with nonconjugated double bonds do not benefit from the resonance energy stabilization of the 1.3-derivatives. This trend is confirmed by the results given in Tables I11 and IV; it must be pointed out, howevcr, that the relatively high amount of 2-methylbutadiene-l,3 (isoprene) is due to its direct formation from thermal degradation of polyisoprenoid compounds present in tobacco. Preliminar:r. results seem to indicate the presence of butadiene-1,2; moreover there is no reason why the allene-type structure hydrocarbons of the Cr series could not be espected to be present in cigarette smoke. Cyclobutadiene is apparently a very unstable substance which has not yet been prepared; cyclopentadiene was tentatively found in small amoLnts. The acetylenic hydrocarbons are usually indicative of a fairly high thermal degradation temperature and are found in relatively
small concentration in cigarette smoke. The conjugated nature of vinylacetylene probably accounts for its presence in a higher concentration than ethylacetylene. The higher boiling temperature and longer retention time of dimethylacetylene, as compared to its isomer ethylacetylene, accounts probably for the lack of identification of this hydrocarbon in the smoke fraction studied in this investigation. LITERATURE CITED
(1) Brooks, B. T., Stewart, S. K., Jr., Boord, C. E., Schmerling, I,., “The Chemistry of Petroleum ‘Hydrocarbons,” 1‘01. 11, p. 63, Reinhold, New York, 1955. (2) Carugno, N., Giovannozzi-Sermanni, G., Proceedings of the 2nd Inter-
national Scientific Tobacco Congress, p. 501, Brussels, June 1958. (3) Fishel, J. B., Haskins, J. F., Znd. Eng. Chem. 41, 1374 (1949).
R. M., Jr., Harlow, E. S., Tobacco Sci. 3, 52 (1959). ( 5 ) Karrer, P., ‘‘Organic Chemistry,” 3rd English Ed., p. 638, Elsevier, Ameterdam, 1947. (6) Norman, V., Newsome, J. R., Keith, C. H., The 17th Tobacco Chemists’ Research Conference, September 22-25, 1963, Montreal, P. Q., Canada. ( 7 ) Osborne, J. S., Adamek, S., Hobbs, M. E., ANAL.CHEM.28, 211 (1956). (8) Patton, H. W., Touey, G. P., Zbid., 28, 1685 (1956). (9) Philippe, R. J., Unpublished data Research Department, Liggett and Myers Tobacco Co., Durham, N. C.,
(4) Irby,
1957. (IO) Philippe, R. J., Hackney, E. J., Tobacco Sci. 3, 139 (1959). (11) Philippe, R. J., Hohbs, M. E., ANAL.CHEM. 28,2002 (1956).
(12) PhiliDDe. R. J.. Moore. Henry, ‘ Tobucco’Sci: 5, 131 (1961). RECEIVED for review October 31, 1963. Accepted January 7, 1964. Seventeenth Tobacco Chemists’ Research Conference, September 22-25, 1963, Montreal, P. Q., Canada.
Spectrophotometric Determination of Ammonia as Rubazoic: Acid with Bispyrazolone Reagent LIDMILA PROCHAZKOVA Hydrobiological I aboratory, Czechoslovak Academy of Science, Prague, Czechoslovakia
b Ammonia reacts with bispyrazolone, 3,3’ dimethyl 53’ dioxo 1,l ’-
-
-
-
- -
-
-
diphenyl-(4,4’-bi-2-pyrazoline), in the presence of Chloramine T at p H 6.0 to produce rubazoic acid, 3-methyl4-(3 methyl 5 oxo 1 phenylpyrazonyliden-4-amino)-!i-oxo- 1 -phenyl2-pyrazoline. This compound, pinkviolet in aqueous !;elution, can b e extracted after acidification into trichloroethylene as the yellow undissociated acid. The absorbance of the extract at 450 mp is a linear function of ammonia in the range 1 to 500 pg. of NH4-N per liter with a The standard deviation of 0.9%. influence of reagents and reaction variables has been studied and a procedure based on the results. The product has been identified by its spectral characteristics and by isolation and comparison with the authentic compound. A reaction mechanism is described.
