Anal. Chem. 1997, 69, 2362-2366
A Reference Electrode with Free-Diffusion Liquid Junction for Electrochemical Measurements under Changing Pressure Conditions Gerrit Peters*
Institut fu¨ r Meereskunde, Du¨ sternbrooker Weg 20, D-24105 Kiel, Germany
A reference electrode for electrochemical measurements under changing pressure conditions is presented. The electrode of the Ag,AgCl|KCl type incorporates a mechanical pressure equalizing system to achieve independence from ambient pressure. An open liquid junction in connection with a controlled electrolyte outflow ensures accurate in situ measurements over periods of up to 10 days. Abrupt pressure changes (0.1 MPa s-1) effected short-time, reversible peak potential changes of less than 1.3 mV. The system was successfully used to monitor gastric pH in diving animals. The advantages of the new design are high tolerance to pressure changes, small size, no memory effects, low susceptibility to junction contaminations, and a long operating life, even under repeatedly changing pressure conditions. Electrochemical methods play an important role in the determination of many ionic or molecular activities.1-5 Despite the great variety of specifically sensitive electrodes, the reference halfcell needed to complete the measurement cell is the same for the majority of applications. This reference system receives much attention, since it essentially determines the overall accuracy of the measurement.6,7 The most frequently used is the Ag,AgCl|KCl system, due to its universal applicability. However, depending on its actual intended purpose, the reference system has been subject to various modifications. Measurements made under high pressure or under weightlessness, for example, make special demands on the reference system: Under elevated ambient pressure (e.g., underwater), a dilution of electrolyte by invasion of the sample solution must be avoided so as to maintain a stable and fast-responding signal. A similar situation occurs where the hydrostatic pressure, normally exerted by the electrolyte column, is lacking due to measurements being made outside a gravitational field (e.g., “Spacelab”).
There are a number of solutions for this problem which can be summarized in three main principles: (1) Electrodes with solidified electrolytes (polymers, gels). These are widely used for applications under slight excess pressure. They work independent of electrode orientation. There is no outflow of electrolyte, but there is a steady dilution by diffusion, which gives the electrodes an unsteady reference potential as high diffusion potentials are developed. This condition is often coupled with pronounced memory effects.7 Electrodes with solidified electrolyte are characterized by a short operational life and are not suited for high-precision measurements. (2) Electrodes with constant excess pressure. This is achieved by introducing a compressed air cushion into the electrode, often in connection with a viscous electrolyte.8 The operating pressure range of this type of electrode is limited by the internal pressure. (3) Electrodes with controlled pressurization. Here, the pressure is controlled by a pressure relay which is interposed between electrode and sample container. These systems are technically complex and bulky and most frequently used in cases where high and changing pressure conditions occur. They are rarely suited for field investigations, being essentially restricted to industrial applications.9 Recently, solid-state electrodes with improved pressure independence have been developed, although they still have their shortcomings.10 This article describes a concept for a reference electrode with a liquid junction which is capable of working under high and changing pressure conditions with high accuracy. Instead of avoiding a liquid junction, the approach was to try to improve this accepted type of electrode by constructive modifications. Initially developed for physiological investigations in diving animals, this electrode should be of use in a variety of other applications. EXPERIMENTAL SECTION Reference Electrode. The basic element of the electrode was a conventional Ag,AgCl|KCl half-cell. The end of the electrode (Figure 1) was connected to a 1 mL disposable syringe (5 mm i.d.) (9) filled with electrolyte (3 M KCl saturated with AgCl). The end of this syringe was sealed by a rubber piston (11), which acted as a movable separator between the electrolyte chamber (12) and the outside. It was pressed toward the electrode tip (3) by the force exerted by a compression spring (8).
