Electrochemically Modulated Liquid Chromatography Coupled On

The third example investigates the electrooxidation of aniline by utilizing an EMLC column as an on-line electrochemical reactor and product separator...
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Anal. Chem. 2000, 72, 2641-2647

Electrochemically Modulated Liquid Chromatography Coupled On-Line with Electrospray Mass Spectrometry Haiteng Deng† and Gary J. Van Berkel*

Chemical and Analytical Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831-6365 Hajime Takano, Daniel Gazda, and Marc D. Porter*

Department of Chemistry, Ames LaboratorysUSDOE, and Microanalytical Instrumentation Center, Iowa State University, Ames, Iowa 50011

Electrochemically modulated liquid chromatography (EMLC) has been coupled to an electrospray mass spectrometer. This combination takes advantage of the ability of EMLC to manipulate retention and enhance separation efficiency solely through changes in the potential applied to a conductive stationary phase, thereby minimizing complications because of possible changes in analyte ionization efficiencies when gradient elution techniques are used. Three examples are presented that demonstrate the attributes of this EMLC/electrospray mass spectrometry (ES-MS) coupling. The first two examples involve the separation of mixtures of corticosteroids or of benzodiazepines, showing the general utility of the union for eluent identification and low-level detection. The ability to identify products from on-column redox transformations is also demonstrated using the benzodiazepine mixture. The third example investigates the electrooxidation of aniline by utilizing an EMLC column as an on-line electrochemical reactor and product separator and ESMS for detection and product identification. A number of reports have described the analytical capability of electrochemically modulated liquid chromatography (EMLC).1 This methodology works by altering the retention of analytes through changes in the potential applied, Eappl, to a conductive stationary phase (e.g., porous graphitic carbon (PGC)). An EMLC column is constructed by fashioning the stationary phase as the working electrode in an electrochemical flow cell that also functions as a LC column. This approach stands in contrast to chromatographic separations in which analyte retention is altered through changes in mobile-phase composition.2 Reports from several laboratories3-23 have demonstrated that EMLC can be * Corresponding authors. GJVB: (phone) 423-574-1922; (fax) 423-576-8559 (e-mail) [email protected]. MDP: (phone) 515-294-6433; (fax) 515-294-3254; (e-mail) [email protected]. † Current address: Laboratory for Macromolecular Analysis and Proteomics, Albert Einstein College of Medicine, Bronx, NY 10461. (1) Porter, M. D.; Takano, H. In Encyclopedia of Separation Science; Wilson, I. D., Adlard, T. R., Poole, C. F., Cook, M., Eds.; Academic Press: London, in press. (2) Weston, A.; Brown, P. R. HPLC and CE: Principles and Practice; Academic Press: San Diego, CA, 1997. 10.1021/ac991461+ CCC: $19.00 Published on Web 05/04/2000

