Polarographic Determination of 3-Nitropropanoic Acid MICHAEL M. FRODYMA, LESTER H. MURAMOTO,' and DONALD J. WILLIAMS Department o f Chemistry, University o f Hawaii, Honolulu 7 4, Hawaii HIROMU MATSUlv\OTO Department of Agriculfural Biochemistry, Hawaii Agricultural Experiment Station, Honolulu 7 4, Hawaii
b Recent investigations involving the plant, creeping irldigo, point up the need for a method for the determination of its toxic constituent, 3nitropropanoic acid, which is more rapid and precise: and less complex than procedures currently in use. Optimum conditions for the polarographic determinution of the acid have been defined b y studying the effects of pH, buffer capacity, and temperature upon the diffusion current arising from the reduction of the nitro group. A linear relationship was observed between the diffusion current and the amount of purified acid in the to concentration range 3.5 X
3.5
C
x
10-5~.
indigo (Indigofera spicata Forsk.), a tropical legume introduced from the O r i x t into Hawaii and Latin America some years ago as a forage and cover crop, has considerable economic potential. Currently found in Hawaii, the contin1:ntal United States, and such tropical areas as Ceylon, Puerto Rico, and Venezuela, its value as a forage crop h,ts been lessened by the presmce of a toxic constituent 1%hich has been ic entified as 3-nitropropanoic acid by Morris, Pagan, and Karmke (9). The ebtablishment of tolerance limits for this toxic conitituent, the development of nontoxic varieties of the legume, and similar investigations h a w pointed up the need for a n analytical method for the determination of the acid \\hich would he more precise, fabler, and less complex than the methodb currently in use. Typical of such methods is the modified Cooke procedure ( I ) , currently cmploj ed ai, the H a n aii dgricultural Experiment Station, i%hich is bascd on the determination of the colored speciea formed by the interaction of nitrites with the Grie.sIlosva-? reagent. The nitrite ion results from the alkaline hydrolysis of a n extract of the nitro vompound obtained, in turn, from an iqueoua extract of plant material. Tarious studies ( 4 ) of RELPIKG
Present address, lndustrial Test Lahoratory, Pearl Harbor Naval Shipyard, Pearl Harbor, Hawaii
the polarographic behavior of aliphatic nitro compounds suggested the feasibility of using the electroreduction of the nitro group as the basis for a method which mould offer considerable advantageb over procedures no\\ used. APPARATUS A N D MATERIALS
A Leeds & Northrup Type E Electrocheniograph \vas employed throughout to obtain and record the polarographic data. The polarizing cell assembly was one described by Furman and Sorton ( 2 ) and included the S.C.E. as the reference electrode. The capillary used had constants of m = 1.96 nig. per second and t = 3.72 seconds a t 25" C. in 0.1S KC1 solution n i t h the circuit open and the m e r c q - column a t 34.6 cm. The resistance of the polarographic cell was of the order of 500 ohms, indicating that I R corrections were negligible. Except for the temperature study, all polarograms were recorded a t 25" =I= 0.2" C. The buffers utilized were prepared by adding 6 X NaOH to a 0.431 solution of the acid or major component until the p H of the mixture equaled the pKa of the acid. so as to provide maximum buffer capacity. The rewlting buffer.. nhich were 0.2-11 nith respect to both components, nere diluted 1 to 1 in the preparation of solution, for 110larographing, ab n-ere the 0.2X 11) drochloric acid and 0.2X SaOH d u t i o n \ employed to provide media of lon and high pH, reapectively. A11 pH measurements were made with a Beckman Model G p H meter. Table I include. 3 summary of the data regarding the this study was synthesized froiii ppropiolactone and sodium nitrite accord-
Table 1.
