Anal. Chem. 1003, 65,2497-2501
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Solid-state Field Decoupler for Off-Column Detection in Capillary Electrophoresis Wim Th. Kok' and Ytiksel Sahin Laboratory for Analytical Chemistry, University of Amsterdam, Nieuwe Achtergracht 166, 1018 WV Amsterdam, The Netherlands
Palladium metal has been used to construct a robust and easy-to-handle field decoupler for capillary electrophoresis (CE) for use with offcolumn detection. The tubular Pd electrode is applied as part of the wall of the capillary system. The capacity of the decoupler to pass the electrolytically generated hydrogen is high enough to accommodate the electric currents as usually found in CE. The use of the palladium decoupler imposes some demands on the buffer to be used as the background electrolyte. Simple monovalent buffer ions are allowed. With multivalent buffer ions such as phosphates, pH shifts during the separation are observed in the separation capillary. The zone broadening in the decoupler, equivalent to a dead volume of 15 nL, restricts the number of theoretical plates that can be obtained toapproximately 100 000 with the capillary system used in this study. Further improvements in this respect are therefore necessary. It is shown that, with the decoupler as developed,electrochemical detection (ECD) is easily combined with CE. Detection limits for catecholic compounds with ECD were in the low fentomole range, 1 order of magnitude or more below those obtained with oncolumn UV detection. INTRODUCTION The inherent high separation efficiency of capillary electrophoresis (CE) has provoked a strong interest in this separation technique in research laboratories in the last six years.' However, ita application in routine laboratories is still limited. One of the bottlenecks for the application of CE is the lack of suitable detection methods. Since the sample mass capacity of CE is low compared to other separation methods such as liquid chromatography, extremely sensitive detectors have to be available before CE will be regarded as a serious alternative in the analytical practice. With most commercial instrumentation only a (on-column)UV detector is available. Because of the small detection volumes allowed in CE, which are in the order of 1-10 nL, the detection limits are usually not below 1V mol L-1.2 Considerably lower detection limits can be obtained with laser-induced fluorescence (LIF) detection.gg Apart from the disadvantage that derivatization will be necessary for most analytes to make (1) Perret, D.; Ross, G. Trends Anal. Chem. 1992, 11, 156. (2) Bruin, G. J. M.; Stegeman, G.; van Asten, A. C.; Xu, X.; Kraak, J. C.; Poppe, H. J. Chromatogr. 1991,559, 163. (3) Gaasmann, E.; Kuo, J. E.; Zare, R. N. Science 1985,230, 813. (4) Gozel, P.; Gaasmann, E.; Michelsen, H.;Zare, R. N. Anal. Chem. 1987, 59, 44. (5) Chung, Y. F.; Dovichi, N. J. Science 1988, 242, 562. (6) Nickerson, B.; Jorgenson, J. W. J. High Resolut. Chromatogr. Chromatogr. Commun. 1988,11,878. 0003-2700/93/0385-2497$04.00/0
LIF detection possible, the high cost of such a detector will limit its application in routine analyses. As has been shown before in liquid chromatography, a small detection volume is not necessarily a disadvantage for an electrochemical detector. Miniaturization of the cell volume may lead to an improved mass transfer toward the electrode surface, while with a decreased electrode surface area, improved signal-to-noise ratios can be obtained. Several research groups have succeeded in applying electrochemical detection (ECD) in CE, with carbonlG18 or metal electrodes.lQ920 Although the development of the CE-ECD coupling is still in its initial phase, detection limits in the order of 10-8-10-7 mol L-1 have already been reported. One of the major problems in CE-ECD is the interference of the high electric field used for the separation with the detection, where very small currents have to be measured. Ewing et al.lsJ8 have shown that end-column ECD can be applied with narrow (2-25 pm) separation capillaries. With wider capillaries off-column detection has to be applied. The high electric field has to be decoupled before the detector, and a second piece of capillary has to be used to transfer the solution with the separated zones from the field decoupler to the detector. We have developed and tested an electrochemical cell with a 1-nL volume and shown in preliminary experiments that it can be used for off-column detection in CE.21 However, it became apparent that with the application of off-column detection new problems arise. The flow of the solution through the second detector capillary is laminar and therefore accompanied by zone broadening. Moreover, the back pressure from the detector capillary disturbs the flat electroosmotic flow profile in the separation capillary. This causes zone broadening in this capillary too. As a result, plate numbers reported for CE with off-column detection are usually below 100 OOO, although with narrow capillaries higher plate numbers have been realized."-13 In a previous paperz2we have shown how the extra zone broadening with off-column detection can be (7) Chung, Y. F.; Wu, S.; Chen, D. Y.; Dovichi, N. J. Anal. Chem. 1990, 62, 496.
