Anal. Chem. 1988, 58, 1251-1254 (8) Jinno, K.; Hlrata, Y.; Hiyoshi, Y. HRC C C , J . High Resolut. Chromatogr. Chromatogr. Commun. 1082, 5 , 102-103. (9) Hirata, Y.; Jinno, K. HRC CC, J . High Resolut. Chromatogr. Chromatogr. Commun. 1983, 6 , 196-199. (10) Jinno, K. Anal. Lett. 1884, 17, (AlO), 933-943. (1 1) Hirata, Y.; Jinno, K. In "Microcolum Separations";Novotny, M., Ishll, D., Eds.; Elsevier: Amsterdam, 1985; p 45. (12) Smit, H. C.; Lub, T. T.; Vloon, W. J. Anal. Chim. Acta 1980, 122, 267-277. (13) Laeven, J. M.; Smit, H. C.; Kraak, J. C. Anal. Chim. Acta 1983, 150, 253-258. (14) Lewis, T. G.; Payne, W. H., J . Assoc. Comput. Mach. 1973, 2 0 , 456-468.
(15) Carney, Daniel P. Ph.D. Dissertation, Southern Illinois University, Carbondaie, IL, 1984.
' Permanent address:
Materials Science, Toyohashi University of Technology, Tempaku-cho, Toyohashi, 440 Japan.
1251
'Current address: BristoCMyers, Analytlcai Research, Evansville, IN 47721.
Kiyokatsu Jinno' Daniel P. Carney2 J o h n B. Phillips* Department of Chemistry & Biochemistry 'IJthern Illinois University Carbondale, Illinois 62901
RECEIVED for review August 5, 1985. Resubmitted January 21, 1986. Accepted January 21, 1986. This work has been by the u*s* Department Of Energy under cooperative agreement number DE-FC22-83FE60339.
Electrochemical Modulator for Liquid Chromatography Sir: All varieties of chromatography require that a modulation signal of some kind be imposed at the head of the column (1). Conventional injection valves do this and, therefore, are modulators. Certain chemical devices may also serve as modulators. A flow through electrochemical cell is particularly attractive as a liquid chromatography modulator because it can, in principle, rapidly change the composition of a very small volume of liquid entering the column. The change in composition is equivalent to a small volume injection and results in a chromatogram at the end of the column. Chemical modulators have no moving p a or seals to cause problems. Instead, chemistry complicates their behavior. This chemistry is also an opportunity to apply them in ways not possible with simple mechanical valves. Several examples of chemical modulators have been described for gas chromatography (2-6), but only two have been used for liquid chromatography (7,8). Smit (9,10)described the use of valves as modulators in liquid chromatography. The requirement for high-pressure operation makes the design of modulators technically more difficult for liquid than it is for gas chromatography. But, since a greater variety of chemical processes can be applied to a liquid than to a gas stream, chemical modulators should be more valuable for liquid than for gas chromatography. A sample stream flowing continuously through an electrochemical cell may be modulated in concentration by application of a varying potential. In the simplest case, the sample passes through the modulator unchanged at one cell potential while at another potential the sample substance is removed by an electrochemical reaction. An electrical pulse starting at the removal potential, rapidly changing to the pass potential, holding there for a short but definite time, and then rapidly returning to the removal potential generates a sharp pulse of the sample substance at its analytical concentration. Such modulation is equivalent to that of an injection valve and could be used to obtain a chromatogram in a similar manner. Since the sample stream is continuously flowing through the cell, the modulation signal form is not limited to a single potential pulse but may consist of a long sequence of potentid pulses or even a continuously varying potential. Such long modulation signals give a much greater sample troughput but result in a multiplexed detector output signal that requires complex computation to recover the chromatogram (1). Ideally, an electrochemical modulator for liquid chromatography should completely electrolyze the sample a t one potential, allow it to pass through completely unchanged a t 0003-2700/86/0358-1251$01.50/0
another potential, and be able to change between the two potentials instantaneously. However, useful analytical data can be obtained if the fraction of sample electrolyzed is simply reproducible a t the two potentials and if the difference in concentration is sufficient to produce an observable signal in the chromatogram. Modulator speed should be fast enough so that pulse duration does not contribute significantly to the variance of the chromatographic peak. EXPERIMENTAL SECTION Equipment. A Model 6000A solvent delivery pump and a Model 440 fixed-wavelength detector at 254 nm from Waters Associates (Milford, MA) were used. A 25 cm X 4.1 mm i.d. column packed with pBondapak CI8was obtained from Alltech Associates (Deerfield, IL). The mobile-phase flow rate was 1.0 mL/min for all experiments. A previously described laboratory computer generated the modulation signal, recorded the detector output signal, computed the chromatogram by cross-correlation, and plotted the results (1).
