Anal. Chem. 1987, 59, 2131-2135
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Bicarbonate-Sensitive Electrode Based on Planar Thin Membrane Technology U r s Oesch, Elzbieta Malinowska,' and Wilhelm Simon*
Department of Organic Chemistry, Swiss Federal Institute of Technology (ETH), Universitatstrasse 16, CH-8092 Zurich, Switzerland
A bicarbonate electrode Is made of a planar assembly of a thln (10-25 pm) neutral carrier-based hydrogen lon selective solvent polymeric membrane and a thln (12 pm) unbuffered internal electrolyte layer on top of a planar sllver/sllver chloride reference element. The electrode responds In a Nernstlan manner to bicarbonate and has a detection limit of -0.2 mY. Response and recovery tlmes (to,%) as low as 30 8 have been determined. The selectlvitles toward Inorgank physiological anions as well as toward acetate, lactate, ascorbate, and sallcylate at pH 7.4 were tested, and no Interference was observed at thek physbiogkalor therapeulkal levels. The llfetlme of the electrode Is >1 month. The electrode Is proposed to be suitable for direct cllnlcal blood bicarbonate determinations.
A direct sensing of bicarbonate especially in clinical chemistry is of utmost relevance (1,2). Although there have been several claims for bicarbonate-selective liquid-membrane electrodes (3-9), no report concerning a realistic practical application of such a system in a direct assay of HC03- has come to our attention. One liquid-membrane electrode praised for HCO, (4) was later shown to have high CO2- selectivity. A sensor for HCO, has been described (6, 7) which is based on a hydrogen-ion-selective liquid membrane, and which simultaneously exhibits high permeability for COz. A superficially identical electrode type was described earlier but the proposed response mechanism appears to be different and is not corroborated by experiments (IO). One of the bicarbonate-selective liquid-membrane electrodes using a tridecylammonium salt as the selective component (5) is probably based on a similar response mechanism. A severe drawback of such devices utilizing gas-permeable polymeric membranes is their low speed of response. Initially typical response times were around 5-15 min (6). By reduction of the membrane thickness from about 200 pm (6) to about 35 pm, the response time was decreased to slightly less than 2 min (7). For clinically relevant applications, response times as well as recovery times of about 30 s are required. Here we report on a bicarbonate-selective electrode obtained by using thin solvent polymeric membranes. In view of a possible integration with the monolith and field effect transistor (FET) technology (11) we have chosen a planar sensor configuration. EXPERIMENTAL S E C T I O N Membrane Materials. High molecular weight poly(viny1 chloride) (PVC), potassium tetrakisb-chloropheny1)borate (KTpClPB), tridodecylamine (TDDA), decane-1,lO-diyl bis(1butylpenty1)glutarate (ETH 469 (12))have been obtained from Fluka, Buchs, Switzerland. Membrane Preparation. The membrane components (1.0 wt % TDDA, 0.7 wt % KTpClPB, 30 wt % PVC, and 68.3 wt % ETH 469) totaling 200 mg were dissolved in 2 mL of freshly distilled tetrahydrofuran (THF). To obtain thick membranes the On leave from the Department of Analytical Chemistry, Technical University, U1. Noakowskiego 3, PL-00-664 Warsaw, Poland. 0003-2700/87/0359-2131$01.50/0
whole solution is cast into a 30-mm4.d. glass ring resting on a glass plate. After overnight solvent evaporation, a membrane of about 200-pm thickness is formed in the glass mold. In order to obtain thin membranes, the whole amount of the solution is first poured into the glass ring and subsequently withdrawn again by pipetting. A thin coating of membrane solution within the glass mold remains. After overnight drying the membrane can be peeled off from the glass plate by a tedious and patient lifting-off of the glass ring. The thin and highly flexible membrane is obtained suspended in the glass ring. In this manner membranes with thicknesses down to 2 pm have been obtained. Membrane Thickness Determination. Thickness of glass ring suspended membranes of less than approximately 50 pm can conveniently be determined from interference of light transmission by using any conventional W-vis spectrophotometer;for further details