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(5) K. K. S. Pilhy, C. C. Thomas, J. A. Sondei, and C. M. Hyche, Anal. Chem.,
43, 1419 (1971). (6) C. Feldman, Anal. Chem., 46, 1606 (1974). (7) J. N. Bishop, L. A. Taylor, and E. P. Neary, "The Determination of Mercury in Environment Samples", Ministry of the Environment, Ontario, Canada,
1973. (8) L. W. Jacobs and D. R. Keeney, Environ., Sci. Techno/.,8, 267 (1976). (9) "Methods for Chemical Analysis of Water and Wastes", U S .Environmental Protection Agency, Cincinnati, Ohio, 1974, p 134-138. (IO) A. A. El-Awady, R. B. Miller, and M. J. Carter, Anal. Chem., 48, 110 (1976).
(1 1) K. I.Aspila and J. M. Carron, "Inter-Laboratory Quality Control Study No. 18-Total Mercury in Sediments", Report Series, Inland Waters Directorate Water Quality Branch, Special Services Section, Department of Fisheries and Environment, Burlington. Ontario, Canada.
RECEIVED for review June 28, 1977. Accepted October 19, 1977. Mention of a product name does not imply endorsement by the Central Regional Laboratory, U S . Environmental Protection Agency, Region V.
Enzymic Substrate Determination in Closed Flow-Through Systems by Sample Injection and Amperometric Monitoring of Dissolved Oxygen Levels Ch-Michel Wolff' and Horacio A. Mottola" Department of Chemistry, Oklahoma State University, Stillwater, Oklahoma 74074
Repetitive determinations using injection of the sample containing the sought-for species (substrate, S) into a conbuffer), is tinuously circulated reagent mixture (enzyme described. The glucose oxidase-catalyzed oxidation of 8o-glucose has been chosen to illustrate methods of substrate
+
+
+
determinatlon based on: S H20 O2 5 P -I-H202,with P = oxidation product(s), E = enzyme. Oxygen depletion is monitored by a three-electrode amperometric system allowing determination rates as high as 700 determinationslh, and with due precautions even about 1700 determinations/h, and relative standard deviations (population) of less than 2 % . The method was compared with the "Beckman Oxygen Rate Analyzer" technique, which requires about 1 min per determination. The correlation factor between the two methods was found to be r = 0.97 (for 44 samples of human blood serum). The proposed approach allows continuous use of the enzyme and more than 10000 serum samples have been injected into the same reservoir solution without any observable interference.
Reagent recirculation in closed flow-through systems has been shown to be a useful ancillary device in repetitive determinations by sample injection (1-3). Enzymes, because of their catalytic nature, suggest themselves as main reagents to be recirculated in the implementation of such procedures. Several enzymic methods are based on the general scheme illustrated by the equation below:
S (substrate) + H 2 0 + O2 5 Product(s)
+ H 2 0 2 (1)
in which E is the appropriate enzyme. Determination of glucose, uric acid, galactose, and D- and L-amino acids can be cited as examples of substrate determinations currently done with methods based on Equation 1. Both equilibrium and kinetic methods are available in which the HzOzproduced is measured by coupling reaction 1 with a second reaction in which the H202oxidizes an organic compound whose color P e r m a n e n t address, Laboratoire de Chimie Physique e t d'Electroanalyse, Ecole National Superiure de Chimie de Strasbourg; 1, r u e Blaise Pascal, 67008 Strasbourg, France. 0003-2700/78/0350-0094$01.00/0
change(s) or fluorescence is monitored. Methods based on the measurement of oxygen decrease (as a result of reaction l),mainly amperometrically, with membrane-Clark typeelectrodes or a platinum disk covered with an immobilized enzyme layer acting as a thin reaction zone ( 4 , 5 ) ,can also be found in the literature, and are both useful and popular. Details of these and similar procedures can be found in monographs dedicated to enzymic determinations (6, 7) and recent reviews on the subject (8). Specificity and regeneration (through the catalytic cycle) are, perhaps, the most significant properties of an enzyme as an analytical reagent. The first of these properties is widely recognized and used; the second, however, has not yet been thoroughly exploited. To our knowledge only a few papers report the re-use of the enzyme and attention has been mainly focused on immobilized enzymes ( I , 9). An interesting exception to the use of immobilized enzymes is a Letter to the Editor recently published by Case and Phillips ( I O ) , when the work reported here was well under way, reporting recycling of the enzyme solution in the Beckman Glucose Analyzer. The major objective of the work reported here was to effect recycling of the enzyme solution with sample injection techniques in continuously flowing streams with amperometric monitoring of changes in oxygen levels as a result of a reaction of the type illustrated in Equation 1. The advantages of enzyme recirculation are obvious and have been briefly discussed above; sample injection affords the use of small sample volumes and in conjunction with flow-through systems allows processing of a large number of samples per unit time. To take advantage of the latter, fast detection is needed, and in this paper we describe the application of the three-electrode nonmembrane system reported previously ( 1I ) . Because of the relatively low price of glucose oxidase, the determination of glucose in:
P-D-GlUCOSe + 0 , + HzO D-Gluconic acid + H 2 0 z in which GOD = glucose oxidase, was chosen as a model to illustrate the application that is the subject of this paper. The method illustrated here is another example of the analytical use of transient signals generated by series reactions and/or processes ( 2 , 3 , 12). As such it involves signal measurements made under dynamic conditions in a system approaching '01977 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 50, NO. 1, JANUARY 1978
L J
95
Recorder
Figure 1. Schematic diagram of closed flow-through system. WE, working electrode (Pt wire); Cf,counter electrode [Pt/NaCl(aq.) 7.0 g/L]; RE, reference electrode (SCE). Arrows indicate the direction of flow. For details regarding the detection system see Ref. 77. The mixing coil was a glass tube of 30.0-cm total length, comprising 13 turns with elliptical cross section (1 X 2 mm). The recording system was a Sargent SRG strip chart recorder or a Tektronix 564 storage oscilloscope with plug-ins
equilibrium (kinetic method of determination). T h e baseline corresponded t o oxygen saturation; reaction 2 produced a sudden decrease in signal, and restoration to baseline signal was the result of the imposed flow and the bubbling of air into t h e solution in t h e reservoir vessel. Determination of glucose in human blood serum obtained b y t h e technique reported here was compared with results independently obtained by use of the Beckman Glucose Analyzer.
EXPERIMENTAL Apparatus. Determinations were carried out with the instrumental setup illustrated in Figure 1. Details on the construction and performance of the three-electrode electrometric cell can be found in a previous communication (11). Since the amperometric response of the platinum electrode to oxygen is very sensitive to changes in flow rate, the flow system was divided in two hydrodynamically independent sections (Figure 1). Because of difference in elevation of points A and B , the solution flows by gravity (at constant rate) from A to B. A peristaltic pump (Masterflex with SRC Model 7020 speed controller and 7014 pump head) takes the solution back from B to the reservoir. The flow between B and C (0.5 to 0.6 mL/s) is almost twice the flow rate between A and B (0.20 to 0.25 mL/s) so as to aspirate air and bubble it through the reservoir solution to keep a constant oxygen level responsible for the baseline signal. In the setup of Figure 1, the reservoir solution, separating branches AB and BC, also acts as a “pool” whose large volume dissipates electrostatic charges generated by the friction of pump rollers on the plastic tubing transporting the solution. If very small differences in current need to be measured (100 nA or less), complete elimination of these charges can be accomplished by grounding the reservoir solution. The potential applied to the working electrode in glucose determinations was -0.60 V vs. the SCE. Reagents and Solutions. Glucose oxidase was Purified Type I1 from Aspergillus niger and supplied by Sigma Chemical Co. (St. Louis, Mo.). All other chemicals were AR grade. Deionized water was found satisfactory for solutions preparation. Typical reservoir solutions consisted of 200-mL volumes containing 7.0 g/L of sodium chloride (electrolyte at the level found in human serum), a phosphate buffer (pH 7.00, 0.10 M total phosphate concentration), and 50 units of enzynelml. One unit of enzyme corresponded to that amount needed to oxidize 1.0 pM of glucose per minute a t pH 5.1 and 35 OC. The glucose oxidase in 200 mL reservoir solution contained 1.1 X lo5 units of catalase impurity
Figure 2. Oscilloscopic traces of typical transient signals obtained by repetitive injection of three 10-pL aliquots of a D-glUCOSe solution containing 10 g/L of the sugar. Y axis: current, 1 div = 2 FA. X axis: time, 1 div = 2 s
(1 unit = amount of catalase decomposing 1.0 @molof H202 per minute a t pH 7.0 and 25 “C, while the H202concentration falls from 10.3 to 9.2 pmol/mL of reaction mixture). Determination of the glucose oxidase units was accomplished following the supplier’s indications (13). Procedure for Repetitive Determination of Glucose. Sample volumes of 10 to 20 pL were injected (as indicated in Figure 1) exactly a t the beginning of the mixing coil by means of a Teflon needle (0.025-inch i.d. bore11 located at. the center of the tube constituting the coil. Although injection for the results reported here was manual and done with the help of a Hamilton gas-tight syringe and a Hamilton PB600-1 repeating dispenser (Hamilton Co., Reno. Kev.),other injection devices are equally amenable to use, such as the forced-flow injection system reported in ( 1 4 ) . The amount of glucose in the injected sample was evaluated by reference to a working curve or by application of the standard addition procedure. Because of the relative nature of the measurements involved, no neemd for therrnostating the solution in the cell was required when standard addition, or comparison of the results with occasional injection of standard, was used.