Cyclic voltammograms were obtained with a BAS-100 electrochemicalanalyzer from Bioanalytical Systems (West Lafayette, IN). Electrochemical Modulator. A Model 5020 guard cell obtained from Environmental Sciences Associates (Bedford, MA) was used as a concentration modulator. The internal volume of this cell was 5 pL, and its pressure limit was 6000 psi. The cell contained a high-surface-area porous graphite test electrode, a counter electrode, and a reference electrode of proprietary material. The controlling couple for the reference electrode was the H2/H+ ion couple and had a potential approximately 0.20 V less than a Ag/AgCl reference electrode. The cell was placed in the high-pressure stream immediately before the column replacing the injection valve. The cell potential was controlled by the computer through a Tecmar (Cleveland,OH) Model S-100 digital-to-analogconverter and the potentiostat circuit shown in Figure 1. Modulation Signal. At the beginning of each data acquisition period a random number was generated (11)and used to decide whether or not to pulse the cell potential. A specified probability determines the pulse sequence and, thus, the modulation signal. During periods in which no pulse was generated,the cell potential was maintained at its low value. In all cases, the pulse duration was the same as the data acquisition period. Solvents and Reagents. Chromatographygrade acetonitrile and methanol from Fisher Scientific (Fair Lawn, NJ) were used to prepare mobile-phase solutions. Reagent grade aniline, also from Fisher Scientific,was used to prepare test samples. Distilled water was additionally purified by use of a Cole Pqmer (Chicago, IL) ion exchanger. Aqueous supporting electrolyte and buffer solutions were prepared by dissolving the appropriate amount 0 1986 American Chemical Society
1252
ANALYTICAL CHEMISTRY, VOL. 58, NO. 6, MAY 1986
COMPUTER
CELL
Figure 1. Schematic of the potentiostat used to control the electrochemical modulator: R1 = 1.0 kO, R2 = 56 0, R3 = 1.0 kO, 134 = optional reslstor included only if cell current Is to be measured: C1 = 0.22 hF; OAl and OA2 = operational amplifiers (741 type).
I
2
4
2
4 6 8 RETENTION TM IE [ M~NUTES1
__
10
I
Figure 3. Chromatogram resultlng from a 34-min electrochemical modulation slgnal. The moblle phase was 50% acetonitrile, 40 % 0.1 M phosphate buffer, and 10% methanol wlth 0.115 ppb aniline. Pulse probabillty was 0.25. Data acquisition period and pulse duration were 250 ms.
Table I. Dependence of Normalized Signal Amplitude on Pulse Durationa
I
il
I
6
8
15
signal amplitude, arbitrary units
pulse duration, ms
0.01 1.00 0.81
250 500
0.75
1000
125
"The mobile phase was 50% acetonitrile, 40% 0.1 M phosphate buffer, and 10% methanol with 1.15 ppb aniline. Pulse probability was 0.25. Data acquisition period and pulse duration were 250 ms.
RETENTION TIME 1 MINUTES 1
Figure 2. Chromatogram resulting from a single 1-s electrochemical pulse from 200 to 800 mV and returning to 200 mV. The moblle phase was 50% acetonitrile, 40% 0.1 M phosphate buffer, and 10% methanol with 1.15 ppm aniline.
of sodium acetate or sodium dihydrogen phosphate in water and adjusting the pH with dilute sodium hydroxide. Mobile-phase solvent was vacuum filtered through a 0.2-rm nylon-66 membrane for particle removal and degassing.