* Phone: Germany 431-597-3939. Fax: Germany 431-597-3994. E-mail:
[email protected]. (1) Covington, A. K., Ed. Ion-selective electrode methodology, 1st ed.; CRC Press, Inc.: Boca Raton, FL, 1980; Vols. I and II. (2) Orio, A. A., Kaldova, R., Nu ¨ rnberg, H. W., Eds. The assessment of electrochemical sensors for water analysis; UNESCO Workshop, Venice, Italy, 4-8 Oct, 1982; special issue of Sci. Total Environ. 1984, 37, 1-128. (3) Oehme, F. Ionenselektive Elektroden: Grundlagen und Methoden der DirektPotentiometrie; Dr. Alfred Hu ¨ thig Verlag: Heidelberg, 1986. (4) Nelson, A., Valenta, P., Hamilton, E. I., Eds. Electrochemical methods; special issue of Sci. Total Environ. 1987, 60. (5) Wang, J. Analytical electrochemistry; VCH Publishers, Inc.: New York, 1994. (6) Wang, J. Analytical electrochemistry; VCH Publishers, Inc.: New York, 1994; p 112f. (7) Galster, H. pH measurement: Fundamentals, methods, applications, instrumentation; VCH Publishers: Weinheim, 1991; Chapters 3.2., 3.3.
(8) Bu ¨ hler, H. W.; Bucher, R. (Ingold AG, Urdorf). Sterilisierbare pH-Messkette zur U ¨ berwachung mikrobiologischer Prozesse. German Patent DE 3 702 501, 1987; ICP G01N 27/56. (9) Oehme, F. Ionenselektive Elektroden: Grundlagen und Methoden der DirektPotentiometrie; Dr. Alfred Hu ¨ thig Verlag: Heidelberg, 1986; p 131. (10) Jerman, R.; Tercier, M.-L.; Buffle, J. Anal. Chim. Acta 1992, 269, 49-58.
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S0003-2700(96)01275-9 CCC: $14.00
© 1997 American Chemical Society
Figure 1. Top and side view of the electrode assembly with PTFE tube, opening to the outside (1), glass microelectrode (2), glass capillary (3), titanium cap (4), Ag,AgCl wire (5), thread, for connection with the titanium housing (6), epoxy (7), compression spring (8), disposable syringe (9), PTFE tubing (10), rubber piston (11), electrolyte chamber (12), space for electronics (enclosed by the titanium housing) (13), refilling unit (acrylic block with bores, threads, and O-ring fittings) (14), acrylic stopper (15), and needle valve (16). Shaded areas indicate the electrolyte-containing space.
Distal to the spring, the end of the syringe led to a silicone grease-filled plastic tube (1) which opened to the outer medium, allowing the external pressure to be communicated to the inside of the electrode. The individual parts of the reference electrode were connected with PTFE tubing (2 mm i.d.) (10), and the whole system was cast in epoxy resin (7). With this system, external pressure acting on the liquid junction was automatically balanced by the pressure acting on the oposite end of the electrode. In this way, no pressure differences could build up across the junction when external pressure changed. Additionally, the spring pushing the piston produced a permanent excess pressure inside the electrode, effecting a controlled outflow of electrolyte that was independent of ambient pressure and electrode orientation. Thus, neither should sample solution be pressed through the junction into the electrode as
external pressure rises nor should electrolyte be forced out of the electrode as external pressure falls, even if the electrolyte was not totally free from air bubbles. The continuous outflow of electrolyte further reduced the risk of electrode contamination and prevented electrolyte depletion due to diffusion. In the present application, the reference half-cell was used in connection with a glass electrode ((2) in Figure 1, Type MI-506, Microelectrodes, Inc., Bedford, NH) to monitor gastric pH in freeranging penguins. It was, therefore, incorporated together with a datalogger in titanium housing so as to obtain a pressure-tight autonomous stomach probe. Liquid Junction. The liquid junction of the reference electrode was achieved with a glass capillary (0.13 mm i.d., length 10.0 mm) instead of the commonly used diaphragms. The superior performance of liquid junctions of this free-flowing, freediffusion type has been demonstrated by Dohner et al.11 Such junctions possess several advantages over common porous ceramic junctions. The relatively low surface-to-volume ratio of a capillary minimizes the effects of selective diffusion due to ionic interactions with the tube material, which can produce the undesirable ζ potential.7 Furthermore, open junctions do not suffer from obstructions, which occur frequently in conventional restrained-flow junctions when measuring in media with a high content of proteins or other substances such as sulfides which can react with the electrolyte to form insoluble precipitates.7 The capillary design helps to prevent this without the need of an electrolyte bridge. Electrolyte. Open junctions usually suffer from high electrolyte consumption and susceptibility to pressure differences. The relatively high cross-sectional area of the pore and the force of the spring producing an internal excess pressure necessitated the use of a gel-stabilized electrolyte. This limited excessive flow rate and would have precluded the mixing of sample solution and electrolyte within the capillary, or the adjacent part of the electrode, in the event that