262
Anal. Chem. 1907, 5 9 , 262-266
(10) Rubinstein, I . Anal. Chem. 1984, 56, 1135-1137. (11) Schulthess, P.; Shijo, Y.; Pham, H. V.; Pretsch, D.; Ammann, D.; Simon, W. Anal. Chim. Acta 1981, 131, 111-116. (12) Oyama, N.; Hirabayashi, K.; Ohsaka, T . BUN. Chem. SOC.Jpn. 1986, 59, 2071-2080. (13) Guilbault, G. G.; Durst, R. A.; Frant, M. S.;Freiser, H.; Hansen, E. H.;
Light, T. S.;Pungor, E.; Rechnitz, G.; Rice, N. M.; Rohm, T. J.; Simon, W.; Thomas, J . D. R . Pure Appl. Ch8m. 1976, 48, 127-131.
RECEIVED for review July 9, 1986. Accepted'September 24, 1986.
Computer-Assisted Rapid-Scan Cyclic Staircase Voltammetry in Normal-Phase High-Performance Liquid Chromatography H a r i Gunasingham,* B. T. Tay, and K. P. Ang Department of Chemistry, National University of Singapore, Kent Ridge, Singapore 0511
The use of a large-volm waU+t detector for cy& stalrcase voltammetry fokwlng normal-phase HPLC separation Is described. Bath reductive and oxldakre ranges are investigated. The detection H m b of the technlque (of the order of tens of plcomoles) are comparable to other scanning voltammetry techniques foilowlng reversed-phase llqukl chromatography. The technlque b tested for some oxy and nltro derivatives of polyaromatlc hydrocarbons, catecholamines, and oestrogen sterolds.
In order to improve selectivity, several workers have combined high-performance liquid chromatography (HPLC) or flow injection analysis (FIA) with scanning and transient electrochemical techniques ( 1 , 2 ) . Under steady-state conditions where the electrode reaction is limited by convective diffusion and the scan rate is slow, the technique is referred to as hydrodynamic voltammetry (HDV). For fast scan rates (>0.5 V s-l), convective diffusion ceases to be the limiting factor; under such conditions, if the potential scan is done in the forward and reversed directions, traditional cyclic voltammetric (CV) conditions prevail (2). The voltammogram then takes on the characteristic peak profile caused by the decline in the mass transfer rate to the electrode surface as the working potential passes the formal potential Eo' because of the depletion effect. In conjunction with HPLC (and FIA), HDV or CV (depending on the compromise selected between scan rate and flow rate) can afford additional qualitative information about the electroactive species being monitored. As the solute band passes through the electrochemical detector, the potential is scanned rapidly across the range in which the solute is electrochemically active. The detector response may then be represented as a three-dimensional surface of the current against potential and time ( I ) . A number of HPLC-rapid-scan voltammetry (RSV) studies have been published (I,3-8). Caudill et al. (3) have described the use of a normal pulse potential waveform which was applied to a channel type electrochemical detector. The working electrode material was made of glassy carbon or carbon fiber. The hydrodynamic voltammograms so obtained afforded the identification of various neurotransmitters which had been separated by reversed-phase HPLC. However, detection limits were found to be higher than those in amperometric detection. More recent applications of HPLC-RSV, have also been applied to reversed-phase HPLC. Ploegmakers et al. (6) reported on a HPLC-RSV technique for the study of Eptoside and Teniposide (anticancer agents). Also, White e t al. (7) have described a scanning microvoltammetric detector for opentubular HPLC. 0003-2700/87/0359-0262$01 S O / O
The application of cyclic voltammetry to FIA was first reported by Thorgeson et al., where a HMDE was used as the working electrode (2). The study, however, was not carried out at high sensitivites. Wang and Dewald (9) described the application of differential pulse voltammetry to FIA which maintained high sensitivity. This was achieved with a large sample volume (200 pL). Fast scan ac voltammetry at a mercury electrode has been described by Trojanek and de Jong (10) for the better resolution of overlapping chromatographic peaks. Sammuelsson et al. have reported on the application of square-wave RSV in HPLC, again using a mercury electrode as the indicator electrode. The sensitivity of this technique was hampered by a high noise level caused by flow pulsations. Solid electrodes are generally preferred for electrochemical detection in flowing streams. The reasons are greater stability, ease of construction, and well-defined hydrodynamic