A three component mixture of 6-D-glucose, D-galactose, and sucrose can be analyzed using three enzymes: glucose oxidase, galactose oxidase, and invertase. Analysis is possible because of the specificity built into these enzymes. Some results obtained in the analysis of such mixtures are given in Table X. It was found that concentrations of galactose up to 100 times that of glucose did not interfere in the determination of the latter, and vice versa. Three component mixtures of glucose, galactose, and sucrose were analyzed for all three components with an accuracy and precision of about 1.5 %. One aliquot (A) was analyzed for glucose using glucose oxidase, another (B) for galactose using galactose oxidase in procedures as described above in the experimental section. A third aliquot
(C) was analyzed for sucrose by addition of invertase to liberate glucose, followed by a determination of total glucose with glucose oxidase. The amount of sucrose present was calculated by subtracting the glucose found in A from that found in C. ACKNOWLEDGMENT
The authors thank Nelson Richtmeyer of the National Institutes of Health for samples of two sugars used in this study. RECEIVED for review February 12, 1968. Accepted April 3, 1968. Work supported by National Science Foundation Grant No. GB 6325.
Use of Metal Ion Catalysis in Detection and Determination of Microamounts of Complexing Agents Autoxidation of 1,-Ascorbic Acid as an “Indicator” Reaction Horacio A. Mottola,’ Martha S. Haro, and Henry Freiser Department of Chemistry, University of Arizona, Tucson, Ariz. 8572I A method for the determination of microamounts of metal complexing agents based on their interaction with metal ion catalysts for oxidation-reduction reactions is presented. The rate of decrease in absorbance of L-ascorbic acid at 265 mp due to atmospheric oxidation catalyzed by Cu(ll) is the basis of a convenient method for the detection and determination of certain Cu(ll)-complexing ligands in aqueous media at pH -6.4. Trace amounts of cysteine, 2-aminoethanethiol, salicylic acid, EDTA, 1,lO-phenanthroline, and ethylenediamine have been determined by this means.
IN THE SEARCH for increasingly sensitive analytical reactions, chemists have employed reagents that would give intensely colored or fluorescent products. This approach, though fruitful, is limited finally by the requirement that the compound of interest be stoichiometrically related to the product. Substances that have catalytic activity or which can modify the action of catalysts, in principle, can be detected at much lower concentrations because the reactants or products of the catalyzed reaction (indicator reaction) will be present at relatively high concentrations, so that changes in these can be readily measured. Provided the uncatalyzed reaction is sufficiently slow, increase of reaction time will further increase the sensitivity of catalyst detection. Both “coordination chain” ( I ) and metal ion-catalyzed oxidation-reduction reactions (2) may be used as indicator reactions (that which provides the time-dependent signal) (3). In both cases the ‘Present address, Department of Chemistry, Oklahoma State University, Stillwater, Okla. 74074 (1) D. W. Margerum and D. K. Steinhaus, ANAL.CHEM., 37, 222 (1965).
(2) H. A. Mottola and H. Freiser, ibid., 39, 1294 (1967). (3) K. B. Yatsimirskii, “Kinetic Methods of Analysis,” Pergamon Press, New York, 1966, p 1.
analytical determination is based on the modification of reaction rate by complexation. A great advantage of such methods is the ease of monitoring the reaction rate. Because the indicator reaction components are in relatively high concentration, a suitable reaction parameter is easily found: absorption or emission of radiant energy, heat of reaction, or any other physical property that would change regularly with the course of the reaction. The application of a metal ion-catalyzed redox reaction to the determination of traces of ethylenediamine-NNN’N’-tetraacetic acid (EDTA) has been reported recently (2), using as indicator system, the oxidation of Malachite Green by periodate ion in which manganese(I1) acts as catalyst. The relatively high values of formation constants of Mn(I1) with EDTA and some of its analogs make the system selective since most other N-containing ligands will not complex Mn(I1) under the experimental conditions employed. This illustrates one of the first steps involved in selecting a suitable reaction system: one in which the metal ion catalyst forms complexes of sufficient stability with the ligand(s) of interest so that formation occurs at low ligand concentrations. If the metal ion concentration required for measurable catalysis is about 10-6M and a change in rate is detectable for a 10% change in metal ion concentration-i.e., [ML]/[M] = 0.1 (assuming the 1 :1 complex to predominate)-it follows that at least lO-7M of the agent is required for formation. Assuming that an additional lO-7M agent is required to ensure complex formation according to mass action considerations, this would give a response whose sensitivity is 2 X 10-7M for the agent. I t can be estimated that the minimum required stability of the metal-agent complex under these conditions would be Ki’
=
0.1 [ML]/[M][L] = 10-7
=
106
where Kl’ is the conditional stability constant.
