Anal. Chem. 1995,67, 1339-1345
On=LineElectrochemical Detection for Supercritical Fluid Chromatography Shawn F. Dressman and Adrian C. Michael* Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260
A self-containedelectrochemical cell consisting of a working and a quasi-referenceelectrode coated with a thin film of a conductive polymer was used as a postcolumn detectorfor supercriticalfluid chromatography(SFC-EC). Cyclic voltammetry was used to obtain chromatogramsof ferrocene, anthracene, p-benzoquinone, and hydroquinone after their elution from a CIS packed column with unmodified, acetonitrile-modified,or methanol-modified C02. In unmodified C02, the quantitative performance of the SFC-EC detection of ferrocene was similar to that obtained with a downstream FID with respect to detection limit, response linearity, and peak shape. The baseline signal of the EC detector remained &it during density gradient elution while a noticeable drift occurred in the baseline signal of the FID. Furthermore, whereas the FID was totally inoperable in a mobile phase containing -3% (v/v) of either acetonitrile or methanol, the EC detector functioned well in the modified fluids. Thus, the EC detector is compatible with both unmodified and modified C02 mobile phases. A valuable feature of these newly developed SFC detectors is their compatibilitywith a very large fraction of the range of separation conditions that are important to SFC. The goal of this study is to demonstrate that electrochemical (EC) detection will be a valuable new detection mode for supercritical fluid chromatography (SFC) systems that use COT based mobile phases. Achieving this goal is important because there is a tangible need for new SFC detectors. SFC is most often performed with COZbecause it possesses a variety of physical properties that make it an ideal mobile phase.' The major limitation of neat COZ,however, is that it is not a particularly effective eluant for polar compounds. The approach commonly adopted to promote the elution of polar compounds is to add a polar modifier to the mobile phase.' Unfortunately, modified mobile phases are incompatible with many available SFC detectors. In addition, while both conventional and capillary SFC are of interest, few available SFC detectors are well suited to both.2 Thus, the "truly ideal" detector for SFC, i.e., one that is highly sensitive, broadly applicable, compatible with unmodified and modified COz, and compatible with conventional and capillary columns, does not yet exist. The coupling of EC detectors to chemical separations that employ conductive mobile phases has been highly suc~essful.~-~ (1) Lee, M. L.; Markides, IC E. Science 1987,235, 1342-1347. (2) Janssen, H.; Cramers, C. A J. Chromatogr. 1990,505,19-35. (3) Cooper, B. R.; Jankowski, J. A; Leszczyszyn, D. J.; Wightman, R M.; Jorgenson, J. W. Anal. Chem. 1992,64, 691-694.
0003-2700/95/0367-1339$9.00/0 0 1995 American Chemical Society
The main focus has been on EC detectors for reversed phase liquid chromatography and capillary zone electrophoresis. One important strength of EC detectors is their low detection limits, which are competitive with those of the most sensitive detectors available. The.extremely low detection limits are, in part, a consequence of the compatibility of microelectrodeswith capillary columns? Furthermore, considerable benefit is derived from the selectivity of EC detectors. Their selectivity has led to the widespread application of EC detectors to samples of biological origin. Electrochemical detectors are not readily compatible with nonconductive mobile phases, such as those used in normal phase chromatography,gas chromatography,and SFC. Some attention has recently been paid to coupling EC detection to normal phase10-12and gas phase13 chromatography. Extending the benefits of EC detection to SFC would also be technically useful and important. Several recent reports on voltammetry in COTbased f l ~ i d s ' ~ - ~ ~ suggest that EC detection is well suited to the needs of SFC. Furthermore, the literature reveals that SFC is frequently applied to compounds that are electrochemically detectable. Early attempts at voltammetry in COZinvolved adding electrolyte to the fluid.18-20Despite the fact that voltammograms could be recorded in this way, it was eventually determined that the experiments did not actually involve dissolved electrolyte. Rather, conductivity was provided by a film of molten salt that formed on the (4) Huang, X.; Zare, R N.; Sloss, S.; Ewing, A. G. Anal. Chem. 1991,63, 189192. (5) Oates, M. D.; Jorgenson, J. W. Anal. Chem. 1989,61, 432-435. (6) Kennedy, R T.; Jorgenson, J. W. Anal. Chem. 1989,61,436-441. (7) Wallingford, R A; Ewing, A. G. Anal. Chem. 1988,60, 258-263. (8) Knecht, L. A; Guthrie, E. J.; Jorgenson, J. W. Anal. Chem. 1 9 8 4 , 5 6 4 7 9 482. (9) Ewing, A. G.; Mesaros, J. M.; Gavin, P. F. Anal. Chem. 