ANALYTICAL CHEMISTRY, VOL. 51, NO. 11, SEPTEMBER (27) Fisher Scientific Co. Bull. No. 441/7-028-10, Fisher Phenol Analyzer. (26) Williamson, J. A. "Rapid Determinationof Phenol Content of Water", paper Dresented at International Water Resources Assoc. Seminar on Water Resources Instrumentation", Chicago, Ill., June 1974. (29) Buck, R. P.;Sirghadeja, S.; Rogers, L. B. Anal. Chem. 1954, 26, 1240-42. (30) Kolthoff, I.M.; Beicher, R. "Volumetric Analysis", Vol. 111; Interscience: New York, 1957; p 113. (31) Buttschell, R. H.; Rosen, A. A,; Middleton. F. M.; Ettinger, M. B. J . Am. Water Works Assoc. 1959, 51, 205-14.
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(32) Grimley, E. B.; Gordon, G. J . Phys. Chem. 1973, 77, 973-78. (33) Murphy, K. L.; Zaloum, R.; Fulford, D. Water Res. 1975, 9 , 389-96. (34) Gkbsz, U. Chem. Stosowana, Ser. A . 1966, 10,211-20,; Chem. Abstr. 1967, 66, 3 1 8 4 4 ~ . (35) Prescott, A. 6.; Johnson, 0. C. "Qualitative Chemical Analysis", 10th ed.; Van Nostrand: New York, 1933, pp 479, 480, 522, 531, 533.
RECEIVEDfor review April 2, 1979. Accepted June 4, 1979.
Catalytic Determinations of Enzymes and Metal Ions by Sample Injection in Closed-Loop Flow Systems S. M. Ramasamy, A. Iob, and Horacio A. Mottola" Department of Chemistry, Oklahoma State University, Stillwater, Oklahoma 74074
Catalysts (enzymes or metal ions) can only be determined in closed-loop systems if, after signal detection, they are physically removed from the system or rendered inactive by an inhibitor. Successful removal of the enzyme glucose oxidase (by physical adsorption on phenoxyacetylcellulose traps) and copper ions (by controlled potential electrodeposition) are described as examples leading to the determination of these catalysts by sample injection in closed-flow systems. The catalytic determination of copper combines a unique electrochemical removal of catalyst and slmultaneous regeneration of the monitored species.
Interest, among analytical chemists, in continuous-flow analyses has increased steadily since 1975 as a result of work with unsegmented continuous-flow analyzers ( I ) . This trend has recently received an adequate forum in internationally attended meetings ( 2 , 3 ) . Several ancillary techniques, when implemented, add to those advantages already recognized in the use of unsegmented streams (4). Typical examples of these ancillary techniques are the use of closed-loop flow-through systems and main reagent regeneration (5-81, whenever their implementation is feasible. The main thrust of this paper is to demonstrate that catalytic determinations, either of enzymes or metal ions, are possible in closed-loop, continuous-flow analyzers. In open systems, in which the catalyst, unreacted reagents, and products are sent to waste after detection, no logistic difficulties can be expected for the determination of catalysts. On the other hand, in the use of continuously circulated reservoir solutions (5-8), accumulation of injected catalyst in the system results in fast deterioration of base line and fast consumption of reservoir solution, rendering the use of a closed system unattractive. Physical removal of the catalyst after signal detection or chemical inhibition (under kinetically controlled conditions) permits the catalytic determinations by sample injection into unsegmented closed-loop flows. As examples of this implementation, the repetitive determinations of the enzyme glucose oxidase (monitoring oxygen consumption from the oxidation of glucose) and the metal ion catalyst copper(I1) (by monitoring the Fe(III)-S2O3*-indicator reaction) are presented here.
DETERMINATION OF GLUCOSE OXIDASE ENZYME Apparatus. An experimental setup basically identical to the one shown in Figure 1 of reference 6 has been used for deter0003-2700/79/0351-1637$01.00/0
mination. The square sponge traps of phenoxyacetylcellulose were positioned in the vessel indicated as point B in the figure and kept suspended in the solution by adequate stirring. The flow rate from reservoir to detection zone was 23 mL/min, and the pumping rate was about 45 mL/min. The potential applied to the working electrode in glucose oxidase determinations was 4.60 V vs. the SCE. Reagents and Solutions. Glucose oxidase was Purified Type I1 from Aspergillus niger and catalase was purified powder from bovine liver, stock No. (2-40. Both were 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 about 100 mL containing 20 g/L of D-glucose and 7 g/L of NaCl in a phosphate buffer of pH 7.00 (0.10 M total phosphate). The injected sample contained the glucose oxidase enzyme and 2.2 X lo4 units of catalase per mL. Typical size of the injected sample was 10 wL. One unit of glucose oxidase corresponded to that amount needed to oxidize 1.0 r M of glucose per minute at pH 5.1 and 35 "C, determined following the supplier's indications (7). One unit of catalase corresponds to the amount of enzyme decomposing 1.0 Mmol of H202per minute at pH 7.0 and 25 OC, while the H202 concentration falls from 10.3 to 9.2 mM of reaction mixture. Procedure. Samples were injected at the beginning of the mixing coil by means of a Teflon needle (0.027-inch i.d. bore) located at the center of the tube constituting the coil. Injection was manual and effected with the help of a Hamilton gas-tight syringe and a Hamilton PB600-1 repeating dispenser (Hamilton Co., Reno, Nev.).