Such K values
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1263
8.0 -
10.0
.-E
‘c
6.0 -
roc X F 4,O
-
2.0 -
8 5.0 4.0
6.0 5 IO TIME, MINUTES Figure 1. Typical first order plots in presence of different amounts of ethylenediamine(en) Ascorbic acid concentration: 4.01 X 10 -6M. Catalyst: 1.98 X 10-6M Cu(I1). A . Ascorbic acid alone; B, C, D,E, and F. ascorbic acid plus catalyst. C. 8.47 X 10-6Men, D. en, F . 2.11 X 10-6 en 4.23 X 10-5Men, E. 8.46 X
can be expected with metal cyanides, iodides, sulff des, and many chelates. The value of the pH, which affects the concentration of ligands having proton affinity, and thereby the value of the conditional constant, can affect the selectivity of the method (2). For complexes having high formation constants to begin with, pH reduction can proceed further without excessive KI’ reduction, providing the basis of greater selectivity. As an example, the iodide-catalyzed reaction of Ce(1V)As(III), inhibited by trace levels of Hg(I1) and Ag(1) can be used as a rather selective “indicator” system for the determination of sulfur-containing ligands such as mercaptoacetic acid, 2-aminoethylthiol, thioacetamide, and dithioxamide (4). The formation constants of Hg(I1) complexes with sulfurcontaining ligands, but not with most nitrogen or oxygen ligands, are high enough to permit operating at a pH of 1.0. Even though selectivity is highly desirable, an indicator reaction of wide applicability can be very useful provided sensitivity is not lost. This paper introduces an indicator reaction of wide applicability, inasmuch as ligands containing oxygen, nitrogen, as well as sulfur, as donor atoms may be detected and determined. The indicator reaction in this system is the Cu(I1)-catalyzed atmospheric oxidation of Lascorbic acid which is quite convenient because its rate can be readily adjusted cia pH control to permit runs at room temperature. The signal (decrease in ascorbic acid concentration) can be easily followed photometrically in the UV region (5). The reaction can be run at low hydrogen ion concentrations facilitating complexation. Further, because it involves dilute solutions, the danger of reagent contamination is decreased.