I
- -
1947 Epstein (E?)first reported the use of pyrazolone( [) in combination with pyridine and bisDyrazolone(I1) to determine cyanide ion. I n 1953 Kruse and Mellon ( 8 ) ,who had observed a strong interference o f ammonia in this method, worked out a procedure for the determination of ammonia based on the N
fact that ammonia reacted in the presence of Chloramine T a t pH 3.7 with a mixture of pyrazolone and a pyridine solution of bispyrazolone to produce a pink-violet color which was proportional to the concentration of ammonia and could be extracted into cc14 as a yellow product. Strickland and Austin subsequently modified the method and used i t for the determination of ammonium ion in sea water (16). Unemoto et al. ( I 7) used pyridine-bispyrazolone reagent for the determination of ammonia in relatively high concentrations in small volumes. This method is not applicable to very dilute solutions of ammonia. Kruse and Mellon assumed that the reaction was of the Koenig type ( 6 ) , with the pyridine ring opening to form the glutaconic aldehyde, which then reacted with the active methylene group of the pyrazolone. The function of the individual reactants-Chloramine T, pyridine, and pyrazolone-was not explained. In a subsequent study, Lear and Mellon (9) offered several schemes to explain the mechanism of the reaction, but could neither explain the experimental facts nor completely define the mechanism. These workers had substituted a number of other primary and secondary amines for pyridine, only to find that the color was no longer
proportional to the concentration of ammonia. When pyrazolone was replaced by various ketones and aldehydes-benzophenone, acetophenone, salicylaldehyde, and the like-the mixtures gave no color. The authors concluded that the reaction was not created by the active methylene group of the pyrazolone nor by a Koenig-type ring opening. The chemical properties of the principal component, pyrazolone, are of interest in connection with this reaction. Pyrazolone is soluble in acids, bases, and ethanol. It is an amphoteric substance existing in tautomeric equilibrium between the keto and enol forms, I a and Ib. The presence of the enol form was confirmed by the reaction of
Ib
oH
diazomethane to produce the methyl enol ether and by acylation to form the corresponding enol ester (4, 16). Mild oxidation-e.g., refluxing with phenylhydrazine-gives bispyrazolone, a small amount of which is added to the pyrazolone reagent used by Kruse and Mellon. Stronger oxidants convert the bispyrazolone to Pyrazolone Blue (6). VOL. 36, NO. 4, APRIL 1964
865
Table 1.
Spectral Characteristics of Pyrazolone and Related Compounds
Compound Pyrazolone(I ) Bispyrazolone(11) Pyrazolone Blue(II1) Rubazoic acid(1V) Pyrazoldione(V)
Solvent CHIOH CHaOH CCl, CHC13 0 . 1 N NaZCOa
CHC4
H20
Log molar absorptivity in (
Am*,, mcLa 24814.13). 28nm 7n)
255ii:iij;295{4:Gj
330(4.13), 600-610(3.20) 373(4.07), 450(4.54) 245(4.33),' 345(4.32), 540(4.00) 275(3.53), 300(3.35), 430(1.57) 230(4.02), 285(3.36), 345(3.87)
).
Reagents. PYRAZOLONE A N D RECOMPOUNDS.Pyrazolone, 3methyl - 5 - oxo - 1 - phenyl - 2 - pyrazoline(I), bispyrazolone, 3,3' - dimethyl5,5'-dioxo - 1 , l ' - diphenyl - (4,4'- bi2-pyrazoline) (11),and Pyrazolone Blue, 3,3' - dimethyl - 5,5' - dioxo - 1,l'diphenyl ( A 4 * 4 ' - bi- 2 - pyrazoline)(III), were prepared as directed in the literature (6). In those experiments in which the presence of bispyrazolone was undesirable, the pyrazolone was dissolved in ethanol, the insoluble residue removed by filtration, and the purified pyrazolone obtained by vacuum distillation after removal of the solvent (b.p. 191" C./17 mm., m.p. 127' (2,). Rubazoic acid, 3-methyl-4-(3-methyl5 - oxo - 1 - phenylpyrazonyliden - 4amino) - 5 - oxo - 1 - phenyl - 2 pyrazoline(IV), was prepared by oxidation of 1 - phenyl - 3 - methyl - 4 - amino - 5oxo-2-pyrazoline with ferric chloride and also by condensation of pyrazoldionewith aminopyrazolone according to literature directions (3, 5 ) . The acid exists in two crystalline modifications (m.p. 186' C. from ligroin, and m.p. 190' C. from the melt). The literature reports m.p. 181-4' C. (uncorr.). Pyrazoldione, 3-methyl-4,5-dioxo-lphenyl-2-pyrazoline(V) , was prepared by hydrolysis of the corresponding ani1 LATED
The action of oxygen or bases a t elevated temperatures causes condensation of the pyrazolone carbonyl group with active methylene groups (6). The N-methyl derivative of pyrazolone, antipyrine, reacts with ammonia in the presence of hypobromite to produce a brown-red color which can be used for the detection of ammonia in moderate concentrations (1) . I n general, these and other reported reactions which are not directly pertinent to the present problem indicate that the reactive part of pyrazolone is the methylene group in the Pposition. Since several discrepancies in the experimental behavior were observed for this reaction, product identification a n d definition of the reaction conditions were needed to derive a more satisfactory method for the determination of ammonia. EXPERIMENTAL
Apparatus. Spectrophotometric measurements in the ultraviolet and visible regions were obtained with either the SF-4 (USSR) or the Universal Zeiss-A (VEB, Carl Zeiss, J e n a ) spectrophotometers. Spectrophotometric measurements in the study of reaction variables were measured with a Pulfrich photometer (VEB, Carl Zeiss, Jena) using the 5-45, S-53, or S-61 'filters. Infrared spectra were recorded with the UR-10 (Carl Zeiss, Jena) spectrophotometer. Melting points were determined with a Boetius micromelting point apparatus and all melting points are corrected. The pH measurements were made with a pHmeter (Mikrotechna, Czechoslovakia), using a glass electrode. All spectrophotometric data were recorded with cells of 0.5-cm. pathlength.
866
ANALYTICAL CHEMISTRY
-
(1.2).