1994,66, 527A537.4 (10) T i t , R J.; Bury, P. C.; Finnin, B. C.; Reed, B. L.; Bond, A M.Anal. Chem. 1993,65, 3252-3257. (11) Gunasingham, H.; Tay, B. T.; Ang, K. P. Anal. Chem. 1987,59,262-266. (12) White, J. G.; St. Claire, R L,III; Jorgenson, J. W. Anal. Chem. 1986,58, 293-298. (13) Parcher, J. F.; Barbour, C. J.; Murray, R. W. Anal. Chem. 1989,61,584589. (14) Sullenberger, E. F.; Dressman, S. F.; Michael, A (2.1. Phys. Chem. 1994, 98,5347-5354. (15) Sullenberger, E. F.; Michael, A C. Anal. Chem. 1993,65, 3417-3423. (16) Sullenberger, E. F.; Michael, A. C. Anal. Chem. 1993,65, 2304-2310. (17) Dressman, S. F.; Garguilo, M. G.; Sullenberger, E. F.; Michael, A. C.J Am. Chem. SOC. 1993,115,7541-7542. (18) Niehaus, D. E.; Wightman, R M.; Flowers, P. A. Anal. Chem. 1991, 63, 1728-1732. (19) Di Maso, M.; Purdy, W. C.; McClintock, S. A.J. Chromatogr. 1990,519, 256-262. (20) Niehaus, D.; Philips, M.; Michael, A; Wightman, R M.J Phys. Chem. 1989, 93, 6232-6236. (21) Michael, A. C.; Wightman, R M. Anal. Chem. 1989,61, 2193-2200. (22) Michael, A. C.; Wightman, R M. Anal. Chem. 1989,61,270-272.
Analytical Chemistry, Vol. 67, No. 8, April 15, 1995 1339
electrode.20 The formation of the film was revealed by spectroscopic data which showed a virtual absence of dissolved electrolyte in the fluid, and by visual inspection of the film itself. These observations were important because they implied that EC detection in COz might not require the addition of electrolyte to the mobile phase. Eliminating the need to add electrolyte to the mobile phase greatly simplifies the coupling of EC detectors to SFC. Since the solubility of electrolytes in C02 is low and since electrolytes are unlikely to dissociate in C02, they actually do little to promote the conductivity of the fluid. Additionally, concern about the impact of the electrolyte on the separation process is obviated, and knowledge of the phase behavior of electrolyte/COz mixtures is no longer needed. The latter point is important considering the availability of an accurate equation of state for pure C0223and the availability of extensive information on binary mixtures in C02?4325 Finally, it would certainly be diflicult to maintain the flow of electrolyte-laden C02 through an SFC system. Blockage of outlet restrictors due to precipitation of the electrolyte, for example, would undoubtedly hamper the routine use of SFC-EC. Several approaches to voltammetry in COz without added electrolyte have been devised.14-17.21,22 These approaches are based on self-contained electrochemical which consist 01 working and counter electrodes mounted side by side in a dual electrode probe and coated with a conductive film. The coated probe is bathed in the fluid to be analyzed, which in this work will be the effluent from a chromatographiccolumn. Components of the fluid are electrochemicallydetected after they permeate the film and reach the underlying electrodes. While the selfcontained electrochemical cells are effective, they do introduce some important factors that must be considered during the development of detectors for SFC. First, the partitioning of solutes between the fluid and the film must be considered, particularly since the partition coefficient will be a function of the elution conditions. Second, because the self-contained cells operate on a limited “ion budget,”27voltammetric rather than the simpler amperometric detection will be advantageous. Voltammetric detection decreases the fraction of time during which faradaic electrochemistry takes place and also allows for the reversal of current flow through the cell. Both of these factors facilitate the maintenance of electroneutrality in the self-contained cell. So far it has been shown that electrodes modified with a molten salt film can be employed for chromatographic detection in unmodified C0z.l8 Flow injection analysis with electrochemical detection has also been demonstratedin water-modified COZusing electrodes coated with ionomer films.21v22The requirement for water-modified fluid was a drawback because water is rarely employed as a modifier in SFC and it also imposes a limited potential window on the voltammetry. Recently, we showed that electrodes coated with films based on poly(ethy1ene oxide) (PEO) can be used to study the voltammetry of several analytes in stationary COz-based fluids both with and without polar modifiers.l4-I6 Now we show that these electrodes are also (23) International Thermodynamic Tables of the Fluid State, Carbon Dioxide; Angus, S., Armstrong, B., deRueck, K. M., Eds.; Pergammon Press: New York, 1976; Vol. 3. (24) Roth, M.J. Phys. Chem. 1991,95, 8-9. (25) Chrastil, J. J. Phys. Chem. 1982,86, 3016-3021. (26) Reed, R. A; Geng, L.; Murray, R. W.; J. Electroanal. Chem. 1986,208, 185-193. (27) Jemigan, J. C.; Chidsey, C. E. D.; Murray, R W.J. Am. Chem. SOC.1985, 107,2824-2826.
