R., Johnson, D. C., Anal. Chem., 46, 865 (1974). (31E) Gaines, P. R., Peacock, V. E.. Johnson, D. C., Anal. Chem., 47, 1373 (1975). (32E) Fujishima, A., iwase, T., Watanabe, T., Honda, K., J. Am. Chem. SOC.,97, 4134 (1975).
Stripping Voltammetry (1F) Colovos, G.. Wilson, G. S.,Moyers, J. L., Anal. Chem., 46, 1045 (1974). (2F) Colovos, G.. Wilson, G. S., Moyers, J. L.. Anal. Chem., 46, 1051 (1974). (3F) Kinard. J. T., Propst, R. C., Anal. Chem., 46, 1W 6 (1974). (4F) Laitinen, H. A,, Watkins, N. H., Anal. Chem., 47, 1352 (1975).
(5F) Csejka. D. A,, Nakos, S. T., DuBord, E. W., Anal. Chem., 47, 322 (1975). (6F) Cox, J. A,, Cheng, K. H., Anal. Len., 7, 659 (1974). (7F) Copeland, T. R., Skogerboe, R. K.. Anal. Chem., 46, 1257A (1974). (8F) Batley, G. E., Florence, T. M., J. Electroanal. Chem., 55, 23 (1974). (9F) Stojek, Z., Kublik. 2.. J. Electroanal. Chem.. 60, 349 (1975). (1OF) Anokhin. E. A,. Ignatov, V. I . , Zh. Anal. Chem.. 29, 1046 (1975). (11F) Clem, R. G., Sciamanna, A. F., Anal. Chem., 47, 276 (1975). (12F) Clem, R. G.. Anal. Chem., 47, 1778 (1975). (13F) Andrews, R. W., Johnson, D. C., Anal.
Chem., 47, 294 (1975). (14F) Laser, D., Ariel, M., J. Electroanal. Chem., 49, 123 (1974). (15F) Miguel, A. H., Jankowski, C. M., Anal. Chem., 46, 1832 (1974). (16F) Roux, J. P.. Vittori, O., Porthault, M., Analusis, 3 , 41 1 (1975). (17F) Luong, L., Vydra, F., Collect. Czech. Chem. Cornmun., 40,2961 (1975). (18F) Copeland. T. R., Osteryoung. R . A,, Skogerboe. R. K., Anal. Chem., 46, 2093 (1974). (19F) Baranski. A.. Fitak, S.,Galus. Z., J. Electfoanal. Chem., 60, 175 (1975). (20F) Marshall, R . A. G., J. Electroanal. Chem., 56, 311 (1974). (21F) Williams, T. R . , Foy, D. R . , Benson, C., Anal. Chim. Acta. 75, 250 (1975).
Ion Selective Electrodes Richard P. Buck The William Rand Kenan, Jr., Laboratories of Chemistry, The University of North Carolina, Chapel Hill, N.C. 275 14
The history of ion selective electrodes (ISE’s) in the past decade shows the typical behavior of expansion followed by consolidation. The early rapid growth of new electrodes for ion activity measurement, new formats, and new materials of construction has given way to more introspective research on “how’s and why’s” of the functioning of various electrodes and to extensive application studies, uses of ISE’s as instrumental components and uses in diverse fields, particularly in clinical and environmental chemistry. I am restricting this review to topics which deepen our understanding of ISE’s or show new and potentially important applications for ISE’s. The extensive bibliographies previously included in the reviews (49, 51) which were intended to be comprehensive, have been omitted. Nevertheless, the actual body of published material on ISE’s is as large as or larger than ever. While risking appearance of chauvanism, with few exceptions, references were selected from journals readily available in the USA. For the first time, a computer search of Chemical Abstracts tapes was used for the initial survey. Since selections were based on titles and key words, important papers involving ISE’s, but not specifically called out, may have been overlooked. For someone seeking a book on the principles and applications of ISE’s, the most comprehensive volume in English is “Ion-Selective Electrodes” by Koryta (198). It is a small volume, 207 pages, but covers basic principles, observed responses, and many applications in tabular form. The book is an expansion of his 1972 review and contains literature references only through 1973. Three important, authoritative reviews have been prepared by Covington, and Moody and Thomas (74, 75, 252a). The first contains information on reversible electrodes and emphasizes new results on glass and neutral carrier membrane electrodes, while the second and third cover historically all types of ISE’s. One other extensive review in Polish is mentioned because it contains 305 references (346). Sollner, who has contributed many reviews previously, has outlined the basic electrochemistry of fixed site ion exchange membranes (339). Riande’s review on the same topic contains 305 references (302). Recommendations for nomenclature, particularly symbols to be used for the selectivity coefficient, kAB (rather than the previously used term “potentiometric selectivity coefficient”, Kpp‘) have been discussed in a new IUPAC Information Bulletin (178). Nomenclature for ISE’s of conventional types and enzyme and gas-sensing electrodes is included. IUPAC tentative conventions for symbols and terminology in the general electrochemical field are given
by Parsons (277). In Ref. 178, both the two-point, bi-ionic (or separate solution) method and the mixture method (or fixed interference method) are briefly described and advocated for measurement of kAB. In this review, 1 will use a compromise symbol k, for the selectivity coefficient. Buck ( 5 4 ) has summarized t i e numerous advantages of the mixture methods, and the indispensible features possessed by mixture methods when dealing with concentration-dependent selectivity coefficient measurements. The nearly unavoidable flaws in the two solution method (discussed below) make it an undesirable technique. The thrust of this paper (54) is not just a history of the various techniques of selectivity coefficient measurement, but rather an experimental and theoretical justification for the occurrence of activity ratio and absolute activity-dependent selectivity coefficients. Although not all cases have been diagnosed, permselective systems for common valence ions are activity ratio dependent, while kinetically controlled (irreversible) systems are activity dependent. Buck advocates plots of log k,, vs. log aJa, to distinguish these cases. Only the mixture methods are capable of showing up these commonly occurring effects. The general procedure suggests computation of hi, (app.) from experimental data for common valence ions according to In ki;(app.) = In (exp[(Eij- &)/SI - 11 - ln(aj/ai) (1) where E,, is the a,,aJ mixture response and E, is a corresponding pure a, response. For the special case of irreversibility manifest only as different slopes, S, and S I , Mohan and Rechnitz (249)suggested:
+
E,, = Elo S,ln(a, + k,,
a,SI’Si)
(2)
Rather than plotting a family of k,,(app.) values for each a,, one can readily correct k,, (app.) to k, through Ink,, = In k,,(app.)
