Anal. Chem. 1980, 52,375-377
and its iodinated products (or, for that matter, any other derivative) compared to similar products of a suitable precursor.
LITERATURE CITED (1) Aue, W. A,; Paramasigamani, V.; Kapila, S. Mikrocbim. Acta 1978(I), 193.
(2) Paiamasigamani, v.; Aue. W. A. Anal. chim. Acta 1978, 98, 393.
375
(3) Paramasigamani, V. Ph.D. Thesis, Dalhousie University, 1979.
RECEIVED for review August 13, 1979. Accepted November 1,1979. Material taken fr;m doctoral thesis ofthe first author. This research was supported by NRC grant A-9604 as well as by a Killam Scholarship and a Dalhousie RDF grant for one of us (v.P.).
Microanalytical Techniques with Inverted Solid State Ion-Selective Electrodes. I. Nanoliter Volumes G. L. Vogel,’ L. C. Chow, and W. E. Brown American Dental Association Health Foundation Research Unit, National Bureau of Standards, Washington, D.C. 2’0234
Several recent papers have demonstrated that solid state fluoride electrodes can be adapted for use with small volumes. However, the methods employed are somewhat cumbersome and require samples of 1-5 KL or greater (1-3). The major cause of these difficulties is the utilization of a standard reference electrode which requires that the microsample be sandwiched into a thin film spread across the entire fluoride-sensing element. This may result in excessive contamination of the specimen and necessitates the application of several aliquots of the same sample to the electrode to obtain a single reproducible reading. A procedure is described here which obviates these problems by utilizing a reference microelectrode brought into contact with a hemispherical microdrop of specimen deposited under mineral oil on the surface of an inverted electrode (Figure 1). Since the tips of such reference microelectrodes, in common use in biophysics, are typically less than 1 pm in diameter, the minimum volume required for a determination is limited only by the ability to make contact with the specimen without contaminating it. By using a micromanipulator to move the reference electrode while observing the specimen with a microscope, nanoliter size samples can be measured successfully. Furthermore, since many samples can be equilibrated on the surface of a single fluoride electrode simultaneously, the method is rapid. Two micromanipulators were used to position the electrodes. One micromanipulator held the reference microelectrode and served primarily to raise and lower it. The inverted fluoride electrode was clamped to the second manipulator which provided x,y horizontal movement for this electrode. All visual observations were made with a stereo zoom microscope with a variable magnification, 10-70X, equipped with a measuring reticule and cross hairs. An electrometer [Keithley model 6161 equipped with a recorder was used for all potentiometric measurements. The reference microelectrodes were manufactured by standard techniques ( 4 , 5). An Orion fluoride electrode, (94-09-00) was adapted for use in the inverted position by carefully filling it with fluoride reference solution so as to exclude air bubbles. After filling, the fluoride electrode was fitted with a rim to form a cup which holds the oil covering the electrode surface. This rim was constructed by coring out the center of a polyethylene bottle cap with a cork borer (Figure 1). Water saturated mineral oil was prepared by shaking mineral oil with distilled water for 2 min, followed by centrifugation. The fluoride standard buffers were made to contain 50% Orion Total Ionic Strength Adjustment Buffer and had a p H of 5.5. In the following discussion p[F] refers to the negative log of the fluoride concentration (moles per liter).