1340 Analytical Chemistry, Vol. 67, No. 8, April 15, 1995
4 electrode
/
Pt d‘sk
I\
fused silica Pt tube
IJ to FID
Figure 1. Schematic of the EC detector. The stainless steel body of the electrode was fitted with a stainless steel ferrule for reproducible placement of the electrode in the cavity of the tee fitting. The column was connected to the electrode compartment by a short length of stainless steel tubing. The FID was connected to the electrode compartment by a fused silica restrictor. Not drawn to scale.
extremely effective as detectors for SFC-EC. The operation of an EC detector in both modified and unmodified COZ without added electrolyte has not been achieved before. EXPERIMENTAL SECTION
Reagents. SFC-grade COZ with a He headspace (Scott Specialty Gas, Plumsteadville, PA), H2 (Valley Welding, Evans City, PA), and zero-grade compressed air (Valley Welding) were used as received. Poly(ethy1ene oxide) (MW = 600 000; Polysciences, Inc., Warrington, PA), anthracene (Aldrich Chemical Co., Milwaukee, WI), acetonitrile (Mallinckrodt, Paris, KY), methanol (Fisher Scientific,Fair Lawn, NJ) ,p-benzoquinone (Eastman Fine Chemicals, Rochester, NY) , dichloromethane (Mallinckrodt), hydroquinone (Aldrich), and lithium triflate (Aldrich) were used as received. Ferrocene (Aldrich) was purified by sublimation. Tris(2,2’-bipyridyl)ruthenium(II) hexafluorophosphate(Ru(bpy)3(PF& was prepared as described before.16 SupercriticalFluid Chromatography. All experimentswere performed on a supercritical fluid chromatographysystem (Model MPS225, Suprex, Inc., Pittsburgh, PA) equipped with a syringe pump, sample injection valve, column oven, and FID. Injections were made onto a Nucleosil c18 packed column (100 mm x 1 mm, 5pm particle diameter) using a 1-pL sample volume with dichloromethaneor acetonitrile as the injection solvent. Unmodified C02 was used as the mobile phase in most experiments described below. When acetonitrile- or methanol-modified fluid was used, 6 mL of modifier was added directly to the syringe pump, which has a total volume of 250 mL. The pump was partially filled with COz, and the pump piston was moved up and down several times to promote mixing of the modifier with the C02. Finally, the pump cylinder was filled with C02 and pressurized to either 100 or 120 atm, such that the resultant mobile phase initially contained -3% modifier by volume. Electrode Design and Fabrication. The two-electrode cell design is shown in Figure 1. A 10-pm-diameter Pt wire (Goodfellow, Malvern, PA) working electrode was sealed into the lumen of a fused silica capillary (i.d. = 250 pm, Supelco, Bellafonte, PA) using 14%(w/w) m-phenylenediamine (Sigma Chemical Co.) in Epon 828 epoxy (Miller-Stephenson Co., Danbury, CT). The
capillary was sealed with the same epoxy into the bore of a Pt tube (0.d. = 600pm, i.d. = 500 pm, Goodfellow), which was used as a quasi-reference electrode (Pt-QRE). Finally, the Pt tube was sealed with epoxy into a 1/16 in. 0.d. glasslined stainless steel tube such that the Pt tube extended -2 mm from the end of the steel tubing. The tip of the assembly was polished on a Nylon pad using 15and 1-pm diamond polish (Bioanalytical Systems, West Lafayette, IN) to produce a miniature ringdisk electrode. Between experiments, the electrode assembly was polished with a 0.3 pm alumina slurry on glass and then sonicated in ultrapure