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Anal. Chem. 1980, 52, 1893-1896
Anion-Selective Electrode Based on Oleophilic Anion Exchange Resin Membrane Toshihiko Imato,' Akinori Jyo, and Nobuhiko Ishibashi Department of Applied Analytical Chemistiy, Faculty of Engineering 36, Kyushu University, Fukuoka 8 12,Japan
Oleophilic anion exchange resin membranes based on homogeneous cross-linked polystyrene membranes are described. The anion exchange membrane prepared was swollen with water-immiscible organic solvents such as nitrobenzene and was used as the ion-selective electrode membrane. The membranes wetted with the organic solvents showed good selectivity and could be used as sensitive membranes of anion-selective electrodes such as nitrate and perchlorate ion selective electrodes. The selectlvity of the membrane electrode can be adjusted by selecting an impregnating organic solvent.
Ilani e t al. and other workers have pointed out that some polymer membranes with fixed ionic groups a t low capacities showed high ion selectivity wetted with a water-immiscible organic solvent (1-5). This suggests t h a t synthetic ion exchange resin membranes will also show good ion selectivity if wettability of the membrane for organic solvents can be enhanced (6). Gregor et al. have prepared ion exchange resins of oleophilic property in order to obtain ion exchangers useful for nonaqueous systems (7). The oleophilic ion exchange resins have a fixed ionic group similar to ion exchange sites in liquid ion exchangers and could be wetted even with nonpolar solvents, although usual ion exchange resins with a hydrophilic ion exchange site, such as trimethylbenzylammonium, are hardly swollen with nonpolar solvents. I n a previous communication (8),we briefly reported that oleophilic anion exchange resins are wetted with nitrobenzene in aqueous surroundings and the resin particle impregnated with nitrobenzene can perform the role of the membrane of anion-selective electrodes. The resin particle, however, makes it inconvenient t o fabricate a practically useful electrode. In this work, we have developed oleophilic anion exchange resin membranes by chloromethylation of the cross-linked polystyrene membrane and by subsequent quaternization of t h e chloromethylated membrane with tri-a-octylamine, one of typical ion exchange sites in liquid anion exchangers. This paper will present properties of the developed membrane and performances of anion-selective electrodes based on the membrane impregnated with water-immiscible organic solvents. EXPERIMENTAL SECTION
Preparation of the Ion Exchange Resin Membranes. The oleophilic anion exchange resin membrane was prepared according to procedures for the preparation of homogeneous ion exchange resin membranes developed by Asahi Chemical Co. (9) and for the preparation of oleophilic anion exchange resin (7). One part (by weight) of the styrene-divinylbenzene mixture (100:5 by weight) was polymerized in the presence of polystyrene (0.13 part) and dimethyl phthalate (0.75 part) in a sealed glass tube for 24 h a t 80 "C. The cylindrical copolymer block (diameter 1.5 cm, length ca. 5 cm) was obtained. This was sectioned into sheets (0.24.5 mm thickness) with a microtome. Copolymer sheets were Present address: Department of Applied Chemistry, Faculty of Engineering 36, Kyushu University, Fukuoka 812, Japan. 0003-2700/80/0352-1893$0 1.OO/O
chloromethylated by the conventional method (10). They were washed with ethanol and then with acetone in a Soxhlet extractor. After drying, chloromethylated membranes were immersed in the benzene solution of tri-n-octylamine (26%) for 1 day and were refluxed for 20 h a t 80 "C. The resulting anion exchange resin membranes were washed with benzene and then ethanol for a few days by using a Soxhlet extractor. The membranes were alternately conditioned with 1 M HC1 and 1 M NaOH, and the membranes in the chloride form were washed with water until washings were free of chloride ion. The membranes in the chloride form were stocked in ethanol. In the conversion of the membrane from the chloride form to another anionic form, the membrane in the chloride form was immersed in the ethanol solution of lithium salts of appropriate anions (0.51.0 M). The ethanol solution was changed two or three times until the chloride ion exchanged could not be detected. The membrane was washed with ethanol until washings were free of lithium ion. Determination of Capacity. The stocked membranes in the chloride form were dried in a vacuum desiccator and were weighed. The membranes were swollen with ethanol and placed in the