sample solution entered the system. For this reason, the electrolyte solution was prepared with the addition of 0.8% (w/v) agar. Refilling and Adjustment of Flow Rate. For repeated use, the system could be refilled through a bore which was closed under operation with an O-ring-sealed stopper. The molten electrolyte (after cooling down to 50-70 °C) was injected against the pressure of the spring, with the aid of a syringe, while carefully avoiding the introduction of air bubbles. To ensure that the electrolyte was not pressed backward upon refilling by the force of the spring, a needle valve was inserted, which had to be briefly closed before removing the syringe and finally applying the stopper. Additionally, this valve facilitated a fine adjustment of the electrolyte outflow. The seals of needle valve and stopper were designed to resist pressure differences of at least 10 MPa. Flow Rate. Electrolyte outflow rates were determined by examining remaining electrolyte stores over time. Furthermore, since the force of the compression spring employed in the pressure balancing system was known, it was possible to predict accurately changes in flow rate over time and thus derive the operating life of the electrode. Electrical Resistance. The electrical resistance of the liquid junction was calculated from the dimensions of the capillary (for 3 M KCl). It was additionally controlled by direct measurements (11) Dohner, R. E.; Wegmann, D.; Morf, W. E.; Simon, W. Anal. Chem. 1986, 58, 2585-2589.
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according to Spiegler12 using an ac bridge at 60 Hz and Ueff ) 5 V. Corrosion Tests. Although it was filled with silicone grease, it was conceivable that the spring side of the pressure balancing system could come into contact with the sample solution as the spring expanded during operation. Therefore, the corrosion resistance of the spring material was tested under the influence of hydrochloric and sulfuric acids at room temperature. Weight loss over time was monitored using an analysis balance (BP 210 D, Sartorius AG, D-37070 Go¨ttingen, Germany). Memory Effects. The memory effects of the electrode were tested with two identical reference electrodes. One of them was maintained in a polyethylene jar containing ∼250 mL of test solution (100-10-5 M KCl and NaCl solutions) to allow a stable potential to develop, while the other was placed in solutions of differing concentrations for 15 min before being retransferred to the test solution. Measurements were performed at 21-22 °C on a grounded worktop using a voltmeter with high input resistance (Fluke 87 True RMS Multimeter, John Fluke Mfg. Co., Inc., Everett, WA). Readings were taken over 15 min at intervals of between 5 s (initially) and 1 min (at the end). All solutions were prepared from freshly deionized water with a conductivity of less than 5 µS m-1. Stirring Experiments. The influence of stirring on the junction potential was tested with a similar system. Both electrodes were placed in the same rectangular jar, separated from each other by a perforated polyester partition. The test electrode was additionally placed in a perforated polyester ring. This setup ensured electrical conductance, while stirring only affected the test electrode. The solution in the polyester ring was stirred by a magnetic rod, with the stirrer set at ∼400 rpm. The electrode tip was placed at a distance of 10 mm from the stirring rod. Flow rates of the electrodes were adjusted to ∼2 µL h-1. Measuring under Elevated Pressure. The mechanical pressure resistance of the unit was tested in a pressure tank and under field conditions. Stability of the reference potential under changing pressure was tested in a modified ultrafiltration chamber (Amicon, Model 402). The potential of the reference electrode was measured against a pressure-insensitive electrode prepared from a plastic tube (1.5 mm i.d., length 50 mm), filled with agar-stabilized electrolyte. A chlorinated Ag wire was inserted into this openended tube. Both electrodes were placed freely hanging in a beaker containing ∼50 mL of a 0.01 M KCl solution. Pressure was increased with nitrogen in 0.1 MPa steps, the transition time between two subsequent pressure levels being 1-2 s. Materials and Reagents. The reference electrode was manufactured from the following materials: chlorinated silver wire (Microelectrodes, Inc., Bedford, NH); glass capillary (Hilgenberg, D-34323 Malsfeld, Germany); noncorrosive spring, molybdenum steel alloy (DIN 17440, X5 CrNiMo 1812, Hensel & Partner, D-25474 Bo¨nningstedt, Germany); disposable syringe (Trans-coject, Hamburg, Germany); refilling unit with needle ventile and stopper, custom-made from polymethacrylate in our institute’s workshop; titanium (Kienzle Titanverarbeitung, D-71149 Bondorf, Germany); housing turned in the workshop; PTFE tubing (Polzin Laborbedarf, D-24105 Kiel, Germany); O-rings, bunanitrile rubber (Mordhorst and Bockendahl, D-24113 Kiel, Germany); epoxy resin (Epoxy-glosscoat, Vosschemie, D-25436 Uetersen, Germany); (12) Spiegler, K. S. J. Electrochem. Soc. 1966, 113 (2), 161-165.