+ (1- S,/S,)ln a,
(3) The method has been used by Hakoila, Lukkari, and Mannonen (145). The basis for well known activity standards for the more common ISE’s, previously published by Bates and Alfenaar (17), is the assumption that the hydration number of an ion is an unalterable property of the species. In contrast, the single ion activity coefficient depends upon the nature of the other ion in simple salt solutions. Bates (18) has reported single ion activities for NaC1, KC1, KI and CaClz to 1 or 2 M, using this assumption. A new pH buffer for the ANALYTICAL CHEMISTRY, VOL. 48, NO.
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physiological range 6.6-7.4 has been reported by Roy, Swensson, LaCross, and Krueger (311). I t should be noted that methods for producing metal ion buffers and anion buffers, which are necessary for reliable measurements of ISE responses, have been patented by workers a t the Radelkis organization in Budapest (153). The essential need for labile complex control of metal ion activities used to calibrate ISE’s at activity levels well below values obtained by dilution, has already been pointed out (314). Covington and Robinson have decided upon standard solutions of Ca2+ and K+ which mimic the conditions in blood serum. These solutions can be used for calibrating ISE’s (76). Computer assisted modes of data acquisition and treatment have appeared. A particularly attractive interactive mode using a full complement of equipment, is that by Frazer, Kray, Selig, and Lim (117). An automated burettitration system with ISE detection is interfaced to a minicomputer with CRT display, light-pen, and teletypewriter accessories. As the titration proceeds a t a controllable rate, a running Gran plot and titration curve are displayed. Least square fitting of the Gran plot is used to predict equivalence points. The interesting feature of the technique is the ability to judge the validity of data points at various percentages of completion. Deducing possible response errors due to electrodes or slow homogeneous kinetics makes this method useful in the design of titrations. Zipper, Fleet, and Perone (384) use a minicomputer to control a flow system in which C1- and F- are monitored with ISE’s. A simple electronic system for direct antilog conversion of potentiometric data has been given by Heath (156). A multiparametric curve-fitting procedure for locating the end points where two straight lines intersect was published by McCollough and Meites (244). Although intended for amperometric, spectrophotometric, and other titrations involving line segments, it seems a logical route for analysis of Gran plots. Meites, Stuehr, and Briggs (44, 245) have criticized and commented on a previous computer based method for simultaneous detection of strong acid impurities in weak acids and extraction of pK information for the weak acid. Binder and Ebel (30) have also developed an interactive fitting method for computing pK values of weak acids from titration curves. The determination of pK with standard deviations of f O . O 1 pK unit is possible without knowing the exact weight of substance used.
TIME RESPONSES AFFECTED BY ELECTRODE PROPERTIES Theories and experiments on homogeneous membrane electrode systems suggest that at least four widely separated time constants may be observable; however, this statement does not imply that voltage-time responses after a bathing solution activity step or impulse can be resolved into four exponentials. Not all processes necessarily occur, and there may be an overlapping of time constants. Furthermore, the slower processes involving diffusion-migration within a membrane are not precisely represented by a single time constant, but by a distribution because of the form of response dependent on t-1/2rather than exp( - k t ) . The shortest time constant in the 1-100 MHz region due to dielectric relaxation traceable in the case of liquid electrodes to solvent dipoles and reorganization of dissolved species and to ionic inertia ( 7 ) ,is not expected to be important for ISE’s. However, the next longer time constant is that due to charging the external space charge surface regions coupled to the high-frequency or ac bulk electrode resistance. The capacitance is geometric and the high frequency resistance is that value calculated classically from the concentrations and mobilities of charge-carrying species a t their steady state or equilibrium distributions. This quantity, r m , is the usual high-frequency thicknessindependent space-charge relaxation time in conducting systems. For silver chloride, it is about 0.3 ms at 25 “C, while for glass pH electrode membranes studied by Sandifer and Buck (317), 100-200 ms is typical for tight, low error pH glasses (Beckman E-2) and 2-10 ms for glasses with high alkali ion content (Beckman G.P.). The third time constant that may be observed arises in systems with slow surface rates coupled with a partially or completely relaxed, usually double diffuse space charge 24R
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capacitances at each interface. When surface rates are rapid and reversible, this time constant does not appear. Slow surface rates behave as apparent resistances and can have a significant effect only if the value exceeds the bulk resistance. The slow time response of glass electrodes has been attributed to this source (317) because the time constant can be shortened by surface etching. However, it is possible, and even likely, that the apparent surface resistance is associated with mass transport through the surface film of protonated and hydrolized glass. At 25 OC, time constants as great as 30 s for E-2 glass and 7 s for G P glass were observed. While these measurements were obtained by voltage perturbations across electrodes in homogeneous solutions, the same order of magnitudes values were obtained by activity pulses (234). Markovic and Osburn also cite many other references to long time constant values in the 1- to 10-s range for glass electrodes. These time constants for electrodes with glass surface films are longer than expected (and found) for relaxation of concentration polarization at electrode surfaces without surface films. Further information on this point occurs in the next section. The longest membrane-controlled time constant arises from concentration polarization of charge carriers within a membrane electrode and is observed when a current passes the membrane. This is the Warburg-finite diffusion process and the appropriate resistance is the dc value which is significantly greater than the ac value because only permeable ions carry current at dc. The dc capacitances are large and arise from charge separation due to transport. The Warburg behavior requires a t least two charge carriers, both of which contribute to high frequency current but one ion is partially or completely blocked a t dc. Thus, Warburg behavior is expected for liquid ion exchange electrodes and may possibly be observed for solid and glass membranes if transport involves typically vacancy and interstitial motion, but is not expected for neutral carrier systems, lipid bilayers, or fixed site membranes through which ions of only one sign are permeable and transported by a single mechanism. Time dependences of the electrical properties of ISE’s are becoming better understood theoretically and experimentally. The analysis upon which the generalizations above have been made is linear transport theory with the condition that perturbations correspond to voltage changes less than RTIzF volts. These time constants are expected when membrane electrodes and their bathing solutions are initially in a steady state or an equilibrium state, and are perturbed by applied voltages, currents or small ( f 5 0 %of initial value) external activity changes. Responses correspond approximately to equivalent series or parallel RC networks. Full description is given by Macdonald in four recent papers (225-228).