RESULTS AND DISCUSSION Figure 2 is a chromatogram obtained by applying a single potential pulse to the electrochemicalcell. The cell potential was raised from 200 to 800 mV, held there for 1s, and then returned to 200 mV. The mobile phase contained 1.15 ppm of aniline. The single vacancy in the chromatogram at 3.4 min is due to oxidative removal of aniline during the pulse. The positive peaks at 2.9,4.0, and 5.1 min are due to products of the reaction. With no aniline present, a single potential pulse yields no observable signals at the highest detector sensitivity. Figure 3 is a multiplex chromatogram resulting from a 34-min electrochemical modulation. The cell potential was pulsed at random times to 800 mV from a 200-mV pass potential. The mobile phase contained 0.115 ppb of aniline. The peak a t 3.0 min is one of the oxidation products of aniline, while the peak at 2.5 rnin is due to oxidation of some other mobile-phase component. A blank composed of only the mobile phase yielded a similar chromatogram but with the peak at 3.0 rnin missing. The major differences between the chromatograms in Figures 2 and 3 follow from the very large difference in sample concentration. Signals due to trace impurities in the mobile phase are not present in Figure 2 because their amplitudes are much less than that of the aniline signal. They are not
observable in a single pulse experiment even at the highest detector sensitivity. The aniline signal itself differs in the two chromatograms. At very low concentrations either the mechanism of aniline modulation or the mechanism of chromatographic retention differs from those at high concentrations. There are several possible explanations for this effect, but no data is available to support any one of them. Since the reaction is taking place in a flowing stream, the duration of the potential pulse may affect the amount of sample modulated. Table I contains normalized signal amplitudes taken from a set of four multiplex chromoatograms obtained by using four different pulse durations. All other experimental parameters remained constant. All four chromatograms show a single aniline oxidation product signal with peak shapes and signal-to-noise ratios almost independent of pulse duration. The optimum pulse duration is about 250 ms. Transit time through a 5-rL cell at 1.0 mL/min is 300 ms. A t shorter pulse durations, signal amplitude is probably limited by the time required to charge the cell double-layer capacitance. Longer pulse durations may partially destroy the measured oxidation product by further reaction at the electrode. Pulse Potential. Figure 4 contains four multiplex chromatograms obtained at four different pulse potentials. The lower, or pass, potential was adjusted to vary the potential difference, while the more positive potential was unchanged. All other experimental parameters remained constant. Table I1 contains the normalized signal amplitudes computed from the chromatograms in Figure 4. A 400-mV pulse gives the largest signal with 600 mV only a little less. Both very large and very small modulation potential differences result in significantlylower signal amplitude. A small potential difference is less effective because there is then less difference
ANALYTICAL CHEMISTRY, VOL. 58, NO. 6, MAY 1986
1253
T
50uA
-
D
1
VI
I-*
5
co
ti .om
d z
R
E VI I
2
4 RETENTION TIME
6 [
10
MINUTES I
Flgure 4. Chromatograms resulting from 64-mln electrochemical modulation signals using a varlety of pulse potentials. The mobile phase was 50% acetonitrile, 40% 0.1 M phosphate buffer, and 10% methanol with 1.15 ppb aniline. Pulse probability was 0.25: (A) pulse range = 50-700 mV, detector sensltivity = 0.1 AU full scale, signal attenuation = 1 2 8 (6)pulse range = 100-700 mV, detector sensltlvi = 0.1 AU full scale, signal attenuation = 128; (C) pulse range = 300-700 mV, detector sensitivity = 0.2 AU full scale, signal attenuation = 128; (D) pulse ranbe = 500-700 mV, detector sensltivlty = 0.2 AU full scale, signal attenuation = 18.
Table 11. Dependence of Normalized Signal Amplitude on Initial Potential and Potential Difference" signal amplitude, arbitrary units 0.26 0.94 0.73
0.11 0.20 0.86 0.94 0.93 0.40
0.14 0.71 0.94
1.00
,
initial potential,
potential difference,
mV
mV
500
200 400 600 650 400 400 400 400
300
100 50 50 100 200 300 400
300 300 300 300
400
150 300
450 600
Experimental conditions are the same as in Figure 4. between the fractions of sample oxidized at the two potentials. The cause of less signal at a very large potential difference is not known. An interfering signal that is probably due to a buffer contaminant can be seen in Figure 4 for the two lowest starting potentials at 50 and 100 mV. Large potential range modulation should be avoided to reduce the possibility of modulating undesired mobile-phase components. Table I1 also contains normalized signal amplitudes for five multiplex chromatograms obtained by using different starting potentials but a constant 400-mV pulse potential difference. A broad range of starting potentials gives essentially constant signal amplitude. Above and below this range, however, signal amplitude decreases significantly. Table I1 also contains normalized signal amplitudes for a series of four chromatograms with a constant starting potential of 300 mV and various pulse potential differences. The signal amplitude increases sharply as pulse modulation increases from 150 to 450 mV. Beyond 450 mV, the improvement is small. The optimum modulation pulse for this aniline system using the currently chosen set of operating conditions is from 300 to 750 mV. Ideally, a small potential difference bracketing the sample's half-wave potential should be sufficient. The large 450-mV
E(UOLT)
Flgure 5. Cyclic voltammograms of 115 ppm aniline in 50% acetonitrile, 40% 0.2 M sodlum phosphate buffer at pH 5.0.