OTHER REACTIONREAGENTS. The citrate-phosphate buffers were prepared from reagent grade chemicals according to the directions of Kolthoff ( 7 ) . Chloramine T of commercial grade was purified by repeated crystallization from water until it had 99.5% of the theoretical active chlorine content. Inert salts, NaOH, and Na2COI were of analytical reagent grade. All organic solvents were freshly distilled before use. Glass-distilled water which had been passed through a U'ofatit F ion exchange column in the acid cycle shortly before use was used in preparing reagent solutions. REAGENTSOLUTIONS.Citrate-Phosphate Buffer. Mix 0.1M aqueous citric acid and 0.2M NazHP04in the volume ratio 7.91 to 12.09, and stabilize by addition of several drops of a 1 to 1 toluene-carbon tetrachloride solution. The p H of this buffer is 5.8. Chloramine T , aqueous 1% solution. This solution is stable for a t least 15 days. Bispyrazolone. Dissolve 0.2 gram of bispyrazolone in 40 ml. of 0.5N Na2C03 a t 90" C. and, after cooling, dilute with
80 ml. of 0.5N Na2CO3. This solution should be freshly prepared for each set of determinations. However, if kept under nitrogen a t 0" C., it shows no change in reactivity for a t least 10 days. Pyrazolone. Prepare a 0.25y0 aqueous solution by dissolving the compound in hot water. The solution is stable for a t least a month. Hydrochloric acid, 0.5.V. Procedure. To a n aqueous 50-ml. sample a t pH 6 to 7 containing 1 t o 500 pg. of ammonium nitrogen (WH4-N) per liter, add 5 ml. of buffer and 2 ml. of 1% Chloramine T solution and allow to stand for precisely 5 minutes a t 15" C. Subsequently quickly add 6 ml. of 0.5N Na2C03 solution containing 10 ing. of bispyrazolone to the stirred solution, After 5 minutes add 10 ml. of the pyrazolone solution. When the blue color, if any, has disappeared, acidify the solution by the addition of 2 ml. of 0 . 5 X HC1. Estract once with 10 ml. of trichloroethylene, using a 3-minute equilibration period. Separate the organic phase, removing any turbidity by the addition of 0.5 gram of anhydrous KazS04. Measure the absorbance at 450 mp.
A calibration curve may be obtained from measurements of known NH4+ solutions or from an actual sample to which known amounts of standard X H 4 + solution have been added. I n the latter case results calculated using the usual methods for standard addition procedures agree with expected values within *3%. The maximum absorbance obtained from a sample containing 0.2 mg. of NH4-K per liter is 0.81 after correction for a blank ( A = 0.02) which corresponds to a 76% yield of rubazoic acid. The reproducibility for samples of surface waters and the like is &0.9%. RESULTS
Identification of Reaction Product. The results obtained by Mellon and Lear (9) by the substitution of other bases-Le., substituted pyridines-for the pyridine in the original method indicate that pyridine itself is not incorporated into the reaction product. This is also supported by the fact that aliphatic amines or even hydroxylic bases (triethanolamine, KaZCOs, and NaOH) produce the same color. These observations led to the elimination of pyridine from the reaction solution, in order to remove its spectrophotometric interference. A second correct assumption was that the pyrazolone moiety was preserved in the product. The ultraviolet and visible spectra of a number of compounds which could be presumed to be important reactants, intermediates, or products are recorded in Table I. From these data two salient features emerge. Whenever the pyrazolone structure is present the spectra contain
with a 1% solution of KOH in 96% ethyl alcohol.
I
I
300
400
I
1
5co
600
WAVELfNGTH(mp Figure 1.
Absorption spectra of pyrazolone and related compounds I. 11. 111. IV.
V. VI.
Pyrazolone in CHaOH Birpyrozolone in CH3OH Rubazoic acid in CC14 Rubazoic acid in 0.1 N Na2C03 Rubazoic acid in CHaOH Pyrazolone Blue in CCl4
a n absorption maximum in the region 240 t o 260 mp and, as the exocyclic conjugation with the ring becomes more extensive, the compounds show an absorption maximum in the visible (cf. Pyrazolone Blue, pyrazoldione, and rubazoic acid). However, of all compounds investigated, only four exhibit a sufficient similarity with the reaction product to be of importance; these are reproduced in Figure 1. Pyrazolone and bisp yrazolone are included because they are obviously present in the reaction mixture as reagents. Pyrazolone Blue, with a long wavelength absorption a t 610 mp which results from a perfect conjugation of the ring systems, appear; in the reaction solution as a side product, resulting in a slight interference in the determination. The spectrum of the vxtracted reaction product compares (Figure 2) in all respects with t h a t of rubazoic acid, with the exception of a higher absorption in t h e reaction extract :Lt 300 mp. This is attributed to the presence of excess pyrazolone. Furthermore, the spectra of rubazoic acid and the product change identically with pH. This identity was substantiated by isolation of the product from a large scale reaction mixture. -4 2000-ml. sample containing 10 mg. of nitrogen as KH4+ was subjected t o the treatment described in the Procedure with appropriate scale-up of reagents and volumes, finally resuliing in extraction
with 500 ml. of CC14. After re-extraction with three 50-ml. portions of 1N NaOH and acidification of the aqueous phase with HCI to a p H of 2 at 5' C., the precipitated compound was removed by filtration, washed with 1% HC1, and dried at room temperature under a vacuum of 0.1 mm. of Hg. A yield of 49 mg. was obtained, which was recrystallized from ligroin. The product was also obtained by the alternative procedure of sorption o n an alumina column, followed by elution
m
400
5x7
WAVEL E N G T H h U 1
Figure 2. Absorption spectra of rubazoic acid and extracted product of ammonia-pyrazolone reaction I.