characteristics. However, solid electrodes suffer from a number of limitations which are particularly evident in aqueous solutions. For example, in the case of glassy carbon electrodes, voltammograms can be severely distorted by high capacitance effects, by high residual current (especially at low pH) and by the adsorption of impurities. It is for these reasons that Kissinger reported his reservations about the value of RSV a t solid electrodes (11). In this paper, the use of computer-assisted rapid-scanning cyclic staircase voltammetry (RSCSV) a t the large-volume wall-jet (WJ) detector following normal-phase HPLC is described. Cyclic staircase voltammetry has been described as an attractive alternative to linear sweep voltammetry (12). This is because the technique has reduced charging current interferences and greater analytical flexibility (as changes in the potential waveform can be modified by altering the software). The general advantages of using amperometric detection in nonaqueous eluents have been discussed in a previous paper (13). These advantages can be extended to HPLC-RSCSV; in particular, greater quantitative information with regard to intermediates may be obtained (14). Also, there are a number of compounds which are sparingly soluble in aqueous media, in which case, HPLC-RSV following reversed-phase separation would not be applicable.
EXPERIMENTAL SECTION Electrochemical System. The large-volume W J detector is identical with the one used in a previous study (15). The WJ detector employs a glassy carbon working electrode (5.5 mm diameter) and a platinum discounter electrode. An Ag-Ag' ion electrode was used as the reference. Potential control was afforded by a PARC Model 174A (Princeton Applied Research Corp. Princeton, NJ) potentiostat. Control System. In this work rapid-scan cyclic staircase voltammetry (RSCSV)plots were obtained under the control of a HP 9826 (Hewlett-Packard) 16-bit computer. The computer 0 1987 American Chemical Society
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FRG) was used for the normal-phase elutions. Chemicals and Reagents. All chemicals and solvents used were of analytical grade or better. The nonaqueous media and the normal-phase eluent comprised hexane-ethanol-glacial acetic acid mixtures, with 0.1 M tetrabutylammonium fluoroborate serving as the supporting electrolyte. The test compounds included p-benzoquinone, 9,10-phenanthrenequinone, 9,lOanthraquinone,1-nitroanthraquinone(Tokyo Kasei Kogyo, Tokyo, Japan), oesterone, oestradiol, oestriol, dopamine, noradrenaline, and adrenaline (Sigma Chemicals, St. Louis, MO).
PAR
114A
iill, J WJD
Flgure 1. Computer control system.
was interfaced to the Model 174 A potentiostat through a HP 6942 A multiprogrammer. The multiprogrammer actually serves as an intelligent controller for the transfer of data and control information between the computer and the actual analog-digital (A/D) and digital-analog (D/A) interface. The multiprogrammer, which has ita own local bus, transfers data and control information via the HPIB bus (the Hewlett-Packard implementation of the GPIB parallel bus, having a maximum bandwidth of kilobyte/ second). The interface subsystem included a 12-bitA/D converter card (HP69751A),a buffered D/A system comprising a 4K buffer memory card (HP68790A) and a l2.bit D/A converter card (HP69720A),and a timer card (HP69736A) which served as the external real-time clock. The conversion time of the A/D converter is 25 fis. Figure 1 shows a schematic diagram of the experimental setup including the computer and interface. The staircase waveform was programmed to increase in steps of 5 mV, whereas the time interval between steps was made variable. For example, if the scan rate was 2.5 V s-', then, the step duration would be 2 ms. The corresponding current would be sampled 1 ms before the end of the step, thus allowing time for the capacitance current (or charging current) to decay. For scan rates of 1.25 and 0.625 V s-l, the step duration would be 3.2 ms and 6.4 ms, respectively, with the corresponding current sampled 1.6 ms and 3.2 ms before the end of the step. The RSCSV plots were stored on a HP9134 Winchester hard disk and later retrieved for background subtraction. Plots were dumped from the HP9826 graphics screen to a HP 2714 G graphics thermal printer. The software for the implementation of the reductive and oxidative RSCSV and background subtraction are not included in this paper but are available from the authors. RsCSVs were obtained by cycling the potential over a 2-V range at various scan rates as the compound elutes through the WJ detector. With p-benzoquinone as a model compound, the effects of various hydrodynamic and chromatographic parameters were examined. HPLC System, The HPLC analyses were carried out with a Perkin-Elmer Series 4 (Perkin-Elmer, Norwalk, CT) microprocessor-controlled q-rnary solvent delivery system. A Merck LiChrosorb Si60 lO-pm, 0.46 X 25 cm column (Merck, Darmstadt,