© 2000 American Chemical Society

applied effectively to the separation of a wide range of analytes, including aromatic sulfonates, carboxylic acids, dansylated amino acids, inorganic anions, metal ions, corticosteroids, and benzodiazepines. The ability to separate racemic mixtures has also been recently demonstrated.19 This paper builds on these earlier reports by coupling EMLC on-line with mass spectrometric detection. As with other hyphenated separation/mass spectrometry (MS) methods,24 the combination of EMLC and MS is expected to improve detection specificity as well as enhance detection levels. Both of these analytical figures of merit can be further improved by using detector schemes based on tandem mass spectrometry (e.g., selected reaction monitoring).25 Moreover, the attributes of EMLC may enhance the (3) Ge, H.; Wallace, G. G. J. Liq. Chromatogr. 1990, 13, 3245-3260. (4) Ge, H.; Wallace, G. G. Anal. Chem. 1989, 61, 2391-2394. (5) Nagaoka, T.; Kakuno, K.; Fujimoto, M.; Nakao, H.; Yano, J.; Ogura, K. J. Electroanal. Chem. 1994, 368, 315-317. (6) Nagaoka, T.; Fujimoto, M.; Nakao, H.; Kakuno, K.; Yano, J.; Ogura, K. J. Electroanal. Chem. 1994, 364, 179-188. (7) Nagaoka, T.; Nakao, H.; Tabusa, K.; Yano, J.; Ogura, K. J. Electroanal. Chem. 1994, 371, 283-286. (8) Nagaoka, T.; Fujimoto, M.; Nakao, H.; Kakuno, K.; Yano, J.; Ogura, K. J. Electroanal. Chem. 1993, 350, 337-344. (9) Nagaoka, T.; Fujimoto, M.; Uchida, Y.; Ogura, K. J. Electroanal. Chem. 1992, 336, 45-55. (10) Deinhammer, R. S.; Shimazu, K.; Porter, M. D. Anal. Chem. 1991, 63, 18891894. (11) Deinhammer, R. S.; Ting, E.-Y.; Porter, M. D. J. Electroanal. Chem. 1993, 362, 295-299. (12) Deinhammer, R. S.; Porter, M. D.; Shimazu, K. J. Electroanal. Chem. 1995, 387, 35-46. (13) Deinhammer, R. S.; Ting, E.-Y.; Porter, M. D. Anal. Chem. 1995, 67, 237246. (14) Ting, E.-Y.; Porter, M. D. Anal. Chem. 1997, 69, 675-678. (15) Ting, E.-Y.; Porter, M. D. Anal. Chem. 1998, 70, 94-99. (16) Ting, E.-Y.; Porter, M. D. J. Electroanal. Chem. 1998, 443, 180-185. (17) Abdel-Latif, M. S.; Porter, M. D. Anal. Lett. 1998, 31, 1743-1756. (18) Ting, E.-Y.; Porter, M. D. J. Chromatogr., A 1998, 793, 204-208. (19) Ho, M.; Wang, S.; Porter, M. D. Anal. Chem. 1998, 70, 4314-4319. (20) Fujinaga, T.; Kihara, S. CRC Crit. Rev. Anal. Chem. 1977, 6, 223-253. (21) Strohl, J. H.; Dunlap, K. L. Anal. Chem. 1972, 44, 2166-2170. (22) Ghatak-Roy, A. R.; Martin, C. R. Anal. Chem. 1986, 58, 1574-1575. (23) Lam, P.; Kumar, K.; Wnek, G. E.; Przybycien, T. M. Anal. Chem. 1999, 71, 4272-4277. (24) Niessen, W. M. A. Liquid Chromatography-Mass Spectrometry; Marcel Dekker: New York, 1999. (25) Busch, K. L.; Glish, G. L.; McLuckey, S. A. Mass Spectrometry/Mass Spectrometry; VCH Publishers: New York, 1988.

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applicability of MS detection because conventional separations often require use of gradient elution strategies. A continually changing mobile phase can degrade mass spectrometric detection because of changes in analyte ionization efficiencies.24 The ability to separate a wide range of mixtures isocratically by EMLC therefore presents an intriguing approach to reduce the complications caused by mobile-phase gradients. One of the most used techniques for connecting condensedphase separations on-line with MS is electrospray (ES) ionization.24,26 Electrospray is an atmospheric pressure technique that employs an electrostatic sprayer to assist the transfer of ionic analytes from solution into the gas phase for mass analysis. ESMS is the method of choice for analyzing nonvolatile, polar, and thermally labile compounds as well as higher molecular weight biopolymers and represents a natural starting point for our investigation. EMLC/ES-MS is then an on-line union of electrochemistry (EC) and ES-MS. Indeed, several groups have recently demonstrated the ability to perform EC/ES-MS experiments.27-34 Central to the successful implementation of EC/ES-MS is the chemical composition and concentration of supporting electrolyte. ES-MS functions best with volatile electrolytes (e.g., ammonium acetate) present at concentrations below ∼20 mM. Nonvolatile electrolytes, although tolerated by sampling orifices designed to be less susceptible to plugging, may severely suppress the analytical signal.35,36 The following describes the successful on-line coupling of an EMLC column with ES-MS. We show that it is possible to operate an EMLC column effectively when using solvent/electrolyte combinations that are compatible with ES-MS. This capability is demonstrated by three different sets of separations. The first two examples involve the separation of a mixture of corticosteroids and of benzodiazepines. These investigations confirm the ability to merge EMLC with ES-MS. The separation of benzodiazepines also shows the value of ES-MS as a detection modality by identification of EMLC-generated electrolysis products and by detection of trace levels of eluent. The last example exploits the EMLC column as an on-line electrolysis reactor and as a LC separation column. EC-LC/ES-MS experiments reported to date have used an electrochemical reactor cell and separate conventional HPLC column.31 Our example, which monitors the electrooxidation of aniline, shows that EMLC can be employed both to generate and to separate electrolysis products, followed by ESMS detection and identification.

