244
Anal. Chem. 1907, 59, 244-247
(23) Kwok, K.-S. PhD. Thesis, Cornell University, 1973. (24) Crawford, L. R.; Morrison, J. D. Anal. Chem. lB68, 4 0 , 1469. (25) Jaklin, J.; Krenmayr, P. Int. J. €nv/ron, Ana/, C h m , 1985, 27, 33.
RECEIVED for review May 5 , 1986. Accepted September 17,
1986. This work was supported by “Fonds zur Foerderung der wissenschaftlichen Forschung” “(Projekt 47811, “Oesterreichische Nationalbank” (Proiekt 1938). . , and “Bundesministerium fuer Wissenschaft und Forschung”.
Coulostatic Electrochemical Detection in Flow Injection Analysis S u s a n R. Mikkelsen and William C . Purdy*
Department of Chemistry, McGill University, 801 Sherbrooke Street West, Montreal, Quebec, Canada H3A 2K6
Coulostatic electrochemlcal detectlon for flow Injection analysls and hlgh-performance llquld chromatography has shown promise for the detection of electroactlve eluents In highly reslstlve moblle phases due to the relative knmunlty of the coulostatlc method to the effects of solullon resistance. I n this paper we derive expressions for the total charge consumed by an analyte bolus as lt passes through a thlnlayer flow cell, descrlbe the deslgn and constructlon of a coulostatlc detector, and lnvestlgate the perfqrmance of the detector In a nonconductlng mobile phase. Results show good agrgement with the developed theory. The detectlon llmlt for the determlnatlon of o-amlnophenol In 0.5 M phosphate buffer is approximately 5 pmol InJected. A linear relationship was observed between peak area and concentration for the determlnatlon of ferrocene In an acetonltrlle moblle phase contalnlng 0.000 18 M tetrabutylammonlum tetrafluoroborate. Repllcate Injections of 1 pM ferrocene In the same mobile phase show relatlve standard deviation values of 4.5% (peak helght) and 6.1% (peak area).
Reversed-phase liquid chromatography with electrochemical detection (LCEC) has become increasingly important for the sensitive and selective determination of electroactive molecules (1). The principle on which LCEC is based is the measurable flow of electrons as analyte is oxidized or reduced at a working electrode poised at an appropriate potential. Potential control is usually accomplished with potentiostatic circuitry, which continuously monitors and adjusts the potential difference between the working and reference electrodes. These detectors, however, are not without limitations; a relatively high electrolyte concentration is required in the mobile phase to minimize the effects of uncompensated resistance between the working and reference electrodes of a three-electrode flow cell, or IR drop between the working and counter electrodes of a two-electrode flow cell. These high-conductivity requirements can be difficult to achieve in nonaqueous reversed-phase liquid chromatography, since the dissolution at sufficient concentration of highly mobile electrolytes is often not possible. In addition, the high salt content of electrochemically useful mobile phases can alter the chromatographic requirements from those useful with a UV absorbance detection scheme, making a progression from UV absorbance to electrochemical detection complex. This provided the motivation for our efforts toward the development of a coulostatic electrochemical detection system for liquid chromatography. Coulostatic potential control has shown promise for nonaqueous electroanalytical applications because of its relative
immunity to the effects of solution resistance. Although, to our knowledge, two coulostatic LC detectors have been described (2,3),the utility of these detectors with nonaqueous mobile phases has not been investigated. In this peper we describe the design and construction of a coulostatic detection system for flow injection analysis. In addition, theory relating the flow injection peak area to analyte concentration, flow cell geometry, and mobile-phase flow rate is derived. The feasibility of coulostatic detection in highly resistive mobile phases is also investigated.