ing to the procedure described by Gresham et al. ( 3 ) and purified by repeated recrystallization from chloroform until needle-like crystals having a iiielting point range of 65" to 66.2" C. nere obtained. Standard solutions were prepared by dissolving mighcd amount> of this purified material in known volumes of solution. PROCEDURE
The polarographic solution. were prepared in 50-nil. volumetric flasks by diluting mixtures of appropriate amounts of aqueous 3-nitropropanoic acid stock solution arid 25 nil. of stock buffer solution to volume n i t h distilled water. The-e solutions were then thermostated a t 25' C. for 15 minutea and deaerated for 15 minutes with oil-pumped nitrogen before recording the polarograms. The adequacy of the buffering in tartrate arid acetate buffers R as checked by polarographing 1.0 X 10-3V solutions of %nitropropanoic acid which were 0.050, 0.100, and 0.160J1 with respect t o each of the buffer components. Thib variation in the concentration of the buffer component\ did not appreciably affect the btructurc of the recduction w a ~ c . The effect upon the half-\va\e potential and the tlifiusion current is summarized in Table 11. The extraction procedure iollon ed in ohtaining samples from plant inaterial n as similar to that used by LTatsumoto el al. ( 5 ) . Two-gram samples of leaf meal were digested with 50 i d . of 0.1S hydrochloric acid for 1 hour on a steam bath. After the digest had cooled, it mas centrifugpd at 2900 r.p.m. for 15 minutes to separate suspended material,
Effect of pH on Half-Wave Potential and Diffusion Current
Major component of buffer system Hydrochloric acid. Tartaric acid Acetic acid Potassium dihj-drogen phosphate Boric acid Sodium bicarbonate Sodium hydroxide
inlffer
solution 0.80 :I. 0 1.8 7.2 9.3 10.3 13.0
graphic: solution
id,
pa.
EI!2,
volt
1.1 3.0
3.01
7.2
2.0% 2.86
--0.67 -0.62 -0.78 -0.92
0.98
--0.99
4 7
9.3 10.4 12.6
3.14
... ...
VOL. 35, NO. 10, SEPTEMBER 1963
...
...
14.83
Table II.
Buffer Tartrata Acetate
PH
Concn. of buffer components, M
3.1 3.0 3.0 4.7 4.7 4.7
0.050 0.100 0.160 0.050 0.100 0.160
Table 111.
Effect of Temperature on Diffusion Current (Acetate buffer. pH = 4.7)
Teamc.P.
i d , pa.
15
21.24
25
24.75
35
28.25
45
31.50
55
35.50
1
Temp. coefficient ( % change) (degree) 1.54 1.33
1.10 1.20
Av. 1.29 f 0 . 1 4 Table IV.
Effect of Mercury Pressure on Diffusion Current
h ~cm. ~ hcom, , cm. 54.6 49.6 44.6 34.6 29.7
fusion-controlled nature of the reduction (6). The reversibility of the reaction was checked by plotting Ed.#. us. log i/ (id+ for the reduction of a 1.7 X 121 solution of the acid in acetate buffer. The plot resulted in a straight line whose slope was such that a value of n equal to 0.60 was obtained, indicating that the electrode reaction was irreversible. This result is in accord with those reported in previous investigations involving aliphatic nitro compounds (4). Concentration Study. A straightline relationship was found to exist between the diffusion current and the amount of the purified acid over the concentration range 3.5 x 10-3 to 3.5 x 10-6&i' in acetate buffer. The relative constancy of the i d / C ratio obtained over this range may be seen in Table V. The diffusion current constant, ID = id/Cm2/3t1'6,under these experimental conditions-acetate buffer, 0.1M with respect t o each of its components, a t 25" C.-was 7.23 (10.12) X 10-3.
Results of Buffer Capacity Study M a d e in Tartrate and Acetate Buffers
53.0 48.0 43.0 33.0 28.1
id,
pa.
id/h"*eft
32.50 30.50 28.50 24.72 22.86
4.464 4,402 4.346 4.303 4.312 Av. 4.366 Std. dev. 10.068 Rel. std. dev. A I . 55
and the centrifugate was filtered through Whatman No. 1 filter paper. The polarographic solutions were prepared by adding 20 ml. of this plant extract to 25 ml. of acetate buffer in a 50-ml. volumetric flask and diluting to volume with distilled water.
id,
pa.