TIME RESPONSES OUTSIDE THE LINEAR REGIME In practical applications of ISE’s, measurement often involves large changes in solution activities. Long time responses from 1 to 10 s are typically observed. The voltage changes and accompanying time constants arise from “massive” reorganization of potential determining species. There are three sources of long time constant response. One is a general phenomenon, viz., whenever an electrode is transferred from one solution to another, a film of solution remains on the surface. In the new solution, some time is required for diffusion-migration as the stagnant film layer assumes the new value. This effect is found also when solutions are washed off with distilled water since this film layer adheres and must be equilibrated. A second process is transport through a surface film attached to the membrane, e.g., hydrolyzed glass on a glass electrode, in response to a change in external activity. Mathematically, equilibration of activity in a finite surface film is practically the identical situation to equilibration of activity in an external convection-controlled stagnant solution film. Calculation of the activity equilibration of a finite region of electrolyte (or surface film) of thickness 6, filled from only one side where there is constant activity a, (a)and
Rlchard P. Buck received his BS and MS degrees in chemistry from the California Institute of Technology in 1950 and 195 1, respectively, and his PhD in chemistry from the Massachusetts Institute of Technology in 1954. Since then he has been assistant research chemist, California Research Corp., 1954-57; research chemist, California Research Corp., 1957-61; principal research chemist, Bell & Howell Research Center, 1961-65; senior scientist, Beckman Instruments, 1965-67; professor of Chemistry, University of North Carolina and assistant director of Crystal Growth and Analysis Laboratory, 1967 to the present. His work includes many facets of electrochemistry. Among these are theory and practice of electrode process identification and measurement, coulometric methods for batch and continuous analysis, and construction of ion-selective electrodes and crystal membrane interface and transport studies. He is also currently working in spark source mass spectrometry and atomic absorption spectrometry for trace analysis of nearly pure solid materials. He is the author of several articles on electrochemistry and fuel cells in technical publications. He is a senior member of the American Chemical Society and was a National Science Foundation Fellow in 1952-53 and a Du Pont Fellow, 1953-54. He was cochairman of the 1964 Gordon Research Conference on Electrochemistry and Chairman of the 1965 Conference.
impermeable at the other side (the membrane surface), leads to the well known finite Warburg response. At short times, the activity at the impermeable side changes as t - l / z , while a t long times an exponential dependence is derived. Morf, Lindner, and Simon (258) have treated this case for an external stagnant film with the result a i ( t ) - ai(O) =
[ a i ( m )- ai(O)][l- e - t / T ]
(4)
where a i ( t ) is the time-dependent activity at the membrane surface which determines the potential response. This quantity varies from ai(0) to a i ( m ) with time constant
P/2D (5) where D is a single ion diffusion coefficient in a supported electrolyte, or the salt diffusion coefficient for a binary electrolyte. Thus ‘T
Time variation of potential response depends only on the ratio of initial to final activities, a point observed by Fleet, Ryan, and Brand (110) for Ca2+-sensing electrodes. Even though only one time constant appears in this equation, responses to step increases are more rapid than steps to more dilute solutions. An equation of this form was also found for equilibration of a surface film on glass electrodes (46). Time responses for glass electrodes have also been measured by Karlberg (185, 186), while Blaedel and Dinwiddie ( 3 4 ) observed time constants for mixed sulfide electrodes in very dilute electrolytes. Their results may be caused by air oxidation or slow dissolution of co-precipitated salts relatively more soluble than the membrane substrate. When ions filling the film can pass into the membrane at a slow rate, film filling is slowed down and the short time response form applies. Even though the film filling may be in the exponential regime, the “siphoning off” of activity into the membrane, dependent on t-’l2, dominates the response. Morf, Lindner, and Simon (258) give for this third case
which has been tested using neutral carrier electrodes. Time responses due to transport through external stagnant films are sensitive to stirring since 6 depends on flow rate. For cylindrical tubes in the laminar regime‘
where u is the linear flow rate and k is a lumped constant. The time to any high level of completion, say t95%, is given from Equation 6 as
t95
-
-0.057 In [a, (O)/a,( a ) ]
(10)
and for steps up in activity a, (O)/a,( a ) Lit Thorough study of solvent effects, KBPh, in o-NO, toluene K+ responses. interferences Use in titrimetry KBPh, in bis( 2-ethylhexy1)adipate Determination of K+ in presence of Na+ K+ and PhNO, Responses t o surfactants; long chain Commercial electrode Ca2+ sulfonates, and quaternary amines distort CaZ' responses in a predictable way Use for 10-1 > C > lo-, M in the bis( 0,0'-diisobutyldithiophosphato)Ni2+,Cd2+,Pb2+ presence of alkali and alkaline M(I1) in PhCl earth ions Patents R,N+ A- in R'X; R = alkyl > 4 carbons Response Nernstian, C > lo-, M Anion responsive R = alkyl < 4 carbons; X = halogen electrodes Orthophosphate Ag(1) thiourea derivatives, viz., Ag polythioureaglutaraldehyde phosphate NO,Trialkylbenzylammonium+ C1- in benzylchloride
stressing their composition is given in Table IIA. Another similar list in Table IIB gives membranes based on PVC or other nonporous supports. Porosity of supports is a gray area in dealing with organic liquids and solids. Presumably, the porosity in this context has to do with water permeation of the membrane. Fritted glass disks and Millipore cellulosic supports tend to pick up water upon hours of exposure while low dielectric, polymeric materials, such as PVC, provide a better, longer lasting barrier against water penetration. As far as the principles of functioning are concerned, all electrodes in Table IIA and B can be considered as a single class. However, specific differences arise because the parameters of functioning, activity coefficients, mobilities and complex formation processes, depend on the specific chemical and electrical, mainly dielectric character of the supports. 30R
ANALYTICAL CHEMISTRY, VOL. 48, NO. 5, APRIL 1976
( 14 ) (219 )
(241)
(121) ( 140)
(131)
NEUTRAL CARRIER MEMBRANE ELECTRODES The problem of the mechanism of function of neutral carrier-based, cation-sensing electrodes remains unsolved. A great deal of information is now available on the responses of these electrodes, primarily responses of K+-selective electrodes in terms of carrier types, membrane solvent effects and cation and anion interferences (37, 74, 198, 254-256). Responses of membranes without added carriers, which also frequently show a region of cation selectivity for lipophobic anion salts, connects this field of study with the general problem of junction potentials involving immiscible phases. T h e problem in its simplest terms, is the question of thick membrane responses (actual thickness large compared with the Debye space charge thickness) compared with thin, lipid bilayer responses (where "thin" means ac-
~~~
~
Table 11, B. Membranes Supported by Polyvinylchloride (PVC) and Related Heterogeneous Systems Ca2+,NO,K+ (patent) K+, Ca+ UO,l+ NO,1-
Various anions ClO,-, BF,ClO,-, I-, NO,-, BF,-, C1c10,Alkylbenzene sulfonate Quaternary ammonium cations
Preparation, conditioning, calibration, and selectivity of these two sensor electrodes KBPh, in bis(2-ethylhexy1)adipate and PhNO, in thermoplastic rubber Two electrodes using valinomycin or didecylphosphonate in cellulose nitrate or acetate. Each surface is coated with PVC Various liquid phosphonate ion exchangers are studied to obtain Nernst response lom6< C < 10-1 M UO,’ at pH 3, preferably in C1- solutions Tetradecylammonium NO, Tetradecylammonium iodide Tetradecyldimethylbenzylammonium salt or methyltricaprylammonium salt Brilliant Green - 3 wt % in PhCl supported in natural rubber sheets. Slightly sub-Nernst response, -lo-‘ t o 10-1 M Aliquat 336 response favors all soluble anions in sequence to left Uses Orion ion exchanger < C < lo-*M Ferroin alkylbenzenesulfonate exchanger responses from in the presence of 10-‘-10-2 M SO,z-, PO,3-, NO,- and C140 wt 3’6 hexadecyltrimethylammonium dodecylsulfate responds to 10-5-10-3 M large quanternary cations Rt and can be used to follow titrations of NaBPh,, reineckate, ferricyanide, or Cr,O,-’ with R+ Br-
tual thickness comparable or smaller than the Debye thickness). From theory and experiment, thin membranes which dissolve ions of one sign, give equilibrium responses of Nernstian form:
RT zF
hE = - In (a’””)
(14)
where a’ and a” are the single permeable ion activities in the left and right bathing solutions. One would expect thick membranes to be electrically neutral in the interior and to extract ions of both signs, as long as the single ion extraction coefficients are not too different from each other. Such extraction membranes would be like liquid junctions with possibly reversible interfacial potential components. The expected membrane potential would deDend on relative ionic mobilities.