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Figure 2. Remaining electrolyte stores as a function of time for starting flow rates of 4 (a) and 6 µL h-1 (b). With a starting flow rate of approximately 6 µL h-1, the reference electrode could work for up to 6 days with a final flow rate of 1.5 µL h-1. An initial flow rate of 4 µL h-1 allowed the electrode to work for up to 10 days with a final flow rate of 1 µL h-1 (all independent of external pressure).
epoxy glue (Uhu-sofortfest, Uhu, D-77813 Bu¨hl, Germany); and silicone grease (Wacker-Chemie, Mu¨nchen, Germany). KCl and AgCl, p.a. grade, were obtained from Merck (D-64271 Darmstadt, Germany); Bacto-Agar was from Difco Laboratories (Detroit, MI). RESULTS Flow Rate. The force (F) of the spring effected a net internal pressure within the electrolyte tank of 0.5 MPa at the beginning and 0.125 MPa at the end. Within these limits, a total of 500 µL of electrolyte could be operated. The flow rate decreased linearly, since it is directly proportional to ∆p (where p is pressure, as force per unit area, F decreasing linearly with spring length), according to the Hagen-Poiseuille law. This allowed predictions to be made regarding the operating life of the electrode under different starting flow rates (Figure 2). Electrical Resistance. The electrical resistance of the liquid junction was calculated to be 19 kΩ for 3 M KCl (with resistivity F ) 2.5 Ω cm), whereas determinations using the ac bridge revealed a resistance increased by ∼10%. Corrosion Tests. The spring material was not affected under the influence of 0.1 M HCl and 0.1 and 1 M H3PO4, but 1 M HCl led to a rapid dissolution of the material with a weight loss of approximately 1.1% per day. Memory Effects. A summary of the memory effect tests is given in Table 1. In most cases, electrode potential stabilized within less than 1 min without any apparent memory effect. Stirring Experiments. Stirring had no influence on the junction potential in 100 and 10-1 M KCl test solutions. In the more dilute solutions, however, the potential oscillated quickly, with the amplitude of oscillation increasing with decreasing solution concentration. Potential differences between unstirred and stirred solutions lay between -0.2 and -0.1 mV for 10-2 M, between -0.6 and -0.3 mV for 10-4 M, and between -0.7 and -0.1 mV for 10-5 M KCl. Measuring under Elevated Pressure. The mechanical pressure resistance of the electrode design in use was determined to exceed 3 MPa (i.e., excess pressure inside the electrode with respect to the pressure inside the titanium housing, i.e., atmo-
Table 1. Apparent Memory Effects in Different Combinations of Sample Solution and Preceding Solutiona sample preceding potential stabilizing solution concn (M) solution concn (M) difference (mV) time (s) KCl
100 100 100 10-3 10-4 10-5 10-2 10-1 10-3
KCl
NaCl
10-1 10-4 10-5 100 100 100 10-5 100 100
0 -0.1 0 -0.1 0 +0.1 0 -0.1 +0.2
30 15 15 60 25 120 90 20 240
a Memory effects are given as the difference between the cell potential 3 min after electrode transfer and the previous potential in the same solution. Stabilizing time is defined as the time to reach the final potential (after 15 min) within (0.1 mV.
