Thermal desorption modulator for capillary liquid chromatography

May 1, 1986 - Thermal desorption modulator for capillary liquid chromatography. Kiyokatsu. Jinno, Daniel P. Carney, and John B. Phillips. Anal. Chem. ...
3 downloads 0 Views 590KB Size
1248

Anal. Chem. 1986, 58, 1248-1251

addition procedure shodd be applied. (2) The reported results for a first screening of analytes strongly suggest that quenched peroxyoxalate chemiluminescence has potential as a detection method in HPLC. Presently we are systematically investigating the immobilization procedure for 3-aminofluoranthene on glass, and furthermore the potential of other fluorophores is explored.

ACKNOWLEDGMENT J. Knox (Edinburgh) is greatly acknowledged for the gift of the spherical carbon column material. Registry No. TCPO, 1165-91-9; NO;, 14797-65-0; SO-:, 14265-45-3;thiohydantoin, 5789-17-3;thiourea, 62-56-6; ethenyl thiourea, 1483-58-5;thioridazin, 50-52-2;sulforidazine, 14759-06-9; methimazole, 60-56-0; aniline, 62-53-3; 3-ethylaniline, 587-02-0; 4-isopropylaniline,99-88-7; 3,5-dimethylaniline,108-69-0;N,Ndimethylaniline, 121-69-7;N,N-diethylaniline, 91-66-7;N,N-dipropylaniline, 2217-07-4;N,N-dibenzylaniline, 91-73-6; benzylamine, 100-46-9;a-naphthylamine, 134-32-7;%toluidine,95-53-4; 4-toluidine, 106-49-0;m-methyltoluidine, 108-44-1;p-isopropylaniline, 99-88-7;N-ethyl-m-toluidine, 102-27-2;3-aminofluoranthene, 2693-46-1.

LITERATURE CITED Williams, D. C.; Huff, G. F.; Seitz, W. R. Anal. Chem. 1976, 4 8 , 1003-1 006. Scott, G.; Seitz, W. R.; Ambrose, W. R. Anal. chim. Acta 1 ~ 8 0 775, , 221-228. Van Zoonen, P.; Kamminga, D. A.; Gooijer, C.; Velthorst, N. H.; Frei, R. W. Anal. Chim. Acta 1985, 767, 249-257. Van Zoonen, P.; Kamminga, D. A.; Gooijer, C.; Velthorst, N. H.; Frei, R. W.; Giibltz, G. Anal. Chim. Acta 1985, 774, 151-161. Kobayashl, S . ; Imakl, K. Anal. Chem. 1980, 52,424-435. Sigvardson, K. W.; Blrks, J. W. Anal. Chem. 1983, 55, 432. Weinberger, R. J . Chromatogr. 1984, 314, 155-165. Grayeski, M. L.; Weber, A. J. Anal. Lett. 1984, 77, 1539-1552. Rauhut, M. M.; Bollyky, L. J.; Roberts, B. G.; Loy, M.; Whiteman, R. H.; Ianotta, A. V.; Semsel, A. M.; Clarke, R. A. J . Am. Chem. SOC. 1967, 89, 6514-6516.

