552
Anal. Chem. 1981, 53, 552-554
pressure, and discharge gap distance. The results of these experiments utilizing 1-octanol, m/z 130 (which produces an (M - H)’ ion), as the sample molecule can be seen in Table 11. From these data it can be seen that as the discharge voltage was increased the background ion noise level increased much faster than the sample ion intensity. The reagent ion intensity maximized a t a discharge gap distance of 0.3 mm, the distance separating the electrode from the probe tip. The optimum operating conditions are a discharge voltage of 1200 V with a quadrupole chamber pressure in the range 8-9 X 10-5torr. The results obtained by using these optimum conditions have been replicated on different days with many changes of gap distance and source pressure in the interim. The Townsend discharge ionization probe described in this paper, which is used routinely with oxidizing-~reagent gases, is simple t o build and enables rapid changeover from filament t o discharge mode.
ACKNOWLEDGMENT The authors wish to thank E. W. Pittenger and W. A. Grote for their technical assistance.
LITERATURE CITED (1) Hass, J. R.; Friesen, M. D.; Hoffman, M. K. Org. Mass Spectrom. 1979. 14. 9. (2) Hunt,’ D. F.; Stafford, G. C., Jr.; Crow, F. W.; Russell, J. W. Anal. Chem. 1976, 48, 2098. (3) Carroll, D. I.; DzMic, I.; Stillwell, R. N.; Haegeie, K. D.; Horning, E. C. Anal. Chem. 1975, 4 7 , 2369. (4) Hunt, D. F.; Harvey, T. M. Anal. Chem. 1975, 47, 2136. (5) Hunt, D. F.; Harvey, T. M. Anal. Chem. 1975, 47, 1965. (6) Hunt, D. F.; McEwen, C. N.; Harvey, T. M. Anal. Chem. 1975, 4 7 , 1730. (7) Horning, E. C.; Carroll, D. I.; Dzidic, I.; Haegele, K. D.; Horning, M. G.; Stillwell, R. N. J . Chromafogr. 1974, 99, 13. (8) Hunt, D. F.; Ryan, J. F. J. Chem. Soc., Chem. Commun. 1972, 620. Sml; A. L. C.; Field, F. H. J . Am. Chem. Soc. 1977, 99, 6471 (9)
RECEIVED for review May 23,1980. Accepted November 17, 1980.
Miniature Glass pH Electrode with Nonaqueous Internal Reference Solution R. F. Savlnell Department of Chemical Engineering, University of Akron, Akron, Ohio 44325
c. c. Liu” Department of Chemical Engineering, Case Western Reserve University, Cleveland, Ohio 44 106
T. E. Kowalsky School of Medicine, Temple University, Philadelphia, Pennsylvania 19 122
J. B. Puschett Renal-Electrolyte Division, Allegheny General Hospital and School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 152 12
The miniature p H electrode is an essential analytical tool for biological laboratory investigations. For example, measurements of pH values in the proximal and distal tubules of the kidneys of rats and dogs have been made with micro glass p H electrodes by using micropuncture techniques. (I, 2). The complexities involved with fabricating micro glass pH electrodes are evident in the literature (2-4). The active electrode tips of proper dimensions are obtained by pulling a pH-sensitive glass capillary from beneath an insulating coating (2)or by fabricating an active tip by use of microforge techniques ( 4 ) . In either case, the glass capillary has to be filled with an internal reference solution, namely, known molality HCl solution. This requirement is detrimental to the physical dimensions of the micro glass pH electrode. The extended time of contact between the electrode tip and the internal aqueous reference solution leads to over-hydration of the glass membrane. Over-hydration results in gelling of the electrode tip and consequently renders it unusable for micropuncture applications. Thus, the percentage of failure for making usable micro glass p H electrodes becomes very high. In order to minimize the tip gelling of micro glass pH electrodes, we used a unique approach in dealing with the internal reference solution. Our approach employs a nonaqueous solvent, eliminating the water molecules from the standard inner electrolyte. The experimental studies which substantiate this approach are described in this communication.