Figure 3. Change in the potential of a cell consisting of the reference half-cell and a second pressure-insensitive half-cell (cf. Methods) as a function of abrupt changes in ambient pressure. During steady pressure release, no changes in cell potential were observed. Abrupt changes in pressure from 0.25 to 0 MPa (within less than 1 s, equivalent to a change of depth of >25 m s-1) induced a pronounced potential shift. (A slight drift of 0.2 mV during the course of the experiment was corrected for.)
spheric pressure). Higher pressures were not tested since this range was sufficient for the application in diving birds. The tests of cell potentials in the pressure chamber revealed short-time changes of up to -1.3 mV for a few seconds following the pressure change (Figure 3). The potential stabilized thereafter at the previous level. The extent of this short-time potential shift decreased with increasing pressure (Figure 3). Application in Field Studies. The reference half-cell was deployed more than 40 times to monitor gastric pH in free-living penguins for up to 10 days.13 These birds repeatedly dived to depths greater than 70 m with maxima of more than 100 m. The electrode worked reliably under these highly adverse conditions, with respect to potentiometric measurements. It was not affected by the oscillating pressure conditions (Figure 4), nor was the junction observed to be clogged. The smooth structure of the inner wall facilitated a constant cleaning of the pore with the outflowing electrolyte, always ensuring proper electrode function over the period of deployment. DISCUSSION The potentiometric measurement of pH or other ionic or molecular activities under high and fluctuating pressure conditions (13) Peters, G. Am. J. Physiol., submitted.
Figure 4. Example of gastric pH in a Gentoo penguin (Pygoscelis papua) during phases of different diving behavior. Observed shorttime pH alterations are probably the result of a change in the orientation of the bird during diving.
has a wide range of applications, e.g., in industrial process control, limnomarine sciences, or physiological studies. Often, samples of interest are transferred to atmospheric pressure conditions before analysis so as to overcome the problems caused by the high ambient pressures. In cases where this is not possible due to an induced change of the sample composition (e.g., CO214) or where continuous recording is desired, reference systems should allow reliable in situ measurements. Here, lack of simple, inexpensive apparatus impedes the use of these techniques in field studies. The reference electrode presented in this work might help to remedy this. With the simple pressure-equalizing system employed, the electrode could be operated over a wide range of repeatedly changing pressure regimes without any significant influence on signal stability or on the operating life of the electrode. The electrode design combined the advantages of a solidified electrolyte with the precise reference potential of a free-flowing, free-diffusion liquid junction.11 The use of a gel-stabilized electrolyte in connection with a valve-controlled flow reduction allowed the present system to have a junction with low electrical resistance while maintaining a reduced flow rate. This could be realized because the site of the main flow reduction was inside the electrode rather than at the junction itself. Thus, the electrical resistance could have been further reduced without altering the flow characteristics, by shortening the capillary (to conform with the German standard DIN 1926415). However, in the present study, an extended capillary was preferred in order to minimize the risk of invasion by gastric juice. The spring-driven outflow of electrolyte effectively prevented any inward seeping of sample solution. Nevertheless, the slight short-time shifts in the cell potential which occurred concomittant with abrupt pressure changes should not have been present if there were perfect pressure equalization. Small air bubbles may have been responsible for this if they were present in the anterior part of the electrode and became compressed under the elevated pressure. Since the cross-sectional area of the junction was wider than that of the needle valve, the relevant volume reduction may not have been immediately balanced by electrolyte flux. Flow reversal might thus have occurred for a short period within the (14) Peck, D. V.; Baker, J. R.; Hillman, D. C. Verh. Int. Ver. Limnol. 1988, 23, 903. (15) DIN 19264: pH-Messung, Bezugselektroden; Beuth-Verlag: Berlin, 1985.
