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detection of low concentrations of colored adsorbates in solution. Through exploitation of both the surface enhancement and resonance enhancement mechanisms, SERS+RRS spectra can be acquired for HDz- at a silver electrode surface from M in initial HDz- concentration. solutions as low as
LITERATURE CITED Van1 Duyne, R. P. I n "Chemical and Biochemical Applications of Lasers"; Moore, C. B., Ed.; Academic Press: New York, 1979;Vol. 4, pp 101-185 and references contained therein. Furtak, T. E.; Reyes, J. Surf. Sd. 1980, 93, 351. Cotton, T . M.;Schultz, S. G.; Van Duyne, R. P. J. Am. Chem. Soc. 1980, 102, 7960. Jeaiimaire, ID. L.; Van Duyne R. P. J . €/ec:troanal. Chem. 1977, 84, 1. Hagen, G.; Glavaski, B. S.; Yeager, E. J . Nectroanal. Chem. 1978,
88, 269. Pemberton, J. E.; Buck, R. P. J . Phys. Chem. 1981, 85, 248. Pemberton, J. E.; Buck, R. P. submitted iior publication in J . Phys. Chem . King, F. W.; Schatz, G. C. Chem. Phys. 1979, 38, 245. Matibis, D. E. Ph. D. IDissertation, Universitjt of North Carolina, Chapel Hill, NC, 1978.
(IO) Woodward, W. S.; Rocklin, R. D.; Murray, R. W. Chem., Biomed. Envlron. Instrum. 1979, 9 , 95. (1 I) Pemberton, J. E . ; Buck, R . P. Appl. Spectrosc., in press. (12) Allen, C. S.; Van Duyne, R. P. Chem. Phys. Lett. W79, 63, 455. (13) Bunding, K. A.; Lombardi, J. R.; Birke, R. L. Chem. Phys. 1980, 49,
53. (14) Demuth, J. E.; Christmann, K.; Sanda, P. N. Chem. Phys. Lett. 1980, 76, 201. (15) Pemberton, J. E.; Buck, R. P. J . Nectroanal. Chem., in press. (16) Piersma B. J. I n "Electrosorption"; Qileadi, E., Ed., Plenum Press: New York, 1967;Chapter 2. (17) Moskovits, M.; Dilella, D. P. J. Chem. Phys. 1980, 73, 6068. (18) Math, K. S.;Freiser, H. Wanfa 1971, 18, 435. (19) Mawby, A.; Irving, H. M. N. H. J . Jnorg. Nucl. Chem. 1972, 84, 109.
RECE~VED for review June 24,1981. Accepted August 28,1981. This work was supported by the National Science Foundation Grants CHE77-20491 and CHE77-14547. J. E. Pemberton acknowledges support of this research in the form of a summer fellowship from the ACS Analytical Chemistry Division sponsored by FACSS.
Neutral Carrier Based Hydrogen Ion Selective Microelectrode for Efxtra- and Intracellular Studies Daniel Ammann, Franz Lanter, Rolf A. Steiner, Peter Schulthess, Yoshio Shijo,' and Wilhelm Simon" Depat?ment of organic Chemistry, Swiss Federal Institute of Technology, CH-8092 Zurich, Switzerland
A H'-selective microelectrode based on a neutral ion carrier belonglng to tho class of lipophllic amines is described. The selectivities in respect to Na', K', Mg" , and Ca2' are sufficient for extra- as well as Intracellular measurements of Hf ion activitles 513.2 X IO-' M (pH 5.511 at the typlcal Ionic backgroiinds encountered. Electrodes with tip diameters of around 1 pm have an electrlcal reslstence of about 10" fi and a 90% response Rime of 1 5 s. An example of an Intracellular appllcation Is given.
To date all pM studies in single living cells that proved to be of practical relevance were performed with glass microelectrodes (1-3) There are mainly three types in use. The system introduced by Hinke (4) features an unconveniently long (210 pm) ( 5 ) )pH-sensitive glass membrane tip. This problem has been overcome by recessed tip arrangements (see Figure 17 in ref 1). Unfortunately the response is relatively slow, depending on the volume of the recess (6). Typical electrode resistances are 101o-lO1l D (6). Electrodes with neither protruding nor recessed tips have been realized (7-9). However, the sealing of pH glass membranes to beveled silica pipets poses many technical problems. Response times are as long as 1-5 min (8). In contrast liquid membrane microelectrodes (IO)are easy to prepare and do not suffer from the disadvantages of tip geometry as dictated by the glass membrane electrode preparation technique. A sensor based on a synthetic proton carrier (11) had adequate selectivity both for intra- and extracellular studies but tip diameters had to be above 2 pm to achieve membrane resistances below lo1' D (figure on p 194 'Present address: Department of Environmental Chemistry, Faculty of Engineering,University of Utsunomiya, 2753, Ishii-machi, Utsunomiya 321, Japan.
