Report
Polyion-Sensitive Membrane Membrane electrodes can be used to
quantitatively detect biomedically important polyions such as heparin and protamine under nonequilibrium conditions
P
olyanions, particularly highly sulfated polysaccharides, are widely used in medicine as anticoagulants and in the food industry as thickening agents. Other polyelectrolytes of obvious analytical interest include DNA, polyphosphates, and protamine, a naturally occurring polycationic protein rich in arginine residues that is used at the end of open-heart surgery to neutralize the anticoagulant activity of heparin (1, 2) Quantitative measurement of such polyionic species (Figure 1) in complex samples such as whole blood or food products is difficult because of their natural polydispersitv chemical hprprncrpneitv and lack of significant absorbance or fluorescent properties Although simple solution-phase ionic dye binding assays (3) as well as more sonhisticated ion exchange d CE t h i (4) have been de el oped to quantitate such species in relatively clean samples, rapid detection of polyion concentrations in a milieu as comi - ^ i
Mark E. Meyerhoff Bin Fu Eric Bakker Jong-Hoon Yun Victor C. Yang The University of Michigan 168 A
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plicated as blood remains a formidable i • i i ii analytical challenge. One of the most successful methods for detecting ionic species in complex samples is potentiometry with ion-selective electrodes (ISEs). Indeed, advanced polymer-membrane-based ISEs using poly (vinyl chloride) (PVC) as the membrane matrix are used universally in modern clinical chemistry instrumentation to measure electrolytes (Na+, K+, Li+, Ca2+, Mg 2+ , and Cl~) as well as pH and C0 2 levels in undiluted blood (5) Because variations in analyte ion specificity can be achieved by judicious choice of the lipophilic ionophore or ion-exchanger structures (and ionic additives) doped within the nolvmeric membranes of such devices polvmer-membrane-tvnp ISEs represent a sensing of
Analytical Chemistry News & Features, March 1, 1996
charged species. In conventional membrane electrodes, the ionophores serve to selectively extract the target ion into the organic membrane phase. Equilibration of the analyte ion at the membrane-sample interface yields an electrochemical phaseboundary potential that is logarithmically proportional to the activity of the target ion in the sample phase via the well-known Nernst equation (6) k, = h, + £ i Ina,,(1) i where E is the cell potential measured in volts; E° is the cell constant, which includes the constant EMF contributions from inner and outer reference electrodes as well as the inner-phase boundary potential of the membrane; z, is the charge of the analyte ion; a is the analyte ion activity in the sample; Tis the temperature in K; and R and F are the gas and Faraday constants, respectively. In practice, the polymer membrane is usually held within an electrode body such that it separates the test sample solution (in which the external reference electrode is placed) from an internal reference electrolyte solution (in which the internal reference electrode, typically Ag/AgCl, is placed). The resulting Galvanic cell can be represented by conventional electrochemical notation as: external reference electrode | sample | polymer membrane | internal electrolyte solution | internal refelectrode. It is interesting to consider whether the basic extraction chemistry used so successfully to develop polymer-membranetype electrodes selective for small inorganic and organic ions can also be applied to develop devices for detecting important large polyionic species such as those shown in Figure 1. At first glance, 0003-2700/96/0368-168A/$12.00/0 © 1996 American Chemical Society
Electrodes for Biomedical Analysis such prospects would seem dim. Even if appropriate chemistry were available to preferentially extract polyions from aqueous phase into an organic membrane phase, the equilibrium EMF of any electrochemical cell containing the polymer membrane should exhibit Nernstian response toward the polyion. With very high charge densities, the typical valence on such polyelectrolytes can easily be > 60 (depending on the chain length) For example, heparin has molecular weight of 15 000 and an average valence of 70 and the Nernst equation would predict changes of < 1 mV for every decade change in polyion concentration/activity a response function that would be useless for rit-flptjpol analytical purposes