Chart 1

Chart 2

EXPERIMENTAL SECTION Chemicals. The corticosteroids (Chart 1) and benzodiazepines (Chart 2) were purchased from Sigma (St. Louis, MO) and used (26) Electrospray Ionization Mass Spectrometry; Cole, R. B., Ed.; John Wiley: New York, 1997. (27) Zhou, F.; Van Berkel, G. J. Anal. Chem. 1995, 67, 3643-3649. (28) Bond, A. M.; Colton, R.; D’Agnostino, A.; Downnard, A. J.; Traeger, J. C. Anal. Chem. 1995, 67, 1691-1695. (29) Xu, X.; Lu, W.; Cole, R. B. Anal. Chem. 1996, 68, 4244-4253. (30) Lu, W.; Xu, X.; Cole, R. B. Anal. Chem. 1997, 69, 2478-2484. (31) Iwahashi, H.; Ishii, T. J. Chromatogr., A 1997, 773, 23-31. (32) Pretty, J. R.; Van Berkel, G. J. Rapid Commun. Mass Spectrom. 1998, 12, 1644-1652. (33) Deng, H.; Van Berkel, G. J. Electroanalysis 1999, 11, 857-865. (34) Deng, H.; Van Berkel, G. J. Anal. Chem. 1999, 71, 4284-4293. (35) Tang, L.; Kebarle, P. Anal. Chem. 1993, 65, 3654-3668. (36) Enke, C. G. Anal. Chem. 1997, 69, 4885-4893.

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as received. Aniline (Aldrich, Milwaukee, WI) was vacuum distilled prior to use. Deionized H2O (Milli-RO 12 Plus, Millipore, Bedford, MA), methanol (HPLC grade, J. T. Baker, Phillipsburg, NJ), acetonitrile (J. T. Baker), ammonium acetate (99.999%, Aldrich),

and acetic acid (PPB/Teflon grade, Aldrich) were used in all sample standards, mobile-phase preparations, and supporting electrolytes. EMLC Columns. The design and construction of the column have been described in detail elsewhere.15 Briefly, these columns consist of a Nafion cation-exchange membrane in tubular form (Perma Pure Inc.) placed inside a porous stainless steel cylinder (Mott Metallurgical Corp.). The Nafion tubing serves as a container for the PGC stationary phase (Hypercarbon, Hypersil). The PGC also functions as the working electrode. The porous stainless steel cylinder prevents deformation of the Nafion tubing under the high pressure of chromatographic flow and is also used as the auxiliary electrode. The Ag/AgCl reference electrode (model RE-4, Bioanalytical Systems, Inc., West Lafayette, IN) is placed in an electrolyte reservoir that surrounds the stainless steel auxiliary electrode. Two slightly different columns were used. Column 1 was identical to that previously described (∼7-µm spherical PGC packing, 9 cm long, 0.38-cm i.d.)15 and was employed for the separation of the corticosteroid mixture. Column 2, which had a smaller internal diameter (0.22-cm i.d.), was utilized in all other separations. ES-MS System. All ES-MS experiments were performed on either an API165 single-quadrupole or API365 triple-quadrupole mass spectrometer (PE SCIEX, Concord, ON, Canada) using a TurboIonSpray source. A 30-cm-long, Teflon encapsulated fusedsilica (TEFS) transfer tube (75-µm-i.d. fused silica encapsulated in 1/16-in.-o.d. Teflon (CETAC Technologies, Inc., Omaha, NE) connected a 3.5-cm-long stainless steel ES emitter (400-µm o.d., 100-µm i.d.) to the stainless steel 254-µm-i.d. bore-through bulkhead “splitter-tee” grounding port built into the TurboIonSpray source assembly. The orifice and ring electrode in the atmospheric sampling interface were set to maximize signal and minimize fragmentation, the latter caused by collision-induced dissociation in this high-pressure (∼1 Torr) region. The emitter was held at 4.2-4.5 kV, placed 1.5-2.5 cm from the N2 curtain gas plate aperture (1.0 kV), and angled to spray across the aperture. Nitrogen was used for sample nebulization and for the heated cross-flow “Turbo gas” (typically 8.0 L/min, 200-250 °C). Fullscan spectra were obtained using a 0.1 m/z step size and a 1.0ms dwell time. Conditions for single ion monitoring and selected reaction monitoring experiments are described in the appropriate figure headings. All tandem mass spectrometry experiments were carried out using a laboratory frame-of-reference collision energy of 35 eV and a N2 collision gas thickness of ∼1.8 × 1015 cm-2. EMLC/ES-MS System. Figure 1 is a schematic diagram of the EMLC/ES-MS system, which is composed of a HPLC pump and solvent reservoir, sample injector, EMLC column 1 or 2, potentiostat, ES-MS instrument, and UV detector. The chromatographic portion of the system consisted of a Perkin-Elmer model 250 binary LC pump (Norwalk, CT), a Rheodyne model 7125 injector (1.0-µL loop, Cotati, CA), and an Applied Biosystems model 785A programmable absorbance detector (Norwalk, CT); the latter was fitted with an 8.0-mm path length (12 µL) flow cell. The electrolytic mobile phase, which was sparged with N2 for 15 min and maintained under a nitrogen atmosphere during use, was delivered isocratically at a flow rate of either 1.0 (column 1) or 0.50 mL/min (column 2). The column exit was connected to a grounded bulkhead splitter-tee that was incorporated into the