where a’s are salt activities. The predicted response is subNernstian, but linear in the logarithm of bathing solution activities with slopes depending on the more mobile ion. The role of carrier is simply to provide a high single ion selectivity for a given ion. Surprisingly, experimental results for neutral carrier, potassium-sensing electrodes using valinomycin and actins show a region of cation-dominant response at low salt activities, a maximum in potential, and a decrease in response a t high salt activities (37). The range of cation response is greatest for KF, decreases for salts with increasingly lyophilic anions following the Hofmeister series. No region of cation response can be seen for K+ salts of highly oil soluble anions such as picrate. The maximum in response also shifts to lower salt activities with increasing anion extractability into the membrane. The actual slope in the cation response region is Nernstian for K F and KC1, but decreases for the others. Response range and slope vary with dielectric constant of the membrane, being best a t low values between 3 and about 12. Transference numbers for K+ are nearly unity when the response approaches Nernstian ideality. There are three theories of cation response which include complex formation of cation and carrier, and include ion pairing with anions. The first theory by Ciani, Eisenman, and Szabo (68) is a space change, thin membrane theory whose logic is clear from the still simpler case of thin membrane responses for membranes which dissolve only a single ion in the absence of any carrier. For this simpler case, exact solutions for concentration, fields, and interior membrane potential for a reversible, cation-permeable case, show that the inner surface concentrations track the bathing solution activities; the interfacial potentials are constant, the net Nernstian response arises from the inner potential difference (90). Ciani et al. allow minimal penetration of anions, ignore free anion transport, but allow for ion
pairing. The ion pairing perturbs the free cation profiles, tends to equalize them at the inner surfaces, and reduces the response below Nernstian. The effect becomes important at high bathing activities and at high formation constants for the ion pairing. Full discussion of this theory and experimental evidence are in an essential volume (69,345). Boles and Buck (37)extended the CES theory to thick membranes by allowing for electroneutrality in the membrane bulk a t high bathing activities and also accounted for the transport of free anions as well as cations, cation complexes, and ion pairs. Responses are Nernstian in K+ activity a t low bathing activities, pass through a maximum and decrease in a way depending on both anion mobility and ion pairing. The assumptions were made that space charge control of potential occurs at the low bathing activities and that the maximum and decrease of potential occur in an activity region where the membrane is electroneutral. Wuhrmann, Morf, and Simon (382) treated the electroneutral range and considered the system to be a liquid junction with interfacial potentials. The maximum response occurs by consumption of carrier. In the other theories, in agreement with experimental resistances as functions of carrier and bathing solutions activities, complete consumption of carriers does not occur because of the presumed small formation constants for ion-carrier complexing and ion pairing (211). In the Wuhrmann-Morf-Simon theory, Nernstian response to cations occurs when anions are not mobile, in agreement with the form of the salt junction, Equation 15 above. The conclusion that a low anion mobility was required for ideal response of liquid junction-like electroneutral thick membrane was made as’ early as 1969 (47). There is no a priori reason why anions should have low mobilities, and consequently Boles and Buck explained their anion responses on a modified space charge theory a t low activities and electroneutral model at high activities where experimental responses are not Nernstian to cations. Finally, the possibility that supported neutral carrier membranes contain fixed negative sites in the support was investigated briefly by Boles and Buck and recently by Seto, Jyo, and Ishibashi (328). Indeed, it is possible to assume that cation carrier complexes exchange into a membrane support a t a concentration equal to the fixed sites. The response was computed from the TMS theory and parameters can be adjusted to fit the experimental responses. However, the fact that maxima are observed with liquid membranes in the absence of supports suggests that this interpretation is not general. Morf et al. (255,256) followed up this idea by intentional creation of mobile anion site exchangers, accomplished by addition of an oil soluble ion, tetraphenylborate, to a neutral carrier membrane. Replacement of extracted anions by membrane phase-trapped anions improved Kf responses a t low salt activities, but did not improve the high activity responses markedly, and maxima were still observed. ANALYTICAL CHEMISTRY, VOL. 48, NO. 5, APRIL 1976
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Table 111. Neutral and Neutral Carrier Membranes. A. Suggested Electrodes and Compositions
K+
Review of principles of selectivity of electrodes based mainly on neutral carriers Macrocylic polyether in PVC
K+
Valinomycin in PVC + dibutyl phthalate
K+
Valinomycin in PVC + dioctyladipate or dioctylphthalate
“Bioelectrodes”
Determination of K’ in biological samples Responses and cation interferences reported Studies also polyurethane, silicone rubber, polystyrene, and polymethylmethacrylate supports < Naf < lo-’ M, Sodium response selectivity and dynamic responses Use in. acid-base titrimetry Nernstian responses Titrimetry with BPh Interference by C S + , ~ B, H~g~2 f + Used in acid -b ase titr imet ry Used in acid-base titrimetry
New synthetic neutral carriers in PVC + solvents ‘Ikicresylphosphate in PVC H+ R,N+; R = C,, C,, C,; C, Dioctylphthalate in PVC N,N-dimethyloleamide in PVC Dextromethorphan Sr2+-polyethyleneglycol complex Srz+ Butanol in fritted glass support H+ 1 4 aqueous-immiscible organic liquids H+ in fritted glass supports Selectivities as a function of plasticizer Kf Valinomycin or dibenzo 18-crown-6in PVC with several plasticizers B. Agents “Carriers” for Transport of Ionsin Hydrophobic Media” Review and transport characteristics of ion carriers and ion-binding proteins (65, 88, 94, 103, 136, 137, 155, Na’
Equilibrium ,properties: extraction equilibria and complex formation equilibrium Spectral studies of carrier complexes and ion pairs Steady-state flux, current, conductances to test carrier model of Na’, K’, ”,+ with actin carriers