stirred aqueous solution of NaN03 (1 M). The chloride ion eluted from the membranes was determined by argentometry. The elution with 1 M NaN03 and the titration of the chloride ion were repeated three or more times until the chloride ion was undetectable in the solution. The elution of the chloride ion with the ethanol solution of LiN03 (1M) was also made according to the similar procedure. Determination of Solvent Content. The solvent content of the membrane was measured for water and nitrobenzene. After the stocked membranes were immersed in a large amount of water for several days, they were weighed in a surface-wiped state. The membranes were dried in a vacuum desiccator at 40 OC until their weights became constant. The water content was calculated from the weight difference between the above two weighings. The nitrobenzene content was determined by the similar procedure. Measurement of Specific Conductance. Specific conductances of the membranes impregnated with a water-immiscible organic solvent and equilibrated with the aqueous NaCl solution were calculated from the electric resistance and the dimension of the membrane. The electric resistance of the membrane was measured by the alternating current bridge method (1000 cycle), using a Yanagimoto YM-8 conductometer a t 25 f 0.5 OC. The membrane equilibrated with the organic solvent or an aqueous NaCl solution was wiped with filter paper and was interposed between two halves of a glass conductivity cell with platinum electrodes. Two compartments of the cell were filled with an aqueous NaCl solution. The electric resistance of the membrane was calculated from the difference of the resistances between the cells with and without the membrane. The effective area and thickness of the membrane contacting with a NaCl solution were 0.2 cm2 and 0.3-0.8 mm, respectively. Concentration Potential. Concentration potentials for the membranes in the bromide, nitrate, and perchlorate forms were measured by using a membrane cell shown in Figure 1. Measurements were made for the membranes impregnated with nitrobenzene and the membranes swollen with water. The membrane surface was wiped with filter paper and the membrane was clamped between two halves of the glass cell. The electromotive force (EMF) of the following electrochemical cell was measured with a Takeda Riken TR 8651 Electrometer or an Orion 801 A Digital Ionanalyzer. -SCE/INH,Cl Salt Bridge 11 Solution NaX(') IMembranelSolution NaX(') NH,C1 Salt BridgellSCE+, where X stands for Br-, NO3-, ~
'01980 American
Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 52, NO. 12, OCTOBER 1980
Flgure 1. Cell for measurement of concentration potential: A, glass cell; B, Teflon holder; C, solvent reservoir; M, membrane. - L o g a8;
or Clod-. The concentration of the solution ("1 was fixed at 1 X M for the water-swollen membrane and at 0.1 M for the nitrobenzene-impregnated membrane. In the case of the nitrobenzene-impregnated membrane, dissolution of nitrobenzene into the adjacent aqueous solutions was compensated from the solvent reservoir. During measurement of EMF, aqueous solutions (') and (") were impinged on the membrane surfaces at a flow rate of 40 mL min-' by using a Tokyo Rikakikai MP-1011 microtube pump. Ion-Selective Electrode Assemblies. The membrane in an appropriate ionic form impregnated with an organic solvent such as nitrobenzene, 1,2-dichloroethane, and 1-decanol was fixed to a body of an Orion 92 liquid membrane electrode. The organic solvent was added to the organic liquid ion exchanger compartment of the electrode body. An aqueous solution containing sodium chloride (0.1 M) and a sodium salt of the objective anion (0.1 M) was used as the internal filling solution. The performance of the electrodes was examined by the EMF measurement of the following cell. -SECIINH4Cl salt bridgellsamplelion-selectiveelectrode+
Sodium sulfate was used for an ionic strength adjustment of the sample. R E S U L T S AND D I S C U S S I O N I o n Exchange Capacity a n d Solvent Content. Although the sliced membranes were flexible before chloromethylation, the quaternized membranes were quite hard. This may be due to leaking of the plasticizer dimethyl phthalate out of the membrane during chloromethylation and quaternization processes. T h e presence of dimethyl phthalate is useful in making the sectioning of the copolymer block easy (9). The prepared ion exchange membranes were pale yellow. Ion exchange capacity of the membrane in the water-swollen state was 0.5-0.7 mequiv/g of dry resin in the chloride form, while in the ethanol-swollen state the capacity was in the