RESULTS AND DISCUSSION Kinetic Consideration. After injection, the glucose sample “travels” through the mixing coil and reacts with the dissolved oxygen in the circulating reagent solution producing a “plug” in which the oxygen level is lower than outside the plug. Because of the predominant laminar nature of the flow, this plug retains its boundaries (limited dispersion) and reaches the working electrode that senses t h e oxygen level in the flowing solution. The passage of the :plug before the sensing electrode produces signal changes illustrated in Figure 2. The picture in Figure 2 was obtained with a Tektronix 564 storage oscilloscope with Type 2A63 differential amplifier and Type 2B67 time base as readout, in place of the strip-chart recorder in Figure 1. T h e small positive signal preceding t h e actual peak profile is the result of a mechanical artifact: the sudden change in flow produced by the injection. T h e time between the beginning of this positive peak and the actual signal measures the delay between the beginning of the reaction and the point of sensing; in our case it amounted to about 2 s. T h e height of the negative peak provides the analytical information for substrate determination since it is proportional to the change in dissolved oxygen concentra t’ion. In the determination illustrated in this paper, the reaction occurring in the plug can be pictured as follows:
a-D-Glucose
* P-D-Glucose
(3)
with the reaction of Equation 2 following Equation 3 but preceding t h e following consecutive reaction:
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(4) with CAT = catalase. In summary, reactions 3,2, and 4 occur in this sequence. Under the experimental conditions used in the work reported here, the half reaction time for reaction 2 is in the range of 5 to 10 s, whereas the equilibrium in reaction 3 is very slowly attained and reaction 4 is very fast. Consequently only reaction 2 needs to be considered as kinetically affecting the oxygen level in the detection zone. Crystalline glucose, however, is in the a-form and glucose solutions prepared from reagent-grade glucose were allowed to stand for at least 2 h to reach the equilibrium between the cy anomer (-36%) and the p anomer (-64%) ( 1 5 ) . The mutarotation was assumed a t equilibrium in the human blood serum samples. The overall reaction of concern can be considered as:
3-D-Glucose f
'/2
O2
GOD
+
CAT
(5)
A plot of the log of the term of the right-hand side of Equation 6 vs. time yields a straight line up to about 90% completion of the reaction. This linear relationship was found a t 20.0, 25.5, 30.0, and 40.0 "C and indicates that the overall reaction proceeds as a pseudo-first order process (partly because the oxygen is in excess when compared with the injected glucose). As such we can write:
C,= C,,e-kt
(7)
with k : rate proportionality constant within the 90% reaction. The concentration C, can be written as:
(Co- C,)
The decrease in glucose concentration, Co - C,, is directly proportional to the decrease in oxygen level, A [ 0 2 ] , from t o to t , which in our case corresponds to the difference in average oxygen concentration between the reservoir solution and the solution volume within the plug at time t. It follows then that:
-Ai = peak height a A[O,]
0:
(1- e - k t ) [ ~ - g l u c o s e ] o
I
-