water. Then, the electrode was momentarily dipped into a solution of 9:l (v/v) MeCN/MeOH, which contained 90 mg/mL poly(ethylene oxide), 20 mg/mL lithium m a t e (UCF3SO3), and 4.3 mg/mL Ru(bpy)~(pF~)~, and dried under NZfor 90 min. Elec trodes prepared in this manner will be referred to henceforth as Ru/PEO-Li-coated electrodes. These films typically last for several hours (Le., multiple injections) under SFC conditions. The polymer-coated electrode assembly was mounted into a Swagelok low dead volume stainless steel tee fitting (Crawford Fitting Co., Solon, OH) which formed the detection compartment Figure 1). The internal cavity of the tee fitting was drilled out to 0.0292 in. (0.742 mm) i.d. to accommodate the Pt tube. The placement of the electrode assembly in the detector compartment was fixed by compressing a stainless steel ferrule onto the body of the electrode. The active tip of the electrode assembly was positioned 0.04 in. upstream of the side arm of the tee fitting. An ohmmeter was used to cofirm that the Pt tube was electrically isolated from the body of the tee fitting. The inlet to the compartment was connected to the column outlet by a short length of stainless steel tubing. The outlet of the compartment (the side arm of the tee fitting) was connected to a downstream FID via a tapered fused silica restrictor (Suprex). This arrangement permitted simultaneous collection of EC and FID chromatograms. Voltammetric and Flame Ionization Detection. For electrochemical measurements, the Ru/PEO-Licoated electrode was connected to an E1400 potentiostat (Ensman Instrumentation, Bloomington, IN). The potentiostat was interfaced to a personal computer with a LabMaster board (Scienti6c Solutions, Solon, OH) and controlled with a software package developed in-house that is available through Ensman Instrumentation. Cyclic voltammetry was performed at a scan rate of 25 V/s at intervals ranging from 1 to 4 s. The range of potentials scanned was adjusted for each analyte, as described below. Chromatograms were calculated by averaging the current values recorded during individual voltammograms over a softwareselectable range of applied potential. Background-subtractedvoltammograms were calculated by taking the difference between the average of several voltammograms recorded during an analyte peak and the average of several baseline voltammograms. The signal from the FID was monitored with both a strip chart recorder and with the personal computer via an analog-todigital converter. The detector was operated at 350 "C with air and Hz flow rates of 600 and 100 mWmin, respectively. Occasionally, the FID was cleaned with repeated dichloromethane injections at fluid conditions of 100 "C and 400 atm. RESULTS AND DISCUSSION SFC-EC with Unmoaed COz. The goal of this work is to
establish high-quality electrochemical detection in SFC, without resorting to dissolved electrolytes, in both neat COZand in COZ containing polar modifiers. The establishment of EC detection
A - EC detector
-
B FID
0
100
200
300
400
500
time (s) Flgure 2. (A) SFC-EC chromatogram of 2pg of ferrocene injected in dichloromethane and eluted with unmodified COn at a pressure of 100 atm and an oven temperature of 50 "C.The inset is a background-subtractedvoltammogram from the same injection. (B) The chromatogram obtained during the same injection with a downstream FID.