see ref 13. The refractive index of the membrane has been determined with an Abbe refractometer (Carl Zeiss, Jena, FRG), nD = 1.4788 (22 "C). The thickness of membranes larger than 50 pm are determined gravimetrically by cutting out a precise membrane disk and by using the semiempirically estimated density of the membrane (14). Cell for emf Measurements. Figure 1 shows a cross section of the measuring cell. It consists of four machined cylindrical poly(methy1 methacrylate) (PMMA) parts (a-d, Figure 1). In the center of the bottom piece a cleaned silver wire (4,l-mm diameter) is f'iied axially with a PMMA glue (Agovit 1900, Degussa, Hanau, FRG), stripped, and polished flush with the surface of the PMMA block. The exposed Ag wire has been chloridized electrochemically in 0.1 N HC1 for 10 h with a current of 4 to serve as the internal reference element of the bicarbonate electrode. A disk of a dialysis membrane (Servapor, Serva, Heidelberg, FRG) of 12-pm thickness and 7-mm diameter is soaked 3 times for at least 5 h in 1mL of internal electrolyte solution (typically 10 mM NaCl) to replace the excess of glycerol in which the dialysis membrane is supplied. The soaked membrane is then placed on top of the chloridized Ag wire (4) and the thin organic membrane (still attached to the glass ring) (2, 7) is subsequently placed air-tight and concentrically on top of the dialysis membrane (3). The remaining PMMA parts are then assembled and clamped together. Due to the silicone rubber spacer (6) the PMMA part c is tightened to the organic membrane. A possible leak of sample along this seal would be collected within the drawn-up membrane on the glass ring. Thus, a possible shunt around the membrane to the internal electrolyte layer is inhibited. A custom-made glass capillary reference electrode (11) (Ag/AgCl, 3 M KCl) with a zirconium oxide diaphragm (junction area about 1 mm2) and a miniature combination pH glass electrode (3.5-mm diameter) can be inserted into the sample compartment (1)(16-mm i.d., 6.6-mL volume). Buffers, Calibration Solutions. Bicarbonate calibration solutions have been prepared either by adding solid NaHC03 into a phosphate buffer (A) or by tonometering a phosphate buffer with a certain partial pressure of carbon dioxide (B). Calibration Solutions A . A plain 100 mM phosphate buffer of pH 7.40 (3.618 g NazHP04.2Hz0and 0.644 g NaHZPO4.H20 per 250 mL, purged with humidified nitrogen) was mixed with a 100 mM phosphate buffer of pH 7.40 containing 75.4 mM bicarbonate (3.461 g of Na2HP04-2H20,0.766 g of NaH2P04-Hz0, and 1.680 g of NaHCO, per 250 mL) in varying ratios. The measured pH (by a combination pH glass electrode calibrated with standard buffers of pH 6.88 and 4.00 (15))of these solutions has been within 10.01 pH units of the iteratively calculated pH. For the calculation of the pH and the actual bicarbonate and 0 1987 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 17, SEPTEMBER 1, 1987
9
10
11
12
EMF
[mvl
MEMBRANE INTERNAL ELECTROLYTE
14 5 ,um
12 0,um
150
100
59 2
50
0.1 M PHOSPHATE BUFFER pH 7 4 0 103 mM CHLORIDE
0
Figure 1. Cross section of the cylindrical measuring cell (front view): 1, sample chamber (6 ml); 2, membrane; 3, internal reference electrolyte (soaked Servapor membrane, 12-pm thickness); 4, silver wire
(1-mm diameter, chloridized at the interface to the internal electrolyte film); 5, poly(methy1methacrylate) body (four individual parts a-d); 6, silicone rubber spacer; 7, glass ring; 8, poly(methyl methacrylate)glue; 9, gas inlet; 10, combination pH glass electrode (3.5-mm diameter); 11, reference electrode (2-mm diameter); 12, gas outlet. The thicknesses of the membrane ( 2 ) and the internal electrolyte (3) are not drawn to scale. phosphate concentrations or activities, respectively, the equilibria constants of the following equilibrium equations have been used: K' = (H+)(HP042-)/(HzP04-) (pK'= 7.208 (22 "C) (16)) (1)