range of 1.0-1.1 mequiv/g of dry resin. According to Gregor et al. oleophilic anion exchangers do not swell sufficiently in water and their water contents are less than a half of those of usual hydrophilic ion exchangers (7). It is known that some portion of a n ion exchange resin particle in insufficient swelling remains inaccessible for a counterion from an external solution (11). This behavior used to be observed in ion exchange of nonpolar organic solvent systems for conventional ion exchange resins. In oleophilic ion exchangers, however, situations are rather reversed. A higher capacity was, thus, observed in ethanol due t o a larger swelling ability than in water. T h e observed water and nitrobenzene contents in the chloride form (g/g of dry resin) were 0.17 f 0.01 and 0.57 f 0.03, respectively. Specific Conductances of t h e Membranes. The specific conductances of the prepared membrane equilibrated with aqueous NaCl solutions of 0.1, 0.5, and 1.0 M were observed t o be 0.65 X 2.6 X and 4.7 X 0-' cm-' , respectively. The increase in the membrane conductance with the external concentration implies that the Donnan exclusion of a n electrolyte is not fully effective. On the other hand, the specific conductances of the membranes impregnated with nitrobenzene were essentially constant (5-6 x 0-l cm-I 1, in the same concentration range of the external aqueous so-
-Log avoj
- L o g acto;
Figure 2. Concentration potential for the water-swollen membranes. M. Concentration of solution (") was fixed at 1.0 X
Table I. Performances of Ion-Selective Electrodes Based on Oleophilic Anion Exchange Resin Membrane sen sing ion of electrode
0) NO,-
C10; C10,-
solvent for impregnation nitrobenzene o-dichlorobenzene 1-decanol chloroform 1,2-dichloroethane benzene nitrobenzene o-dichlorobenzene 1-decanol nitrobenzene o-dichlorobenzene 1-decanol chloroform
lower limit of linear response, M 10-4.0
10-4.5 10-4.0 10-4.0
10-4.5
slope, mV/log i'
-57 * 1 - 57 + 1 -57 * 1 -57 * 1 - 58 ? 4 -54 t 2
10-4.0
-57
10-4.5
- 56
10-3.5
10-5.0
10-5.0 10-4.5
10-4.0
-52 -57 -57 -55 -55
t i
1 3 3 2 1
I
f ?
*
?
2 2
lution. This indicates that the membrane impregnated with the organic solvent completely rejects the Donnan invasion of the electrolyte from the aqueous solution. T h e electric resistances of the membranes impregnated with nitrobenzene are 3 orders of magnitude higher than those membranes swollen with water. This means that the ion exchange site strongly associates with its counterion in the membranes impregnated with the organic solvent. Resistances of the membranes impregnated with nitrobenzene were also measured for the bromide, chlorate, iodide, and perchlorate forms. Their specific conductances were all in the range of 0.92 X to 2.3 X lo+ 0-' cm-'. The membrane impregnated with l-decanol had the specific conductance of 2-3 X lo-' 0-' cm-', indicating that the impregnation of the low polar solvent gives a large electric resistance to the membrane, probably due to the increase in the extent of the ion association. C o n c e n t r a t i o n P o t e n t i a l . For the membranes impregnated with nitrobenzene, the concentration potential was observed to have the linear relationship with a near perfect Nernstian slope ( 2 . 3 R T I F ) against the logarithmic concenM up to 0.3 M. This tration of the solution (') from 1 x means that the electrolyte in the external solution is effectively excluded by nitrobenzene contained in the membrane, in accordance with the observation on the electric conductance of the membranes, and the membrane is perfectly anion selective. For the membranes swollen with water, concentration potentials were observed to deviate from the Nernstian response for the considerably lower concentration of the solution ('1 as shown in Figure 2. For the usual ion exchange resin membrane in the aqueous system, i t is deduced from Teorell-Meyer-Sievers theory that concentration potentials should obey the Nernstian equation for the concentration of a n external solution below approxi-
ANALYTICAL CHEMISTRY, VOL. 52, NO. '12, OCTOBER 1980
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Table 11. Selectivity Coefficients of Ion-Selective Electrodes Based on Oleophilic Anion Exchange Resin Membrane selectivity coeff (log ~ i j P O t ) sensing ion j = C1j = Brj = NO,j = C10; j = 1of electrode ( i ) solvent f o r imoregnation NO,-
c10,-
c10,-
nitrobenzene o-dichlorobenzene 1-decanol chloroform 1,2-dichlorobenzene benzene nitrobenzene o-dichloro benzene 1-decanol nitro benzene o-dichlorobenzene 1-decanol chloroform
-1.7 - 2.0 -1.5 -1.5 -1.7 -1.9 - 1.7
-2.1. - 1.1.
