tern was recorded for equal concentration changes under stirred and unstirred conditions. The results are shown in Figure 12 where the potential is plotted vs. time for a concentration change from 1 t o 10 ppm F- in 11'3 TISAB. Curve a represents the response under unstirred conditions, curve b was obtained under vigorous stirring. I t is obvious t h a t the response is much faster in the latter case. T h e influence of stirring on the time response is typical for a film diffusion controlled process. I t can be concluded that the intrinsic response of the Orion 94-09A fluoride electrode is fast enough t o use it as a sensing element in automated potentiometric systems, but that its actual response is limited by a film diffusion process. An appropriate cell module in which a turbulent flow is maintained as, for instance, by vigorous stirring of the internal solution, should allow operation of the analyzer a t sample rates competitive with those of colorimetric systems.
LITERATURE ClTED (1) (2) (3) (4) (5) (6) (7) (8) (9) (10)
P. W. Alexander and G.A. Rechnitz, Anal. Chem., 46, 860 (1974). P. W. Alexander and G. A. Rechnitz, Anal. Chem., 46, 1253 (1974). B. Fleet and H. von Storp, Anal. Chem.. 43, 1575 (1971). B. Fleet and H. Y. W. Ho, Talanfa, 20, (go), 793 (1973). J. Ruzicka and J. C. Tjeii, Anal. Chim. Acta, 47, 475 (1969). R . T. Oliver, G. F. Lenz, and W. P. Fredericks, "Advances in Automated Analysis", Volume iI, p 309, Technicon International Congress, 1969. I . Sekerka and J. F. Lechner, Anal. Lett., 7, 463 (1974). 2s. Feher, G. Nagy, K. Toth, and E. Pungor, Ana/yst (London), 90, 699 (1974). P. Van den Winkel, J. Mertens, G. De Baenst, and D. L. Massart, Anal. Lett., 5, 567 (1972). J. Mertens, P. Van den Winkel, and D. L. Massart, Anal. Lett., 6, 81 (1973) -I J. Mertens, P. Van den Winkel, and D. L. Massart, Bull. SOC. Chim. Belg., 83, 19 (1974). J. Mertens, P. Van den Winkel, A. Henrion-Boeckstijns, and D. L. Mas\
(11) (12)
sart, J. Pharm. Belg., 2, 181 (1974). Presented at the IUPAC Symposium, Cardiff, Wales, April 1973. (13) J. Mertens, P. Van den Winkel, and D. L. Massart, Anal. Chem., 47, 522 (1975). (14) K. Toth and E. Pungor, Anal. Chim. Acta, 57, 131 (1971). (15) K. Toth and E. Pungor, Anal. Chlm. Acta, 64, 417 (1973). (16) G. A. Rechnitz and H. F. Hameka, Z.Anal. Chem., 214, 252 (1965). (17) G. A. Rechnitz and G. C. Kugler, Anal. Chem.. 39, 1682 (1967). (18) M. J. Brand and G. A. Rechnitz, Anal. Chem., 41, 1169 (1969). (19) M. J. Brand and 0.A. Rechnitz. Anal. Chem., 41, 1788 (1969). (20) M. J. Brand and G. A. Rechnitz, Anal. Chem., 42, 478 (1970). (21) G. A. Rechnitz in "Glass Electrodes for Hydrogen and Other Cations", G. Eiseman, Ed., Marcel Dekker. New York, 1967. (22) R. Rangarajan and G. A. Rechnitz. Anal. Chem., 47, 324 (1975). (23) J. Juillard "Etude de la dissociation des acides en solvants organiques et hydroorganiques", Thesis, University of Clermont-Ferrand. France (1968). (24) G. Johansson and K. Norberg, J. Electroanal. Chem.. 18, 363 (1968). (25) B. Karlberg. Anal. Chim. Acta, 66, 93 (1973). (26) B. Fleet, T. H. Ryan, and M. J. D. Brand, Anal. Chem., 46, 12 (1974). (27) P. Van den Winkel, J. Mertens, and D. L. Massart, Anal. Chem., 46, 1765 (1974). (28) P. Van den Winkei, J. Mertens, and D. L. Massart. Collected papers of the Symposium "Chemistry and Life", Ghent University, Belgium, R . Ruyssen and W. Verstraete, Ed., 1973, p 1837. (29) W. Simon, Communication at the "Journee sur les electrodes specifiques et membranes liquides", Sepfember 1974, University of ClermontFerrand, France. (30) R . H. Muller, Anal. Chem., 41 (12). 113A (1969). (31) M. Vandeputte. L. Dryon, P. Van den Winkel. J. Mertens, and D. L. Massart, unpublished work. (32) C. Gavach, Communication at the "Journee sur ies electrodes specifiques et membranes iiquides" September 1974, University of CiermontFerrand, France.