difference required for maximum sensitivity indicates some limitation in the modulation process. Since the guard cell was not designed for use with a changing potential, its electrode potential may not change as rapidly as the applied potential and may not actually reach the final potential. In addition, the potential distribution on the porous graphite electrode may be nonhomogeneous so that a larger pulse difference simply brings a larger portion of the surface to an effective potential during the pulse period. Figure 5 is a cyclic voltammogram obtained from an aniline solution in an electrochemicalcell with a glassy carbon working electrode, a platinum counter electrode, and a saturated calomel reference electrode. The solvent used for this voltammogram was the same as the chromatographic mobile phase except that phosphate rather than acetate buffer was used. The peak potentials in Figure 5 are approximately 200 mV more positive than those that would be observed in the modulator cell because of the difference in reference electrode potential. The difference between the potential of the onset and the peak of the oxidation wave in Figure 5, about 300 mV, is consistent with the modulation efficiency data in Table 11. The +934-mV peak oxidation potential from the voltammogram in Figure 5 is equivalent to the 300-750-mV pulse in Table 11. With a +300-mV starting potential and a +200-mV reference electrode, a 450-mV pulse reaches a +950-mV peak potential on the voltammogram scale. By use of a +300-mV starting potential, the signal first appears at a 150-mV potential difference, which is equivalent to a +650-mV potential on the voltammogram scale. The chromatographic modulation signal appears, increases, and levels off over the same potential range as the cyclic voltammogram anodic signal. Since the two systems use similar electrode materials and have the same measured oxidation potentials, their chemistries are probably the same. It is clear from the voltammogram that the nonideal behavior is due to reaction kinetics rather than problems associated with the glassy carbon electrode. The porous graphite electrode of the modulator cell probably is similarly limited. The reduction peak is much smaller than the oxidation peak in the voltammogram shown in Figure 5 indicating that the oxidation product decomposes on the time scale of the experiment. This is consistent with the single-pulse chromatogram shown in Figure 2 where three oxidation product peaks are observed at higher aniline concentrations. Parameter Optimization. Experimental variables and modulation parameters that have been individually optimized include pH, supporting electrolyte concentration, pulse starting potential, pulse potential difference, pulse probability,
1254
ANALYTICAL CHEMISTRY, VOL. 58, NO. 6, MAY 1986
Table 111. Simultaneous Optimization of Experimental Parameters
adjustable parameters
signal
expt no.
a
b
c
d
e
f
amplitude
1 2 3 4 5 6 7 8 9 10 11 12
5.0 5.0 5.0 5.0 5.0 5.0 5.0 4.0 4.0 4.5 4.5 5.0
0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.1
300 300 200 200 200 200 200 200 300 200 300 200
600 500 600 600 700 600 600 600 600 600 600 600
250 250 250 250 250 250 500 500 500 500 500 500
0.25 0.25 0.25 0.312 0.25 0.195 0.25 0.25 0.25 0.25 0.25 0.25
0.25 0.25 0.50 0.48 0.60 0.45 1.00
Table 1V. Calibration Data from Aniline Oxidation Signal Obtained from a 34-min Modulation Applied to System with pH 5.0, Acetate Buffer Concentration = 0.2 M, Initial Potential = 200 mV, Potential Range = 600 mV, Pulse Duration = 500 me, and Pulse Probability = 0.25"
0.18 0.12 0.35 0.05
Pulse probability.
and pulse duration (8). Each parameter has been optimized by keeping all others constant while varying the test parameter to find its optimum over a range. The possibility exists, however, that some parameters may interact so that the true optimum differs from that obtained by individual optimizations. Table I11 describes a series of experiments performed to test this possibility. Experiment 1 is the parameter set obtained by individual optimization. Additional experiments were performed simultaneously varying several parameters. The chromatogram resulting from the parameters used in experiment 8 was severely distorted at the retention times of interest and the peak was unmeasurable. All other parameter sets gave usable chromatograms. A factor of 4 improvement in signal amplitude was obtained by this simultaneous parameter optimization. There is significant interaction between parameters, and a multiparameter optimization