EXPERIMENTAL Prior to the application of specimens, the electrode surface is wiped with Orion silicone oil as per manufacturer’s instructions, and the surface is conditioned for 15 min with lo4 M fluoride standard so that the sensing element of the electrode is completely covered. The conditioning fluid is then completely sucked off the surface of the electrode with a plastic pipet tip attached to a vacuum source. Water saturated mineral oil is poured into the cup, and samples are deposited on the surface with an Eppendorf type pipetter. Specimens less than 1 pL can be deposited with oil-filled micropipets of the capillary (6) or constriction (7)type. The samples should form hemispheres on the surface and not spread excessively. Excessive sample spreading may require that the surface of the electrode be wiped again with silicone, oil, followed by reconditioning with IO4 M fluoride standard. When the desired number of samples has been placed on the surface of the electrode sensing element, the tip of the reference microelectrode is brought into the center of the microscope cross hairs and lowered close to the surface of the fluoride sensing element. This arrangement makes it possible to easily change specimens by aligning each in turn with the cross hairs, using the x,y movements on the manipulator attached to the fluoride electrode. After measurement, samples may be removed from the surface by vacuuming the surface of the fluoride electrode two times with a plastic pipet tip, adding more mineral oil between vacuumings. Although samples may be completely removed by this technique, fluoride adsorbed on the surface of the electrode can cause serious contamination when a dilute specimen is deposited on a part of the crystal previously occupied by a much more concentrated sample. This difficulty can be overcome by M fluoride standard for reconditioning the surface with a 15 min as before.
RESULTS AND DISCUSSION Figure 2 shows that with specimens more concentrated than -p[F] 5.3 (5 x 10” M), millivolt readings decrease monotonically to an equilibrium value and that with specimens more dilute than p[F] 5.3, the readings increase to a maximum and thereafter decrease. Furthermore, this effect becomes more pronounced with decreasing sample size. This difficulty appears to arise because of the solubility of the sensing element itself, which causes any specimen more dilute than -p[F] 5 to be slowly contaminated by dissolution of fluoride from the electrode surface. Thus, when such a dilute microsample is deposited on the surface of the electrode, the potential will increase as the equilibrium concentration of fluoride a t the surface of the electrode is depleted by diffusion into the bulk of the sample. With time, however, as the concentration gradient decreases, the rate of release of fluoride ions from the surface will exceed the rate of diffusion into the solution, and the millivolt
This article not subject to U S Copyright. Published 1980 by the American Chemical Society
376
ANALYTICAL CHEMISTRY, VOL. 52, NO. 2, FEBRUARY 1980 -REFERENCE MICROELECTRODE
MICROSAMPLES7
Table I. Apparent Fluoride Concentration and Standard Deviation, u, as a Function of Sample Size and Concentration Determined Near the Minimum Measurable Volumea
I N E R A L OIL PLASTIC C U P
p[F] measured at approximate 3x miniminimum minimum mum volvolume, nL volume ume
SENSING E L E M E N l
p[ F]
Maximum readings used. Sample and standards must be similar in volume
MACROELECTROOE ( O R I O N 94-09-00)
6.00
--
/
I--
.- 10 p I
$-I
16C
0
'PI
1
--.----p [F] = 5 p (F] = 5
p [F] = lv1
5
N
/--.--.I .
5.65
U
5.30
18f
170
250
N
Flgure 1. Fluoride electrode assembly
A
5.3
Y
5.89 8
0.25
0.03
5.17 6 0.04
5.21 10 0.04
Minimum readings used. Readings independent of sample volume 1
5.00
4.99 6 0.04 4.12
4.99 6 0.04
N
3
5
0.1
5 0.03
N 5
4.30
0.1
2
5 15f
7
4.31
a Readings of various nanoliter-size samples are compared with those obtained with volumes > 1 p L of the same solution used as standards.