RECEIVEDfor review June 16, 1975. Accepted October 14, 1975. T h e authors thank the Fonds voor Geneeskundig Wetenschappelijk Onderzoek (F.G.W.O.) for financial support. P a r t of this work was presented a t Euroanalysis 11, Budapest, August 1975.
Glass Microelectrode Probes for Routine pH Measurements J. D. Czaban and G. A. Rechnitz* Department of Chemistry, State University of New York, Buffalo, N. Y. 14214
Microbulb glass electrodes have been assembled, using a novel coaxial design, inside small hypodermic needles (23-26 gauge) as dip and spear type probes for routine pH measurements. Rlgld and flexible mlcroproblng assemblies were constructed as simple pH electrodes and as single unit combination electrodes with either a micro salt bridge or Ag/AgCI external reference element. The small size and versatility of these assemblies suggest their use for appllcations In microanalysis and cllnical pH monltoring.
Previously, we have described the fabrication and evaluation of a series of glass microbulb p H electrodes with tip diameters in the 100-500 micron range ( I ) . Such electrodes can be fabricated in a variety of bulb configurations (Figure 1).We now present the details of the incorporation of these microelectrodes into a variety of p H sensors which may have important applications in routine chemical microanalysis and clinical in vivo monitoring. Muscle surface pH monitoring represents a n important advance in the clinical assessment of the well-being of the
critically ill patient ( 2 ) ;however, the need for surgical implantation of bulky conventional electrodes is a serious drawback of the technique. Gebert and Friedman ( 3 ) developed a very small glass microcapillary electrode for implantation in rat muscle, although it too was positioned via a n incision in the animal's skin. A preliminary report by Mackenzie et al. ( 4 ) presented an intramuscular antimony oxide-silver/silver chloride pH combination electrode fabricated from an 18-gauge hypodermic needle. While this electrode can be implanted without a surgical procedure, its large size together with the many problems associated with the antimony electrode (5, 6), e.g., protein poisoning, drift, low accuracy, redox interference, and oxygen sensitivity, are likely to limit its use. Recently, Wakabayashi e t al. (7) described two new electrode devices for the percutaneous monitoring of muscle p H and oxygen pressure, respectively. The authors give no details on the construction of the pH electrode except t h a t it is a solid state device with a diameter of 1 mm and that it can be implanted using a 14-gauge metal cannula. I t is apparent, even from these latest publications, that there is a real need for a micro pH sensing assembly which ANALYTICAL CHEMISTRY, VOL. 48, NO. 2, FEBRUARY 1976
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trode configuration permits the employment of these probes with conventional measurement equipment and without the need for external electrostatic shielding. T h e resulting electrode probes should be useful not only for clinical measurements but also for other microanalytical purposes.
