- Email: [email protected]
ultramicroelectrode detector
115
J. Efecrroanal. Chem, 216 (1987) 115-126 Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands
ELECTROCHEMICAL DETECTION FOR LIQUID CHROMATOGRAPHY USING THE WALL-JET CELL/ULTRAMICROELEClXODE DETECTOR
S.B. KHOO, H. GUNASINGHAM, Department (Received
of Chemistry,
NOtioMI
K.P. ANG and B.T. TAY University of Singapore,
Kent Ridge, 0.511 (Singopore)
23rd May 1986)
ABSTRACT A carbon fibre array and a platinum thin ring ultramicroelectrode were constructed and used in conjunction with a large volume wall-jet cell as an amperometric detector in liquid chromatography. Electrode characterization studies were performed in 5.00 mM K,Fe(CN)s + 1 M KC1 aqueous solution. It was found that the wall-jet cell/ultramicroelectrode combination has much reduceJ flow rate dependence compared to the wall-jet cell/conventional electrode system. LC application in the absence of supporting electrolyte was found to be possible and have good sensitivity using the ultramicroelectrode. Detection limits were 100 pg and 10 ng for the platinum thin ring and the carbon fibre array electrodes respectively.
INTRODUCTION
Electrochemical detection of species under flow conditions is nowadays frequently used in areas such as pollution control and monitoring and liquid chromatographic detection. Tubular, planar and wall-jet cell/electrode configurations have been commonly used. The wall-jet system is increasingly gaining popularity because of its sensitivity and ease of use [1,2]. Recent developments in the area of very small electrodes (typically tens of microns to submicron sizes), known as microvoltammetric or ultramicroelectrodes, have introduced new domains to electrochemical studies [3-g]. These electrodes have high mass transfer rates due to hemispherical rather than linear diffusion under normal experimental conditions. Steady state, sigmoidal shaped voltammograms are obtained even under quiescent conditions. Currents passed are very small, usually of the order of picoamperes to nanoamperes. Consequently, problems with ohmic potential loss are reduced. Novel systems to which ultramicroelectrodes have been applied include electrochemistry of low temperature organic glass [6], electrochemistry at very high scan rates [5], and electrochemistry in the absence of supporting electrolyte [3,8,9].
116
The use of the wall-jet cell/ultramicroelectrode configuration as a detector in flow systems combines the sensitivity and ease of use of the wall-jet cell with the advantageous features of the ultramicroelectrode. The efficacy of the large volume wall-jet cell, using conventional electrodes under hydrodynamic conditions, have been demonstrated in previous studies [l-2,10-13]. Recent investigations have shown that it is possible to perform electrochemistry in the absence of supporting electrolyte using ultramicroelectrodes [3,8,9]. Also, due to the higher mass transfer rate, the current density is expected to be higher as compared to conventional-sized electrodes. These features are particularly relevant to electrochemical detection in liquid chromatography. The elimination of the need for supporting electrolyte means that normal-phase liquid chromatography using organic solvents as eluents is more practical with electrochemical detection. In many cases, this is not possible because of the absence of suitable supporting electrolyte for these solvents. In addition, the use of extra reagents always introduces new sources of impurities. These will increase the background noise levels. The use of supporting electrolyte may interfere with the detection in cases where specific adsorption of the electrolyte ions or ion pairing may occur. Further, the decomposition of the supporting electrolyte frequently limits the potential range available. In the absence of the electrolyte the potential window can be widened significantly [3]. With the higher current density, better sensitivity per unit area is expected for the ultramicroelectrode compared to conventional-sized electrodes. Since the ultramicroelectrode is a relatively recent innovation, there have not been many studies of it applied to liquid chromatographic detection. In one investigation, a channel-type electrochemical flow cell using a carbon microfibre array electrode was used as a detector [14]. One significant finding of this work was that the detector response has a very much reduced dependency on flow rate. This is important because decreased flow rate dependence of the response leads to reduced noise levels arising from flow rate fluctuations. To the best of our knowledge, only one previous study has been reported in the literature using the wall-jet cell/ultramicroelectrode combination as a chromatographic detector 1151. In this study, a large number of fibres (1200 fibres), in the form of a bundle, were embedded in epoxy. For such an electrode, it is difficult to relate the number of active fibres to the limiting current because the currents due to the individual fibres may not be independent of each bther. In the present study, the use of a carbon micro-fibre array electrode and a platinum thin ring electrode in conjunction with a large volume wall-jet cell as amperometric detector in liquid chromatography is investigated. EXPERIMENTAL
Chemicals Analar
grade potassium ferricyanide (Merck), potassium chloride (Fluka) and quinones ( p-benzoquinone, 9,10-phenanthrenequinone, 1-nitroanthraquinone, 2nitrofluorenone) (Tokyo Kasei Chemicals) were used without further purification.