1O-'M rubazoic acid in CCId Product of sample containing 0.25 mg. of NHa-N per liter in CClr b = 1 cm. 11.
The substance obtained by either procedure melted a t 186-7' C. from ligroin and 190-90.5' C. from the melt. The mixed melting point with rubazoic acid was 189.5-90' C. without depression. Elemental analysis gave: C, 66.75%; H, 5.08%. Calculated (L1.T. 359.4) : C, 66.84%; for C20HI7SSO2 H, 4.77%. Infrared spectra in CC14of the compound and rubazoic acid were identical in the fingerprint region for these compounds (500 t o 2000 cm.-l). Effect of Reaction Variables. T o define t h e optimum reaction conditions, t h e reaction was carried o u t according to the procedure of Kruse and Mellon (8) with the changes of reagents, concentrations, temperature, time, and p H described below. The final conditions which evolved from these experiments were incorporated in the Procedure. EFFECTO F PYR.4ZOLONE AND BISPYRAZOLONE REAGENTS. I n preliminary experiments using the method of Kruse and Xlellon with 50-ml. samples containing 10 pg. of KH4-K, the colored compound was extracted into 10 ml. of CCll and the absorbance was measured with the Pulfrich photometer using the S-45 filter and 0.5-cm. cells. Blank corrections were determined by carrying out the proredure in the absence of SH4+. During the first step of the reaction prior t o the addition of the pyrazolone reagent the p H was found to be 3.8; after addition of the reagent it became 6.1. The Kruse and Mellon procedure yielded an absorbance of 0.19, while the modified method of Strickland and Austin gave a n absorbance of 0.25. (The procedure of Cnemoto cannot be directly compared with the others; absorbance for the same concentration is only 0.05, but the yield of the reaction is relatively high.) If the pyrazolone reagent was excluded from the Kruse and Mellon procedure and the resulting Pyrazolone Blue was decolorized by the addition of hydroxylamine, the absorbance was 0.18; if bispyrazolone was omitted, the absorbance was 0.00. REACTIONIN ABSENCEOF PYRIDINE . ~ N D BISPYRAZOLOSE.. 4 50-ml. sample 5 ml. containing 10 pg. of ?;Ha--S, of buffer (pH 5.8), and 2 ml. of Chloramine T solution was mixed and allowed to stand for 5 minutes a t room temperature. -1 mixture of 10 ml. of the pyrazolone solution and 6 ml. of 0.5N Na2C03 was then added. -1fter 5 minutes the solution was acidified with 2 ml. of 0.5Ji HCl and extracted with one 10-ml. portion of trichloroethylene. The resulting absorbance was 0.04. I n this as in the above experiment the pyrazolone reagent used in preparation of the solution was separated from bispyrazolone as described. The normal pyrazolone product obtained from alVOL. 36, NO. 4, APRIL 1964
867
03
O./t
0.1
I
Figure 3. Absorbance yield as function of solution pH during first step of procedure
I
7
I
I
8
9
I
P"
I
1 O / f
1
Q
Figure 4. Absorbance yield of rubazoic acid as function of final solution pH
Filter 5-45
b = 0.5 cm.
ranging from 0" to 30" C.; prior to extraction each mixture was brought to 15" C. The absorbances obtained for cohol-water recrystallization contains extracted into 10 ml. of trichloroethylthe temperatures O", IOo, 15", 20°, and bispyrazolone as an impurity (13). ene. The results are shown in Figure 4. EFFECT OF PH CHANGESIN FIRST EFFECTOF CHLORAMINE QUANTITY. 30" c. were 0.81, 0.81, 0.80, 0.75, and 0.45, respectively. STEP. To 50-ml. samples each conTo 50-ml. samples containing either 10 EFFECTOF TIME INTERVAL BETWEEN taining 10 pg. of NHa-N were added 2 or 25 pg. of NH4--N, 5 ml. of the p H ADDITIONO F -ALKALI AND BISPYRAZOml. of the Chloramine T solution and 5.8 buffer and various volumes of the 5 ml. of a citrate-phosphate buffer of Chloramine T solution were added. LONE REAGENT. The determinations selected pH. After 5 minutes' standing After 5 minutes the color was developed were carried out as described, except a t room temperature (15" C.), 10 mg. by the addition of 6 ml. of the bispyrathat the 6 ml. of 0.5N Na2C03 was zolone solution in Na2C03. The reof bispyrazolone were added in one divided into two parts: (A) 4 ml. of portion to the stirred solution in a sulting solution was further treated as 0.5N NazC03 and (B) 2 ml. of 0.5N volume of I N Xa2C03 sufficient to inpreviously described. Na&03 containing 10 mg. of bispyrazocrease the p H to 10 to 10.1. The neceslone. Solution B mas added after an The absorbances, obtained as a funcsary quantity of Na2C03was determined interval of 0 to 3 minutes subsequent to tion of the quantity of Chloramine T from prior experiments in the absence the addition of solution A. The readded, are shown in Table 11. At higher of bispyrazolone. When the Pyrazolone Blue appeared, it was decolorized by sulting ultimate absorbances were: 0.80 concentrations of Chloramine T--e.g., addition