EXPERIMENTAL Figure 1 . Photomicrograph of microbulb conformations ( 7) (Top)round, (middle)semi-flat, and (bottom)oblong
M L
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ti
rb)
Figure 2. Schematic diagram of early microsensor design ( a ) Dip- and hypodermic-type probes and ( b ) enlarged view of assembled electrode stem. ( A ) Microbulb electrode, (6)silicone rubber, (C) stainless steel, (D)standard hypodermic needle hub, (0miniature coaxial connector, ( F ) 23-26 gauge hypodermic needle, (G) polyethylene sleeve, (4glass electrode stem, (4 AgIAgCI wire, (Jjinternal buffer solution, ( K ) silicone oil, ( L ) epoxy, (nn) Tefzel-insulated wire
can be administered through the skin via a small hypodermic needle, in order to eliminate surgical implantation and associated tissue trauma. In addition, the development of such an electrode system would have important ramifications in the area of fetal monitoring. Currently, there are two widely used techniques for the detection of fetal distress (asphyxia) during labor, Le., continuous fetal heart rate (FHR) monitoring and fetal blood sampling. Clinical studies indicate that continuous F H R is a good guide to the condition of the fetus that has a normal pH or one that is severely acidotic, but that it is less reliable in cases of mild or moderate acidosis. Although the F H R technique may serve to alert the physician t o the possibility of asphyxia, it is rarely possible to determine the significance of an abnormal F H R pattern with confidence unless the fetal p H is known (8). The p H is usually determined by making a small (1 mm) cut in the fetal scalp to obtain a droplet of blood which is then taken to the hospital laboratory for measurement. This may be done as many as twenty times during a high risk delivery. Despite this tedious and slow procedure, p H estimation of fetal scalp capillary blood is a n important technique in fetal monitoring, and, currently, it is the only reliable method for accurately determining the onset and clinical significance of mild or moderate fetal distress. There is no published method for the continuous in vivo monitoring of fetal blood or tissue p H as an indicator of fetal well-being during labor and delivery. Indeed, such a method has been suggested as an important goal of the future ( 9 , l O ) . The electrode probes described in this paper were developed to demonstrate the applicability of microbulb electrodes as components of direct sensors for possible clinical or biomedical use. T h e sensors were assembled inside 23-26 gauge hypodermic needles for use as dip or spear type probes and were found t o have excellent response characteristics over a wide p H range. A novel coaxial elec278
Procedure-Microprobe Assembly a n d Evaluation. Microbulb electrodes, prepared with Corning type 0150 p H sensitive glass, were incorporated into two types of p H probes: a n early experimental design in which the probe body-a stainless steel tubeacted as the electrode shield and a later, superior design in which a novel coaxial electrode configuration was used with t h e shielding a n integral part of the microelectrode stem. Although t h e original design has been replaced by the later style, a brief description of t h e former is included for completeness. When necessary, t h e operations described below were performed with t h e aid of a 25X microscope. As illustrated in Figure 2a, the microbulb electrodes were assembled in dip- and hypodermic-type probe configurations. A pretested microbulb electrode was filled with a buffered chloride solution (0.025 M Na2HP04, 0.025 M KH2P04, 0.11 M NaC1, saturated with AgC1) using a glass microcapillary needle, and subsequently injected with a layer of silicone oil (Figure 26). A small (0.005inch 0.d.) Ag/AgCl reference wire was soldered to a length of Tefzel-insulated wire (0.019-inch o.d.), bent in the "S" shape as shown and inserted into the aqueous solution through the silicone oil. T h e wire was sealed into the electrode with a small amount of epoxy (Araldit-Standard, Ciba-Geigy) and t h e assembly set aside while the epoxy cured overnight. T h e water-immiscible silicone oil protected the electrode seal from attack by the internal salt solution, while t h e shape of t h e reference wire prevented the oil from creeping into t h e epoxy as it hardened. T h e electrode was then epoxied (Araldit-Rapid, Ciba-Geigy) inside a metallic jacket constructed of telescopic sections of stainless steel tubing which had been soldered together. A stainless steel