117
Catecholamines (dopamine, noradrenaline, adrenaline, L-dopa) (Sigma Chemicals) were also used as received. All solutions were prepared fresh daily. Aqueous solutions were prepared using triply distilled water. For the flow rate dependence studies, a 5.00 mM potassium ferricyanide + 1.0 M potassium chloride aqueous solution was used. Solutions were deaerated with oxygen-free nitrogen. All experiments were carried out using a three-electrode potentiostatic system at a temperature of 25 f l°C. The counter electrode was a 5 mm diameter glassy carbon while the reference electrode was a Ag/AgCl, saturated KC1 electrode. Apparatus The PAR Model 273 potentiostat was employed for voltammetric experiments in still solution and also under hydrodynamic conditions. Voltammograms were recorded using a Graph& model WX 4422 recorder. The flow system was an Eyela microtube peristaltic pump (Model MP-3, Tokyo Rikakikai Co Ltd) connected by a plastic tubing to a glass bulb (approximately 50 ml capacity) half-filled with solution which acted as a pulse damper. The other end of the bulb was connected to the inlet of a wall-jet cell made of Teflon. The large volume wall-jet cell design has been described in full detail elsewhere [12]. The inlet diameter of the cell was 0.5 mm. In operation, the electrode was placed 4 mm away from the point of jet inlet to the cell body in order to avoid interference with the hydrodynamic boundary layer
1131.
For liquid chromatographic experiments, a Perkin-Elmer Series 4 chromatograph together with the Perkin Elmer model RlOO recorder were used. The chromatograph has a microprocessor controlled solvent delivery system with a built in pulse damper and solvent chamber for purging with helium. Samples were injected via a Rheodyne 7105s 6 ~1 injector valve (Rheodyne Corp., Cotati, CA) using a 10 ~1 syringe. For the separation of catecholamines the eluent was 0.1 M acetic acid in water which was also the solvent for preparing the catecholamine solutions. The quinones were dissolved in dichloromethane and the eluent used for their separation was 70% acetonitrile and 30% water. In both cases, the column used was a Perkin-Elmer C,, (0.26 X 25 cm) reversed-phase column. For the detection of catecholamines the flow rate was 1.0 ml min-’ and the working eIectrode potential was +0.80 V (vs. Ag/AgCl) while for the quinones the flow rate was 1.0 ml min-’ and the working electrode potential was -0.90 V (vs. Ag/AgCl). Ultramicroelectroa% construction The carbon microfibre array electrode was constructed in the following manner. A 3.0 cm length of glass tubing of external diameter 3.5 mm was coated on the external surface with a thin layer of epoxy. Individual carbon fibres of 8 pm diameter were then stretched lengthwise and parallel onto the epoxy-coated tubing, extending beyond the ends of the tubing. The fibres were placed about 1 mm apart to ensure that the current responses of the individual fibres were independent of each other [16]. The epoxy film was then allowed to air-dry. At the same time, by
118
the same procedure a layer of fibres was placed around a second piece of glass tubing of the same length but of larger diameter (internal diameter 4.5 mm, external diameter 6 mm). The smaller tube was then placed inside the larger one and both of them were put into another glass tubing of 6.5 mm internal diameter. The spaces in between the tubings at one end (working end) were then filled with epoxy. Electrical contact was made by embedding the carbon fibres extending from the other end in silver cement to which was also sealed a copper wire. The electrode, which contained a total of 33 fibres, was heated at 80°C for 3 h to cure the epoxy and the silver cement. In the case of the platinum thin ring electrode, a glass rod of diameter about 3.5 mm and length 6 cm was coated, by dipping, with a thin layer of metallo-organic platinum paint (liquid bright platinum, Engelhardt). The paint was allowed to air dry and then fired in a furnace at 200°C for 3 min and at 650°C for 15 min. The resultant platinum coated rod was then placed in a 5 cm length of glass tubing (internal diameter 4.5 mm, external diameter 6 mm), one end of which was previously sealed. The sealed end, with the rod inside, was slowly and evenly heated, with suction applied at the other end. As a result, the glass tubing collapsed onto the platinum coated rod for a length of about 1.5 cm to form a tight glass seal. External electrical contact was made by soldering a copper wire to the platinum coated rod.
Fig. 1. Electron micrograph of platinum thin ring electrode. Bar represents 1 pm.
119
To expose the carbon and the platinum ring, both electrodes were polished on a No. 600 abrasive paper with a little water, followed by No. 1200 and No. 1500 abrasive papers. Final polishing was done with a 0.05 pm alumina + water slurry on a polishing cloth. For the ring electrode the internal diameter of the ring was found to be 0.345 cm using a travelling microscope. The thickness of the platinum ring was determined using an electron microscope. Electron micrographs were obtained at three positions around the ring. The electron micrographs showed that the ring thickness was quite even and the average thickness was measured as 0.2 pm. Figure 1 shows an electron micrograph of the ring. RESULTS
AND DISCUSSION
Characterization
of electrodes
Figure 2 shows the cyclic voltammogram obtained for the reduction of the ferricyanide ion (5.00 mM + 1.0 M KC1 in water) at the carbon microfibre array electrode, using a scan rate of 20 mV s-l. A sigmoidal-shaped voltammogram was obtained with the limiting current measured as 156 nA. A value of 59 mV was obtained for E,,d - Es,., which is in agreement with the theoretical value of 56.4 mV for a one electron transfer, within experimental error. A plot of log[(i, - i)/i] yielded a straight line with El,* measured as +0.198 V (vs. Ag/AgCl, sat’d KCl). For a microfibre electrode, the steady state limiting current, for a single fibre [14], is given by i, = 4nFDcr
where c is the bulk concentration of the electroactive species (here 5.00 X low6 mol cme3), r the radius of the fibre (4 X 10m4 cm), the other terms having their usual
I
I
I
20 nA
E/V
Fig. 2. Cyclic voltammogram of 5.00 mM K,Fe(CN), +1 M KCl. Scan rate 20 mV s-l; array electrode. Reference Ag/AgCl (sat’d KCl). Counter electrode glassy carbon disk.
