V O L U M E 20, NO. 10, O C T O B E R 1 9 4 8 on the sulfide, thiosulfate, and sulfite determinations using both methods. A reproducibility study was made on the sulfide determination (in absence of mercaptides) by carrying out the determinations on a single sample. I n Table I11 it may be seen that the probable deviation of a single determination is 1.5% based on the average amount of sulfide found. The simplified procedure requires approximately 20 to 30 minutes for the analysip of a single sample; the procedure providing for the removal of the mercaptans will require a somewhat longer amount of time, depending upon how rapidly the zinc sulfide will filter, and this, in turn, is contingent upon the success the analyst has in preraipitating the zinc sulfide in a crystalline state. Multiple determinations can easily be carried out simultaneously by either procedure. LITERATURE CITED
Baldeschwieler, E. L., IND.ENG. CHEM.,ANAL. ED., 6, 402 (1934). 12) Billheimer, E. C., and Reid, E. E., J . Am. Chem. SOC.,52, 433844 (1930). 3) Caldwell. J. R., and Moyer, M.V., Ibid., 57, 2372 (1935). 1)
919 (4) Griffin, R. C., “Technical Methods of Analysis,” 2nd ed., p. 293
New York, McGraw-Hill Book Co., 1927. (5) Kalichevsky, 1’.A., and Stagner, B. A . , “Chemical Refining of Petroleum,” 2nd ed., pp. 155, 195, New York, Reinhold Publishing Corp., 1942. (6) Kolthoff, I. M., ana Harris, W. E., IND.ENG.CHEM.,ANAL.ED.. 18, 161 (1946). (7) Partington, J. R., “Textbook of Inorganic Chemistry,” 5th ed. p. 526, London, Macmillan Co., 1939. (8) Scott, W. W.,“Standard Methods of Chemical Analysis,” 5th. ed., p . 911, New York, D. Van Nostrsnd Co., 1939. (9) Ih;d.. D. 2181. (io)Ibid.: i.2183. (11) Tamele, M.,and Ryland, L., IXD.ENG.CHEX..ANAL.ED., 8. 16 (1936). (12) Treadwell, F. P., and Hall, W. T., “Analytiral Chemistry,” Vol 11, 9th English ed.. p. 617, New York, John Wiley & Sons 1942. (13) Ibid., p. 621. (14) “U.O.P. Laboratorv Test Methods for Petroleum and It8 Products,” 3rd ed., G-107, Chicago, Universal Oil Products GO.. 1947. RECEIVED January 2, 1948. Presented before the Southwest Regional Meeting, i l x ~ ~ r c nCEEYICAL w QocxErr,Houston, Tex., December 12 and 13, 1947.
p-Aminobenzoic Acid and Its Sodium Salt Properties of Analytical Interest C. J. KERN, THOMAS ANTOSHKIW, AND 31. R. MAIESE International Vitamin Division, Zves-Cameron Company, Znc.,New York, A‘. Y . Properties of highly purified p-aminobenzoic acid and its sodium salt were investigated as possible additional criteria of purity and as the basis of new methods for determination of p-aminobenzoic acid after suitable preliminary separation. When p-aminobenzoic acid is titrated with a strong base, a sharp end point is found between pH 6.5 and 8.5. On titrating sodium p-aminobenzoate with a strong acid one obtains a useful equivalence point at pH 3.5. A characteristic absorption spectrum may be obtained in isopropyl alcohol, wave length = 13iO. In water both p-aminobenzoic acid and its maximum = 288 mp E:?, sodium salt show about the same characteristic wave length, maximum = 266 mp and = 1070. Beer’s law is obeyed in both isopropyl and aqueous solutions. In a comparison of the spectrophotometric, titrimetric, and diazo methods, the new- method showed up favorably. All three measurements arc suggested for the complete characterization of pure p-aminobenzoic acid.