(8)Hernandez, L.; Escalona, J.; Joshi, N.; Guzman, N. J. Chromatogr. 1991,559, 183. (9) Swerdlow,H.;Zhang,J.Z.;Chen,D.Y.;Harke,H.R.;Grey,R.; Wu, S.; Dovichi, N. J.; Fuller, C. Anal. Chem. 1991,63, 2835. (10) Wallingford, R. A.; Ewing, A. G. Anal. Chem. 1987,59,1762. (11) Wallingford, R. A.; Ewing, A. G . Anal. Chem. 1988,60,258. (12) Wallingford, R. A.; Ewing, A. G . Anal. Chem. 1988,60, 1972. (13) Wallingford, R. A.; Ewing, A. G. Anal. Chem. 1989, 61, 98. (14) Olefirowicz, T. M.; Ewing, A. G. Anal. Chem. 1990,62,1872. (15) Huang, X.; Zare, R. N.; Sloss, S.; Ewing, A. G. Anal. Chem. 1991, 63, 189. (16) Yik, Y. F.; Lee, H. K.; Li, S. F. Y.; Khoo, S. B. J . Chromatogr. 1991,585, 139. (17) O'Shea, T. J.; Greenhagen, R. D.; Lunte,S. M.; Smyth, M. R.; Radzik, D. M.; Watanabe, N. J. Chromatogr. 1992,593,305. (18) Sloss, S.; Ewing, A. G . Anal. Chem. 1993, 65, 577. (19) O'Shea, T. J.; Lunte,S. M. Anal. Chem. 1993, 65, 247. (20) Colon, L. A.; Dadoo, R.; Zare, R. N.Anal. Chem. 1993,65, 476.
(21)Tiidds. A. J.: Van Dvck. - . M. M. C.:. Pome. _ - H.: Kok. W. Th. Chiomatograjhia, in press. (22) Kok, W. Th. Anal. Chem. 1993,65, 1853.
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diminished to a large extent by pressure compensation. During the separation a pressure is applied on the front end of the separation capillary, balancing the back pressure of the detector capillary, and thereby conserving the flat electroosmotic flow profile. By using a detector capillary with an internal diameter smaller than that of the separation capillary, the zone broadening in this capillary can be reduced also. Plate numbers of approximately 200000 could be obtained even with fairly long (5-10 cm) detector capillaries. A second problem in off-column detection is the field decoupler itself. Joints made of porous glass or graphite16 have been used for this purpose. Others use semipermeable membrane tubes, slipped over the adjoining ends of the two pieces of capillary. The tube material is permeable for the ions in the solution, thus accommodating the electric current, while the bulk of the solution is retained within the capillary system. However, these porous-joint field decouplers are vulnerable, difficult to handle, and therefore hardly suited for routine use. In this paper we describe a robust, easy-to-handle solidstate field decoupler, made from palladium metal. Its limitations in terms of current capacity, buffer composition compatibility, and zone broadening effects have been evaluated. Application of the decoupler for CE-ECD is shown.