THEORY Consider a molecule of analyte, R, entering a flow cell in which working and auxiliary electrodes form opposite walls, separated by a thin spacer. Assume that the working electrode is poised at a potential on the hydrodynamic plateau for the oxidation of R to 0. As R enters the volume between the working and auxiliary electrodes, it experiences the equivalent of a potential step from a value where no reaction can occur to a value where the electrode reaction is diffusion-limited (if laminar flow is assumed). In quiet solution, this situation is described by the Cottrell equation (eq l),which relates the current response to the time after the potential step
I=
nFAD112C (?rt)1/2
(1)
where I is the total current seen at the working electrode, n is the number of electrons involved in the electrode reaction, F is the Faraday, A is the working electrode area, D is the analyte diffusion coefficient, C is the analyte concentration, and t is the time after the potential step. Differentiating with respect to electrode area gives the expression for current density
It is conceptually helpful to relate time in a quiet solution to distance along the working electrode in a flowing stream. Consider now an infinitesimally thin slice of solution, dx, as shown in Figure 1,flowing from the entrance to the exit of the flow cell between the working and auxiliary electrodes. The time taken for slice dx to travel from one end of the working electrode to the other is equal to the flow cell volume divided by the volume flow rate, t = Lbw/ U. The relevant electrode area for this thin slice is equal to w dx. Substituting w dx for dA and dq/dt for i in eq 2 gives (3) Since eq 3 is a differential with respect to two variables, two
0003-2700/87/0359-0244$01.50/00 1987 American Chemlcal Society
ANALYTICAL CHEMISTRY, VOL. 59, NO. 2, JANUARY 1987
245
dx
Flow t=o t=(Lbu)/U Flgurs 1. Dimensions of a thin-layer flow cell: L. electrode length; w , elactrode width. b , channel height dx. thin slice of soluton of widm wand height b.
integrations must be performed (i) integration over the time i t takes the slice to traverse the length of the working electrode, from t = 0 to t = Lbw/U, and (ii) integration over the length of the analyte bolus, from x = 0 to x = (volume injected)/wb. Hence eq 4 is derived, and this describes the total
Q = 2nFC(~olume)(LDw/*bU)'~~
(4)
charge consumed by an analyte bolus as it p m s through the flow cell. Because the Cottrell equation w u m e s semiinfinite linear diffusion, eq 4 is valid only while AD/Ub 5 0.33 ( t = 0.33b2/D). At this point, the diffusion layer thickness is q u a l to the channel height (4) and one of several situations can occur. Mathematically speaking, the simplest of these is one in which analyte is regenerated at a diffusion-limited rate at the auxiliary electrode; if this occurs, steady-state concentrations of 0 and R will prevail between t = 0.33b2/D and t = L b w / U . Other possibilities include no regeneration of analyte at the auxiliary electrode and situations involving coupled chemical reactions; due to their mathematical complexity, these situations are not considered here. With analyte regeneration at the auxiliary electrode, the expression for total charge consumption can he broken down into two components: charge consumed prior to the onset of steady-state conditions, and that consumed in the remainder of the channel with constant concentrations of 0 and R. T h e first component is a constant value and is obtained by integrating eq 3 from t = 0 to t = 0.33b2/D and from x = 0 to I = (volume)/wb
Q = 2nFC(~olume)(0.33/*)~/*
(5)
The value of the second component is a linear function of distance along the remaining electrode surface, given by substituting t = 0.33b2/D into eq 3. Integrating the resulting expression from t = 0.33b2/D to t = Lbw/U, and from x = 0 to x = (volume injected)/wb gives
Q = nFC(volume)(LDw/Ub
- 0.33)
(6)
The sum of eq 5 and 6 describes the total charge consumed in a regenerative cell Q = nFC(volume)(O.32 LDw/Ub) (7)
+
The crucial difference between the nontegenerative result (eq 4) and the regenerative result (eq 7) is found in the flow rate dependence of the peak area. In the quasi-amperornettic, or nonregenerative region, an inverse squate root dependence will prevail, whereas the prediction for the regenerative region is a simple inverse dependence. Both regions can be explored by controlling the mobile phase flow rate experimentally. EXPERIMENTAL SECTION
A simplified schematic diagram of the coulostatic detector circuitry is shown in Figure 2. While the cell is at open circuit, the working electrode potential is monitored against a preset threshold value, E* When it decays below the threshold, charge injection is triggered through the two monostable multivihrators (IC1and IC2) that control the opening and closing of analog switch
c
R E
Flgun 2. Simplified schematic diagam of delemar circuitry: IC1 and IC2, 74121 monostable munhribrators; IC3-IC5. 74LS191 binary counters; OA1-OA4, TL071 operational amplifiers;CP. LM311 comparatw; S1. TL185 ana@ switch; C. 5-nF capacitor; resistor values: R1. R4. 86 kR; R2. R3, 41 kR; R5. 5 kR; R6, R7, 1 kR; R8. 50 kR; R9. R10. 100 kR; R11. 5 kR; R12. 10 k% R13, R14. 250 kR.