(10) McCapra, F. Prog. Org. Chem. 1973, 8 , 231. (11) Schuster, G. B. Acc. Chem. Res. 1979, 72, 366-373. (12) Schuster, G. B.; Horn, K. A. I n "Chemical and Biological Generation of Excited States"; Adam, W., Cilento, G , Eds.; Academic Press, New York, 1982, Chapter 7. (13) Catherall, C. L. R.; Frank Palmer, T.; Cendall, R. B. J . Chem. Soc., Faraday Trans. 2 1984, 80 823-837. (14) Catherall, C. L. R.; Frank Palmer, T.; Cendall, R. B. J . Chem. SOC. Faraday Trans. 2 1984, 80, 637-849. (15) Mohan, A. G.; Turro, N. J. J . Chem. Educ. 1974, 57,528-529. (16) Chang, S. H.; Gooding, U. M.; Regnier, F. E. Chromatogr. 1976, 720, 321-333. (17) Giibitt, G.; Van Zoonen, P.; Gooijer, C.; Velthorst, N. H.; Frei, R. W. Anal. Chem. 1985, 5 7 , 2071-2074. (18) Donkerbroek, J. J.; Veltkamp, A. C.; Gooijer, C.; Velthorst, N. H.; Frei, R. W. Anal. Chem. 1983, 55, 1886-1893. (19) Gooijer, C.; Velthorst, N. H.; Frei, R. W. TrAC, Trends Anal. Chem. (Pers. Ed.) 1984, 3 , (IO),259-265. (20) Hustings, C. R.; Aue, W. A.; Augl, J. M. J . Chromatogr. 1970, 5 3 , 487-506. (21) Herman, P. P.; Field, C. R.; Abbot, S. J . Chromatogr. Sci. 1961, 79, 470-478. (22) Majors, R . E.; Hopper, M. J. J . Chromatogr. Sci. 1974, 72, 767-778

Piet van Zoonen Dik A. Kamminga Cees Gooijer* Ne1 H. Velthorst Roland W. Frei Department of General and Analytical Chemistry F~~~university De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands Gerald Giibitz Department of Pharmaceutical Chemistry University of Graz Universitatzplatz 10, A-8010, Graz, Austria

RECEIVED for review October 4,1985. Accepted January 21, This work was by Dutch Foundation Of Technical Sciences under Grant ll-20-46/79-O,VCh 11.0137.

Thermal Desorption Modulator for Capillary Liquid Chromatography Sir: Extremely narrow bore columns have significant advantages in liquid chromatography. The use of substantially smaller quantities of stationary phase packing and mobile phase solvent reduces costs and allows the use of exotic materials for better performance (1, 2 ) . Higher efficiency is obtained, which improves resolution, sensitivity, and limits of detection. Despite these advantages, microcolumns are not widely used. Injector and detector volumes must be reduced in proportion to the reduction in mobile phase volumetric flow rate. It is quite possible to reduce the injection volume, but unless the sample pretreatment volumes are also reduced, much of the analyte will be lost during sample preparation. It is also possible to reduce the detector volume, but without very precise and expensive instrument design, detector sensitivity will be Iost. The loss of sample at the injector and loss of sensitivity at the detector result in higher than necessary limits of detection at the bottom end of the calibration curve. The upper end of the calibration curve is limited by column sample capacity, which is proportional to the square of the column diameter. For a sufficiently small column diameter, the calibration curve may not exist as the column sample capacity limit becomes less than the limit of detection. The best way to keep injector and detector volumes in proper proportion to the column is to build them into the 0003-2700/86/0356-1248$0 1.50/0

column itself. On-column detectors are an example of this approach (3, 4). As the column diameter is reduced, the detector volume is automatically reduced proportionately. Injection valves, however, are mechanical devices and cannot be conveniently built into the head of a column. The purpose of an injection valve is not just sample introduction. The primary purpose is imposition of a modulation signal on the sample so that chemical identity information may be encoded in the chemical signal passing through the column (5-7). Divorcing modulation from sample introduction allows the application of many more devices as chromatographic column modulators. Some of these modulators are nonmechanical or chemical devices, which can easily be built into the head of a column. Since the sample stream continuously flows through a chemical modulator, the modulation signal form is not limited to a single pulse but may consist of a long sequence of pulses. Such long modulation signals have much greater sample throughput but result in a multiplexed detector output signal and require complex computation to recover the chromatogram ( 5 ) . A thermal desorption modulator has been described for use with open tubular fused silica capillary columns (5). The head of the column is rapidly heated and cooled to generate a derivative form chemical signal as the sample flowing into the column is alternately driven out of and then readsorbed into 0 1986 American Chemical Society