Such predictions are based on the assumption that total extraction of the polyion into the organic membrane can occur, and thus each individual charge or a given segment of charged sites on the polyion does not behave independently— contrary to what occurs in the electrochemistry of polyelectrolytes with multiple redox centers at solid electrodes (7). We recently demonstrated, however, that large, reproducible EMF responses toward polyanionic and polycationic species be achieved if ISEs with appropriately formulated membranes are operated under nonequilibrium conditions (8 9) In this Report we summarize the mechanism by which such electrodes operate and survey the range of biomedical measurements for which curate determination of heparin levels in undiluted blood specimens Principles of EMF response
Consider a polymer film doped with lipophilic cation-exchange sites in the form
of tetraalkyl quaternary ammonium species (Figure 2). Such membranes have been used by electrochemists for many years to prepare ISEs that respond to small anions such as CI", NO3, and ClOj (10), as well as organic anions such as salicylate, benzoate, and naproxenate (11), based on ion exchange at the membranesample interface. Potentiometric anion selectivity of such membranes is governed by the thermodynamic partition coefficient of the given anion between the aqueous and organic phase (6). Similarly, polymer membranes doped with lipophilic cation exchangers (tetraphenylborate derivatives, alkyl/ aromatic sulfonates, etc.) respond potentiometrically to cations. Again, relative re-
sponse is based on the partition coefficients of the given cations. Surprisingly, the EMF response of such membranes toward polyionic species was not explored until recently (8,12,13). As shown in Figure 2a, in the absence of polyanion, the concentration of small counteranions such as Cl~ in the membrane phases is constant throughout the polymer film, and the phase-boundary potential at the membrane-sample interface is governed exclusively by the activity of the bathing chloride activity in the sample solution. By adding a low concentration of polyanion (Paz") that can exchange favorably with the small anion (A~) in the polymer membrane via formation of strong ion pairs with the lipophilic
Analytical Chemistry News & Features, March 1, 1996 169 A
Report anion-exchange species (R+), the following equilibrium reaction begins to occur i the membrane (m)-aqueous (a) sample interface ra (a) + zR (m) + zA (m) _ > K
z"a(m)
+
zA(a)
C
Initially, the surface of the polymer membrane becomes partially depleted of th small counteranion, and a gradient of the polyion exists in the stagnant, aqueou layer adjacent to the surface of the membrane and throughout the outermost laye of the polymer film (Figure 2b). Under such conditions, the phase boundary between the membrane and the sample solu tion is not at equilibrium with the sample. That is, the favorable extraction of polyanion into the organic membrane via the ion-exchange process, coupled witl the low concentration of polyanion present in the sample phase creates a nonequilib rium,seady-state electrochemicallphase boundary potential the magnitude of whicl depends on the flux of polyanion surface of the polymerfilmand the membrane fluxes are eqiial the steadv state notential observed mav be exnressed by tbe follow \x\g ennation for a nlanar membrane piec
trode configuration (9) AEMF =
— \J i _ ,DA
Figure 1 . Repeating subunit structures of important polyionic species. A segment of the amino acid sequence is given for protamine. 170 A
Analytical Chemistry News & Features, March 1, 1996
)m
where AEMF refers to the sseady-state potential change after the polyanion is added into a sample solution that initially contain only small inorganic anions (CI-) and no analyte polyanion; RT denotes the ionexchange site concentration in the membrane phase; D and 5 are the diffusion coef ficients and diffusion layer thicknesses in the membrane (m) and aqueous (a) sampl phases, respectively; and cP b lk is the sample polyanion concentration. Note that the range of polyion concentrations that ca be sensed by this nonequilibrium potentiometric response can be altered by varyin the diffusion coefficient of the polyion in the membrane or the aoueous phase Indeed the use of poorly Dlasticized membranes is preferred to detect lower concentrations
When the polymer membrane is in contact with a sample solution containing higher concentrations of the polyanion (Figure 2c), or when the membrane has been exposed to a solution containing even a low concentration of the target polyelectrolyte for an extended time such that true equilibrium between the two phases is achieved, the interface is also at equilibrium and the Nernst response toward the target polyanion is observed (14) £pa = E Pa + — -
ln(