A final suggestion made informally by Buck (55) is that slow anion interfacial kinetics a t low salt activities permits near-Nernstian responses to cations. Decrease of response at high activities where surface kinetics are more rapid is the normal sub-Nernstian liquid junction response. The logic is based on the distinction between equilibrium and steady state responses. Bi-ionic potentials for fixed site and liquid ion exchange membranes are always steady state, non-equilibrium cases since there are continuous equal and opposite fluxes of ionic charge at zero current. Likewise, neutral carrier membranes are not a t equilibrium because net flux of salt from high bathing solution to low is a steady-state process. These systems never reach equilibrium in a practical case. If the interfacial processes are rapidly reversible, bi-ionic potentials for fixed and mobile site ion exchanger membranes obey the steady-state NicolskyEisenman form. If the anion exchange rate is low relative to the K+ exchange rate (known to be rapid for valinomycin (201)), the apparent anion “mobility” might be small; the K+ transference number might approach unity and a region of Nernstian ‘response might also result for neutral carrier cases. Conversely, if both anion and cation exchange rates were large, the expectation is sub-Nernstian, junction-like response. Whether this hypothesis has any merit depends on unknown anion extraction rate constants. The only comparable situation is the LaF3 membrane electrode where the exchange rate of F- is very large and the rate of La3+ exchan e is so small that the electrode does not respond to La3$ activity directly. Progress in understanding membrane potentials for neutral systems is occurring on a wide front with contributions coming from interdisciplinary efforts. In Table 111, I give a list of recent papers which bear on these problems. The first part is a list of suggested electrodes and compositions. The second is a list of papers dealing with carrier cation properties. Some specific papers involve new results and new techniques. Sundaram has calculated electrostatic, polarization, and dispersion types of non-bonding interactions to predict much greater stability of K+-valinomycin than K+-hydrate (344). Newcomb, Helgeson, and Cram (269) used chiral neutral carriers, which resemble 18crown-6-cyclic ethers, in CHCls in a U-tube “membrane” to transport salts from one aqueous solution, across the membrane to a pure aqueous phase. When these salts are 32R
ANALYTICAL CHEMISTRY, VOL. 48, NO. 5, APRIL 1976
213, 274, 290, 291, 360, 363, 381) (12, 83, 295) (87, 98, 293, 308) (166, 167)
enantiomeric pairs such as PFe- salts of a-phenylethylamine and methylphenyl lycinates, one enantiomer transports faster than the ot er. Presumably specific binding is involved. Prelog, Simon, and co-workers (355) have uncovered the extraordinary fact that differences in mobilities of enantiomeric pair components are sufficient to show up as observable differences in potentiometric selectivity. Thus, using PVC membranes with a chiral carrier, a-phenylethylammoniumsion of the same absolute configuration shows a more positive response than the other enantiomer by the two-solution method. The selectivity coefficient for the less preferred ion is 10%less than unity and this effect is purely from the permeabilit terms. The latter were compared by double labeling with JH and 14C. Petranek and Ryba (281) have extended their work on macrocyclic ethers by synthesizing a long list of new carriers. The latter were tested for potassium selectivity over sodium. Evans, Cussler, and co-workers (59, 67, 324) have set up real passive membranes involving acids and salts wherein membrane-transported H+ neutralizes OH- in one bathing solution. This spontaneous process drives cations the opposite direction to H+ motion (at zero current) and against their own concentration gradient. Benz, Stark, Janko, and Laeuger continued the series of papers on valinomycin-mediated transport in which effects of chain len t h of lipid on transport parameters were measured
i
(247
Benz and Stark (25) have used the methods of the Laeuger group (stationary conductance and relaxation methods) to determine rate constants, partition coefficients, and complex. formation constants in the aqueous phase for characterization of NH4+, K+, and Rb+ trinactin transport across lipid bilayers. Two short journal reviews (13, 202) and two long expositions in books (203, 341) describe the theory of methods for determining apparent rate constants for formation of ion-carrier complexes at aqueous solution/ membrane surfaces, transfer, and unloading steps. Feldberg and Kissel (105) have suggested charge injection (potential-time relaxation measurements) by external capacitor discharge rather than voltage or current steps for the study of ion transport in lipid bilayers. Charge injection can be done very quickly and short time surface processes can be observed. Because the excitation is a pulse, steadystate data can also be extracted by transform methods.
Table IV. Further Examples of Electronic Conductor/Membrane Contact Electrodes
Pt pt
Pt Pt Pt Pt
Pt Stainless steel Graphite Graphit e Graphite Carbon Graphite Graphite Pt
Graphite-liquid ion exchanger for C1-, NO,-, Ca2+ PVC, Aliquat 336 Fe(Cl),- for Fe(II1) PVC, Aliquat 336 p-toluene sulfonate or lauryl benzenesulfonates for sulfactant anion analyses Naphthalene, PhN0,-with benzyldimethyltetradecylammonium didocylbenzenesulfonate < C < lo-‘ M or anion sensitive to this cation < C < l o oM PVC, alkylphosphoric acid ester Ca2+salt for CaZ+ PVC -KBPh,-dibutyl or diisooctyl phthalate or 3-NO2-o-xylene for K’ PVC-valinomycin-di-n-decylphthalate(or a phosphonate) for K + 7,7;8,8,-tetracyanoquinodimethane and salt (Patent) bis(n-octylpheny1)phosphonate Caz+salt for Calf Cu(I1)-dithizone complex in CCl,. For Cu(I1) Ag(1) dithizone complex in CC1, for Ag’ Cetylhexyldimethyl ammonium dodecylsulfate in PhNO, for this anion PVC-cyclohexanone-Bu3P04-thenoyltriflouroacetone for Ca2+;also, cellulose acetate sulfonated, Me,CO, dimethylsebacate and valinomycin for K+ Benzylcetyldimethyl ammonium p-toluenesulfonate, NO,-, I-, C10,- in PhNO, for these anions “Immunoelectrode”; immobilized antibody Conconavalin A on 5 I-( PVC layer, and responses to yeast mannan Kf- valinomycin surface-coated FET
Ion Sensitive Field Effect Transistor (ISFET)
DeLevie has extended his two-paper series on membrane transport theory for thin membranes permeable to ions of a single sign. The third paper (91) considers current-voltage curves determined mainly by the external concentration polarization; although slow first-order extraction rate was specifically considered as a possible boundary condition. Then the admittance was computed for the same system (92).In the final paper (93) is given the membrane admittance (impedance) at zero current for a single ion with neutral carrier. There is only one time constant, the high frequency geometric value in the absence of external concentration polarization. External concentration polarization is excluded. Finally, Brezina, Hofmanova-Matejkova, and Koryta ( 4 2 ) have applied polaragraphy of T1+- and alkali ion-valinomycin complexes to the determination of complex dissociation rate and complex formation constants in acetonitrile solvent. The potassium-valinomycin dissociation rate ‘was about 102/s and the formation constant was about 10’.