hypodermic needle (19 gauge) was cut off about 1 cm from t h e hub which was then inserted into t h e end of the electrode jacket. T h e wire from the electrode was threaded through the h u b and soldered t o t h e center conductor of a miniature coxial connector (Malco, 031-0003-0001) which was then screwed into the needle h u b (previously threaded with a 10-32 tap). Following this, t h e hub was soldered to t h e stainless steel jacket. In t h e dip-type probe, the tip of t h e microbulb electrode extended beyond the last section of the tubing while, in the hypodermic probe, the last section of tubing was fitted with a stainless steel needle and adjusted so that the electrode bulb was exposed in the bevel of the needle. In both configurations, the electrode jacket was connected as an electrostatic shield electrically in common with t h e external reference electrode. Therefore, in order t o ensure a stable reference potential, t h e shield was insulated from the sample fluid. This was accomplished for the dip-type electrode by coating the lower portion of the stainless steel with silicone rubber (General Electric RTV 118) and, for the hypodermic electrode, by inserting a tight fitting polyethylene sleeve between t h e last two sections of the probe body. T h e newer coaxial arrangement was used for a variety of probe designs including combination electrodes having an integral microsalt bridge or Ag/AgCl reference element incorporated within the probe, These coaxial assemblies were based on two general arrangements: electrodes with a rigid glass stem and those with a flexible stem. T h e steps involved in the assembly of the rigid type p H microprobe are depicted in Figure 3. A pretested microbulb electrode with a long stem (-10 cm) was cleaned in chromic acid, thoroughly rinsed, and allowed t o dry. A thin layer of silver epoxy (Acme, ESolder 3021) was smeared around the shoulder of t h e electrode stem and cured for 30 min a t 80 "C. T h e electrode was then placed in a vacuum metallizer (Edwards High Vacuum Ltd., Model 6E-2) and silver coated from t h e silver epoxy t o within a few millimeters of the bulb (Figure 3a). T h e delicate silver plate was protected and insulated with a very thin layer of high strength silicone rubber (General Electric R T V 7100). This two part coating was applied using a spray applicator with hexane as a diluent, followed by a heat cure (110 "C) for a t least 2 hr. T h e electrode was then filled with a p H 7.4 phosphate, 0.025 M NaCl buffer solution and equipped with an internal Ag/AgCl reference electrode as described earlier. In this case however, the Tefzel wire was replaced
ANALYTICAL CHEMISTRY, VOL. 48, NO. 2, FEBRUARY 1976
E
A
B
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D
Figure 5. Enlarged sectional view showing details of connection between flexible electrode and miniature coaxial cable H
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Silver wire from electrode soldered to center conductor of cable, (5) cable insulation slid down over polyethylene sleeve, (C) cable braid silver epoxied to stainless steel tubing, (D) modified hypodermic needle, (0heat shrinkable tubing (A)
i l l (bi
Figure 3. Schematic diagram of rigid type electrode probe ( a ) Application of coaxial silver shield, ( b )assembled electrode mounted in-
l-cm3 plastic syringe barrel, (c)placement of optional Ag/AgCI external reference wire; ( A ) microbulb electrode, (5)silver epoxy, (C)silver coating, (0) high strength silicone rubber (G.E. RTV 7100). ( E ) silicone rubber, (6 l-cm3 syringe, (G) epoxy, (h'~standard hypodermic needle hub, (0 miniature coaxial connector, (4 micro Ag/AgCI wire side
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Figure 4. Enlarged cross-sectional view of flexible microbulb electrode ( A ) Internal ( E ) silicone
buffer solution, (5)air bubble, (C) silicone oil, ( D ) Ag/AgCI wire, rubber, ( A epoxy, (G) polyethylene sleeve, (H) silver epoxy, (0 stainless steel tubing, (4silver wire, ( K )silver coating with a short length of miniature coaxial cable (Belden 8700). After t h e electrode seal was hard, t h e coaxial cable braid was moved down over the electrode and silver epoxied t o t h e original layer of epoxy. T h e coaxial cable was fitted with a miniature connector (as above) and t h e assembly placed inside a l-cm3 disposable syringe barrel (Figure 3 b ) . T h e distance t h e electrode extended from the stem of t h e syringe was adjusted so t h a t t h e electrode tip just protruded into t h e beveled portion