Figure 1. Schematic diagram of the EMLC/ES-MS system (not to scale). The particulate stationary phase and insulating spacer that electrically isolate the PGC stationary phase and auxiliary electrode (i.e., the porous stainless steel tubing)15 are not shown.

source assembly using 7.5-cm-long, 127-µm-i.d., PEEK tubing. A 45-cm length of 127-µm-i.d. PEEK tubing formed the connection to the absorbance detector. The back pressure on the downstream side of the absorbance detector was adjusted so that the flow split provided 100 (column 1) or 50 µL/min (column 2) to the ES source. Absorbance readings were directed to the mass spectrometer computer system and stored by means of a dual-channel PE Nelson model 970A A/D interface (Perkin Elmer, San Jose, CA) and PE SCIEX sample control software (Version 1.4b3). A PAR model 173 potentiostat and model 175 universal programmer (Princeton Applied Research Corp., Princeton, NJ) were used to control the potential applied to the EMLC columns. RESULTS AND DISCUSSION Corticosteroids. One of the earlier reports using EMLC demonstrated the effective separation of the corticosteroids prednisone (1), prednisolone (2), cortisone (3), and hydrocortisone (4) (Chart 1) using a PGC stationary phase and optical detection.14 The challenge in this separation, as described in more general investigations of PGC as a stationary phase,37 is the discrimination between structures containing different numbers of double bonds (e.g., 1 versus 3) and/or small differences in the identity and number of substituents (e.g., 3 versus 4). At bonded reversed phases, compounds with subtle differences in functionality generally exhibit greater differences in retention than those that differ by the number of double bonds. The opposite is often more important for separations at PGC, which possesses π-electron sensitivity superimposed upon reverse phase characteristics. Using a mobile phase composed of 50/50 (v/v) H2O/CH3CN with 0.1 M LiClO4 added as electrolyte, the above study14 found that 1-4 could be readily separated from one another using EMLC and that the retention of each was significantly influenced by Eappl. The changes in retention reflect a mixing of the dependence of the donor-acceptor properties of PGC on Eappl and the competition from the ionic species (e.g., supporting electrolyte) in the mobile phase.14 To adapt this separation for coupling with ES-MS, we increased the concentration of the organic component of the mobile phase (20/80 (v/v) H2O/CH3CN) and replaced the nonvolatile LiClO4 electrolyte with a much more volatile electrolyte combination (i.e., 10 mM ammonium acetate (NH4OAc) and 0.8 vol % acetic acid (HOAc)). In previous EC/ ES-MS work with organic/H2O solvent mixtures, concentrations Analytical Chemistry, Vol. 72, No. 11, June 1, 2000

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Figure 2. Summed ion current chromatograms recorded in selected ion monitoring mode (330 ms dwell time for each ion) for the protonated molecules of prednisone (1, m/z 359), prednisolone (2, m/z 361), cortisone (3, m/z 361), and hydrocortisone (4, m/z 363), separated on PGC as a function of Eappl: (a) +500 and (b) -1000 mV. (c) Plots of log(k′) vs Eappl for separation of a mixture of 1 (2), 2 (1), 3 (b), and 4 (9). Mobile phase: 20/80 (v/v) H2O/CH3CN containing 10 mM NH4OAc and 0.8 vol % HOAc. Flow rate: 1.0 mL/ min with 100 µL/min of effluent split to ES ion source. A total of 25 pmol of each corticosteroid was injected onto the column.