SI. This connects a 0.174 mA current source for 18 ps to the inverting input of OAl, an operational amplifier. Since the auxiliary and working electrodes of the flow cell are in its feedhack loop, OAl acts as a current-bvoltage converter at the auxiliary electrode. Upon charge injection, a sudden potential increase is seen at the auxiliary electrode (since the working electrode is at a virtual ground), after which the cell is again left at open circuit. The signal from the monostable circuit which activates S1 also triggers a 12-bitbinary counter to increase its total count by one. Two monostable multivihrators are used to control the analog switch in order that discrete, nonoverlapping pulses u ~ l lbe created as required. When the comparator output goes low (0V),an 18@, 5-Vpulse is sent to the switch controller from IC1. For this time and an additional 18 ps, IC2 disables further triggering of S1, resulting in a full square wave of period 36 ps being sent to the switch. If the comparator output remains low following charge injection, the cycle is repeated until the comparator output goes high. This prevents pulse lengths of duration greater than 18 ps from being applied to the switch and ensures that each pulse application triggers the counting circuit individually. The 36-ps square wave period corresponds to the minimum time between injections; the maximum injection frequency is therefore approximately 28 kHz. An IBM personal computer acquires data from the counter via an interface designed and constructed in this laboratory to provide acquisition frequencies in the 0.25 Hz to 10 kHz range. It is based on a Motorola 8255 Parallel Peripheral Interface and a Motorola 8253 Programmable Interval Timer (schematic available upon request). The data that are acquired by the IBM PC are in the form of a number of charge pulses vs. time. As analyte flows through the cell, more charge pulses are required to maintain the working electrode potential due to the faradaic process occurring, which discharges the working electrode's double-layer capacitance. Figure 3 shows an exploded new of the flow cell used in these experiments. Working and auxiliary electrodes,fabricated from low-temperature isotropic carbon (LTIC, Carbo-Medics, Inc., Austin, TX), were separated by a Teflon spacer of 25 pm thickness which compressed to 21 pm in the flow cell. The auxiliary electrode (upper) had two holes drilled to allow for entrance and exit of the eluent stream. The working electrode area was 0.569 cm2. The reference electrode was housed in a downstream compartment and was an Ag/AgCl (saturated KCI) reference for the theoretical study and an Ag/AgNO, (0.1 M AgNO, in CH,CN) reference for the nonaqueous work. The full instrumental arrangement for the flow injection experiments consisted of a Waters Model 590 pump, a Rheodyne injector with a 20-pL sample loop, a coiled delay tube made of Teflon in. i.d. X 2 m), and the flow cell which was connected to the coulostatic detection module and hence to the IBM PC. Distilled, deionized water (Barnstead Ultrapure No. D8902) and HPLC grade acetonitrile (Fisher Scientific) were used as
246
ANALYTICAL CHEMISTRY, VOL. 59, NO. 2,JANUARY 1987
Table 11. Calibration of Peak Height against Concentration of o-Aminophenol Outlet
Kel-F Block
LTlC Plate
Teflon Spacer
Copper Tab
concn: pM
peak heightb,pC
0.250 0.500 0.750 1.000 2.000 3.000 4.000
0.068 f 0.031 0.116 f 0.009 0.180 f 0.018 0.251 f 0.014 0.606 f 0.049 0.895 f 0.023 1.23 f 0.01
corr coeff
0.9991 0.317 f 0.042 -0.041 f 0.057
slope, C/M
intercept, pC Figure 3.