EXPERIMENTAL SECTION For the investigation of the applicability of various nonaqueous electrolytes, macro glass pH electrodes were first fabricated and tested. The macro glass pH electrode had a bulb-shaped active
surface area with a diameter of 3.5-9 mm. These electrodes were used in studies designed to compare the performance of electrodes having a nonaqueous internal electrolyte with those having an aqueous internal electrolyte. Finally, micro glass pH electrodes were prepared with the selected nonaqueous electrolyteto evaluate their response to solution pH and also to determine the integrity of the tip profile. Both the macro and micro glass pH electrodes were constructed with pH-sensitivie glass. Capillary Corning 015 glass tubing of 2 mm outside diameter in the length of 10 cm was first cleaned with detergent and methanol and then air-dried. Some of the microelectrode capillaries were insulated prior to tip fabrication. This was accomplished by following procedures similar to Rector et al. (2). A lead oxide insulator coating was prepared by thinning 3 g of PEMCO (Glidden-Durkee Co., Cleveland, OH) in 5 mL of technical grade turpentine. The coating was applied by dipping a capillary in a briskly stirred PEMCO solution. Five coats were applied with intermittent drying. The coating was baked in an air atmosphere at a temperature of 45MOO OC for 0.5-24 h. In the case of the macroelectrode, a bulb was blown at one end of the uninsulated capillary tube, the size of which ranged from 3.5 to 9 mm. For production of a microelectrode tip, either an insulated or an uninsulated glass capillary was mounted in a vertical pipet puller (David Kopf Instruments, Tujunga, CA). The heat and time of pull were adjusted until tips came within the 1-2-pm range. Although tip lengths varied slightly, all microelectrode tips did conform to the profile shown in Figure 1. Each microelectrode was visually inspected with an optical microscope (1OOX). The tips of the glass electrodes were examined for proper diameter, length, and tip closure. In the case of the insulated electrodes, it was noted that little insulation material appeared within 15 pm of the tip apex. The fabricated electrode capillaries were then filled with an internal electrolyte. The pH glass capillaries (secured to a holder) were placed at a 4 5 O angle, bulb or tips down, inside a beaker containing the internal electrolyte. The internal electrolyte was
0 1981 American Chemical Society 0003-2700/81/0353-0552$01.00~0
ANALYTICAL CHEMISTRY, VOL. 53, NO. 3, MARCH 1981 ~~
BNC CONNECTOR
Table I. Resistivities of the Internal Reference Electrolytes Evaluated in this Study, at 25 "C
EPOXY BEAD
3 I N T E R N A L REFERENCE
WIRE
i
Flgure 1. Assembled micro glass pH electrode and tip profile.
gradually heated up to 35 "C and then the beaker with the glass capillaries was placed under vacuum for approximately 1 h. The vacuum was removed, and in the case of the microelectrodes the beaker was then placed in an ice bath cooling to approximately 10 "C for 15 min. The electrolyte was then drawn into the capillary and filled the glass pH electrode. The internal reference electrode was either a silver or a silver/silver chloride wire with a diameter of 0.13 mm having a length of 5 cm. In the m e of a microelectrode, the wire was first soldered to a coaxial connecting pin (Amphenol BNC male connector pin) and then fed into the open end of the capillary. The pin was secured to the capillary with epoxy. The assembled electrode (see Figure 1 for microelectrode) was air-dried for 24 h. The completed electrodes were hydrated before testing. Various hydration procedures were tried, and the best one in our experience appeared to be immersion of the electrode in deionized water for 20 h. Microelectrodes were hydrated at 5 "C in order to slow down the rate of hydration and preserve the tip integrity. Measurementsof the zero-current potential between the pH glass electrode and a standard reference (either calomel or Ag/AgCl) were accomplished by using a Kiethley Model 616 high-impedence electrometer or a Corning Model 12 pH meter. In measurements against the Ag/AgCl reference electrode, the reference solution was 0.1 N HCl, and it was contacted with the buffer test solution through a saturated KC1-agar bridge. The test solution used was standard phosphate buffers prepared commercially by Fisher Scientific. In some tests a high ionic strength buffer was prepared by adding 0.20 M NaCl and 0.20 M KCl to the standard Fisher Scientific buffer. The pH of the modified buffer was measured with a calibrated, commercial type, glass pH electrode.