scheme such as the simplex technique may be useful (12). Calibration Curves. Table IV contains calibration data for the oxidation product signal a t 3.0-min retention. The signal/ pulse was calculated by dividing the total signal, corrected for detector sensitivity and computational attenuation, by the actual number of pulses made during the 34-min experiment. The calibration is distinctly nonlinear as the sensitivity gradually falls off with increasing concentration. The effect is not a simple overloading of the column, since retention times are quite stable and curvature is apparent throughout the range of the calibration. Short-term reproducibility is quite good as can be seen in the Table IV data. The precision of six consecutive determinations of aniline at a single concentrationwas 17% relative standard deviation. Over the longer term, however, calibration is strongly dependent upon the history of the electrochemical cell. The detection limit here is 0.06 ppb of aniline. Chromatograms obtained at this concentration had signal-to-noise ratios of approximately 2. Lower concentrations have been detected but are difficult to quantify because of poor reproducibility and calibration curve problems. The detection limit with a single 500-ms potential pulse is 320 ppb. With a single 30-pL injection, it is 12 ppb. There is better than 2 orders of magnitude improvement in detection limit using multiplex chromatography with electrochemical modulation over what is possible using a conventional single-injection technqiue for aniline. Conversion of aniline to an oxidation product may be responsible for part of this improvement. The single electrochemical pulse does not give as low a detection limit as it should. This is consistent with the lower sensitivity a t
signal,
PPb
AU/pulse
sensitivity
5.09 x 6.26 X 5-71 x 6.56 X 8.25 X 7.03 X 7.53 x 3.07 X 2.85 X 2.93 X 7.53 x 7.88 X 5.58 x 6.62 x 5.84 X
4.4 x 5.4 x 5.0 x 5.7 x 7.2 X 6.1 X 6.5 X 2.7 X 2.5 X 2.5 X 6.5 X 6.9 X 4.9 x 5.8 X 5.1 X
0.115 0.115 0.115 0.115 1.15 1.15 1.15 11.5 11.5 11.5 115 115 1150 1150 1150
'Aqueous buffer pH. *Aqueous buffer concentration (M). 'Initial potential (mV). dPulserange (mV). ePulseduration (ms). f
aniline concn,
a
10-7
10-1 10-7 10-1 10-1
10-1 10-7
10% 10-6 10-5 10-5
103
103 103 103 lo2
lo2 lo2 lo2 lo2 lo2 10' 10' 10' 10' lo1
The correlation coefficient is 0.996.
higher concentrations shown in Table IV. The precision and linearity measures are not as good as those commonly obtained for chromatographic techniques. The multiplex technique itself can be quite precise and linear (1). The electrochemicalmodulator is apparently the source of the imprecision. Drift and imprecision should be expected when working electrodes composed of carbon materials are used (13, 14).
ACKNOWLEDGMENT We acknowledge the assistance of James Cox in obtaining the cyclic voltammograms and interpreting the electrochemical results. We wish to thank ESA Corp. of Bedford, MA, for the loan of a Model 5020 guard cell. LITERATURE CITED Phillips, John 8.; Luu, Derhslng; Pawliszyn, Janusz B.; Carle, Glenn C. Anal. Chem. 1985, 5 7 , 2779-2787. Lovelock, J . E. J . Chromatogr. 1975, 112, 29-36. Laster, W. G.; Pawllszyn, J. B.; Phllllps, J. B. J . Chromatogr. Sci. 1~82,20,27a-282. Valentin, J. R.: Carle, G. C . ; Phillips, J. B. HRC CC, J . High Resolut. Chromatogr Chromatogr . Commun 1982, 5 , 269-27 1. Valentin, J. R.; Carle, 0. C.; Phllllps, J. B. HRC CC, J . High Resolut. Chromatogr. Chromatogr. Commun. 1983, 6 , 621-622. Valentin, Jose R.; Carle, Glenn C.; PhilllDs, John B. Anal. Chem. 1985, 5 7 , 1035-1039. Carney, Daniel P.; Phillips, J. B. HRC CC, J . High Resolut. Chromatogr. Chromatogr. Commun. 1981, 4 , 413-414. Carney, Daniel P. Ph.D. Dlssertatlon, Southern Illinois university, Carbondaie, IL, 1984. Smit, H. C.; Lub, T. T.; Vloon, W. J. Anal. Chim. Acta 1980, 122, 267-277. Laeven, J. M.; Smit, H. C.; Kraak, J. C. Anal. Chim. Acta 1983, 150, 253-258. Lewis, T. G.; Payne, W. H. J . Assoc. Comput. Mach. 1973, 20, 456-468. Morgan, S. L.; Demlng, S. N. Anal. Chem. 1974, 4 6 , 1170-1181. Bratin, K.; Blank, C. L.; Hull, I. S.; Luste, C. E.; Shoap, R. E. Am. Lab. (Falrfleld, Conn.) 1984, 16(5),33-62. Poppe, H. Anal. Chim. Acta 1883. 145, 17-28.
.
Present address: 47721.
.
Brlstol-Myers, Analytical Research, Evansville, IN
Daniel P. Carney' John B. Phillips* Department of Chemistry & Biochemistry Southern Illinois University Carbondale, Illinois 62901
RECEIVED for review August 5, 1985. Resubmitted January 21, 1986. Accepted January 21, 1986.