carbon fibre
120
I
I
I
I
I
I
I
f
1
.@5 +04 l03 +0*2r0.1 c-o-0.1-02-03
EIV
Fig. 3. Cyclic voltammogram of 5.00 m M K3Fe(CN), + 1 M KCl. Scan rate 20 mV s-l, ring electrode. Reference Ag/AgCl (sat’d KCI). Counter electrode glassy carbon disk.
platinum
thin
meaning. The diffusion coefficient for the ferricyanide ion has a value of 0.763 X lOa cm* s-l [17]. Thus, the limiting current for a single fibre is 5.88 nA. The total current of 156 nA for the array electrode therefore corresponded to 27 working fibres. The assumption that the currents are additive is valid because in fabrication, care was taken to ensure that the fibres were more than 6 diameters apart (here about 1 mm apart), so that there will be no diffusional cross-talk between the fibres [14]. The fact that there were 27 active fibres out of a total of 33 could be due to bad or broken electrical contact to the external lead in the process of fabrication. For the same solution and scan rate, the cyclic voltammogram at the platinum ring electrode is shown in Fig. 3. Again, a sigmoidal-shaped voltanunogram was obtained and the limiting current was measured as 960 nA. El,* was found to be +0.264 (vs. Ag/AgCl, sat’d KCl) and application of Tomes’s criterion gave a value of 74 mV for E,, - E3j4. This value is significantly higher than the theoretical value which could mean that, due to the thinness of the platinum ring, some kinetic control was observed. The standard heterogeneous rate constant for the ferricyanide/ferrocyanide couple is not very high and known to be dependent on experimental conditions (e.g. at a conventional-sired gold disk electrode k” has a value of 3.6 X 10e3 cm s-l in 0.1 M KC1 and 2.8 X lo-* cm s-l in 1.0 M KC1 [18]). Therefore, it is quite reasonable that, with the high mass transfer rate of the ultramicroelectrode, some kinetic effect could creep in. For the very thin ring electrode, the equation describing the steady state current has been given [19], by i,, = nEr2Dc(2ri
+ Ar)/ln{ 16[(2ri/r)
+
11}
where ri is the internal radius of the ring and Ar the thickness of the ring, all the other terms having their usual meaning. For the platinum thin ring electrode used here, ri is 0.17 cm and Ar is 0.2 X 10e4 cm. Using the above equation, i, works out to be 1000 nA, which compares well with the experimental value of 960 nA. Flow rate dependence
The logarithmic plots of limiting current against flow rate (Fig. 4) show that there
121
Log(i,/nA)
2.8
I
0.3
a2
I
I
0.1
OD
I
I
0.1 0.2 03 log (V/ml min-‘)
cl4
Fig. 4. (a) Plot of log(il/nA) vs. log( V/ml min-‘) for carbon fibre array electrode. (b) Plot of log(i,/nA) vs. log( V/ml min-‘) for platinum thin ring electrode. Solution for both is 5.00 mM K,Fe(CN), +l M KCl.
is some increase in the current as the fled ranged from 0.44 to 2.10 ml mm-‘. This r analytical liquid chromatography, which is The straight line plots gave a slope of 0.17 f for the platinum ring electrode was conventional-sized electrodes, the limiting c1 i = 1 38 ~FcD2/s~-sPV’/‘~r *
rate increases. The flow rate studied ge covers most typical applications in sually in the region of 1.0 ml min-‘. r the carbon array electrode while that .16. For the wall-jet cell using ent eqn. (1) is given by
112~314
where Y is the kinematic viscosity, a the di eter of the inlet nozzle, V the flow rate and R the radius of the electrode. All th other terms have their usual meaning. Therefore, the log plot i, vs. V should hav a slope of 0.75. This value has been verified experimentally [2]. The values obtai ed for the ultramicroelectrodes repreIm sent a significant decrease in flow rate dependence. Since one of the noise sources in flow systems arises from fluctuation in flow rate [14], the reduced flow rate dependence leads to a reduction in noise.
122
1
I
6
I
1
4
2
10 pA
-i
Time/
min
Fig. 5. Liquid chromatogram of a series of catecholamines using platinum thin ring as detector. Potential set at +0.8 V vs. Ag/AgCl (sat’d KCl). Eluent is 0.1 M acetic acid in water at 1.0 ml ICI-‘. (1) Solvent front; (2) noradrenaline; (3) adrenaline; (4) dopamine; (5) L-dopa.
Liquid chromatography
Four catecholamines (noradrenaline, adrenaline, dopamine and L-dopa) were studied using the constructed ultramicroelectrodes and also glassy carbon conventional-sized electrodes of 5.5 mm diameter. Figure 5 shows a typical chromatogram. Reproducibility of the peaks was found to be better than 5%. In Figs. 6a to 6d, linear plots were obtained for the log plots of electrode response (charge/area) vs. sample size (ng). On the average, the response per unit area for the ultramicroelectrodes was 1.25 log units higher than that for the macroelectrode. This represents about a 20 times increase in sensitivity. The expectation, as mentioned earlier, that the sensitivity for the ultramicroelectrode would be greater compared to microelectrode is confirmed. The increased sensitivity could thus be used to advantage to lower the detection limit. The normalized responses for the four catecholamines were about the same or lower for the carbon ultramicroelectrode than for the platinum ring electrode. The greater response of the ring results from its more uniform accessibility compared to the disk [20]. Based on the criterion that the detection limit is twice the noise level, the calculated detection limit for the carbon array electrode is 10 ng and that for the platinum ring electrode is 0.1 ng. (It should be mentioned that the system used here
123 log (
response/C
mm-‘)
log (response
2-
_’
/C
mm-21
3-
-1
-5
-E 9
-6
-E,-
-7
1
-2
log
log km&le
(response/C
-7
3
1
size/ ng)
log
mm-*)
log
(response/C
(sat-Zple
mmm2
size
/ng)
3
)
1
1
2
log
( sample
size/
Fig. 6. plots of lOg(resPOnWC nuC2) (c) doDamine and (d> L-dona. li1 Macrn
3
1
ng)
log
2 (sample
size / ng
3
1
versus log(sample size/ng) for (a) noradrenaline, (b) adrenaline, darcv
rarhnn
A=,+--L
< < __
.A:“-^.-_.