R E S U L T S AND DISCUSSION Background Subtraction. One of the practical problems of using solid electrodes in aqueous solutions is that, because of the complex surface redox behavior, background scans are not very reproducible; background subtraction, thus, may not be effective as a means of enhancing signal-to-noise characteristics of voltammograms. In contrast, we have found that background currents obtained in nonaqueous media are more reproducible. The improved background performance is presumably due to the reduced activity of carbon-oxygen functionalities on the glassy-carbon surface in nonaqueous media. Background subtraction was performed by subtracting the forward and reverse scans of the sample voltammogram with the forward and reverse scans of the background current, respectively. The background subtracted scans are then combined to give the familiar CV. The computer enables synchronization of the CV scans with the elution peak and CVs could be obtained at any point on the peak profile. Figure 2 shows the effect of background subtraction on RSCSVs (scanned between +0.5 V and -1.5 V) obtained at the WJ detector during the elution of p-benzoquinone. As can be seen, the quality of the voltammograms has been substantially improved. Inlet-Electrode Separation. In the case of the WJ detector, the inlet-electrode separation is a contributing factor to current efficiency. It has been suggested that the nozzle of the jet should be positioned well clear of the hydrodynamic boundary layer (16). If the nozzle is placed too close to the hydrodynamic boundary layer, then there would be a loss in current efficiency. Outside the boundary layer the current efficiency reaches a constant value and the only upper limit to the inlet-electrode separation is that the free jet should not break up. It has been in fact shown that the jet remains intact for separations up to 10 mm (16). The peak current of the RSCSV was found to increase with increasing inlet-electrode separations, reaching limiting value between 5 and 7 mm. The inlet-electrode separation for the large-volume WJ detector was fixed to be a t 5 mm. Effect of Flow Rate. The relationship between the current response and the flow rate has been defined for the WJ detector (16). For a solute band that passes through the detector, the current is given by the instantaneous solute concentration near the electrode surface. In the case of the W J detector, the peak current is related to volume flow rate to the power of 314 (17). Cathodic RSCSVs. One of the advantages of doing CVs in nonaqueous media is the extended cathodic potential range. In general, with adequate degassing, a cathodic potential limit beyond -2.0 V vs. Ag-Ag+ electrode may be obtained. Figure 3 shows the background subtracted RSCSVs of 9,lOphenanthrenequinone, g,lO-anthraquinone, and 1-nitroanthraquinone. The potential range of these reductive RSCSVs was between +0.5 V and -1.5 V. As can be seen, the voltammograms of the compounds are distinct and different. Effect of Potential Scan Rate. In general, the peak shapes in RSCSV depend critically on the potential scan rate (7),especially if the iR drop is uncompensated. Figures 4 and 5 show the background-subtracted RSCSVs for 9,lO-
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 2, JANUARY 1987
I *05
I
I
-1 5
-0 5 E I V vs Ag/Ag*
Flgure 3. RSCSV of (a) 9.6 pg of 9,lO-phenanthrenequinone,(b) 2 pg of 9,10-anthraquinone, and (c) 1.2 pg of 1-nitroanthraquinone. Eluent was 60/40 ethanoVhexane, 0.1 M tetrabutylammonium fluoroborate, and 0.1 M acetic acM. Scan rate was 1.25 V s and flow rate was 1.0 mL min-I. *05
-Os
E I V v s Ag/A{
-lS
la)
Figure 2. Effect of background subtraction: (a) RSCSV of background; (b) RSCSV of 19.2 pg of p-benzoquinone; (c) background subtracted RSCSV of b. Eluent was 60/40 ethanoi/hexane, 0.1 M tetrabutylammonium fiuoroborate, and 0.1 M acetic acid. Flow rate was 1.0 mL min-‘ and scan rate was 1.25 V s-’.