ANALYTICAL CHEMISTRY, VOL. 58, NO. 6, MAY 1986

Figure 1. Block diagram of a capillary HPLC system with a thermal desorption modulator: (a) syringe pump containing sample; (b) 12 mm modulator; (c) 4 cm by 0.35 mm i.d. capillary connecting tube; (d) 10.5 cm packed capillary column; (e) capillary connecting tube 5 cm by 0.35 mm i.d.; (f) detector flow cell 1 cm by 0.35 mm i.d.; (9) 5 cm packed capillary column for back pressure; (h) signal from spectrophotometer; (i) computer; (i) plotter; (k) optically coupled switch; (I) modulation signal; (m) variable transformer power supply.

the stationary phase with the changing temperature. This thermal desorption modulator has been successfully applied to capillary gas chromatography columns and should operate similarly with micro liquid chromatography columns of comparable diameter (8-11). A thermal desorption modulator has been described for standard bore liquid chromatography columns (6). This modulator generates the expected derivative form signals, but because of the large quantity of stationary phase material present a t the head of the column, the modulator operates very slowly and does not offer any improvement in sensitivity or detection limits over what is possible with a single injection technique. Smit e t al. (12, 13) have applied mechanical valves as modulators for multiplex liquid chromatography. They used conventional size columns for which minimizing valve volume is not important.

EXPERIMENTAL SECTION Equipment. The liquid chromatographic system was assembled from individual components. An Azuma Electric Co., Ltd. (Tokyo, Japan), microfeeder, MF-2, was used to deliver solvent at 4-16 pL/min flow rates. The column was a 10 cm by 0.35 mm i.d. fused-silica capillary packed with 7-pm Chemicosorb ODS/H material from Chemco (Osaka, Japan). The detector was a Perkin-Elmer (Oak Brook, IL) Lambda 3 spectrophotometer. Flow and reference cells for the detector were prepared by stripping the polymer coating from empty 0.35 mm i.d. fused-silica capillary tubing. The transparent portions of the flow and reference cells were placed in holders fabricated from cardboard and black tape. These holders were then placed into the light path of the spectrophotometer. The flow cell inlet was connected to the outlet of the analytical column via a 5 cm by 0.35 mm i.d. capillary. The flow cell outlet was connected to a short length of packed capillary column to provide some back pressure in the flow cell preventing the formation of bubbles. A previously described laboratory computer system generated the modulation signal, recorded the detector output signal, computed the chromatogram by cross-correlation, and plotted the results (5). Thermal Desorption Modulator. The modulator was prepared from a 12 mm length of the same packed capillary column materiial used in the analytical column. Heat was applied to the modulator using the same electrically conductive paint previously described for a gas chromatography modulator (5). The current through the paint was controlled by an Opt0 22 (Huntington Beach, CA) Model OAC5P optically coupled switch. A 4 cm by 0.35 mm i.d. fused-silica capillary connecting tube between the modulator and column allowed the mobile phase to cool before reaching the column stationary phase. The current source was a variable transformer whose output voltage was adjusted to generate a temperature just below that required to boil the mobile phase in the modulator. Boiling solvent caused an obvious change in the appearance of the modulator.

2

4 RETENTION TIME

[

8 MINUTES 1

10

1249

i2

Figure 2. Chromatogram obtained from a 64-min multiplex experiment using a thermal desorption modulator. The mobile phase was 9O:lO acetonitrile-water with 25 ppb p-diisopropylbenzene and 106 ppb ethylbenzene. The flow rate was 8 pL/min. The pulse duration was 500 ms, the data acquisition period was 2 s, and the pulse probability was 0.125.

The boiling point of the solvent was 84 "C at atmospheric pressure. The temperature reached by the modulator during one current pulse was calculated to be 70 "C assuming that no heat is lost to the surroundings during the pulse. Figure 1 is a block diagram of the complete capillary HPLC system. Modulation Signal. At the beginning of each data acquisition period a random number was generated (14) and used to decide whether or not to pulse the modulator temperature. The specified pulse probability determines the pulse sequence and, thus, the modulation signal. During periods in which no pulse was generated, the modulator remained at room temperature. Pulse duration was independent of the data acquisition period. Solvents and Reagents. Chromatography grade acetonitrile and methanol from Fisher Scientific (Fair Lawn, NJ) were used to prepare mobile phase solutions. Test samples were taken from a set of aromatic hydrocarbon standards from Alltech Associates (Deerfield, IL). Distilled water was additionally purified using a Cole Parmer (Chicago,IL) ion exchanger. Mobile phase solvent was vacuum filtered through a 0.2-pm nylon-66 membrane for particle removal and degassing.

