Of the sulfur compounds tested, thiourea enhanced chromogen formation the most a t 635 nm, whereas the addition of mercaptoacetic acid or phenyl mercaptan resulted in complete suppression of this chromogen. Thiosemicarbazide addition decreased the chromogen formation a t 635 nm, but accelerated the formation of a chromogen with a maximum a t 340 nm that had an absorbance 8 times greater than that of the reference solution a t 635 nm. From this evidence, we conclude that these sulfur compounds alter the reaction pathway. Stannous chloride did not have any substantial effect on the color formation. This suggests that the action of the -SH group (mercaptoacetic acid and phenyl mercaptan) was probably not due to a reduction process. Boric acid caused an enhancement of absorbance of 1.5 times. We believe the action of this extremely weak acid may be through the formation of a borate complex ( 5 ) which causes a steric alignment of the hydroxyl groups of the carbohydrate. Based upon the information we have presented, many usable formulations for an o-toluidine or other aromatic amine reagent may be devised. Acetic acid, as a solvent and/or proton source, is not unique. Several other weak
acids may be substituted with the appropriate solvents. However, acetic acid formulations have been found superior in stability since there is no solvent reaction, such as that for glycols or alcohols (esterification), and the purity of acetic acid is better. In comparison, citric acid usually has residual amounts of carbohydrate material which may cause formulations containing high concentrations of this acid to achieve a dark green color on standing. Further advantages of acetic acid are its superior solubility in water and low cost. For continuous flow analysis of glucose with an o-toluidine reagent in glacial acetic acid, the pump tubings require frequent replacement (10). To circumvent this problem, the acetic acid content of the reagent may be reduced ( 4 ) . This modification should result in a wider use of this reagent for automated quantification of glucose. Received for review March 15, 1973. Accepted May 17, 1973. (10) H. Y . Yee and E. S. Jenest, in "Advances in Automated Analysis, Technicon 1969 International Congress." E. Barton et ai. Ed.. Mediad, White Plains, N.Y., 1970. pp 69-72.
Ion-Electrode Based Automatic Glucose Analysis System R . A. Llenado a n d G. A. Rechnitz Department of Chemistry, State University of New York. Buffalo. N. Y. 14214
An automated analysis system is described which utilizes a novel flow-through type ion-selective membrane electrode for the enzymatic determination of glucose. The system functions well in conjunction with aqueous, protein-loaded, and serum samples containing glucose in the physiological concentration range at sampling rates of up to 7 0 determinations per hour. The proposed methodology eliminates the need for color development or dialysis steps and successfully overcomes protein interference at membrane electrodes via improved electrode design.
There has been a tremendous growth of interest in the analytical applications of ion-selective membrane electrodes during the past few years ( I ) , especially to bioanalytical problems (2-5). In a recent paper (j),we demonstrated an automated system capable of enzyme determinations based on the continuous flow technique. We have now extended this principle to substrate determinations by taking advantage of enzyme specificity and ion-electrode selectivity to devise a precise and accurate assay procedure for glucose in synthetic and clinical samples. The proposed approach is made possible, in part, by the development of a novel flow-through electrode which appears to be free of protein-poisoning effects. (1 j R . P. Buck. Anal. Chem.. 44 ( S i , 270R (1972). (2) G. G. Guilbault and J. G. Montalvo, J . Amer. Chem. SOC.. 92, 2533 (1970). (3) R . A . Llenado and G. A. Rechnitz, Ana/. Chem.. 43, 1457 (1971). (4) M . M. Fishman and H. F. Schiff. Anal. Chem.. 44 ( 5 ) , 543R (1972). (5) R . A . Llenado and G. A. Rechnitz, Ana/. Chem.. 45, 826 (1973).
The recognized diagnostic value of glucose has fostered intense development of possible assay procedures. ( 4 , 6-8) Both optical (9-12) and electrometric (13-18) methods have been used for the enzymatic glucose determination. Normally, optical procedures use the coupled glucose oxidase-peroxidase enzyme system to catalyze the transfer of oxygen from the hydrogen peroxide produced from the glucose oxidation to a chromogenic oxygen acceptor such as o-toluidine or o-dianisidine. While such methods have proved satisfactory in many applications, there is the problem of day-to-day variation due to the instability and nonspecificity of the color indicators. Electrometric methods (13-18), on the other hand, are indifferent to optical parameters and, hence, provide certain advantages. The potentiometric method we propose (6) G. G . Guilbault, "Enzymatic Methods of Analysis." Pergamon Press, Oxford, 1970, p 114. (7) R . J. Henry, "Clinical Chemistry: Principles and Technlques," Hoeber, New York, N. Y., 1965, p 620. (8) "CRC Handbook of Cllpical Laboratory Data," The Chemical Rubber Company. Cleveland, Ohio, 1968, p 272. (9) L. L. Solomon and J. E. Johnson, Anal. Chem.. 31, 453 (1959). (10) F. W. Sunderman, Jr., and F. W. Sunderman, Amer. J Ciin Patho/.. 36, 75 (1961). (11) W. J. Blaedel and G. P. Hicks. Anal. Chem.. 34, 388 (1962). (12) H. V. Malmstadt and S. I . Hadjiioannou, Anal Chem.. 34, 452 ( 1962). (13) H. V. Malmstadt and H. L. Pardue, Clin. Chem.. 8, 607 (1962). (14) W. J. Blaedel and C. Olson, Anal. Chem.. 36, 343 (1964) (15) S. J. Updike and G. P. Hicks. Nature (London). 214, 986 (1967) (16) R . K. Simon, G. D Christian, and W. C. Purdy, Ciin Chem., 14, 463 (1968). (17) C. J. Sambucetti and G. W. Neff, "Methods in Clinical Chemistry," University Park Press, Baltimore, Md., 1969, p 118. (18) G. G. Guilbault and G. J. Lubrano, Anal. Chim. Acta. 60, 254 (1972).