Table I. Voltammetric Peak Currents ( I , ) and Peak Potential ( E , ) Values of the Various Test Compounds“
amt, Zp, compound
pg
pA
70 RSD
E,,
mV
RSD (&mV)
Cathodic RSCSV p-benzoquinone 9,lO-phenanthrenequinone
9,lO-anthraquinone I-nitroanthraquinone peak Ia peak IIa peak IIIa
19.2 72.4 9.6 30.5 2.0 15.2 1.2 2.3 15.8 15.4
-983
4.3 2.5 2.0
-927 -1192
4 5 3
2.6 2.5 2.6
-496 -1077 -1253
2 2 2
3.2 2.6 2.4 2.8 2.4 2.7
+278 +326 +374 +751 +698 +707
Anodic RSCSV dopamine noradrenaline adrenaline oesterone oestradiol oestriol
2.5 15.5 5.4 13.5 5.8 14.2 16.0 9.5 16.0 7.1 16.0 5.3
5 4
5 6 6 6
“The cathodic RSCSVs were obtained at a scan rate of 1.25 V s-l in 60/40 ethanol/hexane, 0.1 M tetrabutylammonium fluoroborate, and 0.1 M acetic acid. The anodic RSCSVs were obtained at a scan rate of 0.625 V s-l in 40/60 ethanol/hexane, 0.1 M tetrabutylammonium fluoroborate, and 0.1 M acetic acid. Both cathodic and anodic RSCSVs were carried out at a flow rate of 1.0 mL/min. Peaks Ia, IIa, and IIIa are described in text. phenanthrenequinone and 1-nitroanthraquinone a t various scan rates. A t lower scan rates, t h e RSCSVs were more
I
+o 5
I
I
-0 5 EIV
K
Ag/W
-1 5
Effect of scan rate on RSCSV of 9.6 pg of 9.10phenanthrenequinoneat (a) 2.5 V s-’;(b) 1.25 V s-’,and (c) 0.625 V s-’. Eluent was 60/40ethanoVhexane, 0.1 M tetrabutylammonlum fluoroborate, and 0.1 M acetic acid. Flow rate was 1.0 mL min-‘. Flgure 4.
well-defined and the redox coupled more reversible. However, the oxidative peaks of the reduced products were also smaller. This i s because, at lower scan rates, the reduced products have more time t o be swept away before t h e oxidation potential
ANALYTICAL CHEMISTRY, VOL. 59, NO. 2, JANUARY 1987
I
f
1
1
0
1
2
3
Time
265
(rnin.)
Io
--
to5
-35
Ib
IIo
-1 5
E I V vs Ag/Ag*
Figure 5. Effect of scan rate on RSCSV of 1.2 pg of l-nitroanthraquinone at (a) 2.5 V s-’, (b) 1.25 V s-’,and (c) 0.625 V s-‘. Eluent was 60/40 ethanollhexane, 0.1 M tetrgbutyiammonium fluoroborate, and 0.1 M acetic acid. Flow rate was 1.0 mL/min.
Ib
Ub
(ivl IIb
(a1
+d 5
P I
I +
I
0.5
+
I
1.0
-1 5
E N vs Ag/Aq*
110P
- 0.5
I
I
-05
1.5
I
+1.0
0.0 E IV vs Ag/A
80% 22,23-dihydroav-
ermectin B,, (H2Bla, 1) and