I::,
_-I--
P.-
124
T -I__ 2nA
1
I
I
6
4
2
I Time/min
Fig. 7. Liquid chromatogram of a series of quinones: (1) p-benzoquinone; (2) 9,10-phenanthrenequinone; (3) l-nitroanthraquinone; (4) 2-nitrofluorenone. Platinum thin ring electrode. Potential set at - 0.9 V vs. Ag/AgCl (sat’d KCl). Fluent 70% acetonitrile, 30% water at 1.0 ml mir-‘. No supporting electrolyte.
is far from ideal. For the PAR 174A potentiostat, the most sensitive current range is 20 nA full scale with an accuracy specification of 1.5%. This means that the lowest measurable current is 0.3 nA. However, with the size of the ultramicroelectrodes employed in this study and for very dilute solutions, currents of the order of picoamperes could be measured with a p&ammeter. Further, for small current measurements, the ultramicroelectrodes are susceptible to external noise and a Faradaic cage (which was not used in this work) could be employed to reduce the noise levels drastically. Further work will incorporate the improved features to lower the detection limit.) Figure 7 shows the chromatogram for a series of quinones (reversed phase 70% acetonitrile and 30% water eluent) using the thin platinum ring electrode. The corresponding log plots for the response (charge/area) versus sample size are shown in Fig. 8. As opposed to the earlier results (catecholamines), no supporting electrolyte was used. In this situation, the use of a macro electrode was not possible. It is therefore clear that the use of ultramicroelectrodes widen the applicability of
125
-1
log ( response/C
1
mm-*)
1
2 3 4
-3.
-2-
-4’
/
1
log
2 (sample
4 size/ng?
Fig. 8. Plots of log(response/C mme2) versus log(sample size/rig). (1) p-Fknzoquinone; (2) 9,10phenanthrenequinone; (3) 1-nitroanthraquinone; (4) 2-nitrofluorenone. Platinum thin ring electrode.
electrochemical detection in LC. Normal-phase LC at the moment is essentially confined to other forms of detection such as UV and fluorescence, since the supporting electrolyte requirement is a limitation on the type of solvent which can be used. However, with ultramicroelectrodes, normal-phase LC with electrochemical detection is now a possibility. CONCLUSION
It has been shown that the wall-jet/ultramicroelectrode detector is useful in LC applications. Ultramicroelectrodes have the advantage of much reduced flow rate dependence and increased sensitivity over conventional electrodes. Further the range of applicability is widened since no supporting electrolyte is required. Of the ultramicroelectrodes, a ring electrode is preferred over the microfibre array electrode because it has more uniform accessibility and a higher current is obtained. For example, to obtain the same current level as the ring electrode used here, an array electrode of 163 fibres is necessary. Due to the geometry requirements (tubular) of the wall-jet cell, an array electrode with this number of fibres will be more difficult to construct if the minimum 6 diameters separation condition is required. In addition the use of epoxy resin for holding and filling purposes in the case of the array give rise to higher background noise. AKNOWLEDGEMENTS
The authors wish to thank Dr. Chung Mui Fatt (Dept. of Physics, National University of Singapore) for advice and assistance in taking electron micrographs. Thanks are also due to Mrs. Phua Swee Wah and Miss Pang Teng Jar (Dept. of
126
Physics, National University of Singapore) for obtaining the electron micrographs. Madam Toh Soh Lian has been very helpful with her technical assistance. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
H. Gunasingham and F. Fleet, Anal. Chem., 55 (1983) 1409. H. Gunasingham, K.P. Ang and C.C. Ngo, Anal. Chem., 57 (1985) 505. J. Cassidy, S.B. Khoo, S. Pans and M. Fleischmann, J. Phys. Chem., 89 (1985) 3933. R.S. Robinson and R.L. McCreery, J. Electroanal. Chem., 182 (1985) 61. J.O. Howell and R.M. Wightman, Anal. Chem., 56 (1984) 524. A.M. Bond, M. Fkischma~ and J. Robinson, J. Electroanal. Chem., 180 (1984) 257. R.M. Wightman, Anal. Chem., 53 (1981) 1125A. A.M. Bond, M. Fleischmann and J. Robinson, J. Electroanal. Chem., 168 (1984) 299. A.M. Bond and PA. Lay, J. Electroanal. Chem., 199 (1986) 285. H. Gunasingham, B.T. Tay aud K.P. Ang, Anal. Chim. Acta, 176 (1985) 143. H. Gunasingham, K.P. Ang, CC. Ngo and P.C. Tbiak, J. Ekctroanal. Chem., 198 (1986) 27. H. Gunasingham, K.P. Ang, C.C. Ngo and P.C. Thiak, J. Electroanal. Chem., 186 (1985) 51. H. Gunasingham, B.T. Tay and K.P. Ang, Anal. Chem., 56 (1984) 2422. W.L. CaudiU, J.O. Howell and R.M. Wightman, Anal. Chem., 54 (1982) 2532. F. BelaI and J.L. Anderson, Analyst (London), 110 (1985) 1493. T. Guishi, D. Tokuda and H.J. Makuda, J. Electroanal. Chem., 101 (1979) 29. M. Von Stackelberg, M. Pilgram and W. Toome, Z. Electrochem., 57 (1953) 342. K.R. Wehmeyer, M.R. Deakin and R.M. Wightman, Anal. Chem., 57 (1985) 1913. J.S. Symanski and S. Bruckenstein, paper presented at the Electrochemical Society Meeting, Cincinnati, OH, May 1984. 20 A.M. Bond, M. Fleischmann, S.B. Khoo, S. Pans and J. Robinson, Paper presented at the Alectrochemical Society Meeting, Cincinnati, OH, May 1984.