of a hypodermic needle affixed t o t h e syringe. After making this adjustment, t h e electrode was permanently epoxied into the syringe with fast curing epoxy. A small reusable hypodermic needle (e.g., 25 gauge) was positioned over t h e microelectrode and secured t o t h e syringe stem using t h e following simple procedure. T h e syringe assembly a n d t h e needle were placed on a clean glass plate and t h e electrode was slowly advanced into t h e h u b of t h e needle. If t h e microelectrode was not properly aligned so as t o move into t h e needle stem, t h e needle would begin to move. T h e electrode was pulled back, rotated go", a n d another try was made. Although this was a delicate operation, t h e procedure worked well and no bulbs were broken. This new coaxial design not only provided more efficient shielding. it also allowed t h e needle t o be removed a n d replaced with a blunt needle for dip-type applications. Furthermore, because t h e silver coating and insulation were very thin, t h e gauge of t h e needle used was still determined by t h e diameter of t h e bulb. T h e utility of t h e rigid type of electrode design was improved by incorporating a micro-salt bridge inside t h e needle, thus forming a microprobe combination electrode. A very small capillary salt bridge was formed by extruding a hot polyethylene tube. T h e capillary was filled with a hot 1%Agar, 1 M KC1 gel solution a n d , after cooling, was threaded into an appropriately sized hypodermic needle (e.g., 23 gauge). A slot was c u t into t h e locking stem of t h e plastic syringe a n d t h e needle positioned over t h e electrode in t h e usual fashion. T h e salt bridge tubing was placed in t h e slot t o keep it from being crimped by the needle hub. T h e large end of the salt bridge was secured to t h e electrode body with a small rubber band a n d adjusted so t h a t t h e small end was adjacent t o t h e glass electrode in t h e bevel of the needle. This configuration was useful not only because it functioned as a single unit combination electrode b u t also because the leakage rate a n d composition of t h e salt bridge could be tailored t o a particular application. During this study, a custom-made semimicro-calomel electrode was used in conjunction with t h e salt bridge as t h e reference half cell. We also constructed one other type of combination electrode in this series using a Ag/AgCl wire as t h e external reference electrode.
A small (0.003-inch 0.d.) silver wire was etched in hot nitric acid t o a diameter of about 25 p , rinsed thoroughly a n d dipped in molten silver chloride, thus forming a Ag/AgCl microelectrode. T h e wire was cut t o a length of 2-4 m m a n d cemented with silver epoxy just behind the bulb of a n uncoated microelectrode (Figure 3c). T h e electrode was then silver plated and assembled in t h e usual manner as described above. This type of combination electrode may be especially useful for in vivo monitoring where leakage from t h e salt bridge might be undesirable. T h e second series of microelectrode probes, those with flexible stems, were more complicated t o assemble (Figures 4 a n d 5). A microbulb electrode was c u t about 4 m m from the bulb with a very sharp scribe, cleaned in chromic acid, a n d rinsed thoroughly. T h e inside surface of t h e electrode stem was treated with a siliconizing agent (Clay Adams, Siliclad) and dried a t 100 "C for 5 min. T h e end of a silver wire (0.005-inch o.d., 6 cm long) was etched in hot nitric acid until the diameter was about 25 p , chloridized in molten silver chloride, and shaped in a saw tooth pattern as shown in Figure 4. After checking t o see t h a t t h e AgiAgCl wire would fit inside t h e electrode stem, it was set aside until needed. T h e electrode bulb and about 0.2 m m of the stem were injected with internal filling solution (less than 0.1 gl) and a layer of silicone oil was added as usual. T h e electrode stem was rinsed both internally and externally with chloroform t o remove any traces of silicone oil. T h e micro reference wire was inserted into t h e electrode, and sealed in place with a very small drop of epoxy (overnight cure). A thin walled (--0.001-inch) sleeve, prepared by extruding a short length of polyethylene tubing, was positioned over t h e exposed portion of the internal reference wire and the end of t h e glass electrode stem, and sealed in place with silicone rubber (General Electric, R T V 116). T h e assembly was then silver plated as before in the vacuum metallizer. A