K = (H+)(HCO,-)/(CO,) (pK = 6.376 (22 "C) ( 1 7 ) ) (2) The activity coefficients for bicarbonate and dihydrogen phosphate have been determined by the DebyeHuckel formalism (18)and for monohydrogen phosphate by the polynominal approach (18). The activity coefficient for carbon dioxide has been taken as 1. Solutions prepared in this manner have been stored prior to use in 25-mL aliquots sealed in ground glass sleave bottles with minimum volumes of supernantant air enclosed. After opening of a bottle, only the first quarter of the content was used, the rest was disposed of. Calibration Solutions B. A 100-mL aliquot of a 100 mM phosphate buffer has been purged with a humidified gas mixture of carbon dioxide and nitrogen through a stainless steel frit (2-pm pores) with a flow of about 5 mL min-' for at least 1 h prior to the first use and subsequently continuously during further use. In this manner calibration solutions of pH 7.40 have been prepared with gas mixtures of 9.94% C02 (14.618 g of NazHP04.2Hz0,2.466 g of NaH2P04.H,0, and 54.76 mL of 1 N NaOH per 1000 mL), 2.97% COP (17.380 g of Na2HP04.2H20and 0.324 g of NaH,P04-H20per 1000 mL), and 0.97% C02 (15.419 g Na2HPO4.2H20 and 1.845 g of NaH2P04.H20per 1000 mL). The gas mixtures have been certified by the commercial source (Pan Gas, Luzern, Switzerland). The measured pH of the buffers at equilibrium with the supernatant gas have been within kO.01 pH units of the calculated pH. For the calculations of pH and bicarbonate concentrations or activities, respectively, eq 1-4 have been used, (CO,) = [CO,] = (LY/lOOO) pco, (3) pCOz = (% C0,/100)(BP - e ) (4) where a = 0.0479 mol L-' mmHg-' (2, 19) and BP and e denote the actual barometric pressure (mean value for Zurich: 725 mmHg) and the partial pressure of saturated water vapor (19.872 mmHg for 22 "C (15)),respectively (2). Thus the concentrations of bicarbonate in these solutions have been calculated to be 54.8
__1)2
, -5
-4
-3
-2
,
-1
log atKO;
Figure 2. Electrode function for the bicarbonate sensor based on an assembly of thin planar membranes. The internal electrolyte was a cellulose acetate membrane soaked with 10 mM NaCI. Phosphate buffer solutions were prepared with a constant background of 103 mM chloride and solid sodium bicarbonate (calibration solutions A, see Experimental Section). Arrows indicate 95% normal blood bicarbonate activity range.
mM, 16.1 mM, and 5.2 mM, respectively. Solutions containing acetate, salicylate,ascorbate, lactate, and acetylsalicylate have been prepared by adding 100 mM of the corresponding acid to the plain buffer A and adjusting this solution with NaOH to reach pH 7.40. One volume part of it was then mixed with 2 parts of the 75.4 mM bicarbonate calibration buffer A and 7 parts of the plain phosphate buffer A. The concentrations of the free acids in these solutions are then calculated to be 14.5 pM (acetic acid),0.24 pM (salicyclic acid), 3.26 pM (ascorbic acid), 1.87 pM (lactic acid), and 0.80 pM (acetylsalicyclicacid). emf Measurements. The potentials between reference electrode (11)and internal reference element (4) have been recorded by the same equipment as described in ref 20. All measurements have been performed at 22 "C. The reported emf values are not corrected for the diffusion potential ED of the liquid junction of the reference electrode. Calculations of ED based on the Henderson equation (18) show that it amounts to 1.75 mV for the plain phosphate buffer and 1.48 mV for the phosphate buffer containing 75.4 mM bicarbonate. The electrical membrane resistance was determined by the use of the method of potential attenuation by known shunt (14). For a 14.4-pm-thickmembrane it was measured to be 200 kQ.
RESULTS AND DISCUSSION Figure 2 shows a calibration curve for bicarbonate obtained by using the cell and membrane arrangement as depicted in Figure 1. On the basis of the Nikolskii-Eisenman equation (18,21), upper limits of the potentiometric selectivity coefficients for the prevailing sample anions can be determined from the detection limit found in the calibration curve (21)(see Table I). The selectivity coefficients do not represent a selectivity induced by a competitive anion binding of an ionophore at the membrane/sample solution interface. Due to the incorporation of a hydrogen ion carrier into the organic membrane phase, it is hydrogen ion selective (see ref 22), yet permeable to carbon dioxide. When an unbuffered internal electrolyte and a thin organic membrane to favor the carbon
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 17, SEPTEMBER 1, 1987
Table I. Potentiometric Selectivity Coefficients of the Bicarbonate Electrode X
exptl
OHH2P0, HP0:a
log m o $ i required (23)"