J. Efecrroanal. Chem, 216 (1987) 115-126 Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands
ELECTROCHEMICAL DETECTION FOR LIQUID CHROMATOGRAPHY USING THE WALL-JET CELL/ULTRAMICROELEClXODE DETECTOR
S.B. KHOO, H. GUNASINGHAM, Department (Received
of Chemistry,
NOtioMI
K.P. ANG and B.T. TAY University of Singapore,
Kent Ridge, 0.511 (Singopore)
23rd May 1986)
ABSTRACT A carbon fibre array and a platinum thin ring ultramicroelectrode were constructed and used in conjunction with a large volume wall-jet cell as an amperometric detector in liquid chromatography. Electrode characterization studies were performed in 5.00 mM K,Fe(CN)s + 1 M KC1 aqueous solution. It was found that the wall-jet cell/ultramicroelectrode combination has much reduceJ flow rate dependence compared to the wall-jet cell/conventional electrode system. LC application in the absence of supporting electrolyte was found to be possible and have good sensitivity using the ultramicroelectrode. Detection limits were 100 pg and 10 ng for the platinum thin ring and the carbon fibre array electrodes respectively.
INTRODUCTION
Electrochemical detection of species under flow conditions is nowadays frequently used in areas such as pollution control and monitoring and liquid chromatographic detection. Tubular, planar and wall-jet cell/electrode configurations have been commonly used. The wall-jet system is increasingly gaining popularity because of its sensitivity and ease of use [1,2]. Recent developments in the area of very small electrodes (typically tens of microns to submicron sizes), known as microvoltammetric or ultramicroelectrodes, have introduced new domains to electrochemical studies [3-g]. These electrodes have high mass transfer rates due to hemispherical rather than linear diffusion under normal experimental conditions. Steady state, sigmoidal shaped voltammograms are obtained even under quiescent conditions. Currents passed are very small, usually of the order of picoamperes to nanoamperes. Consequently, problems with ohmic potential loss are reduced. Novel systems to which ultramicroelectrodes have been applied include electrochemistry of low temperature organic glass [6], electrochemistry at very high scan rates [5], and electrochemistry in the absence of supporting electrolyte [3,8,9].
116
The use of the wall-jet cell/ultramicroelectrode configuration as a detector in flow systems combines the sensitivity and ease of use of the wall-jet cell with the advantageous features of the ultramicroelectrode. The efficacy of the large volume wall-jet cell, using conventional electrodes under hydrodynamic conditions, have been demonstrated in previous studies [l-2,10-13]. Recent investigations have shown that it is possible to perform electrochemistry in the absence of supporting electrolyte using ultramicroelectrodes [3,8,9]. Also, due to the higher mass transfer rate, the current density is expected to be higher as compared to conventional-sized electrodes. These features are particularly relevant to electrochemical detection in liquid chromatography. The elimination of the need for supporting electrolyte means that normal-phase liquid chromatography using organic solvents as eluents is more practical with electrochemical detection. In many cases, this is not possible because of the absence of suitable supporting electrolyte for these solvents. In addition, the use of extra reagents always introduces new sources of impurities. These will increase the background noise levels. The use of supporting electrolyte may interfere with the detection in cases where specific adsorption of the electrolyte ions or ion pairing may occur. Further, the decomposition of the supporting electrolyte frequently limits the potential range available. In the absence of the electrolyte the potential window can be widened significantly [3]. With the higher current density, better sensitivity per unit area is expected for the ultramicroelectrode compared to conventional-sized electrodes. Since the ultramicroelectrode is a relatively recent innovation, there have not been many studies of it applied to liquid chromatographic detection. In one investigation, a channel-type electrochemical flow cell using a carbon microfibre array electrode was used as a detector [14]. One significant finding of this work was that the detector response has a very much reduced dependency on flow rate. This is important because decreased flow rate dependence of the response leads to reduced noise levels arising from flow rate fluctuations. To the best of our knowledge, only one previous study has been reported in the literature using the wall-jet cell/ultramicroelectrode combination as a chromatographic detector 1151. In this study, a large number of fibres (1200 fibres), in the form of a bundle, were embedded in epoxy. For such an electrode, it is difficult to relate the number of active fibres to the limiting current because the currents due to the individual fibres may not be independent of each bther. In the present study, the use of a carbon micro-fibre array electrode and a platinum thin ring electrode in conjunction with a large volume wall-jet cell as amperometric detector in liquid chromatography is investigated. EXPERIMENTAL
Chemicals Analar
grade potassium ferricyanide (Merck), potassium chloride (Fluka) and quinones ( p-benzoquinone, 9,10-phenanthrenequinone, 1-nitroanthraquinone, 2nitrofluorenone) (Tokyo Kasei Chemicals) were used without further purification.