THEORY In fused-silica capillaries as used in CE, the electroosmotic flow is usually toward the negative electrode. This electrode is then placed at the detector end of the separation capillary. A t the negative electrode a cathodic electrode reaction takes place, which for aqueous solutions is
2H20 + 2e-
-
H,
+ 20H-
In on-column detection as well as in off-column detection when a porous joint is used, this reaction is irrelevant since it occurs in a buffer vial outside the capillary system. However, when the field is to be decoupled through an electrode directly in the capillary itself, the hydrogen gas production will be a serious problem. The production rate of hydrogen atoms \ k is~ proportional to the electrophoretic current in the separation capillary, which is determined by the field strength E and the conductivity y of the solution in the capillary:
where d, is the capillary diameter and F the Faraday constant. Hydrogen is only slightly soluble in aqueous solutions (7.8 X 10-4 mol L-1 at 1atm and 25 "C). Gas bubbles will develop when the production rate exceeds the rate of removal of hydrogen from the electrode surface with the solution by the electroosmotic flow, which is given by
where C H ~ ~isQ the hydrogen concentration in the aqueous solution. Simple calculations show that, for instance in a solution of pH 7, when the osmotic mobility is approximately 7 X 10-4 cm2 s-1 V-1, the solution conductivity should not exceed 10"' W cm-l to avoid bubble formation. This is much lower than what is usual in CE. A possible alternative way to dissipate the hydrogen produced is through the electrode material itself, when a tubular electrode is used as part of the capillary wall. Metals of the platinum group, and especially palladium, are known for their ability to absorb and pass hydrogen and can therefore be suitable as electrode material in this application. The rate of hydrogen dissipation through a tubular electrode,
which is part of the capillary system, can be calculated when the concentration of hydrogen in the metal at the electrode surface and the diffusion coefficient in the metal are known. From this the maximum current before gas bubble formation starts is found, using Fick's diffusion equation for a cylindrical geometry:
(3) where DH is the diffusion coefficient and c g d the concentration of hydrogen atoms in the Pd metal a t 1atm and Lel, Rout,and R h are the length, outer radius, and inner radius of the tubular electrode, respectively. At low hydrogen pressures, the equilibrium concentration of hydrogen in palladium metal is approximately given by X = 0.04 P H ~ J , , where X is the H/Pd atom ratio and p~~ the partial gas pressure in bar.23 Above a critical value of the hydrogen pressure, which is, depending on the temperature, between 0.01 and 0.1 bar, a hydride is formed with the metal. The H/Pd atom ratio can increase to approximately 0.6 at this pressure.%~25This value is equivalent to 0.07 mol cm3 H. An appreciable higher uptake of hydrogen requires large overpressures. Therefore, 0.07 mol cm3 is the maximum concentration of hydrogen atoms a t the electrode surface before gas bubble formation occurs. The dissipation of absorbed hydrogen through Pd metal by diffusion is relatively fast. For the diffusion coefficient of hydrogen atoms DHand its dependence on the temperature, different values are given in the literature.26 A reasonable estimate Of DH at 25 OC is 3 X lo-' cm2s-l. With these data substituted in eq 3 the maximum current for a particular electrode geometry