RESULTS AND DISCUSSION The cumulative effect of a catalyst in a continuously circulated reservoir solution can be seen in traces A of Figure 1. Base-line deterioration and depletion of one (or more) of the reactants soon makes determination difficult and the advantages of sample injection into closed-loop flow systems vanish. Successful determination of the catalyst requires the removal or inhibition of the catalyst; for obvious reasons physical removal appears more attractive. Considering enzyme-catalyzed reactions, removal of the enzyme catalyst by the so-called "immobilization" of the enzyme can be used to accomplish such a removal. As principal methods of immobilization we can cite: (1)containment by membrane, (2) entrapment, (3) covalent bonding, and (4) adsorption (9). The first three of these approaches are both too involved and too slow for implementation in continuously circulated systems. Adsorption (physical) is left as the only feasible solution to the problem. After evaluation of several possibilities, the best medium found for removal of the enzyme glucose oxidase was small square sponges of cellulose with their surfaces chemically modified to have adsorption walls of phenoxyacetylcellulose. 0 1979 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 51, NO. 11, SEPTEMBER 1979
3.4pA
PUMP
A
a-
-.-
F-l I
MIN
Typical signal traces for glucose oxidase determination. (A) No sponge traps in the system; (B) 0.40 g of phenoxyacetylcellulose sponges, 5 X 5 X 5 mm in size. Each injection represents 3.2 U of enzyme. Other experimental details as in text Figure 1.
The chemical modification followed the directions published by Butler in 1975 ( I O ) . These directions were, however, modified as follows: small, cubic pieces (5 x 5 x 5 mm) of white cellulose sponge (of the type used for household cleaning) were washed in boiling water and then in a basic solution of ethylenediaminetetraacetic acid (EDTA) about M. After thorough rinsing with distilled water, the cellulose sponges were allowed to dry at approximately 60 O C overnight. The sponges were then wetted with a 1:l mixture of dimethylformamide and pyridine (about 17 mL of mixture per gram of cellulose) and phenoxyacetyl chloride was added dropwise with vigorous stirring. The phenoxyacetyl chloride was added in considerable excess of the calculated stoichiometric ratio (about 2 mL per gram of cellulose). After about an hour and a half of reaction a t room temperature, the mixture was heated a t 70 O C for 1 h and then left overnight a t room temperature. After decanting the solvent and unreacted reagents, the sponges were thoroughly washed with 95% ethanol until no pyridine could be detected in the washes. The resulting product was finally allowed to stand and dry at room temperature overnight, before use. The effect of these sponge cubes when present in the closed-loop flow system as indicated above can be seen in traces B in Figure 1. Without the presence of the enzyme trap, deterioration of base line to a point that further use of the system became impossible occurred after 25 injections each corresponding to 3.16 U. The height of the transient signal also diminishes with increasing injections as O2 is being continuously removed by reaction faster than the bubbling in the reservoir can replenish it. In the presence of 0.40 g of phenoxyacetylcellulose sponge, the same effect appeared after 425 injections, each also corresponding to 3.16 U (population relative standard deviation = 2.7%). This represents an adsorption capacity of this particular batch of sponge cubes of 63.8 mg of protein per gram of sponge. General considerations regarding the chemistry and kinetic aspects of determinations based on the reactions responsible for the type of signals shown here have been discussed in detail elsewhere (7). After use, the enzyme can be stripped off the sponge by washing with a surfactant such as Triton X-100 in 100 mL of p H 7.00 containing 0.605 g of tris(hydroxymethyl)aminomethane, “Tris”, and sufficient HCl to adjust the pH). The recovered enzyme retains most of its catalytic activity. Three washings with this solution were
