Amperometric and Differential Pulse Voltammetric Detection in High Performance Liquid Chromatography Dennis G. Swartzfager Central Research and Development Department, E. I. do POnt de Nemours and Company, Wilmington, Del. 19898
An electrochemical detector for high performance liquid chromatography (HPLC) is described and characterized. The detection limit, sensitivlty, precision, and linearity of response of the detector are reportedfor Operation in the amperometrlc mode. The flowrate dependence of the sensitivity is reported for the amperometrlc, pulse, and differential pulse modes of detection. The enhanced selectlvlty which can be obtained when employing the differential pulse mode is illustrated for a test solution of p-aminophenol, 3,4-dihydroxyphenylalanine, and sulfanilamide.
The applicability of electrochemical detectors to high pressure liquid chromatography (HPLC) (1-4) is limited by the fact that the species of interest must be electroactive and the mobile phase must be electrically conductive. The latter problem can be circumvented by post-column addition of a suitable solvent of high dielectric constant containing a supporting electrolyte. The former restriction can be relaxed by prior or post-column derivatization or in-stream reactive coupling ( 4 ) .The inherent selectivity of the electrochemical detectors can be advantageous in many applications. This selectivity, when coupled with the high sensitivity of the detector greatly enhances the utility of the technique for applications such as the trace analysis of known species in a complex matrix. The response (i.e., the measured current) of any electrochemical detector is dependent on the rate of mass transfer to the electrode surface. The convective diffusional mass transfer rate is dependent on the hydrodynamic condition created in the flowstream. The general relationship between the limiting current ( i l ) , the bulk concentration of the electroactive species ( C O ) , and the volume flowrate (Fr) is given by Equation 1 (1,5,6). il =
KnFCOFr"
(1)
where n is the number of electrons, F is the faraday, and k is a constant dependent on the kinematic viscosity of the fluid, the diffusion coefficient of the electroactive species, the geometry and area of the electrode, etc. The exact value of the exponent a , of the volume flow rate (Fr) is determined by the specific nature of the hydrodynamic conditions. For a stationary electrode in a flowing stream, a will generally be between and y~ (6). The fluctuation in the measured current Ail associated with a flow rate change AFr is given by Equation 2.
(i.e., a 2% increase in the flow rate will cause a (200%change in the measured current). The flow rate dependence of both the measured current and the flow noise (Ail) places some restrictions on the pumping system employed. It is also somewhat inconvenient to have the detector response dependent on a chromatographic variable but this should not be a great problem. A recent study (7) on the use of time-dependent applied
potentials (Le., pulse and differential pulse techniques) for a convective mass transport system has shown that convection has little effect on the resulting currents provided that the Nernst diffusion layer is small in comparison to the convective shear layer. Therefore, application of pulse techniques may remove or sharply decrease the flow rate dependence of the measured current from an electrochemical detector. Furthermore, the use of the differential pulse technique offers the possibility of tuning the detector to a specific species, which may considerably enhance selectivity. The present paper describes the design and characteristics of a sensitive and selective electrochemicaldetector for HPLC. The detection limit, sensitivity, precision, and linearity of response of the detector are reported for operation in the amperometric mode. The further enhancement of the selectivity obtained by operating the detector in a differential pulse mode is also illustrated. The flow rate dependence of the detector is reported for constant potential, pulse, and differential pulse modes of operation.