117
Catecholamines (dopamine, noradrenaline, adrenaline, L-dopa) (Sigma Chemicals) were also used as received. All solutions were prepared fresh daily. Aqueous solutions were prepared using triply distilled water. For the flow rate dependence studies, a 5.00 mM potassium ferricyanide + 1.0 M potassium chloride aqueous solution was used. Solutions were deaerated with oxygen-free nitrogen. All experiments were carried out using a three-electrode potentiostatic system at a temperature of 25 f l°C. The counter electrode was a 5 mm diameter glassy carbon while the reference electrode was a Ag/AgCl, saturated KC1 electrode. Apparatus The PAR Model 273 potentiostat was employed for voltammetric experiments in still solution and also under hydrodynamic conditions. Voltammograms were recorded using a Graph& model WX 4422 recorder. The flow system was an Eyela microtube peristaltic pump (Model MP-3, Tokyo Rikakikai Co Ltd) connected by a plastic tubing to a glass bulb (approximately 50 ml capacity) half-filled with solution which acted as a pulse damper. The other end of the bulb was connected to the inlet of a wall-jet cell made of Teflon. The large volume wall-jet cell design has been described in full detail elsewhere [12]. The inlet diameter of the cell was 0.5 mm. In operation, the electrode was placed 4 mm away from the point of jet inlet to the cell body in order to avoid interference with the hydrodynamic boundary layer
1131.
For liquid chromatographic experiments, a Perkin-Elmer Series 4 chromatograph together with the Perkin Elmer model RlOO recorder were used. The chromatograph has a microprocessor controlled solvent delivery system with a built in pulse damper and solvent chamber for purging with helium. Samples were injected via a Rheodyne 7105s 6 ~1 injector valve (Rheodyne Corp., Cotati, CA) using a 10 ~1 syringe. For the separation of catecholamines the eluent was 0.1 M acetic acid in water which was also the solvent for preparing the catecholamine solutions. The quinones were dissolved in dichloromethane and the eluent used for their separation was 70% acetonitrile and 30% water. In both cases, the column used was a Perkin-Elmer C,, (0.26 X 25 cm) reversed-phase column. For the detection of catecholamines the flow rate was 1.0 ml min-’ and the working eIectrode potential was +0.80 V (vs. Ag/AgCl) while for the quinones the flow rate was 1.0 ml min-’ and the working electrode potential was -0.90 V (vs. Ag/AgCl). Ultramicroelectroa% construction The carbon microfibre array electrode was constructed in the following manner. A 3.0 cm length of glass tubing of external diameter 3.5 mm was coated on the external surface with a thin layer of epoxy. Individual carbon fibres of 8 pm diameter were then stretched lengthwise and parallel onto the epoxy-coated tubing, extending beyond the ends of the tubing. The fibres were placed about 1 mm apart to ensure that the current responses of the individual fibres were independent of each other [16]. The epoxy film was then allowed to air-dry. At the same time, by
118
the same procedure a layer of fibres was placed around a second piece of glass tubing of the same length but of larger diameter (internal diameter 4.5 mm, external diameter 6 mm). The smaller tube was then placed inside the larger one and both of them were put into another glass tubing of 6.5 mm internal diameter. The spaces in between the tubings at one end (working end) were then filled with epoxy. Electrical contact was made by embedding the carbon fibres extending from the other end in silver cement to which was also sealed a copper wire. The electrode, which contained a total of 33 fibres, was heated at 80°C for 3 h to cure the epoxy and the silver cement. In the case of the platinum thin ring electrode, a glass rod of diameter about 3.5 mm and length 6 cm was coated, by dipping, with a thin layer of metallo-organic platinum paint (liquid bright platinum, Engelhardt). The paint was allowed to air dry and then fired in a furnace at 200°C for 3 min and at 650°C for 15 min. The resultant platinum coated rod was then placed in a 5 cm length of glass tubing (internal diameter 4.5 mm, external diameter 6 mm), one end of which was previously sealed. The sealed end, with the rod inside, was slowly and evenly heated, with suction applied at the other end. As a result, the glass tubing collapsed onto the platinum coated rod for a length of about 1.5 cm to form a tight glass seal. External electrical contact was made by soldering a copper wire to the platinum coated rod.
Fig. 1. Electron micrograph of platinum thin ring electrode. Bar represents 1 pm.