Y
P
141 I
I
I
I
200
13f T I M E (MINUTES)
Flgure 2. Response of the inverted electrode as a function of con-
v)
centration and sample size
0
readings will reach a maximum and thereafter decrease. For specimens larger than 1pL, the dissolution rate of the crystal appears to be slow enough so that reasonably consistent results can be obtained by taking the maximum reading. Table I demonstrates this effect by presenting the apparent fluoride concentration and standard deviation in the repetitive determination of nanoliter volumes of fluoride standards measured near the minimum measurable volume. Maximum millivolt readings were taken for those samples that showed an initial increase (p[F] I5.3), while minimum readings were taken for samples which showed decreasing initial readings (p[F] I5). These values were then compared with those obtained for the same standards with sample volumes I1 pL, and the apparent fluoride concentration of the nanoliter samples was calculated assuming 55 mV per p[F] unit as the average sensitivity of the fluoride electrode in this region. Clearly, samples more dilute than p[F] I 5.3 must be compared with standards of approximately the same volume if a high level of precision is to be obtained. However, for samples more concentrated than p[F] 5 5.0 the measured potentials appear to be independent of sample size, so that samples less than 1nL can be determined successfully with this technique. Figure 3 presents two standardization curves for the reference microelectrode-fluoride electrode system. In the lower curve 1-pL samples were determined in triplicate using a reference microelectrode as depicted in Figure 1. The agreement between determinations at all levels of fluoride is approximately 1 mV; and the standard deviation of the measured values from the line, as obtained by linear regression analysis, between p[F] values 3 and 5, is 0.015 p[F] unit. For seven repeat determinations at p[F] 5, the lowest fluoride
5 150
P
50
r 3
0
INVERTED ELECTRODE 1 pl SAMPLES
A
UPRIGHT ELECTRODE 10 ml SAMPLES
4
5
6
PPI
Figure 3. Electrode response to fluoride concentration using (1) the inverted fluoride electrode with 1-pL samples as described in the text, and, (2) a conventional fluoride electrode with 10-mL samples. The
reference microelectrode was used in both series of determinations concentration independent of volume effects, the standard deviation was 0.5 mV, which implies a precision of 0.01 p[F] unit. This probably represents the nominal limits of accuracy of the method. The slope of this line is 58.01 compared to 59.1, the theoretical value. In the upper curve, the same microreferencefluoride electrode system was used with both electrodes (clamp upright) in their usual positions, 10-mL standards were used without stirring, and a 30-mV offset was applied to separate the curves. It can be seen from this graph that the two curves are essentially parallel, except in the p[F] 5 to 6 range, where the upper curve falls off slightly less rapidly (44 mV per p[F] unit as compared to 37 mV per p[F] unit in the lower curve). M, repeataWith nanoliter samples more dilute than bility of measurements depends greatly on the ability to transfer such small specimens without contamination. Such
Anal. Chern. 1980, 52,377-379
377
electrodes with no change in equipment. For example, Figure 4 shows a standardization curve for an Orion chloride electrode used in the inverted mode with a reference microelectrode as in Figure 1.
LITERATURE CITED
1
2
3
4
P [CII
Figure 4. Electrode response to chloikle concentration using the chloride
electrode assembly described in the text samples should be run in duplicate or triplicate. Such contamination can usually be eliminated by reconditioning the electrode surface, or recleaning the micropipets. Carryover from the reference microelectrode is rarely a problem, but it can be eliminated entirely by passing the tip of this electrode through a small drop of distilled water on the electrode surface before bringing it into contact with the next specimen. By comparison to other methods (1-3) of modifying solid state electrodes for use with microsamples, the method described here has numerous advantages; it is faster, uses much smaller volumes, and can be used with most solid state
(1) A. S.Hallsworth, J. A. Weatherell, and D. Deutsch, Anal. Chem., 48, 1660 (1976). (2) D. H.Retief,'J. M. Navia, and H. Lopez, Arch. OralBiOl., 22, 207 (1977). (3) A. Venkateswarlu, Anal. Chem., 46, 878 (1974). (4) L. A. Geddes "Electrodes and the Measurement of Bioelectric Events", Wiley-Interscience, New York, 1972. (5) "Physical Techniques in Biological Research", W. L. Nastuk, Ed., Academic Press, New York, 1972. (6) D. J. Prapr, R. L. Bowman, and G. G. Vurek, Science, 147, 606 (1965). (7) H. 0.Lowry, N. R. Roberts, K. Y. Leiner, M. Wu, and A. Farr, J. Biol. Chem., 207, l(1954).