in neat COZhas been the most challenging aspect of this work, since neat COz does not provide the necessary solvation of the ions involved in electrochemicalsystems. Previously, we reported that Ru/PEO-Li films provide sufficient conductivity for the purposes of voltammetry with microelectrodes when exposed to unmodified C0z.l6 The conductivity of the film appears to originate from a plasticizing effect of C02 on the PEO backbone, which we attribute to the complementary Lewis acidity of COZ and basicity of the ether oxygens of PEO. The bulky Ru complex is added to the film to prevent formation of crystalline domains in the PEO, thereby enhancing the ability of COZto plasticize the film.'6 Although the Ru complex is electroactive, its role in electrochemical detection appears to be limited to promoting conductivity of the film in COZ. Figure 2 shows the results of an SFC experiment in which 2 pg of ferrocene was injected onto the column in dichloromethane and eluted with COZat a constant pressure of 100 atm (measured at the pump head) and at a constant oven temperature of 50 "C. The EC chromatogram in Figure 2A shows that a single peak was observed at 386 s, which, by injection of a blank, was confirmed to be due to ferrocene. The chromatogram was generated by recording cyclic voltammograms every 2 s over a potential range of -300 to +lo00 mV vs the Pt-QRE. During individual cyclic voltammograms, data points were digitized at nominal intervals of 4 mV on the applied potential. The data points in Figure 2A were obtained by calculating the average of the 50 current values recorded between 400 and 600mV on the positive going potential sweep of each voltammogram. The inset of Figure 2A shows a so-called background-subtractedcyclic voltammogram extracted from the data collected during the elution of ferrocene. The voltammogram was generated by taking the difference between an ensemble average of the 10 voltammograms centered Analytical Chemistfy, Vol. 67, No. 8, April 15, 1995
1341
-
A EC detector 6.53min
3oo -0
Table 1. Comparison of the Properties of the Ferrocene Chromatographic Peak Obtalned by Electrochemical and Flame Ionization Detection.
3.30min cI
2.23min
mode of detection*
width retention at halfpeak timeC(s) heightc (s) asymmd
EC detector 383 i 3 FID (with EC detector) 383 f 2
100 atm 110 atm 120 atm
B - FID
Figure 3. Chromatograms obtained with (A) an EC detector and (B) an FID for 2 p g of ferrocene injected in dichloromethane and eluted with unmodified COn at 100, 110, and 120 atm and a constant oven temperature of 50 "C.
calibration linearitye
30 f 0.4 1.9 f 0.1 0.9969 f 0.0009 29 i 0.7 1.7 f 0.2 0.9965 f 0.0001
FID (no EC detector) 457 f 2 33 i 1.9 1.5 f 0.1 0.9994 FID (with EC detector) 410 i 17 33 1.1 1.9 f 0.2
*
Chromatograms were obtained with a pure COz mobile phase at a constant pressure of 100 atm and a constant oven temperature of 50 "C. Comparisonsbetween the data in the first two rows and the last two rows are unwarranted because of changing column conditions. FID (no EC detector): the column outlet was connected directly to the FID via a fused silica restrictor. FID (with EC detector): the column outlet was connected to the EC detector compartment via stainless steel tubing and the outlet of the EC detector compartment was connected to the FID via a fused silica restrictor. Means from five replicate injections, except for retention time data in the first two rows, which are the means from three replicate injections. The ratio of the tailing and leading half-widths measured at 10%of the peak height. average of two Mean of at least three replicate injections. "e
correlation coefficientsfrom calibration curves obtained by measuring the peak height from five injections of 20-1000 ng of ferrocene in 1 p L of dichloromethane, except for the FID (no EC detector) value, which was obtained in a single five-injection experiment.
at 386 s and an ensemble average of the 10 voltammograms just before the start of the peak. The result, the voltammogram of width at half-height, peak asymmetry, and linearity of response the analyte, resembles that obtained before for ferrocene in static for ferrocene obtained simultaneously with the EC detector and fluids at Ru/PEO-Lkoated microdisk electrodes. The separation the FID in unmodified COZat 50 "C and 100 atm. The bottom between the oxidation and reduction peak potentials in the two rows compare the performance of the FID with and without voltammogram is 240 mV, which is larger than the 59 mV peak the upstream EC detector in place. When the EC detector was absent, the column outlet was connected directly to the FID via separation expected. The peak separation indicates that the the fused silica restrictor. The data in the top two rows of Table voltammogram is somewhat distorted, most likely due to ohmic 1cannot be directly compared to the data in the bottom two rows resistance in the polymer film. Distortion of this magnitude can because the condition of the column changed substantially be considered minor for the intended purpose of these electrodes, between collection of the two data sets. namely, detection in SFC. The top two rows of Table 1 show that the elution profiles One of the detectors frequently employed for SFC is the FID. obtained simultaneously with the two detectors are essentially Figure 2B is the FID chromatogram recorded during the same identical. The limits of detection provided by each detector are ferrocene injection used to obtain the EC chromatogram in Figure also very similar, with both detectors exhibiting a detection limit 2A. The major difference in the two chromatograms is the of -20 ng of ferrocene injected in dichloromethane. The bottom appearance of a large solvent front in the FID