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AMI. Chem. 1981, 53, 78-80
Symmetrical Two-Electrode Pulsed-Flow Detector for Liquid Chromatography W. J. Blaedel’ and Joseph Wang‘ Department of Chemistry, Unhwsitv of Wisconsin-Madlson, Madlson, Wisconsin 53706
An electrochemical flow detector whh two carbon electrodes h a thin-layer conflguatkm is described. The elhnlnatlon of the conventional reference electrode results in minimal potentlal ( I R ) drop and very simple deslgn. The cell k evaluated for amperometrlc measurements by using the s e d l v e putsebfkw tednlque. welcdeflned curent-potentlal w e 8 are obtalnable. The low pulsed-flow background current pwmlts measurements at the namndar concentratkm level. Applicatbns are lndkated for conthruous flow analysk, Llqdd chromatography, and flow lnjectlon anaiyysls.
T h e use of hydrodynamic thin-layer amperometry as a means of detection in flowing streams is growing rapidly (1). Thin-layer transducers have been extensively used in chromatographic detection and other types of analytical flow systems. A variety of thin-layer flow cells, differing mainly in the type and position of the electrodes, have been designed for various purposes (e.g., improvement of voltammetric behavior by reducing the well-known problems of ZR distortion and nonlinear behavior). These include cells with the following electrode configurations: two-electrode cells with the reference electrode mounted downstream (I);three-electrode configurations with both the reference and the auxiliary electrodes mounted downstream (I);the auxiliary electrode o p p i t e the working electrode and with the reference electrode located downstream (2); both reference and auxiliary electrodes incorporated in the flow pattern of the detector (3). Fourelectrode cells, with two counterposed working electrodes, have been also described, primarily to improve the selectivity (4) and to study electrode rate processes (5). In this paper, the design and operation of an easily constructed and inexpensive thin-layer amperometric detector is described. Two wax-impregnated graphite electrodes are used in a thin-layer configuration. The elimination of a conventional reference electrode (i.e., silver-ilver chloride or calomel electrodes) gives a considerably simplified design and also permits simplified operation. For example, cell volume can be made very low, bridges can be eliminated, and cell resistance is minimized. Since amperometric detection usually employs limiting current data, measurements are made a t constant applied potential, corresponding to the plateau of the voltammogram of the species of interest. The pulsed-flow technique is designed to discriminate against the major components of the background current, which results in high sensitivity. This approach has been exploited by Blaedel and co-workers for continuous-flow analysis using an open tubular (6) and a porous carbon electrode (7). Until recently, the long cycling times (10-30 s) of the pulsed-flow technique have precluded its use as a detection mode in liquid chromatography and flow-injection analysis. However, a recently described rapid pulsed-flow technique (8) is adaptable for these purposes. The thin Present address: De artment of Chemistry, New Mexico State University, Las Cruces, M 88003.
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channel of the present cell reduces the distance through which the diffusion boundary layer has to relax when the flow is pulsed, resulting in short response times. Primarily this paper describes the operation and applications of the two-electrode pulsed-flow detector.