2.85 2.78 2.74 2.80 2.75 2.64
-0.64 -0.61 -0.61 -0.80 -0.78 -0.76
ions, whereas the sudden drop in wave height observed above pH 7 can be ascribed to the tautomeric conversion of the reducible nitro form of the electroactive group to the irreducible aci form. In view of these results, it was decided to carry out the determination in the acid range where the reducible nitro form predominates and where there is no polarographically detectable shift in the nitro-aci equilibrium with time (8). Characteristics of Electrode Process. T h a t the reduction is diffusioncontrolled became apparent when a 1.7 X 10-3M solution of the acid was polarographed in acetate buffer (pH 4.7) over a 15' to 55' C. range a t 10' intervals and a linear relationship between temperature and diffusion current was found (Table 111). The average temperature coefficient of the current thus obtained, +1.3%, is of the order expected for a diffusion-controlled process (7). This conclusion was substantiated by studying the effect of varying the mercury pressure on the diffusion current obtained with a solution of the same composition. The fact that the quantity id/h1/2,ffwas constant within experimental error over a wide range of mercury pressures (Table IV) is likewise indicative of the dif-
Table V.
Effect of Concentration on Diffusion Current Concn. of acid added, idlC x 10-4~ id, pa. x 104 0.510 1.47 0.347 1.04 1.63 1.57 2.55 1.47 1.73 2.43 3.60 1.46 5.30 1.53 3.47 10.4 14.9 1.43 25.0 1.44 17.3 34.5 1.40 24.3 34.7 50.0 1.44 Av. 1.47 Std. dev. 3 ~ 0 . 0 5
.,il
RESULTS AND DISCUSSION II
pH Study. When solutions which were 1.0 X 10-4M with respect to 3nitropropanoic acid were polarographed a t the pH's provided by the buffer systems listed in Table I, a single, welldefined wave resulted in all but the bicarbonate and NaOH solutions (Figure 1). Table I shows the manner in which the half-wave potential became more negative and the diffusion current decreased-especially in the alkaline range-as the pH increased. The data are substantially those expected in light of the findings reported in investigations of the polarographic behavior of other aliphatic nitro compounds. The shift of the half-wave potential to more negative potentials is characteristic of the electroreduction of organic compounds involving hydrogen 1404
0
ANALYTICAL CHEMISTRY
L
____/'
PHOSPHAlt B O R R
BICARBONATE
-c--
-
Na OH 0.1 VOLI
Figure 1.
Effect of pH on reduction curve of 3-nitropropanoic acid All curves begin at 0.00 volt
VI.
S.C.E.
When an extraci; of a composite 2gram sample of creeping indigo leaf meal containing approximately 0.3% of 3-nitroprop:tnoic acid (6) was polarographed in acetatt: buffer, the single well-defined wave which resulted differed from that obtained with the pure acid only in t h a t its slope was less and the half-wave potential was more negative. That ths linear relationship between concentration and diffusion current persisted in the plant extract was shown by adding increments of the acid stock solution and polarographing the resulting mixtures, which ranged to 3.5 X lO+M with from 5.4 X respect to the added acid. When an extract of alfalfa meal which did not contain any 3-nitropropanoic acid ( 5 ) was accorded the E)ame treatment, the wave resulting from the reduction of the added acid was similar to that obtained with the creeping indigo extract. Data obtained in this manner, less a blank value of 1.09 X ll)-4M, provided the standard curve used to determine the concentration of acid in creeping indigo. As may be seen in Table VI, which summarizes the results of this plant extract study, the idiffusion current resulting in these instmces was consistent with that obtained with purified acid for comparable amounts of electroactive material. The polarographic determination of 3-nitropropanoic acid in 14 samples of creeping indigo meal which had been dried for 36 hours a t 58” C. gave values ranging from 0.171 to 0.4710/,. These
Table VI. Plant Extract Study Concn. of x i t i added, id/(? x 10-4111 i d , fia. x lo4 EL,z,volt
Creeping Indigo 0.346 1.72 2.39 3.08 4.06 5.38
0.51 2.44 3.47 4.36 5.81 7.70
1.48 1.42 1.45 1.42 1.43 1.43 Av. 1.44 &
-0.86 -0.90 -0.90 -0.90 -0.91 -0.90 0.02
Alfalfa Meal 0.346 1.03 1.72 2.39 3.08 4.06 5.38
0.52 1.49 2.54 3.55 4.61 6.09 8.09
1.50 -0.97 1.45 -0.96 1.48 -0.95 1.48 -0.94 1.50 -0.94 1.50 -0.94 1.50 -0.94 Av. 1.49 f 0.01