chromatogram two rows of Table 1 show that the ferrocene retention time which is absent in the EC chromatogram. The solvent front obtained with the FID was several seconds shorter when the EC observed at the FID is over 2 orders of magnitude larger than detector was on-line. This was simply a consequence of differthe ferrocene peak, even though a relatively large quantity of ences between the flow restriction that occurred with and without ferrocene (2 pg) was injected. Figure 3 exhibits chromatograms the electrochemical detector in place. Apart from the change in of ferrocene showing that the solvent front can interfere with the retention time, the elution profile at the FID is otherwise little quantitation of early-eluting peaks. Ferrocene (2 pg) was eluted affected by the presence of the upstream electrochemical detector. at COZ pressures of 100, 110, and 120 atm, respectively, with the These comparisons between the EC detector and the FID show column oven at 50 "C. As the mobile phase pressure was that the EC detector contributes negligibly to band broadening. increased, the ferrocene retention time decreased. At an elution In particular, this shows that the polymer film used to provide pressure of 120 atm, the ferrocene peak obtained with the FID conductivity between the electrodes of the EC detector neither was a poorly resolved shoulder superimposed on the solvent front. produces a degradation of the temporal response of the EC In contrast, the ferrocene peak obtained with the EC detector was detector itself nor affects the capacity factors obtained with the superimposed on a completely flat baseline. As the elution packed column. Both these points show that the thickness and pressure increased, however, the amplitude of the ferrocene peak the volume of the Ru/PEO-Li film are too small to affect the obtained with the EC detector decreased. The retention time and separation efficiency. the peak amplitude at the EC detector decreased for the same The potential window that is accessible with these PEO-based reason: as the elution pressure increased, the tendency of films in COZ is sdciently large that both oxidizable and reducible ferrocene to partition into the chromatographic stationary phase compounds can be detected. Figure 4 shows the EC chromatoas well as the polymer film of the EC detector decreased. This gram and background-subtracted voltammogram obtained followexplanation is consistent with the previously reported dependence of the sensitivity of polymer-coated electrodes on fluid d e n ~ i t y . ~ ~ J ~ing the injection of 1pg of p-benzoquinone in dichloromethane. pBenzoquinone was eluted with a constant COZpressure of 110 The top two rows of Table 1compare the retention time, peak 1342
Analytical Chemistry, Vol. 67, No. 8, April 15, 1995
A - EC detector
anthracene
1.'5-0.65V
1.4-1.6V
I
B - FID
, 0
I
1
I
I
I
100 200 300 400 500
time (s) Figure 4. Chromatogram obtained with an EC detector for 1 ,ug of pbenzoquinone injected in dichloromethane and eluted with unmodified COn at a pressure of 110 atm and an oven temperature of 50 OC. The inset is a background-subtracted voltammogram of p benzoquinone.
120 atm I
atm and an oven temperature of 50 "C. Cyclic voltammograms were recorded over a potential range of 0 to -1.2 V vs Pt-QRE. Current values were averaged from -0.7 to -0.91 V to generate the chromatogram. The voltammogram is consistent with data obtained previously for pbenzoquinone in static COz using a Ru/ PEO-Iicoated electrochemicalcell. The voltammogram contains only a single irreversible reduction wave due to the combined effects of both ion pairing between the p-benzoquinone reduction products and Li+ in the film and carboxylation of the pbenzoquinone reduction product.I4 Figures 2A and 3 are the first SFGEC chromatograms obtained in unmoditied COz with a polymercoated microelectrode. Previous EC detection in a COZ flow system with polymercoated electrodes required the use of water-modified fluid.21~22 Here, the requirement for m o d ~ e r shas been eliminated by identifying a polymer tilm that is sufficiently conductive for voltammetrywithout modifiers. The performance of the Ru/PEO-Licoated electrode under the flow conditions of these experiments is not significantly different from that reported before in a static fluid.I6 The chromatographic peaks reported in Figures 2A and 3, as well as those described below, are typical of packedcolumn SFC. They are characteristic of rather moderate chromatographic efficiency and show considerable peak tailing. Table 1 clearly shows, however, that these nonideal features of the chromatographic peaks are not a consequence of the EC detector. Optimization of the chromatographic separation would undoubtedly improve both the efficiency and the detection limits obtainable with this system. In this work, however, we have mainly been concerned with the EC detector itself and have not paid much attention to optimizing the chromatography. The largest possible gains in efficiency and detection limits demand the coupling of EC detectors to capillary SFC. SFC-ECDetection During Density-GradientElution with Unmodified C 0 2 . Figure 5 shows the EC and FID chromatograms obtained following the injection of a standard solution containing 1 pg each of ferrocene and anthracene in dichloromethane. The oven temperature was held constant at 50 "C. The elution was started at a constant pressure of 100 atm. At 6 min
0
1
I
200 400 time (s)
1
600
Figure 5. Chromatograms obtained simultaneously with (A) the EC detector and (B) the FID for 1 ,ug of both ferrocene and anthracene injected in dichloromethane and eluted with unmodified Con. A density gradient was applied as shown in the figure, and the oven temperature was maintained at 50 "C.