short length of fine stainless steel tubing was positioned over the end of the polyethylene sleeve a n d connected t o t h e silver coating with silver epoxy. T h e electrode assembly was then sprayed with a dilute solution of silicone rubber (General Electric, R T V 116) t o form a thin sheath of insulation over t h e silver plating. T h e central conductor of a short length of miniature coaxial cable was butt soldered (Figure 5) to t h e internal reference wire and the cable insulation pushed forward over the polyethylene sleeve. T h e cable braid was then moved down over t h e insulation a n d cemented t o t h e stainless steel tube with silver epoxy. A short piece of heat shrinkable tubing was placed over the exposed braid, contracted by gentle heating, and t h e opening around the electrode sealed with a small amount of silicone rubber. T h e miniature cable was then fitted with a small connector to complete t h e electrode assembly. If the electrode was t o be housed inside a hypodermic needle, a slight modification was necessary. T h e needle h u b was cut off and a short ( 2 m m ) telescopic section of stainless steel tubing soldered over the blunt end of t h e needle. T h e electrode assembly was threaded into t h e needle, positioned so t h a t t h e tip was exposed in the bevel of t h e needle, and secured with fast curing epoxy. Care was taken to ensure t h a t the needle was not in contact with t h e shield of the coaxial cable. T h e exposed braid and t h e end of the needle were then covered with a section of heat shrinkable tubing. T h e needle could now be bent a t any point along its length beyond the first 5 m m after t h e bevel, provided i t was not severely crimped so as to damage t h e electrode insulation. Almost an identical procedure was used t o construct flexible pH-Ag/AgCl Combination microsensors. However, before t h e electrode was silver plated, a micro Ag/AgCl wire was silver epoxied t o t h e glass electrode stem in the region between the end of t h e polyethylene sleeve and t h e microbulb. After plating, electrical continuity was verified between t h e AgiAgCl wire and t h e silvered sleeve, a n d t h e assembly completed a s just described. Although each of the microbulb electrodes was pretested over the p H range 2 to 12 before use, the final probe assemblies were
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Figure 6. Photograph of rigid type microelectrode probe assembled inside l-cm3 plastic syringe barrel
Figure 8. Photoaraoh of flexible OH electrodes with and without 23gauge needle
Flgure 7. Photomicrograph of pH-salt bridge combination electrode inside 23-gauge hypodermic needle also tested under a variety of conditions. The pH and pH-salt bridge electrodes were evaluated at room temperature over the pH range 4 to 9 while the pH-AglAgCl combination electrodes were tested at 25 and 36 'C over the physiologically significant pH range of 6.8 to 1.7. In the later ease, known phosphate buffers were used with a background concentration of 0.10 M NaCI. Electrode potentials were measured using the preamplifier described previously (1) in conjunction with a standard research model pH meter.
RESULTS AND DISCUSSION Early experiments, using the original microsensor assemblies depicted in Figure 2a, were concerned with the determination of the p H gradient along acrylamide gels used for isoelectric focusing studies. The gradient was determined in a matter of minutes simply by positioning the p H probe and a glass micropipet salt bridge, using a micromanipulator, a t 5- or 10-mm intervals along the gel and reading the pH. This technique was not only more convenient and accurate than the conventional slicing method, it was also nondestructive, thus allowing other measurements, such as radioactive counting, to be performed on the same gel. As mentioned earlier, the original probe design was replaced with the newer, more versatile construction, shown as a completed unit in Figure 6. This unique design had a number of important features: a) The novel coaxial arrangement of the electrode shield was very effective in eliminating electrostatic interference. The electrodes were tested under normal laboratory conditions (Le. without a Faraday cage) and were found t o exhibit stable potentials with no response t o nearby motion. h) The electrodes were used as either dip- or hypodermic-type probes with replacement of the needle accomplished quickly and conveniently. c) The electrode stem can he sterilized using chemical, gas or irradiation