up to 10 mM for NH4OAc were found to only marginally suppress analyte signal. Furthermore, the addition of up to ∼1% (v/v) HOAc enhanced the solution conductivity as well as the analytical signal for basic analytes.32-34 Although 100% H2O can be sprayed at 50100 µL/min with pneumatic or heat-assisted ES, analyte signals are generally greater in magnitude when a large fraction of an organic solvent (∼50% (v/v) or more of CH3OH or CH3CN) is used. Under these new mobile-phase conditions, each of the corticosteroids was detected by ES-MS as its protonated analogue, i.e., m/z 359 for (1 + H)+; m/z 361 for (2 + H)+ and (3 + H)+; and m/z 363 for (4 + H)+. The data in Figure 2a and b are the summed ion current chromatograms obtained for the four steroids at two extremes in Eappl (i.e., +500 and -1000 mV) using selected ion monitoring. Figure 2c summarizes the chromatographic data obtained over the full potential range through plots of log(k′) versus Eappl. At the more positive values of Eappl, the elution order is 1 < 2 < 3 < 4, and the four analytes are partially but not fully resolved. 2644 Analytical Chemistry, Vol. 72, No. 11, June 1, 2000

Shifts in Eappl to more negative values affect the retention of each analyte differently. As Eappl becomes more negative, 2 and 4 undergo a general decrease in retention, whereas 1 shows a small increase and 3 first increases and then decreases. These differences in retention lead to the strong overlap of the elution bands for 3 and 4 at Eappl of ∼0 mV, and for 1 and 2 at -200 mV. Importantly, these differences result in an effective separation of the mixture at large negative values of Eappl (e.g., -1000 mV). Some general characteristics of the separations in Figure 2 are similar to those reported previously,14 while others are markedly different. For example, the changes in elution order over the full range of Eappl for Figure 2 follow the same dependencies found using mobile phase with a higher water content and a nonvolatile electolyte.14 The extent of changes in retention (i.e., changes in k′ with respect to changes in Eappl) in the more aqueous mobile phase used previously was, however, larger than observed here. This difference is attributed primarily to the greater organic content of the mobile phase used in the EMLC/ES-MS separations in Figure 2. Further experiments are needed to delineate the mechanistic aspects of these separations in greater detail.38 Together, these findings demonstrate a critical attribute of EMLC as a separation technique when coupled to ES-MS. That is, EMLC has the ability to manipulate separation efficiencies without employing conventional gradient elution techniques, which should facilitate detection via ES-MS. These chromatograms, along with those not shown for the separations at intermediate values of Eappl, also indicate that 1-4 are electrochemically stable throughout most of the tested range of Eappl. However, at +500 mV, a broad elution band at m/z 363 (i.e., the same m/z as for (4 + H)+) superimposes itself across the elution bands for 1 and 2. We do not as yet understand the origin of this band. Nonetheless, we note that coupling EMLC and ES-MS may be particularly well suited for investigating mechanistic aspects of electrode reactions. Indeed, after showing the application of EMLC/ES-MS to the separation of a mixture of benzodiazepines in the discussion below, we address this possibility by utilizing ES-MS for the detection of EMLC-generated electrolysis products. Benzodiazepines. Another recent report described the EMLC separation of five benzodiazepines (Chart 2), viz., oxazepam (5), temazepam (6), desmethyldiazepam (7), diazepam (8), and nitrazepam (9), at a PGC stationary phase with conventional optical detection.18 Using 53/47 (v/v) H2O/CH3CN as the mobile phase and 0.1 M LiClO4 as the supporting electrolyte, the retention of 5 was found to decrease as Eappl became more negative, while that of 6-9 increased. The relative elution order of the benzodiazepines 5-9, in contrast to the corticosteroids 1-4, did not change over the tested range of Eappl (+500 to -300 mV). Thus, changes in Eappl have the unusual effect in this separation of stretching both ends of the chromatogram as Eappl becomes more negative. We note that the negative limit of Eappl was -300 mV because of the apparent reduction of 9 at more negative values. (37) Bassler, B. J.; Hartwick, R. A. J. Chromatogr. Sci. 1989, 27, 162-165. (38) The data in Figure 2a and 2b also show the presence of a low-abundance, unidentified peak at m/z 363 that was not detected in previous experiments using optical detection. This component of the mixture elutes with the solvent front (∼1 min) at an Eappl of +500 mV and elutes with increasing retention time as Eappl becomes more negative. While uncertain as to the origin of this band, the sensitive, m/z-specific detection afforded by ES-MS provides for the detection of this species that went undetected previously.