of 10 ml. of the aqueous pyra(0 minute), 0.43 (1 minute), and 0.33 50 mg. per sample-the initial color is eolone solution. The mixture was then (3 minutes). normal. This, however, rapidly fades to acidified and extracted as described EFFECTOF QUANTITY OF BISPYRAZOa yellow with a resulting lower absorbabove. The resulting absorbance values LONE. The procedure was carried out ance a t 450 mp. This effect, which as a function of the preliminary p H are with 50-ml. samples containing 25 may be caused by oxidation or chlorinaplotted in Figure 3. pg. of NH4--N in the usual fashion, tion of rubazoic acid, was not further EFFECTOF PH DURING FORMATION except that the quantity of bisinvestigated because solutions conOF RUBAZOICACID. The basic propyrazolone added in 6 ml. of 0.5N taining lesser quantities of Chloramine cedure is described above. The initial buffer added was of p H 5.8. BisNa2C03 was varied from 1 to 20 mg. T did not exhibit this phenomenon. pyrazolone mas added together with The final absorbances for each level of EFFECT OF REACTION TIME IN FIRST various volumes of IN Na&03 or 0.2N STEP. To 50-ml. samples containing NaOH. After decolorization of the either 10 or 20 pg. of NH4-N 5 ml. of Pyrazolone Blue with the pyrazolone pH 5.8 buffer and 2 ml. of Chloramine solution, the sample was acidified with T solution were added. The mixtures 0.5M HCl to a p H of 7.0 & 0.1 and were allowed to stand a t 15" C. for 1.2 selected time intervals prior to further treatment as described above. g 1.0 Table II. Effect of Quantity of Chloramine I Added on Yield of AmmoniaPyrazolone Reaction
Chloramine
"I-N, Irg.
10
868
T,
A
mg.
2.5
0.33
5.0 10.0
0.60
20.0
0.80
0.79
50.0
0.51
50.0
1.16
ANALYTICAL CHEMISTRY
The results are given in Figure 5. The optimum time interval for maximum reaction yield is shifted slightly in the direction of longer times a t the higher concentration. However, a time of 5 minutes is optimal with respect to linearity of the calibration curve in the range 1 to 500 pg. of "4-N. INFLUENCE OF TEMPERATURE.A series of experiments was carried out as described immediately above, except that the reaction time was 5 minutes. For each experiment all solutions and the reaction mixture were maintained a t a constant preselected temperature
9 0
$0.8 9
T
0.5
0.1 I
YO
I
15
Time (min)
20
I
30
Figure 5. Dependence of reaction yield on time of reaction prior to addition of bispyrazolone NH4-N 1. 0.4 mg./liter 11. 0.2 mg./liter
EFFICJENCYOF
EXTRACTION BY
10- -.
OTHERSOLVENTS.I n the normal procedure used in these experiments the extraction of rubazoic acid is quantitative with a single extraction using 10 ml. of CCla or trichloroethylene up to a maximum NH4--N concentration of 25 pg. per 50-ml. sample. The procedure was repeated as described, with
10
Time (mid
100
Figure 6. Alkaline decomposition of rubazoic acid in N a O H I. 0.01N 11. 0.1r.I Ill. 1N Filter
S-53
added reagent were: 1.10 (1 mg.), 1.50 (2 mg.), 1.85 (5 mi;.), 2.00 (10 mg.), and 2.10 (20 mg.). Since the increase in absorbance between the last two increments is small, 10 mg. of bispyrazolone per sample were added in all following experiments. ALKALINE DECOMPOSITION OF RUBAZOIC ACID. Rubazoic acid decomposes in alkaline solution to yield a yellow compound whose identity is not now known. The rate of decomposition was studied in order tc determine if the loss in this manner wa:, significant prior to acidification. The r,ite was measured by the decrease in atlsorbance at 533 rnp for solutions containing a constant concentration of the Ecid in 0.01, 0.1, and 1N NaOH at room temperature. The results are p1ott:d in Figure 6. This decomposition apparently does not represent the entire effect of alkali because the product of the analytical procedure seems to be more sensitive to alkali, as shown by a decrease in reaction yield a t p H 11. INFLUENCE OF PH o v FORMATION OF PYRAZOLONE BLUE. The influence of p H on the formation of Pyrazolone Blue was investigated to determine if the extent of the side reaction which produces this compound could be minimized. Conditions approximating its formation in the determination were achieved by carrying out the procedure in the absence of NH4+. To 25 ml. of distilled water were added 1 ml. of buffer and/or a n appropriate volume of 0.5N Na2C03,1 ml. of Chloramine T, and 1 ml. of 0.1N Na2C03 containing 5 mg. of bispyrazolone. The Pyrazolone Blue was extracted after 10 minui,es with 10 ml. of CCl,. I n solutions more alkaline than ca. pH 7, i t was necessary to salt out the compound by the addition of solid KaCI. The absorbance of the extract was measured with the S-61 filter, which has a transmittance maximum close to absorbance maximum of Pyrazolone H u e 619 and 610 mp, respectively. Results ar? plotted in Figure 7 .