agreed substantially with values obtained for parallel determinations carried out on the same samples according to the method of Matsumoto et al. ( 6 ) . I n this range the standard deviations found for the polarographic and spectrophotometric methods were 0.0031 and 0.0071, respectively. The standard deviation for a set of replicate polarographic analyses carried out on an alfalfa meal sample containing 0.026% of added Bnitropropanoic acid was 0.0025. The relative simplicity and rapidity of the polarographic method become apparent when one considers that the analysis can be carried out
directly on the buffered and deaerated plant extract in approximately 5 minutes. The spectrophotometric method, on the other hand, entails heating the buffered plant extract in the presence of formaldehyde for an hour to displace the nitro group of the 3nitropropanoic acid and then going through a rather involved procedure to determine the nitrite ion formed. LITERATURE CITED
( 1 ) Cooke, A. R., Arch. Biochem. Biophp. 5 5 , 114 (1955). ( 2 ) Furman, N. H., Norton, D. R., ANAL. CHEM.26, 1111 (1954). (3) Gresham, T. L., Jansen, J. E., Shaver,
F. W., Frederick, M. R., Fiedorek, F. T., Bankert, R. A., Gregory, J. T., Beears, W. L., J . Am. Chem. SOC.7 4 , 1323 (1952). ( 4 ) Kolthoff, I. ,,M., Lingane, J. J., “Polarography, 2nd ed., Vol. 11, p. 746, Interscience, New York, 1952. (5) Matsumoto, H., Unrau, A. M., Hylin, J. W., Temple, B. P., ANAL. CHEM.33, 1442 (1961). (6) .Meites, L., “Polarographic Techniques,” p. 52, Interscience, New York, 1955. ( 7 ) Ibid., p. 56. ( 8 ) Miller, E. W., Arnold, A. P., Astle, M. J., J. Am. Chem. SOC. 70, 3971 (1948). ( 9 ) Morris, M. P., Pagan, C., Warmke, H. E., Science 119, 322 (1954).
RECEIVEDfor review October 2, 1961. Resubmitted May 13, 1963. Accepted July 1, 1963. Abstracted from a dissertation presented by Lester H. Muramoto to the Graduate School of the University of Hawaii in partial fulfillment of the requirements for the degree of master of science, June 1961.
Coulometric Efficiency of Anodic Deposition and Cathodic: Stripping of Chloride at Silver Electrodes H. A. LAITINEN and
ZUI-FENG LIN1
Department of Chemistry, University of Illinois, Urbana, 111.
b Deposition at ccmstant current from relatively high (0.1 to 1 .OM) concentrations of chloride followed by cathodic stripping at constarit current gave current efficiencies (CE) of essentially zero at extremely low current densities (CD), increasing hyperbolically toward unity with increasing CD. This behavior could be attributed to the formation of AgC12- complex. At low chloride concentrations ( l o w 3tl3 lO-*M), the apparent CE increased with increasing CD, attained a maximum, and then decreased. This behavior could be attributed to dissolution of AgCl at low CD and formation cif free Ag+ at high CD. At 0.01M chloride, the low solubility and low rate of dissolution permit a CE of very nearly unity over a wide range of CD. A coulometer based on
this finding is suggested. Deposition at a constant potentiat, followed by cathodic stripping, gave roughly quantitative recovery of chloride, with a relative standard deviation of *2.5?& and a 1.6%, at chloride concentrabias of tions of 1.76 to 17.6 fig. per ml.
+
T
HE ELECTROLYTIC behavior of chloride and its application to microdeterminations have been studied by many authors. Lingane and Small (14) used a controlled anode potential of 0.25 f 0.01 volt us. SCE to determine 0.25 to 3.0 mg. of C1- coulometrically. Shain and Perone (19) made a limited study of C1- determinations in concentrations down to 4 X 10-6M in 80% ethanol by collecting C1- on a minute
Ag electrode a t 0.30 volt us. SCE followed by linear voltage scanning stripping. Several analytical methods have been proposed for the determination of traces of chloride, based on the anodic formation of films on mercury rather than on silver (1, 4, 10). As far as we can ascertain, however, none of these methods is based on the quantitative deposition of chloride and therefore the precision and accuracy are limited by the reproducibility of the deposition step. Moreover, these investigations involving mercury are not directly pertinent to the present study.
* Present address, Chemistry Department, National Taiwan University, Taipei, Taiwan (Formosa), China. VOL. 35, NO. 10, SEPTEMBER 1963
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