EXPERIMENTAL SECTION Apparatus. The cell design is shown in Figure 1. The body
consisted of two 4-cm X 5cm Plexiglas blocks (Rohm and Haas Co.,Philadelphia, PA), one 1.2 cm thick and the other 0.6 cm thick. A 0.62-cm diameter hole was machined in the center of each block to contain the paraffin-impregnatmi graphite rods (1/4 in. diameter, EG&G, Princeton Applied Research, Princeton, NJ). The rods were cemented to the blocks by using a nonconducting epoxy (Epotek 349, Epoxy Technology, Watertown, MA). The p i t i o n of the electrodes in the blocks WBB arranged so the electrodes were opposite one another when the two blocks were bolted together with stainless steel mews at each corner. For the sample solution inlet and outlet, one of the Plexiglas blocks was drilled to accept Tygon tubing (1/32 in. i.d., 3/32 in. o.d.), press fitted, and sealed with epoxy (Epotek 349). The two blocks were separated by a spacer of Teflon sheet. The hole cut in the spacer thin (0.005in.) (with a razor blade) extended from the inlet hole to the outlet hole and defined the electrode area; this resulted in a working volume of about 7 &. The graphite disks were first rough polished with silicon carbide papers (No. 400 and W),followed by a O.l-pm alumina slurry. In systems containing the supporting electrolyte the resistance between the two electrodes was only 10-15 Q. Such a resistance did not affect the amperometric measurements which were independent of applied potential difference in the plateau region. In continuous flow measurements, the sample solution was stored in a 250-mL beaker. Solution flowed from the beaker to the cell by gravity or by a reciprocating piston pump (“minipump”, Milton Roy Co., Riviera Beach, FL) via 1 mm i.d. Teflon tubing. The pulsating fluid delivery of this pump provided one mode of flow modulation, giving a frequency of 0.5 Hz. Another mode of flow modulation was provided by turning on and off a rotary valve that was inserted between the beaker and the cell. The flow injection measurementa were performed with the rotary valve, by alternating periodically, for fixed times, between the carrier solution (the supporting electrolyte) and the sample solution. The liquid chromatographic apparatua consisted of an Altex (Model 110) pump and an Octadecyl C-18 reverse-phase column of 30-cm length and 4 mm i.d. (Varian Associates Inc., Palo Alto, CA). Sample volumes of 20 pL were introduced through an Altex 210 loop injector. The amperometric detector was compared with a UV detector consisting of a Hitachi 100-10 spectrophotometer operated at a wavelength of 280 nm, in conjunction with an Altex spectrophotometric flow cell. Potentials were applied with a simple battery-powered POtentiometer, and currents were measured with a picoammeter (Model 41&, Keithley Instruments, Cleveland, OH). The current output was recorded on a Houston Omniscribe chart recorder. Reagents. Chemicals and reagents ueed have been described previously (9), except as noted. Millimolar stock solutions of L-dopa, dopamine (Sigma Chemical Co., St. Louis, MO), and ascorbic acid (Merck Co., Rahway, NJ) were made up fresh each day. A 0.07 M KHzP04solution, adjusted to pH 3.5 with HsPO, was used as mobile phase for HPLC. Procedure. Freshly polished graphite surface was activated by applying a potential of +1.35 V for 10 h, while running the buffer solution at very low flow rate (about 0.1 mL/min) through @ lS80 Amerlcen chemicel Socbty
ANALYTICAL CHEMISTRY, VOL. 53, NO. 1, JANUARY 1981
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AE,V Figure 2. Hydrodynamlc voltammograms for 12.5 pM K,Fe(CNk (0) and 10 pM dopamine (0)in 0.1 M phosphate buffer @H 7.4), pulsed-tlow measurements of 1.5 mL/min (on)and 0 mUdn (off).
the cell, After the surface was polished and activated, the daily electrode pretreatment consisted of cycling the applied potential difference between +1.35 and -1.35 V for 12 min, allowing 3 min at each potential. Measurements were made for only short times (30 s at micromolar level to 5 min at the nanomolar level) after the working potential was applied. Specific details of the continuous analysis, flow injection, liquid chromatography,and the various pulsing procedures are described in the following section. R E S U L T S AND DISCUSSION Evaluation of t h e Cell. Figure 2 shows stopped-flow hydrodynamic voltammograms for the oxidation of dopamine and ferrocyanide at the micromolar concentration level. The curves were taken pointwise by making 100-mV changes in the applied potential difference and waiting about 15 s before applying the flow pulse. In this way current-potential data were obtained within 10 min. The waves and plateau regions are welldefined. The background stopped-flow current (not shown) is less than 1% of the analyte stopped-flow current under the conditions of Figure 2. Voltammograms with similar shape and potential regions were obtainable for the 4-month time period over which this study was performed. Over the f i t 3 weeks of operation the limiting currents for ferrocyanide increased up to 100% of those obtained a t a freshly polished surface. This aging effect has been observed at many carbon surfaces (9, IO) and appears to be associated with the formation of oxide groups during the anodic oxidation. The voltammograms shown in Figure 2 are similar to those obtained for dopamine and ferrocyanide in other types of flow cells with silver-silver chloride reference electrode (11, 12). Comparable voltammograms were also obtained when either of the graphite disks was operated as the working electrode and upon removal of oxygen from the solution. The latter may indicate that oxygen is not involved in the reactions at
70
30SEC FlgW.3. sensltMtyandpredsbnobtahedforsucceashrepulsed-fiow measuements of 70 nM K,Fe(CNb. Backgcund p&&fkw cwent Is represented by the dlfference between the dotted line and the L w e n t level. CmfMiow 0.1 M phosphate buffer @H 7.4); applled potential of 0.7 V; Row rates of 1.6 W m i n (H) and 0 (L) W m h ; pulshg time of 10 8.