can be found. For a tubular Pd electrode with a channel length of 1mm, an inner radius of 0.1 mm, and an outer radius of 1.5 mm, as used in this study, a value of 0.5 mA is found. This current capacity of the electrode exceeds by far the currents usually found in CE, which are of the order of 10-100 pA. Therefore, dissipation of the produced hydrogen through a Pd electrode is a viable option to decouple the electric field for off-column detection. EXPERIMENTAL SECTION Fused-silica capillaries with an outer diameter of 370 pm and different internal diameters were obtained from Polymicro Technologies (Phoenix, AZ). For UV-absorbance detection, a 2-mm detection window in a capillary was made by burning off the coating. New capillarieswere etched with a sodium hydroxide solution (1mol L-l) for at least 2 h before use, and etching was repeated at the beginning of every day for 30 min. A prototype of the Prince (Lauer Labs, Emmen, The Netherlands) programmable injector for capillary electrophoresis has been used. Sampleswere introduced with the pressure mode. A 30-kV highvoltage source was controlled from the Prince. A CE microcell from Linear (Reno, NV) was mounted in a Spectra 100 variablewavelength UV detector (Spectra-Physics). Amperometric detection was performed with a homemade microcell as described elsewhere.21 It has a 0.25-mm disk-shaped working electrode made of a bundle of carbon fibers glued together and a Ag/AgCl counter and reference electrode. An Amor (Spark,Emmen, The Netherlands) potentiostat/amplifierwas used. The signals were registered with a strip-chart recorder and with a HP-3394A integrator. Palladium rod (3-mm diameter) was obtained from Drijfhout (Amsterdam, The Netherlands). 1-Naphthol (NOH) was obtained from BDH, 1-naphthalenemethylamine ( N U + )from (23) Powell, G. L.; Lever, W. E.; Laesser, R. Z. Phys. Chem. 1989,163, 47. (24) Wicke, E.; Froelich, K. Z. Phys. Chem. 1989, 163, 36. (25) Kandesanny, K.; Lewis, F. A.; McFall, W. D.; McNicoll, R. A. 2. Phys. Chem. 1989,163,41. (26) Kufudakis, A.; Cermak, J. Z. Phys. Chem. 1989, 164, 1013.
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abs. a
ratio
b
t
- - _ L _ _
1.53
,"I
1-
d
Flgure 1. Cmas section of the decwpler: (a)W union: (b) separatlm capillary: (c)detector capillary: (d) connecting channel. Flgure not on
0.5 -
Scale
A
i (uA)
4
Flgura 3. pH-calibrationplot using me absorbance ratio a1 554 and 230 nm of a phenolphthalein solution. Table 1. Shift of the Buffer pH in the Pd Decoupler during Electrophoresis. buffer romp (mol L-'J initial pH final pH ew calc exp wo3 NaOH n.in .... ...
low,, I -
20
I
I
/ ,
I
,
,
,b,
0.06 0.02
0.0 0.0
0
0
10
20
M
V(kV) Figure 2. Dependency of the electrophoratic current on h e applied voltage: (a) Pd decoupler; (b) stainle88-steel decoupler. For experimental details, see text.
Aldrich, 6-hydroxynaphthalenesulfonicacid (HNSA-) from Merck, and 2-naphthol-3,6-disulfonicacid (NDSAZ)from Fluka and used without further purification. Concentratedstock solutions in water or methanol were kept at 4 "C and diluted to 1W mol L-1 with buffer for injection. Catechol (C) was obtained from BDH, L-DOPA and 5-hydroxyindoleaceticacid (HIM) from Sigma, and 3,4-dihydroxyphenylaceticacid (DOPAC) and 3,4 dihydroxybemicacid (DHBAc)from FIuka Concentratedstock solutions in water of these compounds were diluted to 2.1Wmol L-' in degassed buffer solution containing l W mol L-' EDTA shortly before use. Other p.a. grade chemicals were obtained from standard suppliers and used without further purification.