Exploded view of the thin-layer electrochemical flow cell.
Table I. Pulse Charge Content Calibration DataD capacitor number
'Conditions: mobile phase 0.6 M pH 4.4 phosphate buffer; flow rate 1.00 mL/min; working electrode potential +0.60 V vs. Ag/ AgCl. *These values represent the average peak height for four replicate injections. Uncertainties shown are equal to 2s.
potential step height, mV 312 313 317 320 318
f f f f f
14 12 8 2 8
Table 111. Regression of Peak Area against Inverse Flow Rate
U-',min/mL 3.33 5.00 6.67 10.0 12.5 16.7 25.0 50.0
nSee text for conditions. Uncertainties represent 2s (n = 9) for the average value of ten measurements. solvents and were degassed before use and kept in a nitrogen atmosphere during use. All chemicals used were reagent grade, except tetrabutylammonium tetrafluoroborate (Sigma Chemical Co.), which was used as received.
68 f 87 f 106 f 137 f 153 f 181 f 245 f 486 f
corr coeff slope, pC mL/min intercept, pC
RESULTS A N D DISCUSSION
Calibration of the charge content of a single coulostatic pulse was performed by using an 11.0-nF capacitor (Phillips 2% precision polystyrene film)in place of an electrochemical cell in the feedback loop of OAl, creating an integrator a t which, after buffering the output, potential was measured prior to and following a charge pulse. The charge injection circuitry was triggered at a frequency of 1 Hz and the potential step height was measured on an oscilloscope. Table I shows the calibration data for five separate 11.0-nF capacitors, with ten measurements taken for each capacitor. No significant difference in the potential step height was observed for the different capacitors. Averaging all 50 measurements yields a potential step value of 316 mV ( n = 49, 2s = 11 mV) corresponding to a charge content of 3.48 f 0.12 nC/pulse. In order to verify the expected linear relationship between signal magnitude and concentration, various concentrations of o-aminophenol in 0.6 M phosphate buffer (pH 4.4) were injected into the same mobile phase. The results are shown in Table 11,along with a regressional analysis. Signal-to-noise (S/N) studies indicate that S / N increases with concentration, as expected, with the value for 0.25 pM o-aminophenol being 2.2. This was taken as the detection limit, which corresponds to 5 pmol injected, and compares favorably with the reported detection limits of 40 pmol (2) and 3.3 pmol (3). Higher analyte concentrations were investigated by using potassium ferrocyanide in 1M NaCl in concentrations ranging from 12.9 to 260 pM. The slope of the log (peak area) vs. log (concentration) curve was 0.9221 with R = 0.9995. Expected sources of noise include (i) the random nature of the diffusion process bringing analyte to the electrode surface; (ii) the porous nature of the electrode surface (5), which may be incompletely filled with mobile phase (as air is replaced by mobile phase, a change in both electrode area and double-layer capacitance will occur, causing a spike in the response signal); (iii) electronic noise in the potential measurement circuitry; and (iv) fluctuations in the amount of charge injected in each charge pulse, caused
peak area:
pC
2 2 1 4 8 7 5 30
0.9983 8.8 f 1.6 41 f 20
a Values shown represent the average peak area for four replicate injections; uncertainties are equal to 2s. See text for conditions.
B
-k
.
/
'\
,
I
c
-2
I 1
d g ( F o x Rota. m./nln>
Variatlon of peak area dependence on mobile-phase flow rate. See text for conditions. Figure 4.