P
A1L.l-aminobenzoic acid (d) has gained importance in recent years because of its physiological properties. It may be considered a member of the vitamin B complex (1,7‘). The evidence In the literature indicates that sulfa drugs act as an antibiotic in (-ompetition with p-aminobenzoic acid in the microorganism (11, PI,15,16). Successful treatment of the Rickettsia disease has increased interest in this compound (IS). Furthermore, it appears io be important as a means of maintaining high salicylate levelin the treatment of rheumatic fever (6). The reasons cited above indicate the importance of additional criteria for the purity of p-aminobenzoic acid and its sodium salt These properties may be also useful in its determination in various products after suitable preliminary separation. The sodium salt of p-aminobenzoic acid was purified by the addition of sufficient C.P. sodium hydroxide to convert p-aminobenzoic acid t o its sodium salt, treated three times with charcoal, and crystallized three times from aqueous solution. The salt mas washed three times with 95% alcohol and dried at 100 C. T o prepare purified p-aminobenzoic acid, the sodium salt was redissolved in distilled water and enough C . P . hvdrochloric acid v a s added t o convert it to the p-aminobenzoic acid. The purified
s u b s t a p e was washed with water until free of chlorides and dried at 100 The p-aminobenzoic acid thus purified melted sharply at 187” C. and other batches melted at 187” t o 187.5’ C., compared to 186” to 188”C. previously reported for the purified product (S,4)
.
The p H changes during titration of p-aminobenzoic acid with B standard alkali are given in Figure 1. There is the characteristic sharp change in p H beginning at about 6.5, which is desirable in a good titrimetric reaction. The p H of the equivalence point is 7.85. Thus an indicator that changes color between 7.0 and 8.7 is suitable for this titration. The pKa obtained is 4.65 and 4.80, which may be compared to 4.8 ( 4 ) and 4.68 (3). (The pKa was obtained from the relationship pKa = pH at one-half neutralization of weak acid.) The titration curve of sodium p-aminobenaoate, given in Figure 2, is essentially the reverse of that found for p-aminobenzoic acid. The p H of the sodium salt is 7.86 and when treated with one equivalent of hydrochloric (or sulfuric) acid the pH is 3.50. The change in p H at the end point is not so sharp as for the acid. Nevertheless one may obtain results of high precision when titrating to this pH. The high precision that may be
920
A N A L Y T I C A L CHEMISTRY
4 I
5!L
Table I. Comparison of Diazo with Titrimetric and Spectrophotometric Methods of Determining Pure p-Aminobenzoic Acid and Its Sodium Salt (Values expressed in yo of theoretical value) Method~~
I11 Sodium PABA IV PABA V PABA
VI PABA
J0
,io 250
Melting Point, O C.
Titrimetric with 0.1 N HzSO4
,
99.93 100.17
1871187.5
W t h 0.1 N NaOH
187-187.5 187.0
100.29 100.64 100.46
.... . .. ..
100.29
Spectrophotometric 100.18 Used as standard
Diazo (8)
100.18
99.64
100.5 100.4
99.08 99.35 99.12
Used as standard
99.35 99.94
260
270
280
290
WAVELENGTH I N
Figure 2. pH Changes during Titration of Sodium p-Aminobenzoate (2194.8 Mg. in 100 Ml. of Water) with 1.0 N Hydrochloric Acid
obtained by titration for both p-aminobenzoic acid and its SOdium salt is indicated in Table I. It is evident that most impurities would change the magnitude of titration of both products-for example, titrimetrically inert material (such as water, sand, etc.) would decrease the magnitude of the expected titration. In contrast, titrimetrically active impurities would cause a higher or lower titration, depending on the equivalent weight of the contaminant. Even when the concentration of impurities is such that owing to compensation of errors the magnitude of titration is the same, the shape of the titration curve would change in most cases. Isomers of p-aminobenzoic acid may be eliminated by comparison of the shape of the absorption spectrum (8)and the magnitude of the extinction coefficient a t the wave length of maximum absorption given in Figures 3 and 4 and Table 11. Figure 3 shows the absorption spectrum of p-aminobenzoic rtcid in isopropyl alcohol, which was chosen because this compound is fairly soluble in this solvent. Wave-length maximum is = 1366; E = 18,771 (see Table 11). These 288 to 289 mp3E:?,. values may be compared to those reported by Kumler (9), who found wave-length maximum = 288 mp and e = 17,400 in 957, commercial ethanol, and by Doub and Vandenbelt ( 5 ) , who found wave-length maximum = 284 and E = 14,000 in water a t pH 3.75. The values of Kumler are somelyhat higher than those
Sample I Sodium PABA I1 Sodium PABA
do
ML. 1.0 NORMALHCI AGQED
300
MU
Figure 3. Absorption Spectrum of Pure p-Aminobenzoic Acid in 99% Isopropyl -4lcohol 503.4 micrograms per 100 ml. 1.0-cm. ligh8 absorption path
obtained by the present authors in 95% alcohol as shown in Table 11, but lower than those in isopropyl alcohol. The values of Doub and Vandenbelt (5) in water are lower than those obtained by the authors in water for p-aminobenzoic acid but values for the sodium salt (e = 14,900, maximum = 265) compare well with the authors' for both products in water. Figure 4 shows the relation between concentration of p aminobenzoic acid in isopropyl alcohol and optiial density at
Table 11.
