Anal. Chem. 1980, 52,203-205
LITERATURE CITED Giddings, J. C. Sep. Sci. 1966, 1 , 123. Giddings, J. C. J. Chromatogr. 1976, 125,3. Giddings, J. C.;Myers, M. N.; Moellmer, J. F. J. Chromafogr. 1978, 149, 501. Giddings, J. C.;Yang, F. J. F.; Myers, M. N. Anal. Chem. 1974, 46, 1917. Giddings, J . C.; Smith, L. K.; Myers, M. N. Anal. Chem. 1976, 48, 1587. Giddings, J . C.; Yang. F. J. F.; Myers, M. N. Sep. Sci. 1975, IO, 133. Yang, F. J. F.; Myers, M. N.; Giddings, J. C. Anal. Chem. 1974, 46, 1924. Giddings, J . C.;Caldwell, K. D.; Moellmer, J. F.; Dickinson, T. H.; Myers, M. N.; Martin, M. Anal. Chem. 1979, 51,30. See, e.g., Fujita, H. "Foundation of Ultracentrifugal Analysis"; Wiley Interscience: New York, 1975.
203
(IO) Giddings, J. C.; Yoon. Y. H.; Caldwell, K. D.; Myers, M. N.; Hovingh, M. E. Sep. Sci. 1975, IO, 447. (11) Casassa, E. F.; Tagami, Y. Macromolecules 1969, 2 , 14.
Hiroshi Inagaki* Takeshi Tanaka Institute for Chemical Research Kyoto University Uji, Kyoto-Fu 611, Japan RECEIVED August 13, 1979. Accepted October 1, 1979.
AIDS FOR ANALYTICAL CHEMISTS Electrochemical Detector Flow Cell Based on a Rotating Disk Electrode for Continuous Flow Analysis and High Performance Liquid Chromatography of Catecholamines B. Oosterhuis, K. Brunt,' B. H. C. Westerink, and D. A. Doornbos Laboratory for Pharmaceutical and Analytical Chemistry, Research Groups Medicinal Chemistry and Optimization, State University of Groningen, Antonius Deusinglaan 2, 9713 A W Groningen, The Netherlands
Several different instrumental techniques of electrochemical origin have been applied in detectors for high performance liquid chromatography (HPLC). Recently a review paper with more than 70 references concerning electrochemical detection in liquid chromatography has been published ( 1 ) . Amperometric detectors have been especially successful and some designs are already commercially available. Flow cells for amperometric detectors based on different principles have been designed: the thin-layer cell (2),the wall jet cell ( 3 ) ,and the partial electrolysis cell ( 4 ) . Not only the construction of flow cells but also the electronics in the POtentiostat have been subject to the process of development. Blank constructed the dual amperometric detector ( 5 ) while Brunt introduced the differential amperometric detector (6, 7). I n this paper we introduce a new type of flow cell based on a rotating disk electrode (RDE) for amperometric detection, which can be used in combination with continuous flow analysis as well as with HPLC. Wang and Ariel (8) have adapted the RDE for a flow-through cell for anodic stripping voltammetry. But to the best of our knowledge the application of a RDE as detector principle for HPLC and continuous flow analysis has not been described yet.
THEORY The response of an amperometric detector is dependent on the mass transport of the electroactive component from the bulk solution in the flow cell to the electrode, assuming that the reaction rate a t the electrode surface is infinitely fast compared with the rate of mass transfer. According to Fick's law, the rate of mass transfer depends on the concentration gradient of the electroactive components from the bulk solution in the flow cell to the electrode surface. This means that the thickness of the diffusion layer at the electrode surface is a very important parameter concerning the detector response. A decrease in the thickness of the diffusion layer results in an increase in the concentration gradient and subsequently in an increase in the detector response. T h e thickness of the diffusion layer depends mainly on the flow cell geometry and on the flow rate in the detector flow cell. The influence of the flow rate on the thickness of the 0003-2700/80/0352-0203$01 .OO/O
diffusion layer in a thin-layer cell is subject t o some controversy in the literature. Some authors find a square root dependence ( 7 ) ,and others find a cubic root dependence (9). In the wall jet cell, a more complex relationship exists (10). However, the response of both types of flow cells depends on the flow rate. This indicates that fluctuations in the flow rate (due to the pump) cause fluctuations in the detector response and sensitivity. In the design of the presently described flow cell, the first goal was to minimize the thickness of the diffusion layer and the second was to make the thickness of the diffusion layer independent of the flow rate. In order to fulfill these goals, a rotating disk electrode (RDE) has been used as a working electrode. Using a RDE, the thickness of the diffusion layer is mainly determined by the rotation speed of the electrode.