in ref 12). On the basis of the classical hydrogen ion exchanger OCPH (p-octadecyloxy-rn-chlorophenylhydrazonemesoxalonitrile (13)),a liquid membrane pH microelectrode has been realized only recently ( 1 4 ) . The electrode performance unfortunately was not fully specified (15). Limiting factors seem to be the rather long necessary conditioning time of at least 3 h and the high failure rate of the electrodes of approximately 40%. Here we report on a neutral carrier based liquid membrane pH microelectrode with outstanding selectivity and an electric electrode resistance of -loll Q for tip diameters in the range 0.8-1.0 Mm. It, can easily be prepared and instantaneously reaches its best performance for tip diameters 21 pm.
EXPERIMENTAL SECTION Electrode System. Cells of the type
.
Ag.AgCi, KCI (std )/I M CH3COOLi/sampie solution// J
Y
external macro reference electrode membrane//buffer solution pH 7100LM KH2P04, 0023 M NaOH. 0015 M NaCli AaCl Aa
ion-selective microelectrode or
&.AgCl, 3 M KCl/sarnple solution//
Y external microreference electrode
-
membrane//buffer solution pH 7 (OOL M K%POL, 0023M NaOH, 0015 M NaCI), AgCi.Ag V
I
ion-selective microelectrode
were used. The external reference electrode was a double junction
0003-2700/81/0353-2267$01.25/0 0 1981 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 53, NO. 14, DECEMBER 1981 EMf 1rnV
EMF [mv
TRIS/ HCI
,/=
NaT P E 0-NPOE
NaTPB o-NPOE
LIQUID M E M B R A N E MICROELECTRODE
Mi' CaZC
4 mM 06 mM I . ! mM
TRIS/HCI Na' 10
rnM
K'
200
mM
M$:
2
mM
Ca
001 rnM
HCL i N a O H N ~ ' 69 mM 80-: I l h mM
IO CITRATE-
12
10
8
6
4
mM 6.7 mM
2 PH
Flgure 1. Emf response of the microelectrode cell assembly to different pH buffer solutions at constant ion background, upper trace; pH response of a pH glass electrode at the same Ion background, lower trace; experimental values, dots.
silver/silver chloride electrode, Philips R11. For simultaneous pH measurements a glass electrode Philips GAllO was used. I o n - S e l e c t i v e Microelectrodes. Glass micropipets were drawn from single-barreled Pyrex capillary tubing (GC150T-15, Clark Electromedical Instruments, Pangbourne, Reading, England) which has been cleaned as desribed elsewhere (16). The so treated glass tubing was dried at 200 "C for at least 24 h and stored over silica gel. The tips of the micropipets were broken under microscopic observation on the polished surface of a Pyrex glass rod advanced by a micromanipulator. The resulting tip outer diameters were in the range 0.8-1.0 wm. The micropipets covered with a glass beaker, were predried at 200 "C for at least 30 min. Then a small amount of N-(trimethylsily1)dimethylaminewas added to the beaker. The silane vapor was allowed to react with the glass surface at 200 "C for approximately 30 min. The ion-selectiveliquid consists of 10 wt % of the H+-selective ligand tri-n-dodecylamine (TDDA, see Figure l),doubly distilled (0.05-0.07 mmHg, 240-246 "C) and 0.7 wt % of sodium tetraphenylborate (NaTPB) in o-nitrophenyl octyl ether (0-NPQE). This mixture was stored overnight (- 16 h) under a 100% carbon dioxide atmosphere in a desiccator before use. Due to this treatment small crystals of tri-n-dodecylammonium carbonate (17) may appear. By use of a syringe and as fine as possible plastic tubing, a small volume of this light yellow solution was injected into the top of the microelectrode shank (height -5 mm). It is important that no vacuum be used to remove air bubbles in the shank in order to avoid a loss of carbon dioxide out of the membrane phase. The micropipet was then filled from the back with the internal filling solution (see also ref 1). EMF Measurements. The emf measurements were performed at 22 & 0.5 "C (relative humidity -65%) with a FET operational amplifier (AD 515L, Analog Devices, Norwood, MA; input impedance 1013Q/1.6 pF differential, 1015R/0.8 pF common mode; input bias current