RESULTS AND DISCUSSION In this experimental study, the internal reference electrolyte was comprised of ionic solutes in an aprotic solvent. The rationale for choosing aprotic solvents was based upon their relatively high dielectric constants and favorable solvation properties. The solutes used in this study were benzoic acid (-0.2 M) and silver nitrate (0.1 M). Among the aprotic solvents, methanol and ethanol were tried without much success, but acetonitrile proved to work quite well. The performance of electrodes utilizing a nonaqueous internal reference electrolyte (0.2 M benzoic acid and 0.1 M silver nitrate in acetonitrile solvent) and the performance of electrodes utilizing an aqueous internal reference electrolyte (0.01 N HC1) were compared. For electrodes of all three internal reference electrolytes, the relation between the zero-current potential and the buffer pH was linear and the slope Nerstian. Although the response of all electrodes to pH change were the same, the performance of the electrodes did vary somewhat depending on the internal reference electrolyte. For example, the electrodes having a 0.01 N HC1-aqueous internal electrolyte responded relatively fast to a pH change; namely, it required 12 s for the elctrode to come to equilibration. In
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553
internal reference electrolyte 0.01 N HCI-aqueous 0.1 M AgN0,-acetonitrile 0.2 M benzoic acid-acetonitrile
resistivity, n cm 2.3 X 1.6 X 1.3 x
lo2 loz los
addition, these electrodes gave a very stable response. The response of the electrodes having the 0.1 M AgN03/acetonitrile internal electrolyte were stable, but the response times were longer (15-21 s) as compared to the aqueous-filled electrodes, The electrodes having 0.2 M benzoic acid/ acetonitrile exhibited considerably longer response time (-40 s or 4 times the required response time of aqueous containing electrodes) and their stability was only fair. The variation of the response times and stability between electrodes having different internal electrolytes can be partly attributed to the resistivities of the internal electrolyte which, in turn, influences the time constant due to the product of the resistance and capacitance of the electrode. The resistivities of these electrolytes were measured and are reported in Table I. As can be seen, both the 0.01 N HC1-aqueous solution and the 0.1 M AgNO,/acetonitrile solution have moderatley low resistivities while the 0.2 M benzoic acid/acetonitrile solution has a relatively large resistivity. Hence, the observed performance of the electrodes are consistent with the electrical properties of the internal electrolyte. The response of the macroelectrode having the 0.1 M AgN03/acetonitrile internal electrolyte was again evaluated after 55 days of storage in deionized water. Although some sensitivity was lost, the response of this electrode is strictly due to pH changes and not to changes in other ionic concentrations; a test procedure recommended by Carter (5) was performed. In this procedure, the ionic strength of the standard buffers was increased by adding NaCl and KC1. The test electrode gave true pH readings in spite of the difference in the buffer ionic strength, thus indicating the acceptability of this nonaqueous solution as an internal reference solution. Since the 0.1 M AgNO,/acetonitrile solution performed quite well as an internal reference electrolyte in tests with macro glass pH electrodes, this nonaqueous system was then employed for micro glass pH electrodes fabricated in our laboratory. Out past experiences with aqueous filled microelectrodes resulted in a high percentage of unusable electrodes because of the loss of tip integrity when the glass memebrane gelled during the f i i n g procedure. The electrodes prepared with nonaqueous solvents, on the other hand, showed no observable deformation of the glass electrode tip thus accomplishing one of the primary objectives of using a nonaqueous internal reference electrolyte. The response of uninsulated, nonaqueous microelectrodes to pH change was examined. We have found the response of these electrodes to be quite sensitive to the quality of the tip profile. However, for 11 electrodes studied, the relation between the zero-current potential of the micro glass pH electrode and the pH value of the buffers was found to be linear with Nernstian slopes in most cases. Micro glass pH electrodes that have been insulated also were tested for their zero-current potential response to change in buffer pH value. The purpose of insulating the electrodes is to restrict the pH sensitivity of the electrode to within a limited distance (e.g., 100 pm) from the tip apex. In this way, a measurement of pH within a tubule can be made without interference of external fluids. A total of 27 insulated micro glass pH electrodes were fabrication and tested. However, of these only three electrodes exhibited a response greater than 40 mV/ [pH unit] while none exhibited a response greater than
554
Anal. Chem. 1981, 53, 554-555
50 mV/[pH unit]. We suspect one of the reasons that these electrodes responded poorly or failed to respond at all might have been related to the nature of the insulating procedure. The treatment of the pH glass at elevated temDeratures most likely causes an irreversible dehydration of the pH-sensitive glass, resulting in poor performance of the electrode. The results of this study show that the use of a nonaqueous internal reference solution prevents over-hydration and maintains tip dimensions and quality throughout the filling procedure and provides good Nernstian response. Also, this study suggests that the method of insulating the electrode with a lead oxide coating does not produce a micro glass electrode having pH-sensitive tip. Better micro glass pH . electrodes can be reproducibly prepared by using an internal reference electrolyte of acetonitrile-0.1 M AgN03 and following the fabrication procedures outlined by Pucacco and Carter (4). Another route to obtaining high-strength electrodes suitable for micropuncture studies may be by applying the recently developed Pd/PdO electrode described by Liu et al. (6) and by Grubb and King (7).
