Table I.
Sample
True value
Summary of Data on Metals by Atomic Absorption Spectrophotometry
Number of results
Average
Mean error
0.055 0.502 1.030
+0.005 +o .002 $0.030
1 2 3
0.05 0.50
1 .oo
51 48 49
1 2 3
0.05 0.25 1 .oo
57 58 53
0.067 0.271 1.034
+0.017 +0.021 +Os 034
1 2 3
0.05 0.10 0.20
7 7 7
0.055 0.093 0.215
+O .005
Standard deviation Absolute Relative Zinc 0.023 0.041 0.108 Copper 0.031 0.065 0.116
Total error
f
fk
41.8 8.2 10.5
1.55 0.31 1.94
2.009 2.012 2.011
92.0 16.4 21.6
47.3 2.0 11.2
4.14 2.46 2.11
2.003 2.002 2.007
158.0 60.4 26.6
17.5 10.7 7.6
1.32 1.86 2.33
2.447 2.447 2.447
40.0 20.0 17.0
Silver -0.007 +0.015
are subject to a systematic error should be separated from cases where there is no systematic error. The systematic error may be understood to be involved in cases where the absolute value of the mean error statistically differs from zero on a selected significance level. According to criterion (B), results of the determination of Zn, Cu, and Ag by atomic absorption photometry stated in the study (I) would then have to be assessed as shown in Table I. In this table, t k is the critical value of the Student’s t-distribution on the Significance level (Y = 0.05. It is evident that the total error of the determination of Zn and Ag is lower than stated in (I) when the criterion (A) is applied. The mean error here is statistically insignificant on the significance level a = 0.05 and, therefore, it is not included in the total error value da according to criterion (B). The result of assessing the results of the method of determining Cu are the same as in the study ( I ) , since the mean error significantly differs from zero on the significance level of a = 0.05 and, therefore, is not included in the total error. Assessment of the applicability of an analytical method with the use of a criterion which includes the standard devia-
0.010 0.010 0.017
tion should, however, always be based on an approximately equal, and sufficiently large number of determinations. For example, however, in ( I ) the determination of Ag by atomic absorption photometry is based on seven results while assessment of the determination of Zn with the use of the same method is done from 51 results. This means that the relative width of the reliability interval of the standard deviation estimate is 160% in the first case, and only 40% in the second. Under these conditions, however, it is difficult to compare the total error of the determination of two elements by means of the same method. KARELECKSCHLAGER
Institute of Inorganic Chemistry Czechoslovak Academy of Sciences Prague, Czechoslovakia
RECEIVED for review March 29, 1971. Accepted January 6,1972.
Aromatic Hydroxylation as an Analytical Reaction SIR: The phenyl group has not attracted much analytical attention as a functional group, though it offers the possibility of a general approach to the detection and determination of aromatic compounds. Nitration and bromination have found limited use (I). Aromatic hydroxylation is an attractive reaction because the product, a phenol, is amenable to detection and measurement by many methods. Little prior use has been made of aromatic hydroxylation. Benzoic acid can be determined in foods by conversion to salicylic acid (12% yield) upon treatment with a reagent of ferric ion, hydrogen peroxide, and sulfuric acid (2, 3 ) ; the salicylic acid is removed by extraction and is determined colorimetrically. Bartos (4) determined several aromatic compounds colori(1) N. D. Cheronis and T. S. Ma, “Organic Functional Group
Analysis by Micro and Semimicro Methods,” Interscience Publishers, New York, N. Y., 1964, p 443. (2) J. R. Nicholls, Analyst (London),53, 19 (1928). (3) N. L. Allport and J. E. Brocksopp, “Colorimetric Analysis,” Vol. II,2nd ed., Chapman and Hall, London, 1963, p 52. (4) J. Bartos, Ann. Pharm. Fr., 27,759 (1969).
metrically through formation of o-nitrosophenols by means of a cupric ion-hydrogen peroxide-hydroxylamine reagent. Of the several hydroxylating agents that have been proposed (usually as model systems for enzymes), that ofHamilton seemed to provide advantages of simplicity, yield, and speed (5,6). The Hamilton system consists of an aqueous solution of ferric ion, hydrogen peroxide, and catechol (or another enediol). Hamilton et af. (5,6) studied the hydroxylation of some monosubstituted benzenes, measuring the kinetics of the loss of peroxide when the aromatic substrate was in excess. Anisole and nitrobenzene were found to undergo hydroxylation at comparable rates, ruling out an electrophilic substitution as the mechanism. The present paper reports the application of this system to the quantitative determination of some aromatic compounds. ( 5 ) G. A. Hamilton, J. P. Friedman, and P. M. Campbell, J. Amer. Chem. Soc., 88,5266 (1966).
(6) G. A. Hamilton, J. W. Hanifin, and J. P. Friedman, ibid., p 5269. ANALYTICAL CHEMISTRY, VOL. 44, NO. 4, APRIL 1972
879
Table I.