RECEIVED for review June 6,1978. Resubmitted July 31,1979. Accepted November 2,1979. This investigation was supported in part by Research Grants DE04385 and RR05689 to the American Dental Association Health Foundation from the National Institute of Dental Research and is part of the dental research program conducted by the National Bureau of Standards in cooperation with the American Dental Association Health Foundation. Certain commercial materials and equipment are identified in this paper in order to specify adequately the experimental procedure. In no case does such identification imply recommendation or endorsement by the National Bureau of Standards, nor does it imply that the material or equipment identified is necessarily the best available for this purpose.
Microanalytical Techniques with Inverted Solid State Ion-Selective Electrodes. 11. Microliter Volumes Gerald L. Vogel" and W. E. Brown American Dental Association Health Foundation Research Unit, National Bureau of Standards, Washington, D.C. 20234
We reported recently ( I ) how a commercial solid state electrode may be adapted for microvolumes by bringing a glass microreference electrode into contact with hemispherical microdrops of specimen deposited under mineral oil on the surface of the electrode mounted in an inverted position. While this method is fast (20 to 30 specimens per hour), and although specimens of microscopic size can be determined (300 pL and less), it is somewhat elaborate for routine laboratory use with specimens of 1 to 5 FL. In this paper we describe a simple device that will, in a few minutes, adapt most solid state electrodes for the rapid determination of samples in this volume range.
EXPERIMENTAL An Orion fluoride electrode (94-09-00) is adapted for use in the
inverted position by filling it carefully with fluoride reference solution so as to exclude air bubbles. After filling, a thin coat of hot dental wax is applied to the sensing element, and the electrode is clamped in an inverted position. The adapter consists of a nylon cylinder, approximately 12.5 mm in diameter by 5 mm, in which seven 2-mm holes have been drilled (Figure 1). The device is affixed to the sensing element of a solid-state electrode by gently heating it in an oven or on a low-temperature hot plate and pressing it onto the wax-covered electrode. A 2-mm drill bit is heated and pressed blunt end first into the holes in the adapter, and any hot melted wax forced out by this procedure is removed with a tissue. The remaining wax in the holes is carefully removed with a small piece of cotton
wrapped around a toothpick and moistened slightly with chloroform. Gentle removal of the wax is essential to prevent leaks between the wells. A reference electrode for use with the adapter is constructed as follows (Figure 1): (1)Heavy wall borosilicate glass tubing, 8mm 0.d. by 1.3-mm i.d., is pulled into a thin capillary, -0.15-mm id.; a section of this capillary is then pulled to a still smaller size, -0.03-mm inner diameter; and this section is cut with a diamond or carbide scribe to give a capillary of the shape shown in Figure 1. (2) The capillary is inserted into a short section of fine plastic tubing, -1.0-mm i.d., connected to a tapered glass tube attached to the bottom of a small two-neck distillation flask. One neck of this flask contains a rubber stopper through which is inserted a calomel reference electrode connected to the reference input of the electrometer. (3) The capillary tubing and the two-neck flask are filled with a solution of filtered 0.2% agar containing 1M KC1. The agar will prevent the KC1 solution from emptying out of the electrode by gravity. If the agar is too stiff or the capillary too fine, the agar may tend to pull away from the tip of the capillary and thus break electrical contact with the solution. This will result in unstable reading. Raising the two-neck flask relative to the adapter or applying pressure on a syringe inserted through a rubber stopper in one neck of the flask should stabilize the readings immediately. If it does not, the problem is probably not associated with the agar but is caused by a blocked capillary. This can be replaced easily without affecting the measured values. Fluoride standard buffers containing 50% Orion Total Ionic Strength Activity Buffer are deposited in the holes of the adapter with a Pipetman model p-20 pipetter (Rainin Instrument Co.,
This article not subject to U S . Copyright. Published 1980 by the American Chemical Society