for- the CC14. The results, given as absorbances in Table 111, are for single extractions with 10 ml. of solvent from identical samples of approximately 57-ml. total volume. The S-45 filter normally used was replaced by the S-53 filter for the measurement in butyl alcohol, since it had been previously shown that the 450-mp peak in Cc14 or CHC13 was transformed in alcohols into two peaks, one a t 445 mp (log e = 4.15) and the second at 533 mp (log e = 3.82) in methanol. From the alkaline reaction solution butanol extracts the dissociated form of rubazoic acid. INTERFERENCES. The procedure was used with 50-ml. samples containing 10 pg, of KH4--N in addition to ions and substances thought to be interfering in this method. The effect was investi-
Table 111. Relative Extraction Efficiency for Rubazoic Acid of Several Common Solvents
Solventa
Absorbance
cc1,
0.80
oc
CHC12-CHClz 0.78 CHzC1-CHzC1 0.79 CHClz-CHzCl 0.80 CHCl, 0.82 0.80 C6H6 CeHsCH3 0.76 Iso-C~H~I-OCOCH~ 0.76 n-CaHgOH 0 . 55d Single extraction with 10 ml. of solvent from ca. 57 ml. of aqueous mixture. Cell path length, b = 0.5 cm. c Constant concentration of 0.2 mg. NH4--N per liter. Filter S-53 (A, = 533 mp). 5
Table IV.
Zn +2
-
o.6
-
04
-
$1
02 $
Figure 7. Pyrazolone Blue formation as function of solution pH Filter S-61 b = 0.5 cm.
gated a t three mole ratio levels of 1: I, l O : l , and 1 O O : l with respect to NH4+. The results in terms of final absorbances are given in Table IV. The two interferences, Fe+2 and S03-2, were subsequently shown to be removable, by a prior treatment of the sample with excess O.1N KlMnOa a t ca. p H 2; the sample then must be readjusted to pH 6 to 7 before addition of the reagents. The interference of CN-, CXS-, amines, and amino acids a t a mole ratio level of 10 to 1 can be removed by a n increased quantity of reagents (twofold quantity of Chloramine T and eightfold quantity of bispyrazolone). .imides do not interfere even a t high mole ratio levels. DISCUSSION
The identification of the reaction product as rubazoic acid seems clear from the evidence presented above. Kone of the compounds preliminarily investigated bore close resemblance to the product, except the rubazoic acid. Further, the isolation of the product and its comparison with the known acid yielded identical paper chromatographic R , values, melting points, mixed melting point, and infrared spectra. Inspection of the structure of compound IV indicates that its formation must result from a sequence of reactions involving ammonia and two
Effect of Added Ions and Substances
Ion and substance Fe +3 Fe +2 cu+2
0.g
Io
0.80
0.41 0.79 0.79 0.81 0.79
0.80 0.80
0.80 0.80
0.80 0.79 0.79
Absorbance I1 0.80
0.06 0.77 0.78 0.82 0.73 0.10 0.09 .. ..
0.80
0.29
0.80 0.23 0.53
I11 0.81
0 .,oo
... ... 0.01 .
.
I
... 0.81
... ...
0.81 0.05
Molar ratio, ion/NHd-N: (I) 1:1, (11) l O : l , (111)1OO:l. Experiments in these ratios not performed.
VOL. 36, NO. 4, APRIL 1964
869
active methylene groups. Although the detailed mechanism of the reaction is not y e t clear, a t least the number of possible paths may be restricted by examination of the results obtained in the analytical procedure when various conditions are changed. Pyrazolone itself is not a reagent in the reaction, since the absorbance of the mixture omitting pyrazolone was 0.18 when the unmodified procedure of Kruse and Mellon gave 0.19. The fact that the experiment in which bispyrazolone was omitted gave an absorbance of 0.00 while the preceding result under apparently the same condition gave -4 = 0.04 is explained by the difference in alkalinity between the two. Probably the more alkaline conditions of the latter experiment allowed the formation of a small quantity of the bispyrazolone. The presence of pyridine is definitely neither required nor especially advantageous; the original procedure of Kruse and hIellon yields a n absorbance of 0.19, whereas the suggested method using h’a2COs gives 0.81 for the same concentration of ammonia. The p H of the solution is nevertheless of importance as shown in the Results. From Figure 3 it is apparent that the p H of the solution during the first part of the procedure (prior to addition of the bispyrazolone) should be maintained below 6.5 with respect to the final yield of product, yet the maximum yield over-all is found when the pH of the second step is 10 0.1. Further, the results in Figure 4 below p H 9 and Figure 3 beyond p H 7 indicate that not only the procedure but the reaction must proceed in two steps. Since bispyrazolone is the reagent producing the final compound and it is not present during the first step, the first reaction must involve only ammonia, Chloramine T, and the buffer. Whatever this reaction may be, it must consist of two steps with only the first effective. This is clear from the effect of reaction time during the first step (Figure 5 ) . An increase in time beyond 5 minutes leads to a decrease in absorbance. This time effect is also found in the results obtained when the solution is allowed to stand in alkaline conditions for short intervals before addition of the bispyrazolone. These effects, although measured by an over-all reaction yield, are caused principally by changes occurring in the first step of the reaction. Figure 6 shows that the product itself does not undergo any significant decomposition under the reaction conditions. The influence of p H on the side reaction and the formation of Pyrazolone Blue indicate that this reaction is also pH-dependent but that its presence cannot be minimized by adjustment of p H because the yield appears to be practically independent of p H at values