techniques. d ) The probes were assernbled as simple p H electrodes and as Combination electrodes. In the latter case, either a AgiAgCl reference electrode or a micro-salt bridge (see Figure 7) was incorporated within the probe needle. The simple p H microprobes, when coupled with an external semimicro saturated calomel reference electrode, exhibited a near Nernstian p H response (slopes >58.0 mV/pH unit) over the range tested (pH 4.01 to 9.18). These same electrodes also responded well when evaluated with a micro-salt bridge. However, in this case, the slopes were slightly lower due t o small fluctuations in the liquid-liquid junction potential at the salt bridgeisample interface. One way to eliminate the errors of a varying junction potential is to remove the liquid junction, as was done with the pHAgiAgCl Combination microprobes. The AgIAgC1 reference electrode has been widely used in p H cells without liquid junctions and bas been thoroughly discussed by Janz (11). 280
I
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Figure 9. Photomicrograph of flexible pH-AgIAgCI combination electrode without silicone rubber insulation Band and Semple (12) have used the Ag/AgCl reference electrode in their intra-arterial p H assembly and found that physiological variations in the chloride concentration had a negligible effect on the observed pH. The rigid type pH-Ag/AgCI microelectrodes exhibited excellent p H response over the biologically important p H range of approximately 6.8 to 7.7 in phosphate buffer solutions containing a constant chloride ion activity (0.10 M NaC1). Experiments a t room temperature and at 36 "C showed that the electrode slopes were within 1 mV/pH unit of that of a commercial pH-calomel cell. At elevated temperatures, the resistance of the glass electrodes decreased sharply; the resistance a t 36 "C was practically half that a t 25 "C, thus improving the observed response time. (Because of the total input capacitance (CIN) of the preamplifier which must be charged through the source resistance of the glass sensing membrane (RE), the observed response time was partially determined by the RECINtime constant.) The flexible pH and pH-Ag/AgCl electrode assemblies (see Figures 8 and 9) also exhibited slopes in excess of 58 mV/pH unit over the p H ranges 4.01 t o 9.18 and 6.80 to 7.73 (0.10 M NaCl), respectively. A typical combination electrode was found t o accurately detect a sudden shift in p H from 7.36 to 7.15 with a response time of about 45 sec. In clinical situations, a p H change of this magnitude would be indicativs In order f p H responsi sistance (> reference wire ana oom m e sample soiuuon a n a m e coaxial silver coating, Of the many insulating materials with which we experimented, the polyethylene sleeve was t h e only reliable insulator. During our early experiments, the intense thermal radiation from the heating element of the vacuum metallizer would distort this thin walled plastic tubing. However, this was minimized by positioning the electrode far from the source and by using a low heating current. The silver coating often showed a nonuniform bluish discoloration apparently due t o impurities in the polyethylene; however, this did not appear to affect the electrical conductivity. We attempted to use the silver shield directly as the external reference electrode by electrolytically chloridizing it in dilute hydrochloric acid. This approach was abandoned, however, because the silver invariably peeled away from
ANALYTICAL CHEMISTRY, VOL. 48. NO. 2. FEBRUARY 1976
the glass or polyethylene substrate, even a t very low current densities. T h e generally larger diameters of the flexible electrode assemblies limited t h e needle size with which they could be used t o 23 gauge or larger.
C 0N CL U SIO N
It has been demonstrated that glass microbulb electrodes can be fashioned into a variety of rigid and flexible p H sensing assemblies. Dip and hypodermic type microsensors were evaluated in vitro and generally found t o have all the desirable characteristics of ordinary glass electrodes, suggesting their use for both routine and novel analytical measurements. While it was not possible to test the various microprobes in vivo, it was evident t h a t they represent a n important improvement over the devices presently used for such life saving techniques as muscle p H monitoring. Furthermore, the adaptability of the microelectrodes t o new probe configurations should prepare the way for new biomedical techniques such as continuous fetal pH monitoring.