EXPERIMENTAL Flow Cell Design. The working electrode compartment of the flow cell is in principle a wall jet construction with a rotating electrode (Figure 1). Via the inlet the eluent enters the flow cell and impinges normally on the RDE. The solution leaves the working electrode compartment by streaming upward between the RDE and the wall of the vessel, through channel (a),diameter 2 mm, to compartment (A) in which the reference and auxiliary electrode are located. The working electrode is constructed from a Kel-F tube (b), diameter 6 mm, and a brass rod (c). The outer diameter of the lower part of the RDE (d) is 8.5 mm and fits in the working electrode compartment of the flow cell, diameter 9.0 mm, leaving enough space between the RDE and the wall of the vessel for the eluent to stream upward. The hole (e), diameter 6 mm, in the electrode is filled with carbon paste. The carbon paste surface acts as the working electrode. A t the top of the electrode, a mercury contact (0 has been constructed to connect the electrode with the potentiostat. The electrode is mounted in the holder for rotating electrodes, constructed according to Coenegracht (11). This holder is placed with the tapered end in the conical part of the working electrode compartment of the flow cell. The height of the RDE is variable (g).
Three pulleys with different diameters have been constructed at the top of the RDE holder in order to rotate the R D E at different rotation speeds. The pulleys are driven via an elastic C 1979 American Chemical Society
204
ANALYTICAL CHEMISTRY, VOL. 52, NO. 1, JANUARY 1980 deaeratina
1 2 0
waste
buffer
0.32 Y
L
holder
r,
.,
1
Figure 2. Flow diagram of the continuous flow analysis system. Flow rates are given in mL min-'
10
a
i inlet
Figure 1. Flow cell with the rotating disk electrode mounted inI the holder. For details see the text
string by an electromotor. The pulleys and the string are grounded by a sliding contact. The flow cell is in principle the small cone under the carbon paste electrode. The cone has been made deliberately in order to prevent the rotating carbon paste electrode from damage at the bottom of the cell. The effective dead volume of the flow cell is much smaller than the volume of the cone (35 pL) and does not contribute to peak broadening in HPLC (manuscript in preparation). Apparatus. In the continuous flow analysis system, a multichannel Cenco pump was used with plastic tubing and standard fittings (Technicon). A simple potentiostat of Bioanalytical Systems, type LC-2A, has been used in combination with the described flow cell. The rotation speed of the RDE was in all experiments 20 rps and the potential of the working electrode was 650 mV vs. SCE. The liquid chromatograph was constructed from individual parts and consisted of a Spectra Physics pump, model 740, a Valco sample injection valve with a 20-pL sample loop and a Nucleosil R P 18, 10 pm, reversed phase column of 15-cm length and 4.6-mm i.d. Chemicals. All solutions have been made with doubly glass distilled water. The chemicals used were of analytical reagent grade (pro analysi, E. Merck, Darmstadt) and used without further purification. The carbon paste was homemade and consisted of 35% by weight of Dow Corning high vacuum silicone grease and 65% by weight of graphite powder (UCP-1-M, Ultra Carbon, Bay City, Mich.). The surface of the carbon paste electrode was superficially polished on a piece of paper before use. Dopamine (Serva, Heidelberg), noradrenaline (Fluka, Buchs) and 3,4-dihydroxyphenylaceticacid (Fluka, Buchs) have been used as test compounds. The buffer solution in the continuous flow analysis was composed of 2.49 g of citric acid (C6H807.2H20)and 19.51 g of disodium hydrogen phosphate (Na2HP04.2H20)per 100 mL at pH 7.