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Figure 5. Path-dependent distribution (cx/c1) of the sample concentration within the junction for flow rates of 24 (a), 10 (b), 5 (c), and 2.5 µL day-1 (d).
junction which effected the potential shift, either by disrupting the diffusion zone in front of the junction or by inducing an electric field due to electroosomotic flow.16 In support of this, the effect decreased with higher pressures, due to the fact that the absolute volume of a given air bubble decreases inversely with increasing pressure, so the extent of the resulting flow reversal would decrease rapidly during the initial steps of pressure increase. However, the short duration and the low extent of the potential shift observed (a 1 mV potential change corresponds to an error of 4% in the determination of the concentration of a monovalent ion) will render negligible for most applications. The strong memory effects normally associated with reference electrodes containing solidified electrolytes were effectively eliminated by the relatively high outflow rate. At a flow rate of 4 µL h-1, for example, the electrolyte in the capillary proceeded with a speed of 5 mm min-1, which results in a total renewal of the junction volume every 2 min. Even at a flow rate of 1 µL min-1, the outward flux was still high enough to impede any inward diffusion, as can be illustrated by the following calculation. The concentration distribution of sample solution within the liquid junction as a function of the distance from the inside can be well approximated by the formula described by Galster:7
cx exp(qERx/DFEx1) - 1 ) c1 exp(qER/DFE) - 1
where cx is the path-dependent concentration of sample solution, c1 the concentration of sample solution, qE the junction outflow (mL day-1), R the junction resistance (Ω), x the distance within the junction, measured from the inside, x1 the total junction length, D the diffusion coefficient (1.5 cm2 day-1 for aqueous solutions), and FE the electrolyte resistivity (2.5 Ω cm for 3 M KCl). Thus, it follows that, at a flow rate of 1 µL h-1, a significant proportion of sample solution would only invade into the outer few tenths of a millimeter of the junction (Figure 5). Less than (16) Lucy, C. A.; Underhill, R. S. Anal. Chem. 1996, 68, 300-305.
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30% of the sample concentration is found to diffuse into the outer 0.1 mm, whereas only 8% of the sample concentration remains at 0.2 mm, a section which is totally displaced within 10 s under these conditions. This indicates that the observed apparent memory effects are mainly a result of the diffusion potential and reflect the time needed to develop a stable diffusion interface in front of the junction. From these calculations, it can be concluded that electrolyte outflow rates even lower than those used in this study would probably give satisfactory results, unless samples of low ionic strength are to be analyzed. The only inconvenience of the new design presented here proved to be the refilling and the adjustment of the flow rate, which demands some experience. However, as demonstrated above, a particular flow rate may not be critical for the accuracy of the measurement but rather for the operating time. Handling might be further improved by using a different needle valve with a tighter thread. Using preferred flow rates, the electrode could be operated for up to 10 days with one filling. In applications where space is not limited, this may easily be extended to a multiple of that by simply upscaling the present design. The pressure range could be extended to infinity if the electrode were used so that the ambient pressure also acted on the exterior of the embedding resin, a feature that could not be realized in the present design because the electronics had to be kept under atmospheric conditions within the same titanium housing. In conclusion, the reference system proposed seems to be useful for achieving independence from ambient pressure. In contrast to solid-state electrodes, which are among the recent developments in this respect,10 this system is totally free from memory effects, needs no preconditioning, has a long operating life when regularly refilled, and shows only minor short-time potential changes upon pressure alterations in the lower pressure range. In addition, with the thin capillary tip, the system might be used to measure microgradients, such as in sediments, with the reference electrode being near the ion-selective electrode in order to reduce the influence of diffusion potentials. ACKNOWLEDGMENT Thanks are due to Dieter Adelung and Rory Wilson for their advice and support. Gu¨nter Dorn manufactured vital parts of the electrode. G. Hensel from Hensel & Partner produced “minute” quantities of custom-made compression springs. Marc Hebert from Microelectrodes, Inc. and G. Hilgenberg from Hilgenberg GmbH kindly provided free samples of material. Thanks are further due to Rory Wilson for making critical comments on the manuscript and for improving the English. This work was partially supported by the Deutsche Forschungsgemeinschaft (DFG Ad 24/11) and the Volkswagen-Stiftung (Az I/70 122). Received for review December 17, 1996. Accepted March 26, 1997.X AC961275T X
Abstract published in Advance ACS Abstracts, May 15, 1997.