(4) Gail Miller and Martha S. Haro, unpublished results, University of Arizona, Tucson, 1967. (5) R. W. Herbert, E. L. Hirst, E. G. V. Percival, R. J. W. Reynolds, and F. Smith, J . Chem. SOC.,1933, 1280. 1264
0
ANALYTICAL CHEMISTRY
-LOGIO [En] Figure 2. Working curve for determination of ethylenediamine(en) using the slopes of Figure 1 -k
=
2.303 X slope of first order rate plots
Several complexing agents giving stable complexes either with Cu(I1) or Cu(1) have been tested. Ethylenediamine, salicylic acid, 1,lo-phenanthroline, EDTA, cyanide, 2-aminoethanethiol, cysteine can be detected. EXPERIMENTAL Reagents and Solutions. The water used was of the same quality reported before (2). Initial batches of all solutions were stored for several days in borosilicate glass containers and then discarded in order to precondition the containers. All reagents are of AR grade or purified as indicated. BUFFERS.The work reported here was performed using phosphate buffers of pH = 6.40 and p = 0.01 because they are transparent at 265 mp. ASCORBIC ACIDSOLUTIONS.L(+)-Ascorbic acid (Mallinckrodt, AR) was used as received. Stock solutions (wlO-3M) were used within three days of preparation. COPPER SOLUTIONS.A copper stock solution (ylO-3M) was prepared from Cu(C10&.6HzO (G. F. Smith Chemical Co.,), from which’dilutions were made within 8 hours of use. The stock solution was standardized by EDTA titration (6). LIGANDS.L(+)-cysteine hydrochloride (Eastman, white label) and 2-mercaptoethylamine (Aldrich Chemical Co., m.p. 67-70 “C),were used without further purification. A stock solution (app. 10-3M) in 0.10M HC1 was prepared daily and standardized by titration with ferricyanide (7). Salicylic acid (Mallinckrodt, AR, m.p. 156-8 “C) was used without purification in a 10-3M stock solution prepared daily. 1,lo-Phenanthroline (Eastman, white label) was recrystallized twice from warm water and dried at 100 “C to constant weight. The final anhydrous product had a melting point of 117-18 “C. Solutions of EDTA were prepared and standardized as reported earlier (2). Stock solutions (-10-2M) of ethylenediamine (Matheson, Coleman and Bell, 98-100%) standardized by potentiometric
(6) H. A. Flaschka, “EDTA Titrations,” Pergamon Press, New
York, 1959, p 78. (7) H. L. Mason, J . Biol. Chem., 86, 623 (1930).
Table I. Determination of Various Complexing Agents Either by Slope Method [k (Observed First Order Rate Constant) US. Ligand Concentration] or One Point Determination (Absorbance at Constant Time us. Ligand Concentration), Using [Cu2+] = 2 X 10-M Reoroducibilitv data Useful range of 2 ligand concentration Ligand concentration Standard deviation Complexing agent 3.0 x 10-6M (6)b 0.10 x 10-6 1 to 5 x 10-5M 2-Aminoethanethiol 2.0 x 10-6M(6)b 1 to 8 X IO-eM 0.15 x 10-6 Cysteine 7.0 x lO-6M (6)b 2 to 8 X lO-6M 0.09 x 10-6 EDTAa 4.2 x 10-6M(8)b 0.02 to 2.00 x 1 0 - 4 ~ 0.07 x 10-5 Ethylenediamine 1.7 x 10-6M(8)b 0.03 to 1.00 X 10-6M 0.10 x 10-6 1,lo-Phenanthroline 0.14 x 10-7 3.4 X lO-’M (8)b 1.04 X 10-4M(8)5 0.4 to 4 x 1 0 - 4 ~ 0.15 x 10-4 Salicylic acid 4.16 x 10-4M(8)b 0.36 x 10-4 a With [Cut+] = 4 x 10-7M, the useful concentration range can be lowered to 2 to 6 X 10-7M. Number of replicates.