RESULTS AND DISCUSSION Hydrogen Production and Dissipation. InFigure 1the cross section of the palladium field decoupler as developed in our laboratory is shown. It resembles a low-dead-volume unionasusedinliquid chromatographyandincludesstandard fittings to connect the separation and detector capillaries. The channel between the two capillaries is approximately 1 mm in length and 0.2 mm in diameter, giving a dead volume of approximately 30 nL. The electrical contact to ground is made with a metal clamp on the outside of the decoupler. To test the applicability of the decoupler, current-voltage relations were measured in a CE system. A 50-cm separation capillarywasused, with an internal diameter of 1M)pm, filled with a huffer solution made of 0.10 mol L-1 boric acid and 0.01 mol L-' sodium hydroxide (pH 8.0). For comparison, a decoupler of stainless steel with exactly the same geometry was also tested. In Figure 2 the results of these measwements are shown. With the palladium decoupler the current increaseswiththevoltageas expected,while with thestainless steel device only a small (leakage) current can be passed. With shorter and wider capillaries, and a more concentrated buffer,currentsupto280pAcouldbepassedbythepalladium decoupler. At higher applied voltages the current became irregular and finally dropped to zero. However, this may not be due to hydrogen gas evolution but to hoiling of the buffer solution. From the increase of the solution conductivity compared to that at low electric fields it could be derived
Capillary, 50 cm X 103 pm i.d.; applied voltage, 20 kV; current,
21 A
(with the help of tables giving the temperature dependence of the viscosity of water) that the solution temperature was close to 100 "C. Hydroxide Ion Production. Another factor to be considered is the production of hydroxide ions at the electrode surface. Depending on the buffer composition of the solution, a pH shift will result. Therefore,it has to betaken into account that the detection pH is higher than the separation pH. This was studied by collecting the effluent of the CE system. First, a pH-calibrationcurve was constructed using phenolphthalein as an indicator. The ratio of the absorbance of phenolphthalein at 554 and 230 nm was measured in huffer solutions of known pH with a spectrophotometer (see Figure 3). The effluent of the CE system, running continuously without sample introduction, was collected during 2 h in an nonbuffered aqueous solution of the indicator. The absorbance ratio of this solution was measured and the pH of the capillary effluent was determined using the pH-calibration plot. This experiment was performed using borate buffers of different compositionas background electrolytefor the electrophoresis. InTable ItheexperimentallyobservedpHshifta arecompared to the values calculated from the currents measured. Good agreement is found, indicatingthatthemodel for the processes in the decoupler as described above is valid. More prohlematic than the pH shift for detection, however, is the possible migration of the pH shift into the separation capillary. Althoughsuch aneffect may beutilized asavariant of gradient elution, in most cases a changing pH during the separation is undesirable. The hydroxide ion production will result in a local change of the ion concentrations in the solution. As long as the mobility of the produced and consumed ions is lower than the electroosmoticmobility, the pH change will he swept away toward the detector. At pH values ahove 4 or 5 this is usually the case for most singlecharged ions. Only the hydroxide ion itself is able to migrate in the opposite direction. However, when the buffer capacity of the hackground electrolyte is adequate, the hydroxide ions produced will be eliminated from the solution hy the acidic species of the buffer couple. This was shown by experiments in which the current was monitored during the application of the electric field. At the same time anon-columnUV detector wasmonitoringpossible
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 18, SEPTEMBER 15,
1993 3
“i;
\
uv
a
-
5
0
5 141
1
II n
1
10
10
t (min)
5
0
t (min)
Flgure 5. Separationof catecholic compounds with simultaneous oncolumn UV and off-column electrochemicaldetection. Sample: 2 X lo-‘ mol L-’ (1) C, (2) DOPA, (3) HIAA, (4) DOPAC, and (5) DHBAc. For experimental details, see text.