3.4 - 3.7 - 2.1. -3.1. -
mately one-tenth of the fixed ion concentration of the membrane (12). From this reasoning, the membrane potential of the prepared membrane should obey the Nernstian relation up to about 0.3 M, since the molal concentration of the fixed ion of the membrane swollen with water was about 3 m, which was calculated from the capacity of the water-imbibed membrane and its water content. However, the deviation from the Nernstian relation already began a t a lower concentration, lo-* M. T h e deviation is progressively larger in the order of C1< Br- < NO3- < Clod-, in a reverse direction to the lyotropic series of the anions. Even in aqueous solutions, the associations between alkylammonium ions and some inorganic anions have been observed (13-15). I t has been pointed out that ion exchange sites may associate with less hydrated counterions in t h e usual hydrophilic ion exchange resins (16). In the prepared oleophilic membrane, such tendency for the association would be supposedly stronger than in usual anion exchange membranes. This association means the decrease in the fixed ion concentration effective to prevent the Donnan invasion of an external electrolyte which may cause a conspicuous deviation from the Nernstian potential response. Formation of the strong ion association in the oleophilic membrane may be supported from its lowered specific conductance. A p p l i c a t i o n of O l e o p h i l i c A n i o n E x c h a n g e R e s i n M e m b r a n e s t o Ion-Selective Electrodes. T h e nitrate, chlorate, and perchlorate ion-selective electrodes were fabricated, and their performances were summarized with impregnated organic solvents in Tables I and 11. Most of electrodes showed a nearly Nernstian response down to 10-4-10-5 M for the objective anions. In the concentration range where the electrodes shou'ed a linear response, equilibrium potentials were obtained within 2 or 3 min, whereas a response time longer than 5 min was needed in the further lower concentration level. In the case of usual liquid membrane electrodes, a lower limit of detection is governed by dissolution of the electroactive material, the ion exchange site and its paired objective ion, from the membrane into the sample solution (17 , 18). In the case of ion exchange resin membrane electrodes, the sensitivity of the electrodes will be improved since exchange sites are fixed to the resin matrix. This expectation, however, could not be realized as can be seen in Table I. There was no remarkable difference in the lower limit of detection between the oleophilic ion exchange membrane electrode and the usual liquid ion exchange membrane electrodes with a mobile site (19, 22). Some impurities in the ion exchange resin membranes such as amine unreacted or formed by decomposition of the resin may affect the lower limit of detection (20). T h e electrode potential was observed to oscillate within a few millivolts by stirring of samples in the case of an ionic
+ o.:t + 0.l +0.l
+ 0.6 +1.0 + 0.3 +1.2 + 0.5 + 0.9 + 0.6 + 0.7 + 0.5
- 2.:3 - 2.:3 - 1.:t -1.8
- 1.8 - 1.4 - 0.4 - 0.6
-0.6
+0.2
0.8
+o.:t --o.:t
-
- 0.4
- 0.3 - 0.5 - 0.8 - 0.6 - 1.1 - 0.4 - 2.8 -
3.0
- 1.4 - 2.2
+ 0.2 -0.2
0.0 - 2.4 - 2.8 - 1.1 -1.9
strength less than M. This phenomenon could be eliminated by an addition of sodium sulfate as an indifferent electrolyte in accordance with the observation by Srinivasan and Rechnitz on the fluoride electrode (21). Sulfate ion does not interfere with electrode response but is also useful for improvement in a stability of electrode response in low objective ion concentrations. By holding an ionic strength a t 0.3 M by an addition of sodium sulfate, we could lower the limit of the linear response by about 0.5 in the logarithmic expression, compared with that for the samples containing the objective salt alone. Selectivities of the electrodes were examined by the mixed solution method, under a fixed activity of an interferent ion (22). As pointed out by several