870
ANALYTICAL CHEMISTRY
less than 10, whereas that of rubazoic acid passes through a maximum a t p H 10 and both decrease rather similarly beyond that point. The effect of temperature is minimal with respect to expected laboratory conditions; the destructive effect of increasing temperature is not noticeable until a value above normal room temperature is reached (20’ C.). I n practice it suffices to maintain the solutions and reagents slightly below room temperature. Because of the nature of the experiments it is impossible to define the position of the temperature effect, but some indication can be inferred from the magnitude of the effects of other variables on the two parts of the reaction. Unless the second reaction has a much lower activation energy than the first, the principal effect of temperature changes is to enhance the second step of the first reaction. For the most part the reactions cited in the Results were carried out under much the same conditions of time and temperature, unless these were the variables in question. Thus the yields obtained amount to singlepoint kinetic measurements. The increase in yield with increased added bispyrazolone is proportionally less than for the increase in Chloramine T. I n addition, the latter passes through a maximum, whereas the former displays a monotonic increase to a limit. The decrease in yield with p H for the first step in the p H interval 7 to 8 (Figure 3) is much steeper than the increase in yield for the interval 8 to 9 in step two (Figure 4). Thus it appears that the first step of the procedure is the most sensitive to change. Chloramine T or the simpler hypohalite ion is necessary for this reaction. As shown in Table IV, ferric ion, which is known to react with pyrazolone to produce Pyrazolone Blue ( 5 ) , does not interfere in the formation of rubazoic acid. Other oxidants-e.g., hydrogen peroxide-do not form the color. Aside from its oxidizing properties, Chloramine T also reacts with ammonia to form chloramine and dichloramine (IO,14). Dichloramine is not produced from ammonia directly but in a second reaction of the chloramine with Chloramine T. I n comparison to the previously reported reaction of ammonia with hypohalite, these reactions may be written : NHI NH&l
+ Chloramine T + Chloramine T
NHzCI (1) -L NHCl2 ( l b )
-t
Both reactions are pH-sensitive; at p H 6 the predominant species is the dichloramine. As the p H is shifted to more alkaline values, the equilibrium position of the two reactions is also shifted in favor of the monochloramine. Since the results shown in Figure 3 indicate that a n increase in p H beyond
6 for the first part of the procedure leads to low results, it appears that the reactive species is the dichloramine. The second step of the reaction requires a higher pH, yet the time interval allowable at this p H without addition of the bispyrazolone is short. This shows that the equilibrium between the chloramine and dichloramine is shifted in favor of the unreactive monochloramine. This was also demonstrated by a n experiment in which ammonia was added to the reaction mixture at a point (high pH) where the dichloramine could not be formed; this procedure produced no color a t all. I n the light of these observations the second step of the reaction is proposed as the reaction of dichloramine with bispyrazolone in the 4-position to form the chloramino bispyrazolone :
NHCli
CH3
0
This intermediate product loses the elements of HC1 in the alkaline medium to form rubazoic acid in a Stieglitz (11, 18) rearrangement:
CH3 I
0 II
The exact mechanism of these reactions is not determined by the present experiments. However, one reviewer has suggested the possibility that both steps involve the formation of nitrenes as intermediates in a manner similar to the reactions of carbenes: NHClz
+ OH-* : NC1 : NC1
+ H20 + C1-
+ I1
VIa
+
+
IV
VIa
(2a)
(38)
(3b)
It would appear that if this reaction does follow this course, Pyrazolone Blue should also react with the nitrene chloride to form, ultimately, rubazoic acid. This possibility has been tested
and a small quantity of rubazoic acid was formed, but only a t acid or neutral p H during the forma ,ion of chloramine. This fact excludes the addition of NH&1 across the double bond of Pyrazolone Blue. Pyrazolone, which was considered the major reagent in the Kruse and Mellon procedure, has been shown in the present work to function solely as a decolorizing agent for the side product, Pyrazolone Blue. This reactior is probably the addition of pyrazcllone across the reactive 4, 4’ double bond of Pyrazolone Blue:
VI1
t o form l,l‘, l”-tripheny1-3,3’, 3”trimethyl-(4,4‘, 4”-ter-2-pyrazoline)-5, 5‘, 5” -trione (VII) as described by Westoo (19-99). Hidroxylamine and hydrazine, which also decolorize Pyrazolone Blue, do so pimesumably by the formation of analogous compounds. It is not advantageow to add pyrazolone with the bispyrazolone to increase the product yield through direct reaction of the pyrazolone. M‘hen both reagents are present throughout the reaction, the yield is substantially lower. Hence the pyrazolone is added after the second 5-minute interval in the suggested procedure. The formation of Pirazolone Blue is a much slower reaction .han the formation of rubazoic acid and this side reaction does not interfere seriously beyond consumption of reagents. The slight absorbance at 450 nip is effectively removed by the pyrazolone decolorization step. The p H of the second step cannot be raised beyond the opt mum of 10 to 10.1 because the rubazoic acid is hydrolytically decomposed in alkaline solution to It is yellow, unknown products. possible that the product is the phenylhydrazone of acetylglyoxalic acid (6). Hydrolysis of rubazoil: acid, Pyrazolone Blue, and 3-methyl-4, 5-dioxo-1-phenyl2-pyrazoline(V) yields yellow products in each case. However, the spectra of these decomposed compounds do not correspond to that o i the decomposed
reaction solution. This point was not further investigated, since it is of little importance in the determination. Pyrazoldione(V) was tried as a reagent for ammonia in a procedure omitting the Chloramine T, but was abandoned when it was found that the colorless hydrated ketone in water became an extractable red form in the organic phase, thus interfering with the determination. I n contrast to the findings of Kruse and Mellon that CS-, CKS-, Fe+z, Zn+2, C U + ~and , Ag+ were serious interferences, at mole ratio level 1 to 1 in the present procedure only Fe+2 interferes. Fortunately, this interference of Fe +2 as well as of other reducing ions (SO3+) can be readily removed by a prior oxidation with KMn04. Ferric ion does not interfere even if an extensive precipitation of the hydrous oxide occurs. CN-, CKS-, amino acids, and amines diminish the absorbance at high mole ratio levels, since they compete with ammonia for Chloramine T and bispyrazolone. This interference can be removed by increasing the quantity of these reagents. Other ions interfere slightly at high concentrations by a salt effect on the buffer p H which renders adjustment of the p H in the two steps difficult. I n samples containing high concentrations of an indifferent salt i t is necessary to add approximately equivalent amounts of the salt to the standard solutions when preparing the calibration curve. I n extreme cases i t may be necessary to use a buffer of slightly different p H and modify the amount of NanCOaadded to achieve correct results.
LITERATURE CITED
( 1 ) Dehn, W. M.,Scott, S. F., J . Am. Chem. SOC.30, 1422 (1908). (2) Epstein, J., ANAL. CHEM. 19, 273 (1947). (3) Gysling, H., Schwarzenbach, G., Helu. Chim. Acta 32, 1484 (1949). (4) Himmelbauer, R., J . Prakt. Chem. 54, No. 2, 207 (1896). (5) Knorr, L., Ann. 238, 137 (1886). ( 6 ) Koenig, W. J., J . Prakt. Chem. 69, 105 (1904). (7) Kolthoff, I. M., “Der Gebrauch von Farbindikatoren,” 2nd ed., p. 124, Springer Verlag, Berlin, 1923. (8) Kruse, J. M., Mellon, iM.G., ANAL. CHEM.25, 1188 (1953). (9) Lear, J. B., Mellon, M. G., Ihid., 29, 293 (1957). (10) Pascal,‘ P., ‘Tjouveau Trait6 de Chimie Minhrale, Vol. X,. p. 260, Masson, Paris, 1956. (11) Pinck, L. A., Hilbert, E. G., J . Am. Chem. SOC.59, 8 (1937). (12) Sachs, F., Barschall, H., Ber. 35,1438 (1902). (13) Shirai, Hideaki, Yashiro, Tamotsu, Bull. Il’agoya City Univ. Pharm. School, X o . 3, 30 (1955). (14) Sidgwick, N. V., “Chemical Elements and Their Compounds,” 4th ed., Vol. I, p. 705, Clarendon Press, London, 1961. (15) Stolz, F., J . Prakt. Chem. 55, S o . 2, 169 (1897). (16) Strickland, J. D. H., Austin, K., J . Conseil24, 446 (1959). (17) Unemoto, Tsutomu, Tsuda, Yuzuru, Hayashi, Makoto, Yakugaku Zasshi 80, 1089 (1960). (18) Vosburgh, I., J. Am. Chem. SOC. 38, 2081 (1916). (19) Westoo, G., Acta Chem. Scand. 7, 352 (1953). (20) Ibid.. D. 449. ( 2 l j Ihid.; i o , 9 (1956). (22) Ihid., p. 587.
RECEIVED for review February 11, 1963. Accepted Tovember 29, 1963.
Correction Component Analysis of Isoparaffin-Olefin Alkylate Capillary Gas Chromatography I n this article by L. R. Durrett, L. hf. Tavlor. C. F. Wantland. and I. Dvoretzk; [ANAL. CHEM. 35, 637 (1963)], in Table I V on page 639 the alkylate shown in column five was
+
by
+ +
produced from %obutane Pentene-2” and not ‘Tsonentane Pentene-2” as is indicated. Thus the heading above the data should appear as follows:
Isobutane 2-Methyl- 2-,MethylIsobutylene Butene-2 butene-1 butene-2 Pentene-2
Isopentane
+
,
Isobutylene Butene-2
VOL. 36, NO. 4, APRIL 1964
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