by both the timing of the switch control and the magnitude of the current from the current source. These sources of noise will be investigated quantitatively in future studies. In order to verify eq 4 and 7, flow injection peak area was measured as a function of mobile-phase flow rate for injections of 43.4 pM potassium ferrocyanide in 1 M KCl. Figure 4 shows a log-log plot of these variables, where the inflection corresponds to the transition between a nonregenerative and a regenerative detection scheme. Table I11 gives a regressional analysis of peak area vs. 1/ U for the eight slowest flow rates. The measured slope of 8.8 pC (mL/min) is in agreement with the value of 9.04 predicted by eq 7, and the measured intercept is within experimental error of the predicted value of 26.8 pC. Table IV shows the regression of peak area vs. (l/U)ll2 for the nine highest flow rate values. Although the plot shows
ANALYTICAL CHEMISTRY, VOL. 59, NO. 2, JANUARY 1987
Table IV. Regression of Peak Area against the Inverse Square Root of Flow Rate U+,
(min/mL)1/2
peak area:
0.63 0.67 0.71 0.76 0.82 0.89 1.00 1.15 1.41
jd
8.2 f 0.3 9.8 f 0.7 11.5 f 0.7 13.7 f 0.5 17.1 f 0.8 21.1 f 0.4 26.8 f 1.6 33.5 f 0.9 45.5 i 1.6
corr coeff slope, pC(mL/min)1/2 intercept, WC
0.9996 48.7 f 1.0 -22.7 i 0.9
a Values shown represent the average peak area for four replicate injections; uncertainties are equal to 2s. See text for conditions.
d
f
I in
0 0
10
Conc.ntrotion.
15
20
25
"M
Flgure 5. Comparison of Calibration curves obtained for the determination of ferrocene in a mobile phase consisting of 0.000 18 M tetrabutylammonium tetrafluoroborate in acetonitrile: 0,peak height response; A, peak area response. good linearity, it has a nonzero intercept and the experimental value for the slope, 48.7 pC (mL/min)1/2,was found to be significantly greater than the value of 31.0 predicted by eq 4. This is attributed to the turbulent flow of eluent due to the flow cell geometry; neither the inlet nor the outlet to the flow cell is parallel to the electrode surface, so that solution entering the cell is impinging on the working electrode surface. Since it is expected that this will cause greater deviations at higher flow rates, a separate experiment was conducted and
247
at 5 and 10 mL/min a significant positive deviation from the straight line was observed. To determine the performance of our coulostatic detector using mobile phases of higher resistance, acetonitrile was employed as mobile phase, with varying concentrations of tetrabutylammonium tetrafluoroborate as the supporting electrolyte. Conductivity measurements on solutions of this electrolyte in acetonitrile indicated approximately 1 order of magnitude less conductance for the acetonitrile systems compared with the same concentrations of KC1 in water. The lowest electrolyte concentration investigated was 1.8 X M, and the specific conductance of this solution was 3.94 pmho/cm. Six replicate injections of 1pM ferrocene in 1.8 X M tetrabutylammonium tetrafluoroborate showed a relative standard deviation (n = 5) of 4.5% for peak height and 6.1% for peak area measurements. A calibration curve was constructed for this mobile phase and is shown in Figure 5. Linearity is indicated by the peak area vs. concentration plot, for which R = 0.999 91, while the peak height correlation is R = 0.992, showing a curvature downward at the higher concentrations. It was originally considered that this nonlinearity might be due to migration effects, since the transference number of an ionic species-the fraction of the total bulk current carried by that species-is proportional to concentration. If this were the case, however, the transference number and hence the migration current contribution to the total charge would increase with concentration, and curvature would be seen in the opposite direction to that found. It has since been suggested (6) that this deviation may be due to variations in the diffusional properties of ferrocene with concentration as the analyte bolus flows from injector to detector. As an analytical tool, peak area calibrations show better linearity than peak height calibrations at low electrolyte concentrations. LITERATURE CITED (1) Johnson. Dennis C.; Ryan, Michael D.; Wilson, George S. Anal. Chem. 1084, 56, 7R-20R. (2) Barnes, Anthony C.; Nieman, Timothy A. Anal. Chem. 1983, 55, 2309-2312. (3) Last, Thomas A. Anal. Chem. 1983, 55, 1509-1512. (4) Weber, Stephen G.; Purdy, William C. Anal. Chem. 1982, 5 4 , 1757-1764. (5) Lankelma, J.; Poppe, H. J . Chromatogr. 1976, 125, 375-388. (6) Johnson, Dennis C., Department of Chemistry, Iowa State University, Ames, Iowa. personal communication, March, 1986.
RECEIVED for review April 30,1986. Accepted September 22, 1986.