Figure 3. Plots of log(k′) vs Eappl for the separation of a mixture (20 pmol each) of oxazepam (5, b), temazepam (6, 9), desmethyldiazepam (7, 2), diazepam (8, 1), nitrazepam (9, [). The reduction products of nitrazepam (9a and 9b, ]) are first observed at -550 mV along with nitrazepam. At more negative potentials, nitrazepam is no longer observed and the log(k′) values for the reduction products dramatically decrease. EMLC/ES-MS conditions are described in Figure 4.

This separation was adapted for use with EMLC/ES-MS by changing again the electrolyte from 0.1 M LiClO4 to 10 mM NH4OAc/0.8% (v/v) HOAc and by increasing the organic content of the mobile phase (40/60 (v/v) H2O/CH3CN). Figure 3 summarizes the results over the complete potential range investigated through plots of log(k′) versus Eappl. We found that 5-9 are effectively separated at +500 mV. As Eappl moves to more negative values, the separation efficiency for all compounds increases, except for 8 and 9, which begin to coelute at about -300 mV. Excluding the coelution of 8 and 9, these changes in retention with Eappl are generally consistent with those observed previously18 but quantitatively differ because of differences in mobile phase and supporting electrolyte. Importantly, the results also show that 5-9 are electrochemically stable between +500 and -500 mV. The results in Figures 3 also extend the previous study18 to more negative values of Eappl than previously investigated by taking advantage of ES-MS for identification of products from any online redox transformations. Thus, separations at the more extreme negative values of Eappl were first carried out by monitoring the full mass spectrum in order to detect reduction products at all possible m/z. On the basis of the structural differences of 5-9, the observed electrochemical stability of 5-8, and literature on the electroreduction of monosubstituted nitrobenzenes,39 the instability of 9 to electrolysis is attributed to its nitro group. Equations 1 and 2 present the two-step electrolysis of 9 which

R-NO2 (9) + 2e- + 2H+ f R-NO (9a) + H2O (1) R-NO (9a) + 2e- + 2H+ a R-NHOH (9b)

(2)

yields 9a and 9b as the major reduction products (Chart 2).39 Indeed, the ensuing EMLC/ES-MS results confirmed the presence of 9a and 9b by detection of ions at m/z 266 and 268, which correspond to the expected m/z ratios for their respective protonated molecules. (39) Lund, H. Chapter VII In Organic Electrochemistry; Baizer, M. M., Ed.; Marcel Dekker: New York, 1973; pp 315-345.

The data in Figure 4 show the UV detector trace (top) and mass chromatograms acquired in parallel at Eappl of -550, -650, and -700 mV. The reduction products of 9 are first observed above background levels in the mass chromatograms at Eappl of -550 mV. Interestingly, 9a and 9b coelute with 9 at this potential, which we attribute to the low-level generation of electrolysis products at the end of the column. At -600 mV, complete reductive transformation of 9 has taken place as evidenced by the disappearance of any features in the mass chromatogram at m/z 282 (Figure 4b). The relative abundances of the reduction products, as expected, have increased, whereas their retention times have decreased. At -700 mV (Figure 4c), 9a and 9b exhibit a further decrease in retention. In all three cases, 9a and 9b coelute. This situation is a result of the reversibility of the twoelectron reduction of 9a to 9b (eq 2). We attribute the change in retention of 9a and 9b with Eappl to two related factors. Both arise from how changes in Eappl alter the ability of the analytes and PGC to interact through donoracceptor interactions.13 First, a decrease in Eappl increases the donor strength of PGC in terms of its interaction with analytes by increasing the negative charge density at the PGC surface. Second, there is an inherently large decrease in the acceptor strength of 9a and 9b with respect to 9 due to the two-electron reduction processes in eqs 1 and 2. Thus, the extent to which 9a and 9b can interact as acceptors with PGC is much less than that for 9. This decrease, coupled with the increase in the donor strength of PGC as Eappl becomes more negative, results in the observed decrease in the retention of 9a and 9b as Eappl becomes more negative. The data also show that, as Eappl become more negative, there is a change in the relative amounts of 9a and 9b, with the amount of 9a increasing with respect to 9b. We tentatively ascribe this trend to the kinetics of the overall conversion process. However, a more detailed interpretation requires a through kinetic investigation. Detection Levels. The development of EMLC has focused to date mainly on column design and application assessments. There has been little attention to the minimum quantity of analyte that can be injected and still be detected. To investigate this issue, 20 µM 6 was first prepared in CH3OH solution (1.02 mg/mL) and then serially diluted with CH3OH to 10, 1.0, 0.50, and 0.10 µM. This compound was chosen for this assessment mainly because of its relatively short retention time (∼2.5 min at +300 mV). Since the volume of the injection loop was 1.0 µL, 0.10 pmol of 6 was injected into column for the 0.10 µM solution. However, the split ratio, set by the back pressure on the optical detector, reduces the amount of eluted analyte that travels on to the mass spectrometer for detection by a factor of 10. Figure 5 shows the ion signal from selected reaction monitoring (m/z 301 f 255) recorded from three replicate injections of sample blanks and three replicate injections of 6 ranging from 0.10 to 20 pmol onto the EMLC column. From the inset in Figure 5, it is apparent that the detection level for 6 under the specified operating conditions is between 0.10 and 0.50 pmol injected onto the column. Furthermore, the amplitude of the signal exhibits a linear dependence from 0.50 to 20 pmol injected. These detection levels are comparable to those determined concomitantly using the UV data. Analytical Chemistry, Vol. 72, No. 11, June 1, 2000