after the injection, the pump pressure was ramped from 100 to 120 atm at 20 atm/min. The pressure ramp was employed to elute anthracene, which is considerably less volatile than ferrocene, within a reasonable time frame. Cyclic voltammetry was performed over a potential range from 0 to 2 V vs R-QRE.The EC chromatogram was generated by averaging the currents from 0.5 to 0.65 V on the positivegoing sweep for the detection of ferrocene and from 1.4 to 1.6 V for the detection of anthracene. Again, the background-subtracted voltammograms of ferrocene and anthracene closely resemble those recorded before in static f l u i d ~ . ~ ~ J ~ Even the mild density gradient used in this experiment produced a noticeable baseline shift in the chromatogram obtained with the FID. On the other hand, no such baseline shift is evident in the chromatogram obtained with the EC detector. Thus, the data presented so far show that the EC detector is significantly less affected by both injection solvents (vide supra) and density gradients than the FID. The peak height for ferrocene obtained with the EC detector is -6 times smaller than the anthracene peak (Figure 5) because (1) the oxidation of anthracene is nominally a twoelectron process while the oxidation of ferrocene is a oneelectron process and (2) anthracene has a greater tendency to partition into the PEO-based film than does ferr0~ene.l~ It is interesting to note the correlation between the peak height obtained with the EC detector and the chromatcgraphic retention times of the eluted compound. In both Figure 3 and Figure 5, the EC detector delivers a larger peak height for compounds with longer retention times. This trend confirms that the sensitivity of the EC detector is dependent, at least in part, on the partition coefficient of the analyte, as was first suggested by previous work in static fluids.l4-l6The results Analytical Chemistry, Vol. 67, No. 8, April 15, 1995
1343
0
100 200 300 400 500
time (s) 0
100
200
300
400
500
time (s) Figure 6. (A) Chromatogram obtained with the EC detector for 1 pug of ferrocene injected in dichloromethane and eluted with CO2 modified with 3% acetonitrile by volume at a pressure of 100 atm and an oven temperature of 50 "C. (B) and (C) Chromatograms obtained with the EC detector and the FID, respectively,for 1 pg each of ferrocene and anthracene injected in dichloromethane and eluted with Cog modified with 3% acetonitrile by volume at a pressure of 120 atrn and an oven temperature of 50 "C. The break in the baseline in (B) at 200 s is due to a change in the potentials over which currents were averaged.
in Figures 3 and 5 clearly show that partitioning between COz and the chromatographic stationary phase correlates with partitioning between COZand the PEO-based films on the electrodes. This correlation has some beneficial consequences. First, since fluid density in the detection compartment is generally lower than on the column, the fluid density in the detector will generally favor partitioning of sample components into the PEO-based film.Thus, under these conditions it is unlikely that sample components will fail to partition into the electrochemical detector. Second, the EC detector tends to exhibit greater sensitivity toward later-eluting components, which offsets the decreasing chromatographic efficiency that accompanies longer retention times. SFC-ECwith Modified Con. Figure 6 shows chromatograms obtained with the EC detector (Figure 64B) and with the FID (Figure 6C) using a COz mobile phase modified with -3% (v/v) acetonitrile. The chromatogram in Figure 6A was obtained with the EC detector after the injection of 1 pg of ferrocene in dichloromethaneat a pressure of 100 atm and an oven temperature of 50 "C. Voltammograms were obtained every 3 s over a potential range from 0 to 1.2 V, and the chromatogram was calculated by averaging the current from 0.55 to 0.75 V of each voltammogram. The chromatogram is very similar to that obtained in unmodified CO2 at the same temperature and pressure (Figure 2A). Under these conditions, the use of a modified mobile phase has no noticeable effect on the performance of the EC detector. Thus, the operation of an EC detector in both modifed and unmodified COz without the addition of a dissolved electrolyte has been demonstrated for the fist time. Following the generation of the chromatogram in Figure 6 4 the conditions of the experiment were adjusted to permit the generation of the chromatogram in Figure 6B following the 1344 Analytical Chemistty, Vol. 67, No. 8, April 15, 1995
Flgure 7. Chromatogram obtained with the EC detector for 10 ng of hydroquinone injected in acetonitrile and eluted with CO2 modified with 3% methanol by volume at a pressure of 150 atm and an oven temperature of 60 "C.