the counterelectrode. The nature of these reactions is subjed to future studies. The dependence of limiting current upon flow rate was evaluated by using 12.5 pM KJe(CN6, over the range 0.55-2.7 mL/min. A log-log plot of current against flow rate (not shown) was linear with a slope of 0.52. This value is in good agreement with the theoretical hydrodynamic characteristics of the thin-layer flow cell, for which a value of 0.50is expected (13). (There is some controversy in the literature regarding this dependence; a cube root dependence has been found by other authors (14).) The percentage of K,Fe(CNI6 which reacts at the electrode declines from 1.9% to 0.9% as the flow rate increases over the range studied, indicating a low-yield amperometric mode of operation. Analysis i n Flowing Streams. The exploitation of the cell for hydrodynamic amperometry in a variety of flowing systems is based upon operation on the limiting current plateau. For selecting the desired constant-working potential, hydrodynamic voltammograms like those shown in Figure 2 may easily be recorded for the analyte (or analytes in case of LC) in the particular flowing stream. The sensitivity of the detector is demonstrated by the continuous pulsed-flow analysis of 70 nM ferrocyanide (Figure 3). The background pulsed-flow current corresponds to a concentration around 45 nM. The noise level is around 0.4 nA, corresponding to a concentration around 10 nM. The pulsed-flow response times a t this concentration level are 2 s (on) and 5 s (off). A series of 10 successive pulsed-flow measurements of 0.5 pM NADH gave current pulses similar to those of Figure 3, which permitted measurement of the precision (conditions: flow rate (on) 1.5 mL/min, +1.0 V applied potential, 0.1 M phosphate buffer (pH 7.4),10 s pulsing period). The mean pulsed-flow current difference found was 20.6 nA with a relative standard deviation of 1.8%. These data indicate feasibility of monitoring NADH at the submicromolar concentration level (without the need for chemical mediators), which is of interest for the analysis of many substrates and enzymes. Liquid Chromatography. Figure 4 shows two chromatograms of a test mixture of ascorbic acid, L-dopa, and d o p amine at nanogram levels, with the ultraviolet detector (a) followed by the electrochemical detector (b) in series. Because of its high sensitivity, the electrochemical detector was used in the dc mode without pulsing, a t a working potential of 1.05 V. A comparison of the two t r a m illustrates complementary characteristics for the two detectors for the particular chemical system of Figure 4: (1)The electrochemical detector has a much higher signal to noise ratio, and therefore a higher sensitivity. (2) The relative sensitivitiea of the two detectors differ for different compounds. In fact, the electrochemical detector is highly sensitive to an impurity compound (peak D)associated with the dopamine that was UBed to prepare the sample mixture, whereas the UV detector is not sensitive to this compound a t all. (3) Resolution is about the same for the two detectors. (4) The advantage of using two different detectors in series is clear.