EXPERIMENTAL Liquid Chromatograph. A Du Pont Model 840 Liquid Chromatograph was employed throughout these studies. This instrument uses a high pressure gas source to propel the mobile phase and thus provides a pulse-free fiow, thereby eliminating the need for a damping device. The only modification to this instrument was replacement of the standard reservoir with one allowing agitated vacuum degassing. The electrochemical cell was attached to the chromatograph a t the outlet of the uv detector. A Chromatronix HPSV valve fitted with a 25-p1 sample loop was used for injecting the sample. A 1-m column of a strong cation exchanger (Zipax SCX, chromatographic packing) was used with a 0.05 M HzS04 aqueous mobile phase. Prior to these experiments the chromatograph and column were passivated by passing copious amounts of dilute (3 M) nitric acid through the system. This procedure lowered the detector background current by more than an order of magnitude, to 5 nA or less a t a potential of 1.0 V SCE. Electrochemical Equipment. A Princeton Applied Research Model 174 Polarographic Analyzer was used throughout these experiments. Modifications to the timing circuitry of this instrument were made to increase its flexibility when operated in either the pulse or differential pulse mode. These modifications permitted: (1)the pulse time ( P T )to be varied from 40 to 850 ms in seven steps of 40 to 200 ms, (2) a threefold increase in the duration of the current sampling intervals, and (3) a twofold decrease of the tracking time constant in the sample and hold circuits. The IR compensation circuit which is available in the P.A.R. Model 174/50 AC Polarography Accessory was used whenever currents in excess of 1FA were encountered. Detector Cell. Shown in Figure 1 is a schematic of the general cell design. The cell block and electrode holder were machined from Delrin acetal resins. The spacer was cut from ca. 0.178-mm thick Teflon. The width of the flow-channel was approximately 6 mm. A %-in. platinum outlet tube served as the auxiliary electrode. The screw fit of the electrode holder permitted quick replacement of a carbon paste electrode. The uncompensated resistance of the cell was 5 kQ for 0.05 M HzS04 supporting electrolyte. The cell design is readily adaptable to many different working electrode materials, such as carbon paste, glassy carbon, platinum, or gold. Carbon paste electrodes which are easily formulated and have low residual currents were used here. The carbon paste was prepared by thoroughly mixing 6 ml of mineral oil (Nujol) with 10 g of graphite
ANALYTICAL CHEMISTRY, VOL. 48, NO. 14, DECEMBER 1976
2189
I J6
TIME
Figure 3. Amperometric chromatogram of 61 pg (0.56 pmol) of p aminophenol Figure 1. Cell schematic (1) lniet from chromatograph:(2)flow channel In
spacer, (3) l/8-ln. platlnum outlet tube and auxiliary electrode; (4) flow to reference electrode; (5)cavity for carbon paste electrode: (6) platinum wire contact: (7) O-ring seals
I
" ' 0
.PULSE 0
,
10 [Fr(rnl/rnin)]
CURRENTS
S T E A D Y STATE D C CURRENTS
,
15
"3
Flgure 4. Steady state (amperometric and pulse current) dependence
on
TIME Figure 2. Time dependence of the applied potential and resultant cur-
rents E, applied potential: iF, faradaic current: ic,capacitive current; ir = iF resultant current
+ ic, total
(Special Spectroscopic graphite powder, Grade SP-1,National Carbon Company, New York, N.Y.). Pulse and Differential Pulse Operation. When the detector is operated in an amperometric mode, the resultant current is proportional to concentration. In the pulse and differential pulse modes, the potential varies with time as shown in Figure 2 (upper trace). The difference between the pulse and differential pulse modes of operation is the amount of data collected and their subsequent display. In the pulse mode the current is measured during the time interval A (equal to n times 16.7 ms where n is an integer) a t the end of each pulse. In the differential pulse mode the current is measured during both the time intervals A and B and the differential current (Ai = i.t, - i ~ is) displayed. Pulse and differential pulse currents ( i ~ )will be composed of a faradaic (oxidative or reductive) current ( i ~ )and a capacitance (charging) current (ic), (lower three traces in Figure 2). Since the capacitance current is an unwanted component, it is desirable to work with long pulse durations (100-400 ms) where the fast (exponentially) decaying capacitance current is minimized with respect to the slower (-1/t1l2 at short times) decaying faradaic component. 2190
Fr'I3.
In practice the operation of the detector in a pulse mode offers little or no advantage over the amperometric mode. However, the flow rate dependence of the pulse and differential pulse currents should be quite similar (7) and under certain conditions the pulse currents can be treated as the result of a series of individual single potential step experiments which greatly simplifies interpretation of the results.