Flgure 2. Schematic diagram of closed-flow system for Cu(I1) determinations. A: Anode; C: cathode; SCE: saturated calomel electrode. i: Injection port directly into detection zone. ii: Injection port when delay-mixing coil is inserted between points a and b in the loop. Detector: UDI silicone photodiode detector (United Detector Technology, Inc., Santa Monica, Calif.). Amplifier: Tektronix AM502 with Tektronix PS 501-1 Power Supply. Recorder: Sargent SRL. For other details of photometric system see Ref. 11. Pump: Masterflex with SRC Model 7020 speed controller and 7014 pump head. Diameter of delay-mixing coil (made of Teflon): 1 mm, typical length: 4 m
followed with washing twice with 2-propanol and then thoroughly (6 or 7 times) with distilled water. The washed sponges can be used again but with a 50% decrease in adsorption capacity. The adsorption capacity can be restored to the original level by re-treatment with phenoxyacetyl chloride. The immobilization of the enzyme glucose oxidase on phenoxyacetylcellulose sponges has, in this particular case, two helpful features for the purpose of repetitive enzyme determination: it holds the catalyst a t a single point in the flow limiting its contact with substrate and reactive medium and it also reduces the activity of the enzyme by adsorbing it. This decrease in activity helps delay the effect on the base-line level of dissolved oxygen. From a practical viewpoint, and with adequate calibration, repetitive determinations of enzyme activities should be useful in enzyme production (quality control) as demand increases for the use of enzymes as analytical reagents.
DETERMINATION OF COPPER(I1) IONS Apparatus. The spectrophotometric flow system was a custom-assembled unit, details of which are shown in Figure 2. Injection of sample containing the sought-for species and the thiosulfate reactant was accomplished by means of Hamilton gas-tight syringes and a Hamilton PB600-1 repeating dispenser (Hamilton Co., Reno, Nev.) modified to drive two syringes simultaneously. In experiments in which injection took place directly in the detection zone, the reacting mixture in the photometric cell was constantly stirred with a Model 19 heatless-submersible magnetic stirrer (Technilab Instruments Inc., Pequannock, N.J.) and spectrophotometer cell spin-bar (Bel-Art Products, Pequannock, N.J.). The controlled potential electrolysis was performed maintaining the anode potential at 0.60 V with respect to a saturated calomel electrode and with a MP-1026 Potentiostat-Regulated Power Supply operating in the potentiostat mode (PacificPrecision Instruments, Concord, Calif.). Both cathode and anode were platinum gauze cylinders 3.2 cm in diameter and 3.1 cm tall. The photometric unit, detector, and readout have been described elsewhere (11). The current during electrolysis varies from 5 to 10 mA depending on the concentration of electroactive species in the system. Monitoring of the red complex of iron and thiocyanate was done at 480 nm. A constant flow rate of 15 mL/min was maintained through the experiments. Reagents and Solutions. All reagents used were AR grade. The water used during this work was purified by ion exchange and double distillation. Typical reservoir solutions consisted of
ANALYTICAL CHEMISTRY, VOL. 51, NO. 11, SEPTEMBER 1979
A
P
D
Figure 3. Typical signal traces for Cu(I1) determinations, 4-m delay-mixing coil and 30 mL/min flow rate. Experimental conditions as
in text. b: base line. Cu(I1) in ppm: A, 0.0; B, 10.0; C, 20.0; D, 25.0 500 mL containing 8.30 X M Fe(II1) added as nitrate, 1.60 x M SCN- (added as its potassium salt) (12), and 0.20 M KN03 and having the pH adjusted at a value of 3.00 with HC104. At pH's above 4.6 the catalyzed reaction is too slow and if the pH is below 2.0, electrolytic decomposition of thiosulfate yields colloidal sulfur. Typical size of a sample injected was 5.0 p L of 0.10 M thiosulfate solution and sample. Thiosulfate concentrations lower than 0.10 M yield catalyzed rates too small for analytical use. Higher concentrations of this reagent produces base-line drifts.