119
To expose the carbon and the platinum ring, both electrodes were polished on a No. 600 abrasive paper with a little water, followed by No. 1200 and No. 1500 abrasive papers. Final polishing was done with a 0.05 pm alumina + water slurry on a polishing cloth. For the ring electrode the internal diameter of the ring was found to be 0.345 cm using a travelling microscope. The thickness of the platinum ring was determined using an electron microscope. Electron micrographs were obtained at three positions around the ring. The electron micrographs showed that the ring thickness was quite even and the average thickness was measured as 0.2 pm. Figure 1 shows an electron micrograph of the ring. RESULTS
AND DISCUSSION
Characterization
of electrodes
Figure 2 shows the cyclic voltammogram obtained for the reduction of the ferricyanide ion (5.00 mM + 1.0 M KC1 in water) at the carbon microfibre array electrode, using a scan rate of 20 mV s-l. A sigmoidal-shaped voltammogram was obtained with the limiting current measured as 156 nA. A value of 59 mV was obtained for E,,d - Es,., which is in agreement with the theoretical value of 56.4 mV for a one electron transfer, within experimental error. A plot of log[(i, - i)/i] yielded a straight line with El,* measured as +0.198 V (vs. Ag/AgCl, sat’d KCl). For a microfibre electrode, the steady state limiting current, for a single fibre [14], is given by i, = 4nFDcr
where c is the bulk concentration of the electroactive species (here 5.00 X low6 mol cme3), r the radius of the fibre (4 X 10m4 cm), the other terms having their usual
I
I
I
20 nA
E/V
Fig. 2. Cyclic voltammogram of 5.00 mM K,Fe(CN), +1 M KCl. Scan rate 20 mV s-l; array electrode. Reference Ag/AgCl (sat’d KCl). Counter electrode glassy carbon disk.
carbon fibre
120
I
I
I
I
I
I
I
f
1
.@5 +04 l03 +0*2r0.1 c-o-0.1-02-03
EIV
Fig. 3. Cyclic voltammogram of 5.00 m M K3Fe(CN), + 1 M KCl. Scan rate 20 mV s-l, ring electrode. Reference Ag/AgCl (sat’d KCI). Counter electrode glassy carbon disk.
platinum
thin
meaning. The diffusion coefficient for the ferricyanide ion has a value of 0.763 X lOa cm* s-l [17]. Thus, the limiting current for a single fibre is 5.88 nA. The total current of 156 nA for the array electrode therefore corresponded to 27 working fibres. The assumption that the currents are additive is valid because in fabrication, care was taken to ensure that the fibres were more than 6 diameters apart (here about 1 mm apart), so that there will be no diffusional cross-talk between the fibres [14]. The fact that there were 27 active fibres out of a total of 33 could be due to bad or broken electrical contact to the external lead in the process of fabrication. For the same solution and scan rate, the cyclic voltammogram at the platinum ring electrode is shown in Fig. 3. Again, a sigmoidal-shaped voltanunogram was obtained and the limiting current was measured as 960 nA. El,* was found to be +0.264 (vs. Ag/AgCl, sat’d KCl) and application of Tomes’s criterion gave a value of 74 mV for E,, - E3j4. This value is significantly higher than the theoretical value which could mean that, due to the thinness of the platinum ring, some kinetic control was observed. The standard heterogeneous rate constant for the ferricyanide/ferrocyanide couple is not very high and known to be dependent on experimental conditions (e.g. at a conventional-sired gold disk electrode k” has a value of 3.6 X 10e3 cm s-l in 0.1 M KC1 and 2.8 X lo-* cm s-l in 1.0 M KC1 [18]). Therefore, it is quite reasonable that, with the high mass transfer rate of the ultramicroelectrode, some kinetic effect could creep in. For the very thin ring electrode, the equation describing the steady state current has been given [19], by i,, = nEr2Dc(2ri
+ Ar)/ln{ 16[(2ri/r)
+
11}
where ri is the internal radius of the ring and Ar the thickness of the ring, all the other terms having their usual meaning. For the platinum thin ring electrode used here, ri is 0.17 cm and Ar is 0.2 X 10e4 cm. Using the above equation, i, works out to be 1000 nA, which compares well with the experimental value of 960 nA. Flow rate dependence
The logarithmic plots of limiting current against flow rate (Fig. 4) show that there
121
Log(i,/nA)
2.8
I
0.3
a2
I
I
0.1
OD
I
I
0.1 0.2 03 log (V/ml min-‘)
cl4
Fig. 4. (a) Plot of log(il/nA) vs. log( V/ml min-‘) for carbon fibre array electrode. (b) Plot of log(i,/nA) vs. log( V/ml min-‘) for platinum thin ring electrode. Solution for both is 5.00 mM K,Fe(CN), +l M KCl.
is some increase in the current as the fled ranged from 0.44 to 2.10 ml mm-‘. This r analytical liquid chromatography, which is The straight line plots gave a slope of 0.17 f for the platinum ring electrode was conventional-sized electrodes, the limiting c1 i = 1 38 ~FcD2/s~-sPV’/‘~r *
rate increases. The flow rate studied ge covers most typical applications in sually in the region of 1.0 ml min-‘. r the carbon array electrode while that .16. For the wall-jet cell using ent eqn. (1) is given by
112~314
where Y is the kinematic viscosity, a the di eter of the inlet nozzle, V the flow rate and R the radius of the electrode. All th other terms have their usual meaning. Therefore, the log plot i, vs. V should hav a slope of 0.75. This value has been verified experimentally [2]. The values obtai ed for the ultramicroelectrodes repreIm sent a significant decrease in flow rate dependence. Since one of the noise sources in flow systems arises from fluctuation in flow rate [14], the reduced flow rate dependence leads to a reduction in noise.
122
1
I
6
I
1
4
2
10 pA
-i
Time/
min
Fig. 5. Liquid chromatogram of a series of catecholamines using platinum thin ring as detector. Potential set at +0.8 V vs. Ag/AgCl (sat’d KCl). Eluent is 0.1 M acetic acid in water at 1.0 ml ICI-‘. (1) Solvent front; (2) noradrenaline; (3) adrenaline; (4) dopamine; (5) L-dopa.