ACKNOWLEDGMENT The technical assistance given by Daniel Bradshaw was gratefullyappreciated,
LITERATURE CITED (1) Windhager, E. E. In “Molecular Biology and Medlcine Serbs”; A p p k tonCentury-Crofts: New York, 1968. (2) Rector. F. C.; Carter, N. W.; Seldin, D. W. J . Clln. Invest. 1985, 44, 278-289. (3) Puschett, J. B.; Zurback, P. E. KMney Int. 1974, 6 , 81-91. (4) Pucacco, L. R.; Carter, N. W. Anal. Blochem. 1978, 89, 151-161. (5) Carter, N. W. Yale J . B b / . Med. 1972, 45, 349-355. (6) Liu, C. C.; Bocchicchio, B. C.; Overmyer, P. A,; Neuman, M. R. Sclence 1980, 207, 186-189. (7) Grubb, W. T.; King, L. H. Anal. Chem. 1980, 82, 270-273.
RECEIVED for review September 19,1980. Accepted November 3,1980. Support from the Chemical/Petroleum Engineering Department of the University of Pittsburgh and from the Department of Medicine, Renal-Electrolyte Section, Allegheny General Hospital, both in Pittsburgh, PA, are gratefully acknowledged.
Controlled-Rate Evaporator for Thousand-Fold Concentration George D. Price’ Department of Entomology and Nematology, University of Florida, Gainesville, Florida 326 7 7
David A. Carison Insects Affecting Man and Animals Research Laboratory, AR, SEA, US. Department of Agrlcutture, Gainesville, Florida 32604
In the course of a continuing investigation of human skin emanations that attract female mosquitoes (1-4), it was necessary to concentrate quantitatively extracts of those emanations. The concentration was carried out in the evaporator shown in Figure 1. A Buchler rotary evaporator, minus the usual condenser and fixed at a 45’ angle, was fitted a t the top with a rubber stopper pierced by a longitudinally adjustable 1/8 in. stainless steel tube and a disposable pipet; at the bottom was attached an approximately 10-mL glass tube having a 1.7 mm i.d. x 20 mm well. The well was calibrated in 10-pL increments to 50 pL with a microliter syringe. Approximately 600 mL/min of nitrogen was passed into the apparatus through the stainless steel tube and out through the needle during each concentration. The end of the tube, which determined the “turn around” point of the nitrogen, was adjusted to a position just above the no. 7 Ace-Thred joint (Ace Glass Co.). A measured volume of the extract to be concentrated was placed in the 10-mL tube and heated by means of the metal floored constant-temperature water bath, which was held at a temperature 5 O C above the boiling point of the solvent. The sample level was monitored through the transparent walls of the bath. The evaporation rate was a function of four variables: (1) nitrogen flow rate, (2) stainless tube position relative to the refluxing solvent, (3) bath temperature, and (4) solvent surface area. Only the area varied appreciably; it decreased markedly when the solvent level entered the well. The decrease made it relatively easy to stop the concentration at the desired well calibration mark and thereby quantitate the results. Because human emanations were known to contain a mixture of fatty acids, the recovery of propionic, butyric, and 0003-2700/81/0353-0554$01 .OO/O
Table I. Reproducibility of Recovery from Ether Solution Using Evaporator % recovery
trial 1 2 3
mean % % std dev
propionic butyric valeric acetopheacid (bp acid (bp acid (bp none (bp 141°C) 163°C) 187°C) 202°C) 73.0 83.4 70.4 75.7 6.9
70.0 106.4 76.4 84.3 19.4
87.2 85.3 107.8 93.4 12.5
91.6
90.5 109.9 97.3 10.9
valeric acids plus acetophenone added as an internal standard was carried out as follows: One drop of each of the acids and of the acetophenone was dissolved in 10 mL of absolute ether (propionic acid, 12.68 mg; butyric acid, 12.77 mg; valeric acid, 13.09 mg; and acetophenone, 19.49 mg). From the resulting nominal 1pg/pL stock solution were prepared serial dilutions at 0.1,0.01, and 0.0o01 &pL. Twenty milliliters of the 0.0o01 pg/pL dilution was then concentrated to 20 pL, a lO00X concentration. With 100% recovery, 4.5 pL of this solution should have contained the same quantity of solute as 4.5 pL of the 0.1 pg/pL dilution. Quantitation was accomplished by the method of Ackman and Burger (5), using a Varian 2100 gas chromatograph equipped with a flame ionization detector, a glass column (1.8 m X 2 mm i.d.) containing 25% NPGA on Chromosorb W AW (100-120 mesh), and a HewlettPackard 3380A recording integrator. Actual percent recoveries in three trials of the apparatus are given in Table I. The solvent was diethyl ether (bp 35 OC). 0 1981 American Chemical Society