ELECTRODES BASED ON BLOCKED INTERFACES Ordinarily new ion permeable membranes used as ISE’s are first conceived in the “membrane cell” configuration, in which the membrane separates two solutions. One strives to find an inner filling solution which contains an electrolyte with a salt of the most membrane permeable ion, and an ion of opposite charge reversible to the inner reference elbctrode. The membrane permeable ion is also in reversible ion exchange equilibrium a t the inner membrane interface. The solution under test contains an unknown activity of this ion and perhaps some interfering ions. The cell is completed usually with a junction-type external reference electrode. Configurations of this normal type have been demonstrated and the component potential differences specified (47, 74, 198). The reason for use of reversible interfaces a t all points, except the liquid junction, is that equilibrium or steady state thermodynamic responses of the ISE’s can be predicted and compared with experiment. Completely blocked electrodes and blocked interfaces are those across which no ions or electrons pass. These interfaces are electrostatically floating in response to adsorbed dipoles and ions, and there is no significant exchange current. Interfaces may be partially blocked in three senses: 1) Ion exchange membrane interfaces may be reversible to ion transport (high exchange current density for ions) but may be blocked for electron exchange; 2) ion exchange membrane interfaces may be reversible to electron exchange but blocked for ion exchange; or 3) ion exchange membranes may be in the “kinetic domain” where
the interfacial ion exchange is very slow, but non-zero, and passage of current is determined both by interfacial resistance and bulk resistance. Ordinarily, one would not choose to use an interface which is completely blocked because the interfacial potential differences are not easily defined and may not remain fixed. A number of electrode configurations use ionically blocked, but electronically reversible interfaces. These are the “all-solid-state” configurations. When a metal directly contacts a crystal, rapid reversible electron exchange is possible for crystals and pressed pellets of heavy metal halides and sulfides where the electronic band gap is small. Koebel (192) and Buck and Shepard (53) have treated the responses of all-solid-state electrodes and particularly emphasize the values of E o in comparison with electrodes of the second kind based on the same materials. E o values .depend on the membrane’s electron activity, which is identified approximately as the free metal content in the membrane. Buck and Shepard computed the range of E o values for many electrodes in terms of the elemental defect activity referred to pure metal, and they demonstrated the wide range of E o values for AgBr electrodes containing excess Br instead of the more usual case of excess Ag. Koebel emphasized the complications that arise when mixed salts electrodes are used, and he treated the case of multiple phases which are possible, for example, as Cu activity is varied in CuS to yield Cu2S. Electrodes are expected to be completely blocked when electronic conductors (usually inert metals or pure carbon) contact organic liquids or contact polymer films containing organic liquids, in which there are no ions common to, and reversible at, the metal/membrane interface. The interfacial potential is fixed in part by the sheath of solvent dipoles aligned a t the metal/solvent interface. This effect has been discussed long ago by Parsons (276). In addition, there is nonspecific and probably specific adsorption of ions of one sign so that an equivalent dipole sheath is set up consisting of charge on the conductor, the adsorbed ion layer, and the diffuse layer of counter ions about one Debye thickness away. These charged, blocked interfaces are capacitors and the inner potential of the conductor is determined by capacitive coupling to the inner potential of the membrane phase. Low voltage, 10-mV peak-to-peak ac, impedance measurements on PVC coated-Pt, where Aliquat NOa- is incorporated in the PVC, show pure capacitive reactance and no Faradaic current. However, at high applied voltages, -50 V, it is possible to pass current through these interfaces as shown by Carmack and Freiser (60). At these very high voltages, the whole concept of irreversible vs. reversible interfaces breaks down because rates of normally slow processes change drastically a t high voltages and new ANALYTICAL CHEMISTRY, VOL. 48, NO. 5, APRIL 1976
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Table V. Reference Electrode Studies Ref.
AgIAgBr
&I AgOAc
Ag/Ag oxalate Hg 1Hg (I) oxalate Ag/AgSCN AglAg,S Agl AgSeCN Amalgam o f Cd, Pb, Tl, for halides Sr amalgam Cu amalgam Lanthanide and actinide amalgams Bi-Amalgam
Formation, current-voltage dc and ac characteristics in aq. KOH and methanolic KOH Spontaneous formation in 1 N HC1 using C cathode Patent: Preparation from thermal decomposition of Ag,O-binder and anodization Preparation from molten AgCl containing 1%KCI at 470 "C Standard,potentials 0-95 "C in 3.5 M and saturated KCl Study using scanning electron microscopy of porous Ag during conversion t o AgCl in 1 N KCI Standard potentials in formamide (also AgIAgI), 5 4 5 "C Standard potentials in tert-butanol-water mixtures, 25 "C Standard potentials in formamide, 5-45 "C Standard potentials in formamide, 5-45 "C
( 9 6 , 97, 3 2 1 ) (358) ( 133) (242) (20) (348)
Standard potentials, 15-55 "C Responses especially upon addition of cations which form insoluble sulfides Standard potentials, 35-50 "C See text
(204) (18 2 1 (84) (261 1
(86) (190) (267)
(85)
Standard potentials 10-70 "C Standard potentials, 15-26 "C Standard potentials based on polarographic E,,, Study of possibility of use as a phosphate-selective second kind electrode
(282)
Table VI, A. Continuous Monitoring of Watery, Nonbiological Samples Circulating system for monitoring Na', K', Mg2+, and C1Orion Patent Two streams, one monitored and one containing known concentration of reference ion are mixed. Two selective electrodes, one monitoring and one sensing reference ion, provide a difference signal Foxboro Patent Two streams, sample and reagent mix. Treated sample and fresh reagent pass on two sides of a selective electrode Systems for C1- and F- in surface and underground water Systems for monitoring dissolved H,S making use of air-free NaOH solution Cyanide monitor in ore treatment. Study of suitability of Ag,S, Cu,S, and Ni,S, as electrodes, especially sensitivity to xanthates Cyanide monitoring using flowing solution, treated coulometrically with Ag'. Standard Ag' is a second solution. Each is fed t o opposite sides of a Ag,S membrane and difference potential measured Behavior of CuS-Ag,S as a micro flow-through electrode for low activity Cuz+monitoring. Feasibility of monitoring C1-, CaZ+,and Mgz+ in sea water by successive dilutions. Author is optimistic Feasibility of monitoring sulfides in sea water Feasibility of monitoring Cuz+in sea water Use of split crystals to monitor test and reference streams by differential potentiometry Flow-through NO,- ISE with 50-pl dead volume, applied t o monitoring NO,- and NO,- effluents from ion exchange columns Systems for hydrogenating sulfur, C1- and F-containing effluents from GC columns to give H,S, HCl, or HF which are absorbed in electrolytes and detected with small dead volume micro ISE cells
