).
LITERATURE CITED WI
I
Flgure 8. Series dual-electrode chromatogram of gentislc acid. Conditions: 2 ng injected, W1 = +1.35 V, W2 = 4-0.20 V; 25 cm Biophase C18 column; flow rate 1 mL/min.
reduction currents were observed at W2. Current-potential information is especially helpful for confirming the identity of chromatographicpeaks in complex samples. Figure 7 shows the series detection chromatogram of a urine sample obtained 4 h after a normal acetaminophen dosage (two Qlenol tablets). Current-potential data obtained for the peak (designated in Figure 7) with the same retention time as the acetaminophen standard was obtained to confirm the identity of the compound. Several series detection
(1) Shoup, Ronald E., Ed. "Bibllography of Recent Reports on Electrochemical Detectlon"; BAS Press: West Lafayette, IN, 1981. (2) Fenn, R. J.; Slggla, S.;Curran, D. J. Anal. Chem. 1978, 50, 1067-1073. (3) Blank, L. J . Chromafogr. 1976, 117, 35-46. (4) Roston, D. A,; Klsslnger, P. T. Anal. Chem. 1981, 53, 1695-1699. (5) Schieffer, G. W. Anal. Chem. 1980, 52, 1944-1998. (6) MacCrehan, W. A,; Durst, R. A. Anal. Chem. 1981, 53, 1700-1704. (7) Bratln, K. B.; Kisslnger, P. T. J . Ll9. Chromafogr. 1981, 4 , 321-357. (8) Roston, D. A.; Kissinger, P. T. "Liquld Chromatography/Electrochemlstry: Princlples and Appllcations"; Klssinger, P. T., Ed.; BAS Press: West Lafayette, IN, 1982, Chapter 7. (9) Kissinger, P. T. Anal. Chem. 1977, 49, 447 A-456 A. (10) . . Kisslnaer, P. T.: Bruntlett, C. S.: Bratin, K; Rlce, J. R. NBS S w c . Publ. (U.S.yl979, No. 519. (11) Albery, W. J.; Hitchman, M. L. "Ring-Disc Electrodes"; Oxford Unlverslty Press: London, 1971; Chapter-3. (12) Matsuda, H. J . Necfroanal. Chem. 1968, 16, 153-164. (13) Braun, R. J . Elecfroanal. Chem. 1968, 19, 23-35. (14) Gerischer, H.; Ingeborg, M.; Braun, R. J . Elecfroanal. Chem. 1965, 10. 553-567. (15) Charalambous, G.; Bruckner, K. J.; Hardwick, W. A.; Linnebach, A. Tech. Q.-Master Brew. Assoc. Am. 1973, 10, 74-78.
RECEIVED for review October 6, 1981. Accepted November 19, 1981.