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ANALYTICAL CHEMISTRY, VOL. 53, NO. 1, JANUARY 1981 I
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(flowing for 10 s) alternating with a carrier stream of 0.1 M phosphate buffer (pH 7.4) (flowing for 20 s). The pulsing frequency is 0.5 Hz, with the flow rate alternating between 4.6 mL/min (on) and 0 mL/min (off). Study of Figure 5 shows that the 0.5-Hz signal d m not decay to base line in the peak region, caused primarily by the slow mass-transport proceasee when the flow is off. A linear correlation was obtained between the rapid ac response and the analyte concentration. Eight concentration incrementa from 0.625 to 6.875 pM K,Fe(CN)6 yielded a linear plot. (Conditions: average flow rate 1.35 mL/min (2.7 mL/min (on) and 0 mL/min (off)), applied potential and buffer 88 in Figure 5.) A least-squares analysis of the plot gave a slope of 63 f 5 nA/pM (90% confidence limits). Reciprocating piston pumps are of the most useful type in HPLC (15). However, their pulsations do affectthe noise level in most detection systems, and expensive damping systems are used to reduce the pulses. It is known, however, that pulsed flow is generally not detrimental to resolution in a chromatographic separation. For systems in which electrochemical detection is adequate, considerable economy and simplicity might be achieved by constructing and operating the whole HPLC system in the pulsed flow mode.
ACKNOWLEDGMENT 1
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TIME, MIN. Fhps 4. Ulbavklet (a)and electrochemice1(b) chromatogam of 20 pL solution containing 7 pg/mL ascorbic acid (A), 4.7 pg/mL pa (B), and 14 pg/mL dopamine (C). conditions: 3oCm reversed-phase column; 0.07 M KH$'04 adjusted to pH 3.5 with H3P04;flow rate of 2.2 W m l n ; applied potential of +1.05 V.
The assistance of R. H. Barbhaiya in the liquid chromatography experimenta is highly appreciated.
LITERATURE CITED (1) Kisshgsr, P. T. Anel. Chem. 1977, 4 9 , 447A-458A. (2) "Mrrcayec Transducers for Electrochemical Detectkn"; Bbanatytical Systems Inc.: West Lafayette, IN. (3) Hunphrey. D. W.; Gddman, M. E.; Wscox, R. E.; Mdtm, C. K.; Sdth, R. V. k(la0Chem. J . 1980, 2 5 , 186-195. (4) Fenn, R. J.; Siggia, S.; Cwran, D. J. Anal. Chem. 1978, 50, 1067- 1073. (5) Aoki, K. Doctoral Thesie,Tokyo Instttute of Techndogy, 1978. (8) Bteedel, W. J.; I(recson, D. G. Anal. Chem. 1977, 49, 1583-1568. (7) Bteedel. W. J.; Wang. J. Anal. Chem. 1979, 51, 799-802. (8) Btaedel. W. J.; Y h , A. Anal. Chem. 1980, 52, 564-568. (9) Bleedel. W. J.; Wang, J. Anal. Chem. 1980, 52. 78-80. (10) Wfghtman, R. M.; Pak, E. C.; Barnan, S.; Dayton, M. A. Anel. Chem. 1978, 50, 1410-1414. (11) Bkedel, W. J.; Wang, J. AM/. Chem. 1979, 51. 799. (12) Bleedel, W. J.; Wang, J. Anal. Chem. 1980, 52, 1897. (13) Brunt, K.; Bruins, C. H. P. J . ChrOmetOgr. 1979, 172. 37-47. (14) Weber, S. Q.; Pudy. W. C. Anel. CMm. A& 1978, 100, 531-544. (15) Ktsslnger. P. T.; Felice, L. J.; KLng, W. P.; PacMa, L. A.; Rl~gln,R. M.; shoup, R. E. J . Chem. E&. 1977, 54, 50-54.
lmin
Flgwe 5. Repetitive ln/ectkn of 3 pM dopamhe sokrtion: sample Row 10 s;Caniec Row 20 s;average flow rate 2.3 rrVmin (4.6 mL/mh (on) and 0 mumin (off)); sample sire of 0.38 mL, containing 0.22 pg of dopamine; applied potential of 1.O V.
+
Application to Flow Injection Analysis. A reciprocating piston pump may be used to generate the pulses for operation of the electrochemical detector in the pulsed flow mode. Figure 5 gives the response for samples of 3 pM dopamine
RECEIVEDfor review August 19,1980. Accepted September 25, 1980. This work was funded in part by the Graduate School Research Committee of the University of Wisconsin and in part by the University Sea Grant Program under a grant from the Office of Sea Grant, National Oceanic and Atmospheric Administration, US. Department of Commerce, and by the State of Wisconsin.