processes become possible. The principal interests in blocked electrode responses are the questions of the type of response (mV vs. activity) they give, and whether they might be useful as sensors for a species which adsorbs or a species which produces changes in the adsorbed species by replacing it or by ionizing it. An example of the latter is a monolayer of an organic acid on a metal which ionizes in basic solutions (126). Extent of adsorption of dipolar or charged species is governed by an isotherm. Consequent1 one does not expect a priori, a Nernstian response for hocked interface electrodes toward a pertinent species activity. A number of factors would have to occur fortuitously and simultaneously. One does expect potential vs. log adsorber to show an Sshape response limited by the adsorption isotherm such that no adsorption (no response relative to pure solvent) occurs a t low levels while saturation occurs a t high activity levels. Analysis of these interfaces and their potentials depend upon electrolyte activity and the distribution of charge between adsorbed and nonadsorbed states (89).The theory is tested by observing responses (potentials vs. log activity) at constant charge, which can be linear (EsinMarkov effect). However, in sensor applications the elqc; 34R
ANALYTICAL CHEMISTRY, VOL. 48, NO. 5, APRIL 1976
trode charge may not be constant from run to run and so the response may become a complicated function of activity. However, adsorption of negative ions drives the potential of the adjacent phase negative and conversely. With the exception of thinly coated ion exchangers on silicon oxide bases of field effect transistors (259), ISE's with blocked electrodes are thickly coated with polymer, plasticizer, and oil-soluble electrolyte ion exchanger or neutral carrier. Thus, the immediately preceding comments which are based on ideas and results in the field of double layer structure, would not apply directly to blocked coated wire electrodes because, in the latter cases, the film is much thicker than a few monolayers. Thus, the coated carbon and coated wire electrodes reported in previous reviews (49,5 1 ) and in Table IV are thick enough to separate the inner interfacial process a t the wire surface from the outer surface exposed to the test solution where rapid, reversible ion exchange equilibrium occurs. The interfacial potential established by the latter process and capacitively coupled to the metal is expected to dominate the overall response. Coated wire electrodes may indeed be stable despite the illdefined, capacitively-coupled interface process, simply because the inner interfaces are isolated and protected so that
Table VI, D. General Applications
Table VI, B. Analyses of Clinical Interest (in addition to some mentioned in the text) Ref.
SMAC system for direct potentiometric determination of Na’ and Kf in blood serum Electrodes for monitoring Na+, K+,and CaZf in blood serum Potentiometric system for monitoring Ca2+ movement across biological membranes Fluoride determination in plasma for use in clinical laboratories. Based on slope determination by dilution and standard addition Fluoride determination in biological materials: separation techniques and analysis Analysis of NO,-and NO,- reductases using NO,-and NH4+sensing electrodes Serum CO, by a continuous flow method using a CO,,- sensing electrode I- permeability of lipid bilayers followed with ISE’s Cl-analysis in blood serum using an anion exchange membrane (SIEM) Enzymic chloresterol determination using Ielectrode Construction and testing o f enzyme electrodes Use of NH, electrode for determining creatinine and serum urea Use of Ag,S electrodes for protein monitoring and antibody-antigen precipitin reaction monitoring Improved penicillin sensing enzyme on glass electrode Systems for urea analysis Systems for L-aminoacid analysis Systems for Cholesterol analysis
(297, 3 6 7 ) (212) (229)
(11 9 ) (369) (1 7 5 )
(158)
(326)
torted by charged surfactants or other charged materials, it is a fair assumption that the interference is engaging in concurrent ion exchange in the conventional manner. When neutral species cause interference (electrode fouling), the fair assumption is that the potential determining ion exchange process is being blocked either by an area decrease (cutting down exchange current) or by introducing an energy barrier making the rate of ion exchange slower (a real decrease in exchange current density).
Table VI, C. Gas Sensing Electrodes and Systems Ref.
Gas sensing electrodes €or CO,, NH,, Et,”, SO,, NO,, based on pH glass membranes; H,S, HCN, HF, and C1, based on Ag salt membranes Ammonia sensor based on Selectrodes with HgS, Ag2S, or Ag,S-CuS tips Ammonia, SO, and “NOx” sensors based on pH glass membranes. Also a continuous analyzer. Recent applications to boiler feed water, fresh water, blood plasma, Kjeldahl digests, and effluents are referenced Ammonia electrode construction patent Orion patents on ammonia sensor, HCN sensor C0,-sensors of Severinghaus-type C0,-sensor combined with second pH sensor Completely air-gap sensors
Establishment of F-selective ISE as an official AOAC method for feeds Establishment of C1- responses of a solid state ISE in 10-100% iso-PrOH for lo-’ < C1- < 10-3 M Demonstration of titrimetry of H,S with CdCI, monitored by solid state ISE in nonaqueous solvent (aimed at H,S in petroleum fractions) Exploration of suspension effects and inter(154, 2 0 0 ) ferences in ISE responses t o C1- and Ca2+ in aq. soil suspensions Analysis of natural waters for I- and CNAnalysis of natural waters for NO,hocedures for determining fluoride in airborne dusts Procedure for precision (+4%)determination of boron in silicon using BF,- ISE Response of C10,- electrode t o IO,- and automatic reaction rate method for vicinal glycols based on time to consume a fixed amount of IO4Differential end-point potentiometry in Ag+ titration of CN- using Ag,S vs. AgI-Ag,S sensor pair Miniature probe containing sensors for (45) CO,, 0,, H f , K+, or Ca2+
(307)
(8) (115, 3 0 3 ) (114, 3 3 7 ) (207) (107, 315, 31 6 )
conditions do not change with time. In addition, it is possible that redox processes and functional groups on the Pt, Pd, stainless steel, and carbon surfaces (OH groups, COOH groups) permit potential stabilization by ion exchange (faradaic coupling) in those cases that protons are available in the membrane phase. Our experience with coated wire electrodes is that they are prone to solvent (water) penetration, and whatever stabilizing processes occur initially a t the inner interface are soon destroyed. One of the basic hypotheses of interfacial electrochemistry is that nonspecific and specific adsorption have no effect on the interfacial potential of a reversible, ion exchanging interface. Whatever adsorbs presumably adjusts its activity so as to conform with the potential as determined by equality of electrochemical potentials of the potential-determining ion exchanging species. When it is observed that a normally Nernstian electrode response is dis-