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Figure 4. UV detector trace (top) and ion current chromatograms recorded in selected ion monitoring mode (100 ms dwell time for each ion) for the protonated molecules of oxazepam (5, m/z 287), temazepam (6, m/z 301), desmethyldiazepam (7, m/z 271), diazepam (8, m/z 285), nitrazepam (9, m/z 282) and reduction products of nitrazepam (9a, m/z 266 and 9b, m/z 268) separated on the EMLC column at Eappl of (a) -550, (b) -600, and (c) -700 mV. Mobile phase: 40/60 (v/v) H2O/CH3CN containing 10 mM NH4OAc and 0.8 vol % HOAc. Flow rate: 0.50 mL/min with 50 µL/min of the column effluent split to the ES ion source. A mixture of the five benzodiazepines (20 pmol each) was injected onto the column.

Chart 3

Figure 5. Ion current chromatograms recorded in selected reaction monitoring mode (m/z 301 f 255, 200 ms dwell time) for three replicate injections on-column of a sample blank and 0.10-20 pmol of temazepam (6). Eappl, +300 mV. Mobile phase: 40/60 (v/v) H2O/ CH3CN containing 10 mM NH4OAc and 0.8 vol % HOAc. Flow rate: 0.50 mL/min with 50 µL/min of the column effluent split to the ES ion source.

On-Column Oxidation of Aniline and Separation of Its Redox Products. In the previous section, we demonstrated the advantage of coupling EMLC with ES-MS for the detection of electrolysis products (i.e., the conversion of 9 to 9a,b). This section explores this attribute in more detail, recognizing that the ability to generate and to separate redox products by using EMLC in a “dual-purpose” mode would eliminate the need to interface an electrochemical cell on-line with a traditional chromatographic column or to generate products off-line prior to chromatographic separation.31,40 To this end, we examined the electrooxidation of aniline,41-43 a process that forms a conducting polymer of (40) Volk, K. J.; Yost, R. A.; Brajter-Toth, A. Anal. Chem. 1992, 64, 21A-33A. (41) Mohilner, D. M.; Adams, R. N.; Argersinger, W. A., Jr. J. Am. Chem. Soc. 1962, 84, 3618-3622. (42) Yang, H.; Bard, A. J. J. Electroanal. Chem. 1992, 339, 423-449.

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considerable interest in areas such as electronics, energy storage, and electroanalysis systems. Polyaniline is readily prepared electrochemically from aqueous solution; however, the polymerization mechanism continues to be an extensively debated issue. Along these lines, Deng and Van Berkel34 have examined the electrochemical polymerization of aniline by on-line EC/ES-MS and have identified several short n-mers (n ) 2-10) and the structure of the dimers (see monomer (10), dimer (11), and tetramer (12) structures in Chart 3). In these experiments, 2 nmol of aniline (10) was injected into the EMLC/ES-MS system at +600 and +700 mV. A mobile phase of 5/95 (v/v) H2O/CH3CN containing 10 mM NH4OAc and 0.8% (v/v) HOAc was used at a flow rate of 0.5 mL/min. The ion current chromatograms in Figure 6 present the results, monitoring m/z ratios selected on the basis of literature precedents for protonated molecules of 10 and low molecular weight polyaniline oligomers.34 At +600 mV, 10 is partially, but not fully, oxidized upon passage through the column. This transformation is reflected by the oxidation products observed at longer retention times, viz., aniline dimers, (11 + H)+, at m/z 183 and tetramers, (12 + H)+, at m/z (43) Trivedi, D. C. In Handbook of Organic Conductive Molecules and polymers; Vol 2. Conductive Polymers; Synthesis and Electrical Properties; Nalwa, H. S., Ed.; John Wiley: New York, 1997; Chapter 12, pp 505-572.