buv \
5
0
10
t (min)
fered solution and the phosphate buffer shows that the background electrolyteis changing during electrophoresiswith these systems. The passage of a pH front along the on-column detector is clearly visible in the detector signal. With the sodium chloride solution this front is formed by hydroxy ions, with the phosphate buffer by multiple-charged phosphate ions. Stable currents, indicating the absence of a pH shift in the separation capillary,could be obtained with various buffers such as acetate, borate, Tris, and barbital buffers. Zone Broadening in the Decoupler. To determine the zone-broadening contribution of the decoupler, a UV detector was used in the on-column and in the off-column modes. For on-column detection, a 90 cm X 75 pm i.d. separation capillary with the detection window at 75 cm was used. For off-column detection, the separation capillary was 75 cm X 75 pm i.d. A 12 cm X 50 pm i.d. detector capillary was used with a detection window at 5 cm from the decoupler. A compensating pressure of 85 mbar was applied during electrophoresis to eliminate the laminar backflow in the separation capillary.22 Four naphthalene derivatives with different effective charges were used as model compounds (see Table 11). Zone variances were calculated from the recorded electropherograms. To calculate the expected zone variances in off-column detection, the variance contribution in the detection capillary as Calculatedwith the Taylor-Aris equationn*28(0.9 s2) was added to the results obtained with on-column detection. In Table 11, the calculated values of the zone variances are compared with the experimental values obtained with off-column detection. A difference of 15 f 3 nL is found, which may be attributed to the contribution of the dead volume in the palladium decoupler. Obviously, this contribution is still too large to obtain very high plate numbers. An improved construction with a lower dead volume is now being tested. Coupling with an Electrochemical Detector. For the evaluation of the palladium decoupler in CE-ECD, catechol derivatives were used as model compounds. An on-column UV detector (284 nm) and an off-column electrochemical detector (+0.9V vs Ag/AgCl) were used simultaneously. In the separation capillary, with an internal diameter of 100 pm
“ 1
uv
0
5
10 -t
I (min)
Figure 4. Current traces and detector signals during electrophoresis with a background electrolyte of (a) sodium chloride, (b) phosphate buffer (pH 7.0), and (c) borate buffer (pH 9.0). Table XI. Zone Broadening in On-Columnand Off-Column Detection. zone variance f (92) elution time t ( 8 ) compd
onb
offc
NMA+ NOH HNSANDSA2-
241 329 514 805
246 334 527 831
onbexp offccalc offCexp Aa(nL) 0.3 0.4 0.7 2.8
1.2 1.3 1.6 3.7
3.4 3.5 3.3 7.3
15 15 13 19
a For experimental details, see text. On-column detection. Offcolumn detection.
changes in the background electrolyte. In Figure 4, current traces and detector signals obtained with an nonbuffered sodium chloride solution, a borate buffer, and a phosphate buffer are shown. The increase in current with the nonbuf-
(27) Taylor, G. Proc. R. SOC.London, Ser. A 1953, 219, 186. (28) h i s , R. R o c . R . SOC.London, Ser. A 1956,235, 67.
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Table 111. Sensitivities and Detection Limits with On-Column UV and Off-Column Electrochemical Detection. sensitivity mobility uv ECD detection limit (1o-B mol L-1) detection limit (fmol) compd (10-4c m 2 V-18-1) (AU mol-' L) (106A mol-' L) uv ECD uv ECD C 0.15 6.2 9.4 19 0.3 380 6 0.35 6.0 DOPA 5.5 20 0.5 400 11 2.77 16.0 HIAA 3.7 8 0.8 160 16 2.88 12.5 DOPAC 4.1 10 0.7 200 15 3.16 16.0 DHBAc 3.2 8 0.9 160 19 Sample volume approximately 20 nL. For further experimental details, see text.
and a length of 75 cm, a detection window was made at 60 cm. For the coupling to the electrochemical detector cell, a capillary of 50 pm in diameter and 7 cm in length was used. The separation was performed in a buffer composed of 0.02 mol L-1 Tris and 0.01 mol L-1 acetic acid (pH 8.4), with an applied voltage of 21 kV and a compensating pressure of 85 mbar. Approximately 20 nL of sample solution was injected hydrodynamically. Figure 5 shows the simultaneous electropherograms obtained with a standard solution of 2.10-4 mol 1-1 catechol, DOPA, HIAA,DOPAC, and DHBAc. The decreaseof the separation efficiency with off-columndetection is visible in the loss of resolution between the DOPA and catechol peaks. Still, with the off-column electrochemical detector plate numbers of 70-100 OOO are obtained.
In Table 111,the sensitivities and detection limits obtained with the two detectors are compared. With the electrochemical detector, the detection limits are 10-60-fold lower. With an improvement of the working electrode construction, as is now pursued in our laboratory, a further decrease of the ECD detection limits can even )e expected.
ACKNOWLEDGMENT Thanks are due to Mireille Van Dyck and Roelant Harmsen for their participation in the experimental work, and to Henk Lauer for making available the Prince prototype. RECEIVED for review January 6, 1993. Accepted June 7, 1993.