LITERATURE CITED (1) J. D. Czaban and G. A. Rechnitz. Anal. Chem., 47, 1787 (1975). (2) R. M. Filler and J. B. Das, "Muscle pH, pOn, pC02 Monitoring: A Review of Laboratory and Clinical Evaluations", in "Ion-Selective Microelectrodes", H. J. Berman, and N. C. Hebert, Ed., Adv. Exp. Med. Biol., 50, 175 (1974). (3) G. Gebert and S. M. Friedman, J. Appl. Physiol., 34, 122 (1973). (4) J. W. MacKenzie, A. J. Salkind, and S. R. Topaz, J. Surg. Res., 16, 632 (1974). (5) R. M. Bates, "Determination of pH, Theory and Practice", 2nd ed., J. Wiley and Sons, New York. N.Y., 1973, p 300. (6) R. Green and G. Giebish. "Some Problems with the Antimony Microelectrode", in "Ion-Selective Microelectrodes", H. J. Berman and N. C. Hebert. Ed., Adv. Exp. Med. B i d , 50, 43 (1974). (7) A. Wakabayashi, Y. Nakamure, T. Wooley, R. J. Mullin, H. Watanabe, 1. Ino, and J. E. Connolly, Arch. Surg.. 110, 802 (1975). (8) R. W. Beard, Pediafrics, 53, 157 (1974). (9) J. F. Roux, M. R. Neuman, and R. C. Goodlin, Crir. Rev. Bioeng., 2(2), 119 (1975). (10) H. J. Berman and N. C. Hebert, Ed., "Ion-Selective Microelectrodes", Adv. Exp. MedBiol., 50, 188 (1974) (11) G. J. Janz, in "Reference Electrodes", D. J. G. lves and G. J. Janz, Ed., Academic Press, New York, N.Y., 1961, Chap. 4. (12) D. M. Band and S. T. G. Semple, J. Appl. Physiol., 22, 854 (1967).
RECEIVEDfor review September 8, 1975. Accepted October 20, 1975. We gratefully acknowledge the financial support of a grant from the National Institutes of Health.
On-Line, Computer-Controlled Potentiometric Analysis System John M. Ariano and W. F. Gutknecht" Department of Chemistry, Duke University, Durham, N.C. 27706
A computer-controlled, direct potentiometric analysis system has been developed. During a typical analysis, a series of standard additions are made to an unknown solution in a cell containing an ion-selective electrode and a reference electrode. These additions are optimized in that the volume of each is automatically adjusted so as to yield an even distribution of resulting cell voltages. The cell voltages, which are acquired using a computer-optimized sampling technique, and the standard addition data are fit to the Nernst equation using the nonlinear least squares procedure. This system has been used to automatically analyze for potassiM, with um ion over a concentration range of lo-' to the resulting accuracy and precision both being about 2 YO.
Over the past several years, the small digital computer has become an almost commonplace instrument in many analytical laboratories. In most cases, however, the small computer has been used only for calculations, or relatively simple instrumental control and data acquisition (1-4). Little has been done in the way of computer-controlled real-time experimental alteration; that is, having the computer alter a n experiment during the course of that experiment so as to optimize the analytical output. Perone e t al. ( 5 ) have described one such system, however, wherein stationary electrode polarographic measurements were optimized by means of real-time computer control. It is in such applications that the real power of the computer-in terms of its capabilities for rapid calculation, rapid acquisition of high precision data. and control of the instrumental system-is used to the fullest extent.
In the work presented here, a small digital computer has been used for the real-time control of a direct potentiometric analysis system. Several of the computer-controlled operations which are a part of this system were optimized so as to permit automated analyses, with the accuracy and precision of these analyses both being about 2%. In the system, a series of standard additions are made under computer control to a n unknown solution in a cell containing a pair of electrodes (ion-selective and reference). T h e volume of each of the standard additions is optimized under computer control. In general terms, this volume optimization involves increasing the volume of each successive standard addition so as to yield an even distribution of resulting cell voltage values. In addition, the cell voltage readings after each addition are acquired using a unique data sampling and averaging technique. T h e concentration of the unknown is calculated by fitting the standard addition data t o the Nernst equation
after a method described earlier by Brand and Rechnitz (6) In Equation 1, E , is the cell voltage, E o is the standard potential, C, is the concentration of the unknown, Vu is the volume of the unknown, C, is the concentration of an individual standard addition and V , is the volume of a n individual standard addition. Concentration could be used here in place of activity as the changes which occurred in the ionic strength of an unknown solution during analysis were minimized by making all solutions 0.5 M in an indifferent electrolyte, MgSOJ. The three unknowns determined in the fitting process are E " , RTInF, and C,. ANALYTICAL CHEMISTRY, VOL. 48, NO. 2, FEBRUARY 1976
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