titration with hydrochloric acid (8) were found to be stable for at least a week. APPARATUS.Absorbance measurements were made with a Beckman DU spectrophotometer and I-cm (occasionally 10-cm) silica cells. A constant 0.10-mm slit width was used in all cases. A Beckman GS pH-meter with a glass electrode-calomel pair, standardized with Beckman buffers at pH 7.00, was used for pH control. Procedures. In a 25-ml volumetric flask there is added 1.0 ml of phosphate buffer pH 6.4, p = 0.25 and then, in the following order: given volumes of ligand solution, Cu(I1) solution, and 1.0 ml of ascorbic acid stock solution. The flask is shaken vigorously during addition to avoid reaction in a localized zone. The solution is made up to volume with water. The initial time (t = 0) is taken as the instant when ascorbic acid is added to the system. The temperature of the reaction mixture is maintained at 25 =t 1 “ C (controlled temperature laboratory). The absorbance is read every 3 minutes during 15-20 minutes and plotted as log absorbance os. time to establish the slope of first-order plot (Figure 1). The concentration of ligand is found from a working curve constructed from known amounts of ligands such as shown in Figure 2. A less accurate but faster method may be used in which measuring absorbance at a constant time and plotting this as a function of ligand concentration (Figure 3). RESULTS AND DISCUSSION
Autoxidation of ascorbic acid is very slow at pH values below 7 in the absence of metal ion catalysts (9, 10). The presence of traces of copper(I1) provides sufficient catalytic activity for the reaction to proceed at measurable rates at pH values above 5. The major product of the reaction is dehydroascorbic acid (75 %) with oxalic and l-threonic acids comprising the remaining products. At the pH selected for this study (6.40) on the basis of convenience (many ligands of interest have copper complexes with reasonable Kl’ values at this pH), ascorbic acid is present predominantly as the monoprotonated anion (pKal = 4.49) (11) which has an absorption band centered at 265 mp (5). Inasmuch as none of the (8) E. F. Hillenbrandt, Jr. and C. A. Pentz, in “Organic Analysis,” Vol. 111, J. Mitchell, Jr., I. M. Kolthoff, E. S . Proskauer, and A. Weissberger, Editors, Interscience, New York, 1956, pp 142-3. (9) . . E. Guzman Barron, R. H. de Meio, and F. Kiemperer, J. Biol. Chem., 112-625 (1936). (10) V. S . Butt and M. Hallawav. Arch. Biochem. BioDhvs., _ _ 92, . .
.
_
CYSTEINE CONCENTRATION, MICROMOLES/LITER Figure 3. Working curve for determination of cysteine Absorbance measured at 265 rnr after 20 minutes of reaction. Ascorbic acid concentration: 4.16 X lO-5M; catalyst, 2.06 X 10-6M Cu(I1); buffer as reference oxidation products absorb at 265 mp, it is convenient to follow the course of the reaction spectrophotometrically at this wavelength. According to Nord (12) the catalyzed reaction is proportional to ascorbic acid, copper, and dissolved oxygen concentrations and is a function of pH. In our work, temperature control of 1-2 “Cwas found to be sufficient to reduce variation of either rate constant or oxygen solubility below the level of experimental reproducibility desired. Our work confirmed the first order dependence of the rate on ascorbic acid and copper concentrations. No special precautions had to be taken to assure saturation of the reaction mixture by 02. The decrease of the rate of the catalyzed autoxidation by the addition of Cu-complexing ligands attests the inability of these complexes to function as catalysts, an interesting contrast to the catalytic activity some Cu-amine complexes have in analogous oxidations of phenols (13). The extent of decrease in the rate by the ligands tested would indicate by the linear relationship that the ligand combines stoichiometrically with the copper and thereby reduces the amount available for catalysis. A list of the ligands tested and a summary of results is shown in Table I. It will be noted that the copper concentration
I
24(1961). (11) “Stability Constants of Metal Ion Complexes,” compiled by L. G. Sillen and A. E. Martell, The Chemical Society, London, 1964.
(12) H. Nord, Acta Chem. Scand., 9,442 (1955). (13) W. Brackman and E. Havinga, Rec. Trau. Chim., 74, 937 (1955). VOL. 40, NO. 8, JULY 1968
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used in these runs is 2 X 10-6M. The use of higher metal concentrations would undoubtedly extend the range to higher ligand levels. All determinations were performed either by slope (Figures 1 and 2) or one point determinations (Figure 3). Most of the experiments have been performed with 1-cm silica cells. The use of IO-cm cells provides greater sensitivity. It is interesting to note that the useful range of concentration for 1,lO-phenanthroline is an order of magnitude smaller than simple equilibrium considerations based on Cu(I1)phenanthroline data would predict. We have no explanation for this behavior at the present, unless the phenanthroline complex of Cu(1) is more stable than that reported for Cu(1I).
Examples of curves used for determination are shown in Figures 1, 2, and 3. CONCLUSIONS
We have demonstrated the suitability of the Cu(I1)-catalyzed autoxidation of ascorbic acid for the development of a rapid, simple, and sensitive means of determining a wide variety of complexing agents.