workers (23-27), the selectivity of liquid membrane electrodes is essentially governed by a solvent of the membrane as long as no specific interaction such as chelate formation occurs between the ion exchange site and its counterion. In addition, from biionic potential studies on liquid ion exchange membranes (28-33), it is known that nitrobenzene and o-dichlorobenzene as the liquid membrane solvent give highly selective perchlorate electrodes, whereas 1-decanol is not an effective solvent for high selectivity. In the newly developed membrane electrodes, the selectivity is able to be adjusted by selecting an impregnating organic solvent species as shown in Table 11. The electrodes with the membrane impregnated with nitrobenzene, 1,2-dichloroethane, and o-dichlorobenzene are comparable in the selectivity to those of corresponding liquid membrane electrodes (19,22). T h e nitrate and perchlorate ion selective electrodes were fabricated by using membranes after 2 years of storage. Both electrodes showed almost the same performances shown in Tables I and 11. I t is concluded t h a t the newly developed oleophilic anion exchange resin membrane will be useful as the membrane of the ion-selective electrodes responsive to various inorganic and organic anions, by conversion of an ionic form through ion exchange and by selection of an organic solvent to be impregnated. ACKNOWLEDGMENT The authors thank K. Kurusu, Faculty of Dentistry, Kyushu University, for the use of the microtome in this work. LITERATURE CITED (1) Ilani, A . I s r . J . Chem. 1966, 4 , 105 (2) Ilani. A . Siophys. J . 1966, 6 , 329. (3) Shohami, E.; Ilani, A . Siophys. J . 1973, 13, 1242. (4) Kedem, 0.; Perry, M.; Bloch, R. I n "Charged Gels and Membranes Pari 2"; S616gy, E., Ed.; D. Reidel Publishing Co.: Dordrecht, 1976; p 126. (5) Astrorn, 0. Anal. Chim. Acta 1975, 8 0 , 245. (6) Buck. R. P. Anal. Chem. 1978, 5 0 , 17R. (7) Gregor, H. P.; Hoeschele, G. K.; Potenza, J.; Tsuk, A . G.; Feiniand. R.; Shida. M.; TeyssiB, Ph. J . Am. Chem. Soc. 1965. 87, 5525. (8) Jyo, A,; Irnato, T.; Fukamachi, K.; Ishibashi. N. Chem. Lett. 1977, 815.
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(9) Tsunoda, Y.; Seko, M.; Watanabe, M.; Ehara, R.; Misumi, T. Japanese Tokkyo Koho 4143, 1957. (IO) Pepper, K. W.; Paisley, H. M.; Young, M. A. J . Chem. Soc. 1953, 4097. (11) Helfferich, F. "Ion Exchange"; McGraw-Hill: New York, 1962; p 512. (12) Teorell, T. Prog. Biophys. Biophys. Chem. 1953, 3 ,305. (13) Diamond, R. M. J . fhys. Chem. 1963, 6 7 , 2513. (14) Lindenbaum, S.;Boyd, G. E. J . fhys. Chem. 1964, 68,911. (15) Boyd, G. E.; Schwarz, A,; Lindenbaum, S. J . fhys. Chem. 1966, 7 0 , 821. (16) Gregor, H. P.; Belle, J.; Marcus, R. A. J . Am. Chem. Soc. 1955, 7 7 , 27 13. (17) Ishibashi, N.; Kohara, H.; Uemura, N. BunsekiKagaku 1972, 27, 1072. (18) Kamo, N.; Hazemoto, N.; Kobatake, Y. Taianta 1977, 2 4 , 111. (19) Camman, K. "Working with Ion-Selective Electrodes"; Springer-Verlag: Berlin, Heidelberg, New York, 1979; Chapter 3. (20) Buck, R. P. The University of North Carolina, Chapel Hill, NC, private communication. (21) Srinivasan, K.; Rechnitz, G. A . Anal. Chem. 1968, 4 0 , 509. (22) Moody, G. J.; Thomas, J. D. R. "Selective Ion Sensitive Electrodes"; Merrow Publishing Co.: Watford, England, 1971. (23) Baum, G. J . Phys. Chem. 1972, 76, 1872.
(24) B l c k . S.;Sandblom, J. Anal. Chem. 1973, 4 5 , 1680. (25) Morf, W. E.; Amman, D.; Pretsh, E.: Simon, W. Pure Appl. Chem. 1973. 36,421. (26) Reinsfelder, R. E.; Schultz, F. A. Anal. Chim. Acta 1973, 6 5 . 425. (27) Yoshida, N.; Ishibashi. N. Chem. Lett. 1974, 493. (28) Shean, G.; Sollner, K. J . Membr. Bioi. 1972, 9 , 297. (29) Jyo. A.; Torikai, M.; Ishibashi, N. Bull. Chem. Soc. Jpn. 1974, 47, 2862. (30) Jyo, A.; Mihara, H.; Ishibashi, N. Denki Kagaku 1976, 4 4 , 268. (31) Yamauch, A.; Minematsu, T.; Kimizuka, H. Maku 1977, 2, 69. (32) Yoshida, N.: Ishibashi, N. Bull. Chem. SOC. Jpn. 1977, 50, 3189. (33) Shean, G. M. J . Membr. Sci. 1977. 2 , 133.