observed to elute from the column. It is, however, possible that the higher n-mers may have formed, but because of their strong adsorption onto PGC, may not have eluted. This likelihood is supported by the large amount of 10 (i.e., nanomoles) that was required for injection before realizing detectable n-mer signals. The detection of only oxidized n-mers arises because the higher n-mers are more easily oxidized than aniline.41

Figure 6. Ion current chromatograms recorded in selected ion monitoring mode (100 ms dwell time for each ion) for the protonated molecules of aniline (10, m/z 94), aniline dimers (11, m/z 183), and aniline tetramers (12, m/z 363) recorded from the injection of 2.0 nmol of aniline on-column at Eappl of (a) +600 and (b) +700 mV. Mobile phase: 5/95 (v/v) H2O/CH3CN containing 10 mM NH4OAc and 0.8 vol % HOAc. Flow rate: 0.50 mL/min with 50 µL/min of the column effluent split to the ES ion source.

363. Both n-mers elute much later than 10, with 12 being retained more strongly than 11. These differences in retention are consistent with the π-electron sensitivity of PGC as a stationary phase. The small bands that elute with 10, and correspond in m/z to dimers and tetramers, originate from oxidation of 10 in the stainless steel ES emitter capillary.34 At +700 mV, all of 10 is oxidized upon passage through the column. This conversion is evident by the absence of a detectable band in the ion current profile for (10 + H)+ at m/z 94 and the absence of coeluting bands at m/z 183 and 363. At +700 mV, the retention times of both 11 and 12 formed on-column are less than those at +600 mV. We are, as yet, uncertain as to the basis of this trend in retention or on the variations in the shapes and width of the elution bands and are designing experiments to address these issues. The presence of the retained n-mers in the chromatograms reflects the oxidation of 10 to its radical cation, which gradually couples to form larger, less soluble n-mers of polyaniline. The n-mers are depicted in Chart 3 as having a head-to-tail linkage, which is the dominant linkage for aniline n-mers formed by electropolymerization in moderately acidic solutions.34,41-43 Some of the other possible linkage sequences (i.e., head-to-head, and tail-to-tail) may have formed, but are not resolved in these separations. We have not, at this stage, determined whether it would be possible to separate n-mers that have different linkage sequences. Last, we point out that these n-mers are in their fully oxidized state (i.e., imine nitrogens) and that no other n-mers were

CONCLUSIONS The results presented in this paper demonstrate the first successful coupling of EMLC and ES-MS as well as several of the attributes of this on-line union. Importantly, this combination enhances the ability to detect and identify on-column generated electrolysis products. It also provides highly specific (m/z) lowlevel detection. We also established that EMLC can be used to both generate and separate electrolysis products, which can then be characterized by ES-MS. This use of EMLC circumvents the need to use a separate electrolysis cell and a conventional HPLC column in tandem for the same purpose. Under present conditions, the time from product generation to detection will vary from less than 1 min to as much as 10 min depending on both the location of product formation within the column and the retention of the product on the column. Experiments to further exploit and augment these demonstrated capabilities are underway. For example, EMLC has the ability to manipulate separation efficiencies without employing conventional gradient elution techniques. We are investigating this capability to facilitate detection via ES-MS in cases where analyte detection is detrimentally impacted by changing mobile-phase composition. Future work will also include the development of smaller columns for direct coupling with ES-MS (no flow splitting) and for use as preconcentration devices12 and the investigation of various conductive stationary-phase materials to expand the separation capability and adaptability of EMLC. ACKNOWLEDGMENT H.D. acknowledges support through an appointment to the Oak Ridge National Laboratory (ORNL) Postdoctoral Research Associates Program administered jointly by the Oak Ridge Institute for Science and Education and ORNL. ES-MS instrumentation was provided through a Cooperative Research and Development Agreement with Perkin-Elmer SCIEX Instruments (CRADA ORNL96-0458). The work was supported by the Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences, United States Department of Energy under Contract DE-AC05-96OR2464 with ORNL, managed by Lockheed Martin Energy Research Corp. H.T. acknowledges a postdoctoral fellowship through the Microanalytical Instrumentation Center of Iowa State University. The work was supported by the Microanalytical Instrumentation Center of Iowa State University. The Ames Laboratory is operated for the U.S. Department of Energy by Iowa State University under Contract W-7405-Eng-82.

Received for review December 21, 1999. Accepted March 29, 2000. AC991461+

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