RECEIVED for review August 7, 1967. Accepted March 2 5 , 1968. Work supported by Edgewood Arsenal through Contract DA18-035-AMC-744(A).
Use of Metal Ion Catalysis in Detection and Determination of Microamounts of Complexing Agents Catalimetric Titration of Cyanide Ion Horacio A. Mottola’ and Henry Freiser Department of Chemistry, Unizersity of Arizona, Tucson, Ariz. 85721
A new and widely applicable approach in the development of methods for the detection and determination of trace levels of complexing agents by modifying the rate of metal-ion catalyzed redox reactions is presented. The metal ion catalyst is used as a titrant for a solution containing the complexing agent as well as an indicator reaction mixture. Catalysis becomes effective only after the ligand has become complexed so that a dramatic change is observed with a slight excess of the catalytic titrant. Using as indicator reaction the autoxidation of L-ascorbic acid catalyzed by traces of Cu(lI), low concentrations of cyanide ions (lO-7M level) have been determined in a simple and reproducible fashion. The end point i s reached when one mole of copper has reacted with two moles of cyanide ion. The effect of various parameters affecting the titration are discussed. The effect of some interferences has been considered. THEPRACTICAL VALUE of metal-catalyzed oxidation-reduction reactions for the determination of minute amounts of complexing agents has been recently demonstrated ( I , 2). This paper presents a new analytical approach which permits simpler and faster determinations. In this method, readily adaptable to automation, the metal ion catalyst is used as a titrant. The possibility of using catalytic reactions as an end point indication had been first pointed out by Yatsimirskii (3), who coined the term catalimetric titration, but has been practically neglected. The autoxidation of L-ascorbic acid catalyzed by traces of Cu(I1) provides a versatile and sensitive indicator reaction for 1 Present address, Department of Chemistry, Oklahoma State University, Stillwater, Okla. 74074
(1) H. A. Mottola and H. Freiser, ANAL.CHEM., 39, 1294 (1967). (2) H. A. Mottola, M. S. Haro, and H. Freiser, ibid., 40, 1263 (1968).
(3) K. B. Yatsimirskii and T. J. Fedorova, Proc. Acad. Sci. USSR, 143, 143 (1962).
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ANALYTICAL CHEMISTRY
the detection and determination of several complexing agents with different donor atoms (2). The determination of CNat pH 6.5 using the methods described earlier (2), was found to be of limited utility because it was applicable in a very narrow concentration range. These observations, in agreement with earlier observations at higher concentrations and by different techniques (4), suggested the use of a new titrimetric approach in which increments of Cu(I1) solution would be added to the indicator reaction mixture containing CNuntil a [CUIlevel is reached at which the reaction becomes fast enough to be easily followed. The amount of titrant required to reach this point would be proportional to the cyanide content of the sample, provided that the copper cyanide formation is complete and rapid (relative to the rate of the autoxidation). EXPERIMENTAL
Reagents and solutions as well as general precautions have been described earlier ( 2 ) . Most titrations were performed in a carbonate-bicarbonate buffer of pH = L0.50 ( p = 0.01). All determinations were made at room temperature, 23-5 “C. Apparatus. The photometric cell (40 mm x 60 mm x 50 mm) was constructed from 3-mm Plexiglas sheets sealed with epoxy resin. The path length was approximately 42 mm. The cell contained two quartz windows made from 1-mm transparent fused quartz microscope slides (Thermal American Fused Quartz Co.), 35 X 25 mm in size. The cell is similar to one described by Headridge (5). Stirring was accomplished by means of an air-driven magnetic stirrer (Chemical Rubber Co.) located underneath the cell compartment and using a ‘/*-inchTeflon-covered magnetic stirring bar in the cell. The titration assembly was composed of a Beckman DU spectrophotometer equipped with hydrogen lamp and photo-
s. Butt and M. Hallaway, Arch. Biochem. Biopltys., 92, 24 (1961). ( 5 ) J. B. Headridge, “Photometric Titrations,” Pergamon Press, New York, 1961, p 31. (4) V.