Liquid chromatography
Four catecholamines (noradrenaline, adrenaline, dopamine and L-dopa) were studied using the constructed ultramicroelectrodes and also glassy carbon conventional-sized electrodes of 5.5 mm diameter. Figure 5 shows a typical chromatogram. Reproducibility of the peaks was found to be better than 5%. In Figs. 6a to 6d, linear plots were obtained for the log plots of electrode response (charge/area) vs. sample size (ng). On the average, the response per unit area for the ultramicroelectrodes was 1.25 log units higher than that for the macroelectrode. This represents about a 20 times increase in sensitivity. The expectation, as mentioned earlier, that the sensitivity for the ultramicroelectrode would be greater compared to microelectrode is confirmed. The increased sensitivity could thus be used to advantage to lower the detection limit. The normalized responses for the four catecholamines were about the same or lower for the carbon ultramicroelectrode than for the platinum ring electrode. The greater response of the ring results from its more uniform accessibility compared to the disk [20]. Based on the criterion that the detection limit is twice the noise level, the calculated detection limit for the carbon array electrode is 10 ng and that for the platinum ring electrode is 0.1 ng. (It should be mentioned that the system used here
123 log (
response/C
mm-‘)
log (response
2-
_’
/C
mm-21
3-
-1
-5
-E 9
-6
-E,-
-7
1
-2
log
log km&le
(response/C
-7
3
1
size/ ng)
log
mm-*)
log
(response/C
(sat-Zple
mmm2
size
/ng)
3
)
1
1
2
log
( sample
size/
Fig. 6. plots of lOg(resPOnWC nuC2) (c) doDamine and (d> L-dona. li1 Macrn
3
1
ng)
log
2 (sample
size / ng
3
1
versus log(sample size/ng) for (a) noradrenaline, (b) adrenaline, darcv
rarhnn
A=,+--L
< < __
.A:“-^.-_.
I::,
_-I--
P.-
124
T -I__ 2nA
1
I
I
6
4
2
I Time/min
Fig. 7. Liquid chromatogram of a series of quinones: (1) p-benzoquinone; (2) 9,10-phenanthrenequinone; (3) l-nitroanthraquinone; (4) 2-nitrofluorenone. Platinum thin ring electrode. Potential set at - 0.9 V vs. Ag/AgCl (sat’d KCl). Fluent 70% acetonitrile, 30% water at 1.0 ml mir-‘. No supporting electrolyte.
is far from ideal. For the PAR 174A potentiostat, the most sensitive current range is 20 nA full scale with an accuracy specification of 1.5%. This means that the lowest measurable current is 0.3 nA. However, with the size of the ultramicroelectrodes employed in this study and for very dilute solutions, currents of the order of picoamperes could be measured with a p&ammeter. Further, for small current measurements, the ultramicroelectrodes are susceptible to external noise and a Faradaic cage (which was not used in this work) could be employed to reduce the noise levels drastically. Further work will incorporate the improved features to lower the detection limit.) Figure 7 shows the chromatogram for a series of quinones (reversed phase 70% acetonitrile and 30% water eluent) using the thin platinum ring electrode. The corresponding log plots for the response (charge/area) versus sample size are shown in Fig. 8. As opposed to the earlier results (catecholamines), no supporting electrolyte was used. In this situation, the use of a macro electrode was not possible. It is therefore clear that the use of ultramicroelectrodes widen the applicability of
125
-1
log ( response/C
1
mm-*)
1
2 3 4
-3.
-2-
-4’
/
1
log
2 (sample
4 size/ng?
Fig. 8. Plots of log(response/C mme2) versus log(sample size/rig). (1) p-Fknzoquinone; (2) 9,10phenanthrenequinone; (3) 1-nitroanthraquinone; (4) 2-nitrofluorenone. Platinum thin ring electrode.
electrochemical detection in LC. Normal-phase LC at the moment is essentially confined to other forms of detection such as UV and fluorescence, since the supporting electrolyte requirement is a limitation on the type of solvent which can be used. However, with ultramicroelectrodes, normal-phase LC with electrochemical detection is now a possibility. CONCLUSION
It has been shown that the wall-jet/ultramicroelectrode detector is useful in LC applications. Ultramicroelectrodes have the advantage of much reduced flow rate dependence and increased sensitivity over conventional electrodes. Further the range of applicability is widened since no supporting electrolyte is required. Of the ultramicroelectrodes, a ring electrode is preferred over the microfibre array electrode because it has more uniform accessibility and a higher current is obtained. For example, to obtain the same current level as the ring electrode used here, an array electrode of 163 fibres is necessary. Due to the geometry requirements (tubular) of the wall-jet cell, an array electrode with this number of fibres will be more difficult to construct if the minimum 6 diameters separation condition is required. In addition the use of epoxy resin for holding and filling purposes in the case of the array give rise to higher background noise. AKNOWLEDGEMENTS
The authors wish to thank Dr. Chung Mui Fatt (Dept. of Physics, National University of Singapore) for advice and assistance in taking electron micrographs. Thanks are also due to Mrs. Phua Swee Wah and Miss Pang Teng Jar (Dept. of
126
Physics, National University of Singapore) for obtaining the electron micrographs. Madam Toh Soh Lian has been very helpful with her technical assistance. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
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