RESULTS AND DISCUSSION For the determination of copper(I1) by means of the indicator reaction involving S2032-and Fe(II1) (13, 14), the uncatalyzed reaction can be written as: 2Fe(III)
+ 2S2032- = 2Fe(II) + S40G2-
This is not slow enough to provide a long-range base-line stability. T o avoid such effect, one of the reactants, in this case the thiosulfate, is simultaneously injected with the sample instead of being directly incorporated in the reservoir solution. Injection of this reactant in a small volume and in a relatively high concentration creates a temporarily high concentration within the "plug" which is, however, diluted to considerably lower values upon reaching the reservoir and thus keeps the uncatalyzed reaction from proceeding at a noticeable rate. Introduction of the sample and reactant via merging streams (15, 16) can equally well be used for this purpose. The rate of reaction 1, as well as that of the catalyzed reaction in the presence of Cu(II), can be followed absorptiometrically by treating the Fe(II1) with SCN- and forming the red Fe(Hz0)6SCNZ+ complex. The formation constants of the iron complexes (about 100 for Fe(II1) and 10 for Fe(I1)) (17)permit the existence of sufficient free Fe(I1) for successful
1639
anodic oxidation of this species, under controlled potential conditions, and thus the regeneration of Fe(II1) and of monitored species. This oxidation can be conveniently performed if the anode potential is kept at +0.60 V vs. the SCE since it simultaneously allows one to remove the ionic catalytic species of copper by reducing them to Cu(0) in the cathode, and thus in a single, unique operation, remove the catalyst from the system and regenerate the main reagent in the indicator reaction. Electrolysis also helps in diminishing the rate of the uncatalyzed reaction since the Sz02- undergoes decomposition in the electrochemical cell with evolution of SOz(g). Direct injection into the detection zone allows work in only the 100 to 250 ppm range of Cu(II), but intercalation of a 4-m (1.0-mm i.d.) delay-mixing coil (as indicated in Figure 2) permits lowering the limit of detection to at least 5 ppm. Utilization of a smaller volume flow-through cell should allow lowering even more the limit of detection. Use of the coil has also resulted in improving reproducibility of the determinative approach to values of 1.8% relative standard deviation (population). The maximum number of determinations per hour is 250, injecting either into the detection cell or a t the beginning of the delay-mixing coil. Figure 3 shows typical signal profiles obtained with the 4-m delay-mixing coil and at a flow rate of 30 mL/min; the profiles show a positive peak (increase in absorbance with respect to base line) for which the authors lack a convincing explanation. It should be mentioned, however, that in the absence of the necessary amplification to obtain these signals, the positive peak disappears. It may be an electrical artifact due to charge and discharge of capacitors in the series RC networks intercalated in the LF-3dB Point Selector of the Tektronix AM502 amplifier.
LITERATURE CITED (1) Beneridge, D. Anal. Chem. 1978, 50, 832A. (2) Symposium on "Sample Injection Techniques into Unsegmented Flows", "Abstracts of Papers", 176th National Meeting of !he American Chemical Society, Miami Beach, Fia., September 11-14, 1978. (3) International Conference on Flow Analysis, Amsterdam, The Netherlands, September 11-13, 1979. (4) Ruzicka, J.; Hansen, E. H. Anal. Chim. Acta 1978, 99,37. (5) Bergmeyer, H. U.; Hagen, A. Fresenius' 2.Anal. Chem. 1972, 267, 333. (6) Eswara Dun, V. V. S.;Monola, H. A. Anal. Chem. 1975, 4 7 , 357. (7) Wolff, Ch-Michel; Monola, H. A. Anal. Chem. 1978, 50, 94. (8) Nikoielis, D. P.; Mottola, H. A. Anal. Chem. 1978, 50, 1665. (9) Konecny. J. I n "Survey of Progress in Chemistry", Vol. Ba; Academic Press; New York, 1977; p 206. (10) Butler, L. Arch. Biochem. Siophys. 1975, 171, 645. (11) Chlapowski. E. W.; Mottola, H. A. Anal. Chim. Acta 1975, 76, 319. (12) Sandell, E. B. "Colorimetric Determinations of Traces of Metals", 3rd ed.; Interscience: New York, 1959; Chapter XXII. (13) Yatsimiirskii, K. B. "Kinetic Methods of Analysis"; Pergamon Press: Oxford. 1966; pp 120-121. (14) Khalifa. K.; Doss, H.; Awadallah, R. Analyst (London) 1970, 95,207. (15) Bergamin Filho, H.; Reis, B. F.; Zagatto, E. A. G. Anal. Chim. Acta 1978, 9 7 , 427. (16) Bergamin Filho, H.; Zagatto, E. A. G.; Krug, F. J.; Reis, B. F. Anal. Chin?. Acta 1978, 101, 17. (17) Ringbom, A. "Complexation in Analytical Chemistry". Wiley-Interscience: New York, 1963; p 310.
RECEIVED for review April 16,1979. Accepted June 14,1979. This work was supported by the National Science Foundation (Grant CHE-7681587-A01)and it was in part presented at the 1978 Symposium on Sample Injection Techniques into Unsegmented Flows, 176th National Meeting of the American Chemical Society, Miami Beach, Fla., September 14, 1978.