RESULTS AND DISCUSSION Figure 2 is a representative chromatogram obtained by applying a 64-min modulation signal to the thermal desorption modulator while pumping solvent containing 25 ppb of pdiisopropylbenzene and 106 ppb of ethylbenzene through the modulator, column, and detector. A total of 825 thermal pulses were applied to the modulator during the period of the experiment. Pulse probability, pulse temperature, pulse duration, data acquisition period, alignment of the cell holders in the spectrophotometer, and length of the connecting tubing between modulator and column have all been optimized to give the largest possible signal-to-noise ratio. The signal at 3.0 min retention is due to ethylbenzene and that a t 4.7 min is due to p-diisopropylbenzene. The concentrations detected are approximately 2 orders of magnitude below those that can be detected with a single 1.0-pL injection of the same substances using the same apparatus and experimental conditions. The total quantity of sample substance passing through the column to the detector is approximately the same for multiplex and single injection techniques and, thus, quantity limits of detection are comparable. In the multiplex technique the sample is diluted with a larger volume of solvent and pumped through the column over a longer time. With a thermal desorption modulator, sample volume is not constrained by the volume appropriate for a single injection

1250

ANALYTICALCHEMISTRY, VOL. 58, NO. 6, MAY 1986

Table I. Retention and Modulator Efficiency Parameters Calculated for p -Diisopropylbenzene from a Chromatogram Obtained under the Same Conditions as That in Figure 2a retention time, s capacity factor, k duration at base line of 1.0-pL injection band, s duration at half height of 1.0-pL injection band, s length at base line of 1.0-pL injection, cm length at half height of 1.0-pL injection, cm duration of thermal modulator signal at base line, s length of thermal modulator signal at base line, cm duration at half height, positive part signal, s length at half height, positive part signal, cm duration at half height, negative part signal, s length at half height, negative part signal, cm intrasignal duration, s intrasignal length, cm

279 2.7 25 19 0.9 0.68 71 2.6 18

0.65 22 0.78 26 0.92 " The mobile phase contained 2.5 ppm p-diisopropylbenzene and 10 ppm ethylbenzene. pulse but may be as large as the entire volume of mobile phase pumped through the column during a determination. For the system described here this difference is typically 500 pL vs. 1pL, a factor of 500. It is quite practical to prepare a sample in 500 pL of solvent and then pump the entire quantity of sample through the modulator and column to obtain a multiplex chromatogram. It is much more difficult to prepare a sample in 1.0 pL of solvent and then inject it all in a single pulse without losing some in the syringe or injection valve. In practice, samples are often prepared in somewhat larger volume than that which is to be injected and only a reproducibly injectable fraction is taken. If these losses during sample preparation are included in evaluating the overall efficiency of an analytical procedure, then the multiplex technique has lower quantity as well as concentration detection limits. The detector used in this work was chosen for convenience. Conventional liquid chromatographic detectors are incompatable with these microcolumns and no ultra-low-volume detector was available. This spectrophotometer used as a liquid chromatography detector is not very sensitive because of the extremely short on-column path length. With a more sensitive detector, both single injection and multiplex techniques would have substantially lower detection limits. The multiplex technique would retain its relative advantage. Alternatively, a less expense detector could be used with the multiplex technique to give results equivalent to a more expensive detector with the single injection technique. The specific on-column detection technique used in this work is not practical for routine use because alignment of the cell holders in the sample compartment is not easily reproducible. Modulation Pulse Duration and Length. The chromatogram in Figure 2 consists of peaks followed by vacancies as expected for a thermal desorption modulator (5). Both signals are somewhat obscured by noise since the concentration of this sample is near the multiplex detection limit and substantially below the conventional injection detection limit. The ethylbenzene signal is broader than the p-diisopropylbenzene signal, especially in its negative part, which overlaps the following p-diisopropylbenzene positive part signal. The ethylbenzene signal may have been broadened by overlap from a contaminant in the mobile phase with approximately the same retention time. The ethylbenzene signal observed at higher concentrations is sharper and smaller relative to the p-diisopropylbenzene signal. This technique responds to modulatable substances present in the mobile phase whatever their source. Because of the sensitivity of the technique, mobile phase contaminants that would not be seen in conventional single injection chromatography may appear in multiplex chromatograms.