A methanol-McIlvaine buffer solution, pH 5.2 (1:20), was used as mobile phase for HPLC at a flow rate of 1.0 mL min-'.
RESULTS AND DISCUSSION Continuous Flow Analysis. Figure 2 shows the manifold used to test the detector flow cell in a continuous flow system. Dopamine was chosen as test compound. In order t o obtain a homogenous mixture of the sample and the concentrated buffer solution, a double mixing coil (DMC) has to be used. T h e buffer was mixed with the sample first to obtain a constant p H value in the solution in the flow cell-the potential of the oxidation wave of dopamine is p H dependent (12)-and
rnin.
8
4
0
Figure 3. Typical recordings of dopamine standards. The numbers indicate the concentration of dopamine in the standard solutions in ng mL-'
second to serve as supporting electrolyte. T h e sample time and wash time were both 40 s and the washing compartment was filled with doubly glass distilled water. Typical recordings of freshly prepared dopamine standard solutions of concentration of 2.5,5.0 and 10.0 ng mL-' are given in Figure 3. A good linear standard curve could be obtained with continuous flow analysis of dopamine standard solutions. Calculation of this standard curve for the concentration range of 1-10 ng mL-' dopamine (12 measurements) by means of the linear least-squares method results in Equation 1 in which nA represents the detector response in nanoamperes and c the concentration dopamine in ng mL-'. T h e squared correlation coefficient r2 = 0.9988.
nA = 0.95 c
+ 0.24
According to Equation 1 the sensitivity of the detector is 0.95 nA ng-' mL. But, as indicated in the flow diagram (Figure 2) the sample is diluted. This means that the real sensitivity of the detector is about 1.25 nA ng-' mL. T h e noise in the base line (Figure 3) is very low (about 0.1 nA). This indicates that the detection limit of the dopamine is in the range of a few tenths of a nanogram. These results are in good agreement with the results obtained by Strohl and Curran (13) who used flow injection analysis with reticulated vitreous carbon flowthrough electrodes for the analysis of L-dopa. High Performance Liquid Chromatography. The flow cell has also to be tested in combination with HPLC using a test mixture of dopamine, noradrenaline, and 3,4-dihydroxyphenylacetic acid. The operation of a detector for HPLC must not significantly alter the separation achieved by the column by mixing the eluent in the flow cell. The influence of rotating the electrode in the flow cell on the performance of the detector system is shown in Figure 4. Rotating the electrode increases considerably the response of the detector while no change in the noise level is observed. Also no peak broadening due to the rotation of the electrode is noticed. Just like in the continuous flow
Anal. Chem. 1980, 52, 205~207
The influence of the rotation speed, flow rate, cell dimensions, etc. on the detector response is currently under investigation. Preliminary experiments show deviations with the ideal behavior of the RDE in which the limiting current is proportional to the square root of the angular velocity. These deviations are probably due to wall effects in the limited volume of the flow cell.
20 r . p s
na da
205
I
ACKNOWLEDGMENT T h e authors thank P. M. J. Coenegracht for his valuable discussions concerning the theory and practice of rotating disk electrodes. The authors are also indebted to J. F. C. Nienhuis of the instrumental workshop of our laboratory (supervisor A. Oosterhoff) for constructing the electrochemical flow cell.
0 r.p.s. I
jnnA
LITERATURE CITED
a
4 MIN.
0
8
4
0
MIN.
Chromatograms of a test mixture composed of 2.5 ng noradrenaline (na),2.5 ng dopamine (da), and 2.5 ng 3,4-dihydroxyphenylacetic acid (dopac),without (0 rps) and with (20 rps) rotation of the electrode Figure 4.
analysis, a linear relationship exists between the peak height in nanoamperes and the sample concentration. When trapped air bubbles are to be removed or when repacking of the electrode is needed, the flow cell can be dismantled within some seconds by lifting the RDE holder.