RECEn'ED for review January 7,1980. Accepted July 17,1980. This work was presented in part at the 26th International Congress of Pure and Applied Chemistry, Tokyo, Sept 1977 (Paper No. 8A610). This work was partially supported by Grants-in-Aid for Special Project Research (Grant No. 011911, No. 111211, No. 210410) from the Ministry of Education.
Automated Continuous-Flow Determination of Serum Albumin by Differential Pulse Polarography P. W. Alexander* and M. H. Shah Department of Analytical Chemistry, University of New South Wales, P.O. Box 1, Kensington, 2033 New South Wales, Australia
A polarographic study of the interaction of serum albumin with potassium titanium(1V) oxalate is reported over a buffer range of pH 4-5. The shift in the half-wave potential of the titanium reagent after reaction with albumin is used to develop a novel automated method for determination of albumin in blood serum. Polarographic analysis in a continuous-flow system is operated in the differential pulse mode at a fixed potential of -0.68 V. Under controlled solution conditions at pH 4.89, albumin is shown to selectively react with the Ti(1V) reagent without serious interference from other serum components including the major globulin and glycoprotein fractions unless they are present in excess of the albumin concentration. The polarographic method for analysis of 15 human serum samples is compared to the bromocresol green spectrophotometric dye-binding method for albumin determination, giving a correlation coefficient of 0.9934.
Although determination of albumin is one of the most common of analytical requirements in clinical laboratories, there are at present no procedures reported for its polarographic determination in blood serum. The polarographic reagents available for determination of serum proteins are Brdicka's hexaaminecobalt(II1) chloride ( I ) and a rhodium(111)-substituted ethylenediamine complex ( 2 , 3). Both of these, however, react with a wide range of serum proteins and cannot be used for specific determination of albumin without a prior separation step. We report here a method using potassium titanium(1V) oxalate as a reagent for polarographic determination of serum albumin in an automated continuous-flow system with differential pulse (dp) operation. T h e most commonly used methods for determination of serum albumin are spectrophotometric dye binding techniques for which bromocresol green, BCG ( 4 ) , HABA (2-(4'hydroxyazobenzene)benzoic acid) ( 5 ) ,or methyl orange (6) 0003-2700/80/0352-1896$01.OO/O
are selective reagents. In this paper, the Ti(1V) reagent is used for d p polarographic determination of albumin in a series of serum samples, and the results are compared with t h e BCG method. Good agreement between t h e results for the two methods is obtained, and the polarographic method is shown to be of comparable sensitivity to the BCG method. We recently reported (7) the use of the same Ti(1V) reagent for polarographic determination of serum glycoproteins in a continuous-flow system in an acidic solution at p H 1.6. Under these conditions, albumin and globulins in serum precipitated out and did not interfere with the glycoprotein determination. In this paper, by further study of p H effects on the response of various proteins with Ti(IV), we show that, a t higher p H values in a phosphatecitrate buffer, the reverse determination is possible. Albumin reacts with the reagent but glycoproteins and globulins have little effect. Study of a range of interferences shows that albumin can be determined selectively a t pH 4.89, and the analysis has been automated in a continuous-flow system.
EXPERIMENTAL SECTION Reagents and Stock Solutions. All reagents used were A.R. Grade, and solutions were prepared in distilled water throughout. A stock 0.1 M solution of the reagent, potassium titanium(1V) oxalate, was prepared after dissolving the salt in warm water. Working reagent solutions were then prepared by appropriate dilution of the stock solution to give Ti02+concentrations in the range (1.0-8.0) X M in a disodium hydrogen phosphate/citric acid buffer. To study the effect of buffer pH in the range pH 4-5, we varied the concentration of disodium hydrogen phosphate between 4.0 X M while the citric acid concentration was and 3.0 X kept constant at 4.0 X M. Values of pH >5.0 were not studied because of precipitation of the reagent. Bovine albumin (fraction V), bovine glycoprotein (Cohn fraction VI), bovine 0-globulin (fraction IV), bovine P-globulin (fraction 111), and human y-globulin (fraction 11),all from Miles Laboratories, Inc., were used in this work. Stock solutions of each (lo00 mg L-I) were prepared in distilled water, and appropriate dilutions S 1980 American Chemical Society