Aromatic Compounds Determined by the Hydroxylation Procedure
Reaction time, hr 1 1 1
OB2t
O'Ob
io
Qo 80 Time (minutes)
40
rbo
IhO
Figure 1. Change of absorbance with time for hydroxylatic of anisole at room temperature in aqueous solution, using the analytical procedure described Initial concentrations: anisole, 3.96 X lO-'M; ferric perchlorate, 5.9 X lO-'M; hydrogen peroxide, 3.6 X 10-3M. 0,110 cyclodextrin; 0,1.27 X 10-3M8-cyclodextrin
Analytical wavelength, nm"
Sample 10-3~.,,b 505 4.5 Benzene 505 1.8 Toluene Ethylbenzene 505 1.4 1 505 3.9 Anisole Benzyl alcohol 1 505 2.8 Benzoic acid 2 495 2.5 2 505 2.1 Benzamide Nitrobenzene 1 395 1.9 2 510 3.9 Acetanilid Methyl benzoate 2 510 1.6 Hydrocinnamic acid 1 505 2.1 a-Methoxybenzoicacid 1 493 3.6 m-Methoxybenzoic acid 2 49 3 2.4 p-Methoxybenzoic acid 2 485 4.2 CY-Meth ylbenzylamine 2 500 4.1 a Color developed by the 4-aminoantipyrine method except with nitrobenzene, which was measured through the nitrophenolate absorption. * Apparent molar absorptivity based upon total sample concentration, regardless of fate, in the final colorimetric solution.
EXPERIMENTAL
The following analytical procedure has been developed: To a 50-ml volumetric flask are added 2.0 ml of 3.0 X 10-aM aqueous catechol, 2.0 ml of 1.5 x 10-aM ferric perchlorate, and enough of the aromatic compound (contained in up to 16 ml of aqueous solution) such that its concentration in the diluted solution will be in the range lo-' to 10-aM. Thirty milliliters of pH 4.0 acetate buffer (total buffer concentration 5 X 10-aM, ionic strength O.lM), containing 1.4 x 10-aM P-cyclodextrin, is added and the solution is diluted to volume with water. Reaction is initiated by adding 0.2 ml of 3 % hydrogen peroxide solution (30 % hydrogen peroxide diluted 1:lO). After 1 to 2 hr at room temperature, a 5.0ml aliquot is pipetted into 5.0 ml of borate buffer (prepared to be 0.4M in total boric acid and 0.2M in sodium hydroxide). This quenches the reaction. The color is developed by adding 0.1 ml of 3% (w/v) aqueous 4-aminoantipyrine followed by 0.2 ml of 10% (w/v) potassium ferricycanide. The absorbance is measured immediately at the absorption maximum against a reagent blank carried through the same procedure. A standard curve is prepared by subjecting known concentrations, bracketing the unknown concentration, of the same aromatic compound to the procedure. RESULTS AND DISCUSSION
As an analytical reagent, the hydrogen peroxide in the Hamilton system must be in excess over the aromatic sample compound. Under these conditions, we have observed that hydroxylation continues beyond the introduction of the first hydroxy group. With the quantitative measurement employed here [oxidative coupling with 4-aminoantipyrine (7-9), which incidentally gives no interfering color with the catechol in the reagent], only the monohydroxy compound appears to be detected. Figure 1, showing the time course of the treatment of anisole with the Hamilton reagent (open circles), reveals the production and then loss of the monohydroxy compound. By reducing the initial ratio of peroxide to aromatic compound to 2.0 or less, stable absorbance values (7) E. F. Mohler, Jr., and L. N. Jacob, ANAL.CHEM.,29, 1369 (1957). (8) H. 0. Friestad, D. E. Ott, and F. A. Gunther, ibid., 41, 1750
(1969). (9) D. Svobodova and J. Gasparic, Mikrochim. Acta, 1971,384. 880
ANALYTICAL CHEMISTRY, VOL. 44, NO. 4, APRIL 1972
were obtained, the yield depending upon this ratio. In a practical analytical situation, this ratio cannot be held constant, and the outcome is the result shown in Figure 1 and a curved absorbance-concentration plot. The destruction of the monohydroxy product by rapid subsequent hydroxylation therefore renders the Hamilton system ineffective as an analytical reagent. This problem has been overcome by incorporating a cyclodextrin in the reaction mixture. The hypothesis was formulated that in the presence of cyclodextrins [ring compounds of 1,Clinked D-glucose polymers (10, I ] ) ] , inclusion complexes of aromatic compounds will be formed, and that selectivity may be achieved on both an equilibrium basis (different extents of complexation by the aromatic substrate and the phenolic product) and a kinetic basis (inaccessibility of sites for further hydroxylation of a complexed phenol). Figure 1 (filled circles) shows that P-cyclodextrin indeed stabilizes the initial hydroxylation product. Both processes, that is, the formation of monohydroxylated product and its subsequent further hydroxylation, are slowed down; at least part of the stabilizing effect is believed to result from the concurrent decomposition of the Hamilton system, the cyclodextrin merely delaying the attainment of the maximal initial product concentration long enough for the incursion of the subsequent hydroxylation to be prevented by degradation of the reagent. The mechanism of the cyclodextrin effect is being studied further. There is no evidence for specific catalytic effects by the cyclodextrin, as has been noted with hydrolyses of phenyl esters (12) and with aromatic chlorination (13). a-Cyclodextrin and P-cyclodextrin show similar behavior in the hydroxylation system. Table I lists the compounds that have been successfully treated by this procedure. Linear working curves of absorb(10) D. French, Aduan. Curbohyd. Chem., 12,189 (1957). (11) J. A. Thoma and L. Stewart in "Starch: Chemistry and Technology," Vol. I, R. L. Whistler and E. F. Parschall, Ed., Academic Press, New York, N. Y., 1965, p 209. (12) M. L. Bender, R. L. Van Etten, G. A. Clowes, and J. F. Sebastian, J. Amer. Chem. Soc., 88,2318 (1966). (13) R. Breslow and P. Campbell, ibid., 91, 3085 (1969).