Table 11. Calibration Data for p -Diisopropylbenzene in 9010 Acetonitrile-Water Solvent" concn, ppm 12.6b 6.3b 2.52 1.26 0.252 0.126

signal, arbitrary units

sensitivity, arbitrary units

420 390 300

33 62 120 200 160 54 210 34 270 " Experimental conditions are the same as in Figure 2. Correlation coefficient is 0.989. Slope is 110. Intercept is 30. bThesedata were not included in calculation of correlation coefficient because the column is overloaded at these concentrations. Table I contains some measures of signal duration and band length along the column calculated from a chromatogram obtained under the same conditions as that in Figure 2 but at a higher concentration and better signal-to-noiseratio. The basic signal generation mechanism is the same as that previously described for capillary gas chroniatography and the significance of these measures is similar to those previously reported (5). Void volume delay in this very small column is substantially less than that in the connecting tubing. Both retention time, t R , and void volume delay, to, have been corrected for connecting tubing delay before computation of the capacity factor. At 2.6 cm vs. 0.9 cm the overall length of the signal generated by the thermal desorption modulator is substantially longer than that generated by a 1.0-pL injection pulse. Resolution is limited by the modulator rather than the column. The positive and negative parts of the modulator signal when measured individually, however, are comparable in length to the 1.0-pL injection. As was found in the capillary gas chromatography case, it is the modulator interband length rather than the lengths of the two parts which dominates the overall signal length (5). The generated chemical concentration signals are pseudoderivatives, that is, the positive and negative parts of the signal are farther apart than they would be if this signal form was a true derivative of a Gaussian. A deconvolution computation could be applied to the pseudoderivative signal form to bring its resolution up to that of the conventional pulse signal form. Ultimately, both techniques should be column limited. Calibration Curves. Table I1 contains calibration data for the p-diisopropyl signal a t 4.7 min retention. Above 3.3 ppm, the calibration curve bends over. At this concentration some component of the chromatographic system, probably the column, is overloaded. The detection limit at the lower end of the calibration curve depends mostly upon the detector sensitivity. In this particular set of calibration experiments it was about 126 ppb. Detector sensitivity, however, is strongly dependent upon fabrication and alignment of the flow cell in its sample compartment. Substantially lower detection limits have been obtained in some additional experiments (15),for example, that shown in Figure 2.

LITERATURE CITED Jlnno, K.; Fujlmoto, C. J . L i q . Chromatogr. 1084, 7 , 2059-2071. Jinno, K. HRC C C , J . High Resolut. Chromatogr. Chromatogr. Commun. 1982, 5 , 364-367. Yang, F. J. HRC CC, J . High Resolut. Chromatogr. Chromatogr. Commun. 1083, 6 , 348-358. Yang, F. J. J . Chromatogr. 1982, 236, 265-277. Phillips, John B.; Luu, Derhsing; Pawliszyn, Janusz E.;Carle, Glenn C. Anal. Chem. 1985, 57, 2779-2787. Carney, Daniel P.; Phllllps, J. B. HRC CC, J . Hlgh Resolut. Chroma togr. Chromatogr. Commun. 1081, 4 , 413-414. Phillips, J. B.; Valentin, Jose R.; Carle, Glenn C. In "Toxic Materials in the Atmosphere: Sampling and Analysls"; American Society for Testing and Materlals: Philadelphia, PA, 1982; ASTM STP 786.

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 electrochemical analyzer 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-analog converter 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