A N A L Y T I C A L C H E M I S T R Y , VOL. 45, NO. 13, NOVEMBER 1973
2165
.
. incubation
Enzyme
water
.c
Iodide
m.,
b Oxygen
1.00:
Acid
0.60:
bath
'
0
1.20;
d
'
..
0-:
to waste
..
-
PH/,V
debubbling e
I
I I
(
E
electrode
orsembly
'
1.60:
;-,
'
recorder
c L /
)
I Schematic diagrams of flow-through electrode assemblies. Electrode C and cap D were constructed in our laboratory A , reference electrode: 6, Orion model 94-53A iodide electrode; C, newly designed flow-through electrode: D , flow-through cap: E , stirrer: F , debubbler: G , inlet for flow stream; H, to waste: I, iodide selective membrane (AgI/Ag2S) Figure 2.
in this paper not only functions well in protein-loaded or synthetic aqueous samples, but also has the advantages of eliminating the need for color development, substituting an inexpensive molybdate catalyst for the expensive peroxidase enzyme, avoiding the need for dialysis or deproteinization, and reducing equipment and data handling costs. The electrode based procedure can be used in either an equilibrium or kinetic mode. However, for technical reasons (19, 20), we favor a fixed time kinetic approach based on the reaction sequence: Glucose
+
H20
+
glucose
0, -------* oxidase
Gluconic acid
H,O,
+
21-
+
2H'
Mo(V1)
I,
+
+
H,O,
2H,O
(1)
(2)
Glucose is, therefore, oxidized for a fixed time under con(19) H. V. Malmstadt, E. A. Cordos. and C. J . Delaney. (121, 26A (1972). (20) H. 6 .Mark, J r . , Talanta. 19, 717 (19721.
2166
Anal. Chem..
44
trolled conditions to produce an amount of hydrogen peroxide proportional to the initial concentration of glucose. The hydrogen peroxide, in turn, reacts with iodide in a stoichiometric manner and the change in iodide concentration is measured with the flow-through electrode to produce the analytical signal.
EXPERIMENTAL Apparatus. The continuous analysis system for glucose consists of an automated sampler, a proportioning pump, a water bath, a flow-through iodide electrode plus a reference electrode, and a high impedance recording pH/mV meter as shown in Figure 1. Technicon samplers and proportioning pumps were intentionally used since they are readily available in most clinical laboratories. An Orion iodide (Model 94-53A) electrode fitted with a cap served as one flow-through electrode version and a locally constructed novel flow-through electrode served as the other (Figure 2). In both electrode arrangements an Orion Model 90-01 electrode served as reference and the potentiometric output of the electrodes was continuously recorded with a Beckman Model 1055 pH recorder.
A N A L Y T I C A L C H E M I S T R Y , VOL. 45, NO. 13, N O V E M B E R 1973
nr
T 1
ni
Figure 4. Typical g l u c o s e response of the analytical system Preparation of Flow-Through Electrode Assembly I. The active ingredients for making the iodide-selective membrane are prepared by bulk coprecipitation of AgI/AgZS as described elsewhere ( 2 1 ) with minor modifications. Compressed membranes are roughened on all sides and then imbedded in bioplastics (Ward's Natural Science Establishment, Inc., Rochester, S . Y . ) (22) to give a transparent plastic disk 30 mm in diameter and 15-20 mm thick with the active membrane a t the center. A drill press is used to punch a hole (1-3 m m ) perpendicular to the crystal membrane and another hole through the side of the membrane to provide the reference contact, i,e., 0.1M AgNO3 solution and a silver wire ( 2 2 ) .The resulting flow-through electrode is shown in part I of Figure 2. A typical response recording of this electrode under continuous flow analysis condition is given in Figure 3. To construct the flow-through assembly 11, a commercially available Orion Model 94-53A iodide electrode is simply fitted with a cap machined to fit snugly while leaving a small dead volume of solution (22. 2 3 ) . Electrode assembly I (flow channel diameter = 3 m m ) is used for precipitate and protein-bearing flow streams which tend to clog the narrow channels of electrode assembly I1 (inlet/outlet port diameter =