ance against concentration are observed for most compounds up to concentrations of 8 X lW4M. Fading of the color occurred with methyl benzoate and benzaldehyde, so the absorbance values were extrapolated to time of color development. (Nitrobenzene also exhibited this fading, which may be associated with electron-withdrawing groups; with this substrate, the color development was omitted and the nitrophenolate absorption was measured.) Benzaldehyde gave a standard curve with marked negative curvature. Pyridine could not be determined; it is not yet known whether hydroxylation of pyridine does not occur or whether hydroxypyridines do not respond to the color development. In the determination of 6 X 10-4M acetanilid, little or no interference was caused by 10-3M of methanol, acetonitrile, acetone, or ethyl acetate; N,N-dimethylformamide caused a 20 % decrease in response and dioxane, a 40% decrease. Under the conditions of the color development, phenol itself gives a molar absorptivity of 1.23 X lo4; the yield of phenol from benzene is therefore about 3 7 x . Yields from other substrates are probably similar, the final absorbance
depending upon the yield of monohydroxylated product, the isomer distribution, and the molar absorptivities of the coupling products. Work is continuing on the generality of this method, modifications such as alternate finishes, the mechanism of the stabilization by cyclodextrins, and applications to particular problems. In addition to the general analytical method described here, the concept of developing analytical selectivity by means of complex formation with cyclodextrins may be worth further exploitation. KENNETH A. CONNOEU KENNETH S. ALBERT School of Pharmacy University of Wisconsin Madison, Wis. 53706 RECEIVED for review November 29, 1971. Accepted January 31, 1972. This work was supported by the Research Committee of the University of Wisconsin.
Nuclear Magnetic Resonance Determination of tert-Butyl Hydroperoxide SIR: Having used for some years the conventional iodometric procedure (1) for determining tert-butyl hydroperoxide in our commercial source of the material, it seemed highly desirable to switch to the more rapid NMR method recently described by Ward and Mair (2). The 60-MHz spectrum of the commercial mixture (K & K Laboratories) is shown in Figure 1. This material, sold as 7 0 x hydroperoxide, usually assays a little higher when freshly received and decreases slightly in titer with time. The sample shown gave an iodometric titer corresponding to 68 hydroperoxide. According to Ward and Mair (2), the low-field peak is due to the methyl protons of the hydroperoxide, the middle peak is due to tert-butyl alcohol, and the upfield peak is due to di-tert-butylperoxide. Clearly this assignment is not consistent with the independent analytical assay and the areas under the curve in Figure 1. Here it is the central peak (area 7 0 x of whole) which appears to correspond with the correct amount of hydroperoxide. Support for this contention is offered also in Figure 1 where the results of two incremental additions of tert-butyl alcohol are shown. In each case the ratio of areas for the middle and upfield peaks remained as in the initial sample. Similar results (not shown) were obtained on adding known di-tert-butylperoxide to the initial sample; the upfield peak being enhanced as expected from the literature assignment. In conclusion, the use of NMR to determine tert-butyl hydroperoxide is advantageous as initially contended (2); however, it is the central peak which should be assigned to the hydroperoxide. These results also confirm the necessity of running known mixtures when using the NMR technique for such determinations.
x
(1) R. Hiatt, T. Mill, K. C. Irwin, and J. K. Castleman, J. Org. Chem., 33, 1421 (1968). (2) G . A. Ward and R. D. Mair, ANAL.CHEM., 41, 538 (1969).
Jk
Figure 1. NMR spectrum of a commercial sample of tert-butyl hydroperoxide in deuterochloroform (1 :10 VIVI. Chemical shifts in ppm from TMS at 60 MHz. Known tert-butyl alcohol was added in portions to produce the middle and upper scans, respectively.
I
I 1.3
1.2
Chemical Shift WILLIAMB. SMITH
Department of Chemistry Texas Christian University Fort Worth, Texas 76129RECEIVED for review November 17, 1971. Accepted January 28,1972. Appreciation is hereby expressed to The Robert A. Welch Foundation for support of this work. ANALYTICAL CHEMISTRY, VOL. 44, NO. 4, APRIL 1972
881