injection of a mixture of ferrocene and anthracene (1 pg each). The elution pressure was increased to 120 atm, and the range of the voltammetric potential sweep was extended to 1.8V. The EC chromatogram in Figure 6B was generated by averaging the currents from 1.1 to 1.3V for the f i s t 200 s of the chromatogram and from 1.5 to 1.7 V for the final 160 s of the chromatogram. The EC chromatogram contains well-defined peaks for both sample components, but the overall performance of the EC detector is slightly poorer under these conditions. First, the baseline in Figure 6B slopes more than before. Inspection of individual voltammograms near the beginning and the end of the chromatogram shows that the sloping baseline is due to instability of the Ru(bpy)3(PF& salt in the PEO film. The extended potential range of the voltammetry occasionally causes this instability. Therefore, the use of the Ru complex to promote the conductivity of the PEO film,while adequate for many chromatographic conditions, does have some limitations. Presently,we are examining other polymer formulations to alleviate this limitation. In addition to the sloping baseline, the ferrocene peak in Figure 6B is noticeably less symmetrical than the peak in Figure 6A. Whether the asymmetry is due to the retention process or the EC detector itself is unclear at this point. Nevertheless, despite the poorer performance of the EC detector under this particular set of conditions, the EC detector continues to operate under conditions with which the FID is incompatible. The chromatogram in Figure 6C was obtained simultaneously with Figure 6B using the downstream FID. Figure 6C clearly illustrates the incompatibility of the FID with modifed mobile phases. The only peak obtained in the FID chromatogram is that of the injection solvent. The amplitude and the noise level of the background signal generated by the FID in the presence of the modifer are so large that the ferrocene and anthracene peaks are totally obscured, even with a large amount of material injected. Figure 6 demonstrates that the EC detector is compatible with modified mobile phases. As a result, we expect SFC-EC to be suitable for compounds which are so polar that their elution from a packed column requires a modified mobile phase. We have used hydroquinone to test this expectation. Hydroquinone is
sufticiently polar that we were unable to elute it from the C18 column with unmodified COZat pressures up to 300 atm. Figure 7, however, shows the EC chromatogram obtained following the injection of 0.01 pg of hydroquinone in acetonitrile. The elution was carried out with a mobile phase containing -3% methanol by volume at a pressure of 150 atm and an oven temperature of 60 "C. A chromatogram could not be obtained with the FID because the FID is incompatible with methanol-mod~edCOz. The EC chromatogram was calculated by averaging the current from 1.2 to 1.8 V on the forward sweep of individual cyclic voltammograms which were collected every 2 s. The peak centered at 234 s corresponds to the elution of hydroquinone. The peak is broad and exhibits signifcant tailing (peak asymmetry,BIA = 3.0),but we have not yet attempted to optimize the chromatographic conditions for hydroquinone. Nonetheless, the sensitivity of the electrochemicaldetector toward hydroquinone is greater than for any compound we have studied so far. In fact, the amount of hydroquinone injected in Figure 7 (10 ng) is even less than the
limit of detection we obtained for ferrocene (20 ng, vide supra). This observation is consistent with the premise that the sensitivity of the EC detector is dependent on the partitioning of solutes between the fluid and the film. Hydroquinone, being polar, partitions extensively into the film resulting in highly sensitive detection. This has a very important consequence, in that the EC detector is especially well suited to those compounds which are most difficult to detect in SFC, Le., those which require modified mobile phases. ACKNOWLEWMENT This work was supported by the Central Research and Development Fund of the University of Pittsburgh. Received for review June 3, 1994. Accepted January 29, 1995.@ AC9405821 Abstract published in Advance ACS Abstracts, March 1, 1995.
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