RESULTS AND DISCUSSION Detection Limit, Sensitivity, Precision, and Linearity of Response. Where applicable, amperometry is one of the most sensitive means of detection for HPLC. Shown in Figure 3 is an amperometric chromatogram of 61 pg (5.6 X l O - l 3 mol) of p-aminophenol. The detection limit for this compound is estimated to be about 5 pg with this relatively inefficient column. It should be noted that this result was obtained on a commercial (all metal) chromatograph but is comparable to previously reported values obtained on systems constructed from glass, Teflon, and Kel-F. The sensitivity (Le., peak current per nmol), precision, and linearity of response of the detector were studied by injecting 2 5 - ~ samples 1 of 3,4-dihydroxyphenylalaninea t 11different concentrations ranging from 5.14 X M to 1.02 X M (12.83-0.0256 nmol). The data taken from 30 injections over a period of nearly 7 h gives sensitivities of 0.1297 f 0.0009 (0.69%RSD)and 0.1286 f 0.0022 (1.71% RSD),respectively, for the highest (12.83-0.513 nmol) and lowest (0.385-0.0256 nmol) concentration ranges studied. The precision is excellent at high concentrations. The standard deviation (1.71%) at the
ANALYTICAL CHEMISTRY, VOL. 48, NO. 14, DECEMBER 1976
2
100 -o ,-o-a
00-
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t
i
n
20 r
lOnA
4 -
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z
,
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+0.5 c0.6 +0.7 +0.8 t l . l E (S.C.E 1
+1.2
+1.3
u
:t O
53
A E ; +5OmV MIN _)1
-5
1
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Flgure 5. Current-potential curves
Er
n
*
20nA
$ -
(0)paminophenol, (0)3,4-dihydroxyphenylalanine, and (A)sulfanilamide
5 4 *
E,: + l 10 AE=*50mV
-
20nA
TIME
E = t0.46
Figure 7. Differential pulse chromatograms, each optimized for the selective detection of one of the three components Upper trace: paminophenol, Middle: 3,4-dihydroxyphenylaianine, Lower: SUI-
fanilarnide E= b-
* 0.70
z
w a
a u 3
E = + 1.20
I \
Figure 6. Amperometric chromatograms, each optimized for the se-
lective detection of one of the three components Upper trace: paminophenol, Middle: 3,4-dihydroxyphenylalanine,Lower: SUIfanilarnide
lower level (-0.1 nmol or 20 ng) is nearly identical to that reported by Adams and co-workers ( 2 ) ,1.9%at the 15-ng level (dopamine). The linearity of response as measured by the constancy of the sensitivity is within the precision of the measurements. Flow Rate Studies. The flow rate dependence of the amperometric and pulse currents was studied by monitoring the respective currents for the oxidation of 3,4-dihydroxyphenylalanine at a carbon paste electrode. The detector was connected directly to a large reservoir containing the test solution M of 3,4-dihydroxyphenylalanine, 0.05 M H2S04) (9.90 X and provided gravity flow rates of up to 3 ml/min. The amperometric current was determined a t +0.90 V (SCE); the initial and final potentials in the pulse n o d e were +0.35 and 0.90 V, respectively. The pulse frequency was 0.20 pulsesls and sampling interval was 16.8 ms. This rather low pulse frequency allows the pulse current to be interpreted as the result of a single potential step experiment. The flow rate dependence of the amperometric current was determined by a least squares fit of the data which gave a value of a of 0.33. This value is identical to that obtained by Blaedel for a tubular platinum electrode (8).Figure 4 shows a plot of both the pulse and amperometric currents vs. Fr1I3. ‘The
number heading in the pulse current plots is the time (PT in ms) after the application of the pulse, when the measurements were made. The pulse currents are nearly independent of the flow rate a t low flow rates and/or at short pulse times. Under these conditions the relationship between the pulse current and the time is given by the Cottrell equation. A plot of i vs. t-1/2for the slowest (0.183 ml/min) flow rate was linear with a zero intercept while the same plot for the fastest flow rate (3.00 ml/min) deviated significantly from this behavior. These results are very similar to those reported by Osteryoung (7) for pulse currents a t a rotating disk electrode. The flow rate dependence of the differential pulse current was studied by repeated injection of 25-pl samples of 4.1 X M 3,4-dihydroxyphenylalanine onto the chromatographic column. The initial potential was 0.525 V (SCE);the pulse amplitude was 100 mV. The pulse width and frequency were 212 ms and 2.0 pulses per s. The sampling interval was 51.1 ms. The peak height (PA) from the electrochemical (EC) chromatogram was divided by the peak height (absorbance units) from the uv detector which was simultaneously monitored. This procedure corrects for the flow rate dependence of the column parameters. The corrected currents were found to be nearly independent of the flow rate, which is the expected result based on pulse current experiments. Small deviations (less than 6%) from this behavior were observed at low flow rates (