(1) Brunt, K. fharm. Weekbl. 1978, 113, 689-698. (2) Kissinaer, P. T.: Refshauae. C.: Dreilina, R.; Adams, R. N. Anal. Len. 1973,-6, 465-477. (3) Fleet, B.; Little, C. J. J . Chromatogr. Sci. 1974, 12, 747-752. (4) Bollet, C.; Oliva, P.; Caude, M. J . Chromatogr. 1978, 149, 625-645. (5) Blank, C. L. J . Chromatogr. 1976, 117, 35-46. (6) Brunt, K.; Bruins, C. H. P. J . Chromatogr. 1978, 161, 310-314 (71 I , Brunt. K.: Bruins. C. H. P. J . Chromatoor. 1979. 172. 37-47. (8) Wang, J.: Ariel,-M. Anal. Chim. Ac& 1578, 99; 89-98. (9) Weber, S. G.; Purdy, W . C. Anal. Chim. Acta 1978, 100, 531-544. (10) Yamada, H.; Matsuda, H. J . Nectroanal. Chem. 1973, 44, 189-198. (11) Coenegracht, P. M. J. fharm. Weekbl. 1972, 107, 769-782. (12) Sternson, A. W.; McCreery, R.; Feinberg. B.; Adams, R. N. J . flectroanal. Chem. 1973, 46, 313-321. (13) Strohl, A. N.; Curran, D. J. Anal. Chem. 1979, 51. 1045-1049. ~~
RECEIVED for review June 18,1979. Accepted September 10, 1979.
Microcomputer Interfaced Spectrophotometer for Kinetic Studies C. S. Nichols, J. N. Demas," and T. H. Cromartie" Department of Chemistry, University of Virginia, Charlottesville, Virginia 2290 1
Detailed kinetic studies routinely require collection and reduction of great amounts of data which is generally a manpower-intensive process. With the advent of the minicomputer, computerized data acquisition and reduction has reduced these manpower requirements. Initial interfaces between recording spectrophotometers and computers were inconvenient because of the need for direct connections to the internal circuits of the spectrophotometer ( 1 - 4 ) . These interfaces were not widely adopted because of expense, complexity, and difficulty of fabrication. Modern spectrometers are generally equipped with T T L compatible digital communication lines which can greatly simplify interfacing. For example, for signal averaging, an infrared spectrometer has been interfaced to a minicomputer using its standard spectrophotometer interface plug ( 5 ) . We describe here a n inexpensive microcomputer interface for the common Beckman 25 absorption instrument and a Processor Technology SOL-20 microcomputer. The same interface would, however, work with any microcomputer having one 8-bit input and one 8-bit output port. This system permits data acquisition a t any rate from 1 s per point to >6500 s per point. Data acquisition may be terminated a t any time by the operator, and the acquired data may then be reduced by the method of initial rate for kinetics done under zeroth-order conditions or by a linear least-squares fit of the semilogarithmic data plots for first-order kinetics. Data reduction is complete within seconds of termination of the 0003-2700/80/0352-0205$01.OO/O
kinetics run. The interface does not interfere with operation of the spectrophotometer recorder so that graphs may be obtained concomitant with computer-controlled data acquisition.
INSTRUMENTATION AND SOFTWARE The Beckman 25 is one of the many absorption instruments currently available which has a digital voltmeter display of the absorbance; the binary coded decimal (BCD) data for the digital readout is brought out to a rear connector along with several control lines. These TTL compatible output and control lines were intended for interfacing with the manufacturer's data logging printer but, with a minimal interface, they can be used for transferring data into a dedicated 8-bit microcomputer for logging and later reduction. The format and pin basing for the Beckman interface plug is shown in Figure 1 (6). The output of the analog-to-digital converter (ADC) is four digits. The range on the absorbance scale is 0.ooOto 2.8 absorbance units, but the spectrophotometer is only rated t o 2.000 absorbance units. Underranges are indicated by a 9 in the left-most digit. The decimal point is always fixed with three digits to its right. A concentration range mode is also available, but this cannot be used conveniently with the interface described here. The remaining spectrophotometer pins have the following functions. When the Convert Input (CI) pin is high, the spectrophotometer is free running; but when this input goes low, the current conversion is stopped and no new ones ace initiated. After CI has been 0 for at least 150 ws, a 0 1 transition on the CI
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1979 American Chemical Society