R E F E R E N C E ELECTRODES Murray and Aikens (261) have made an important contribution in their study of Cd, Pb, and T1 amalgam electrode potentials in propylene carbonate containing halides of these metals and potassium halides. In addition to stability and response to micropolarization, the point is made that the potential-determining solid phases are not necessarily simple divalent halides, but may be solvates or double salts. This possibility has not been stressed in using junctionless membrane cells bathed in two different solvents on each side and may be a source of apparent asymmetry. A Pb/PbClZ reference electrode has been suggested and tested for use in liquid ammonia (143). Stelting and Manahan (342) point out the fact that complexing agents can change the E o for a reference electrode when the activity of water is significantly altered. Their example is the LaF3-F- electrode in water when complexing agent acetonitrile is present a t concentrations in excess of 0.1 M. Ferrocene is stated to provide a reference electrode in wet sulfuric acid solvent, while polynuclear hydrocarbons form a reference a t high sulfuric acid contents (39). The problem of extrapolating measured cell potentials to infinite dilution to obtain E o values for cells with associated electrolytes has been analyzed by Lilley and Briggs (215). Their procedure is variational using formation constant and ion pair distance as adjustable parameters. Other studies are reported in Table V. APPLICATIONS An increasingly important area of application for ISE’s is continuous monitoring of water based solutions. There have been six recent reviews which are relatively short and historical (9, 72, 169, 233, 246, 334). Particular papers are listed in Table VI. The procedures are mainly potentiometric. However, one continuous coulometric approach uses ISE’s (262). Continuous coulometry is a procedure in which four electrodes are used: two for generation of titrant ANALYTICAL CHEMISTRY, VOL. 48, NO. 5, APRIL 1976
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and two for sensing. The sensor pair triggers generation of titration when a sample appears in the stream. By using low concentration streams, samples can be completely titrated very rapidly as they pass through the cell. Generator current is proportional to concentration. In this case, (262), a more subtle procedure is followed: Ag+ is generated with a large triangular current pulse. Chloride is locally titrated to an end point and excess Ag+ formed. During the declining side, another end-point potential is crossed. The spacing in time between end-point potentials can be related to the C1- concentration. Their method is closely related to the gradient addition technique proposed by Fleet and Ho LITERATURE CITED
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ISE's in clinical analysis is a natural field for application, once unsuspected problems of interferences are worked out. There have been quite a few reviews written on this topic already. Those by Guilbault (138, 139) and Rechnitz (300) emphasize their nearly unique contributions to the field. The other reviews are more nearly introductions to ISE's as they apply to clinical analysis (108, 301, 305). ACKNOWLEDGMENT The author recognizes the assistance of David Gutterman.
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Electrophoresis R.
D. Strickland
Veterans Administration Hospital, Albuquerque, N.M. 87 108
This review extends the coverage of a previous one (137) by citing articles published between mid-1973 and mid1975. METHODS AND APPARATUS
Particle Electrophoresis. Particles such as cells and cell fragments carry charges when they are dispersed in a fluid. If the particles differ with respect to their ratios of charge to mass, they can be separated by electromi ration. Almost all electrophoresis of particles has been I fone on this rudimentary basis. This is surprising in view of the possibilities for improving separations by using more sophisticated approaches. One might, for example, use a viscous suspensions medium that would discriminate between particles with different hydrodynamic profiles. Another variation might be to impose electromigration against a density gradient; this would separate particles having the same mobility if the differed in density. One departure from simple electrop rloresis, isoelectric focusing, has been used to demonstrate subpopulations of lymphocytes in the peripheral blood of both humans and rabbits (89). Even by simple electrophoresis, it has been possible to separate immuno lobulin-bearing and immunoglobulin-free lymphocytes ecause the latter migrate more rapidly (54). The observation that thrombocyte mobility in an electrical field diminishes in the preclinical phase of coronary artery disease (62, 134) is profoundly interesting; if the predictive value of this phenomenon is as high as is claimed, it will enable pro hylactic treatment of this disease. The cause of the re&ced thrombocyte mobility is not known, but it may be related to diminished phosphate or carboxylate groups on the cell surfaces (135, 136). Male and female sperm cells from humans can now be separated efficiently and without damage to the cells (128). Electrophoresis has proved to be a useful technique for preparing intact nuclei from tissue homogenates (139). Mobility measurements have shown that rat brain contains a t least two distinct classes of nuclei and that nuclei from human brain tumors migrate faster than those from normal tissue (7).
%
The uses of electrokinetic techniques for studying formed blood elements (86) and microorganisms (118) have been reviewed. An experiment with particle electrophoresis was done on Apollo 16. Two sizes of polystyrene particles were separated. The absence of thermal convection and sedimentation effects permitted sharper separations than were obtainable with the same apparatus on earth (129). Much more ambitious projects involving electrophoresis in space are now in progress according to a recent newsletter (Universities Space Research Association, P.O. Box 5127, Charlottesville, Va. 22903). Improved apparatuses for observin the electrokinetic behavior of cells have been described f67, 1 1 1 ) . Electroosmotic back flow can be reduced and made uniform by coating the walls of the capillary that serves as a migration chamber with agarose and plugging its ends with the same material (149). Laser doppler spectrometry has been used to measure the mobilities of blood cells; results are obtained uickly and agree with those found by direct microscopic aservation (147). Immunoelectrophoresis. The proteins of erythrocyte membranes, solubilized with Berol EMU-043 or other nonionic detergents, can be resolved into about 20 components by crossed immunoelectrophoresis in a arose gels that contain the solubilizing detergent (9,10). many as five antigens in a com lex mixture can be quantitated simultaneously if a higR titer antiserum is available; the antiserum need not be nonspecific (511. A new apparatus uses one gel for separations and another for specific reactions; it permits the analysis of proteins in dilute solutions, e.g., cerebrospinal fluid, without preliminary concentration (58). A technique called affinity immunoelectrophoresis makes use of four contiguous gels to display the characteristic zone electrophoresis pattern, the pattern following reaction with Sepharose-coupled antihuman serum albumin, the pattern in Sepharose alone, and the immunoelectrophoretic interaction of each of these patterns with free antibodies (19). A somewhat simpler version using the same principles has been used to investigate the interaction of concanaval-
1s
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