Extinction Coefficient and X Maxima
Product PABA 5
Solvent Isopropanol
PABA 6
Isopropanol
99%
PABA,
X Max.
El% 1 om.
288-290
1380
18,910
t
%
PH
Mg.
...
0.5035
288-289
1343
18,400
.. .
0.5034
PABA (no Isopropanol caustic treat99% ," ment) PABA 5 .Purified isopropanol 99% PABA 6 Puplfied isopropanol
288-289
1349
18,490
...
0.5032
289-290
1385
18,980
,
..
0.5032
288-289
1355
18,570
PABA 5
287-288
1186
16,250
287-288
1151
15,760
99%
PABA 6 PABA 5
99 %
Commercial ethanol 95%
Commercial ethanol 95%
Distilled 266 water PABA (no Distilled 266 caustic treatwater rnent) Sodium PABA Distilled 266 2 water Sodium PABA Distilled 266 3 water PABA 5 Distilled 266 water PABA 5 Distilled 266 water and 1/z equivalent of NaOH PABA 5 Distilled 266 water and 1 equivalent of NaOH PABA 5 Distilled 266 water and 2 equivalents of NaOH a Calculated on PABA content in Na 6 In t e r m of mg. % N a PABA.
1088
14,900
1093
14,980
107W 929 1072a 924 1099
0.5025
.
... ... .. . ...
0.5041 0.5041 0.4008 0.4070
15.060
3:59
0.4027b 0,3470 0.4047 b 0.3487 0.4038
1102
15,090
4.78
0.4021
1092
14,960
9.19
0.4040
1088
14,900
PABA.
14,770 14,690
11.58 0.4027
V O L U M E 20, NO. 10, O C T O B E R 1 9 4 8
921 methods are compared to the U.S.P. method in which p-aminobenzoic acid is titrated by diazotization (14) and depends on the amino group present. EXPERIMENTAL
.
The spectrophotometrio data were obtained with a Beckman quartz spectrophotometer, Model DU, equipped with the ultraviolet accessorv set. Tho wave-length scale setting was WAVELENGTH IN MU checked using several of the Figure 5. Absorption Spectrum of hydrogen lines emitted by the Pure Sodium p-Aminobenzoate in hydrogen discharge lamp. Distilled Water The DH data were comoiled using Leeds & Sorthrup po402.7 micrograms per 100 m l . 1.0-em. light tentiometer, Catalog KO. 7661, absorption path and a Beckman pH meter, Model G. The p-aminobenzoic acid samples were assayed by titrating accurately weighed samples, dissolved in distilled water, with 0.1 A; sodium hydroxide, using phenolphthalein test solution as the indicator. One milliliter of 0.1 sodium hydroxide is equivalent t o 13.71 mg. of paminobenzoic acid. PABA IN MICROGRAMS P E R - M . The sodium salt samples were assayed by potentiometrically titrating accurately weighed samples, dissolved in distilled water Figure 4. Relation between Absorption at with 0.1 N sulfuric acid until a pH of 3.45 \vas reached. One Maximum (288 mp) and pAminobenzoic milliliter of 0.1 ,V sulfuric acid is equivalent to 15.9 mg. of sodium 4cid Concentration in 99% Isopropyl Alcohol p-aminobenzoate. 1.0-om. light absorption path Samples of both p-aminobenzoic acid and its sodium salt were assayed spectrophotometrically through the uqe of selected samples of each which were used as standards. '288 mp (which is the wave length of maximal absorption). As may be readily observed, Beer's law holds and thus the quantitative usefulness of this set of conditions is indicated. Figure 5 shows the absorption spectrum of the sodium salt of 7 p-aminobenzoic acid in water; wave-length maximum = 266 I I I I I I mp; = 1075 (based on p-aminobenzoic acid content); E = 14,730. This shift in the maximum to the further ultraviolet is apparently due to the solvent and not to use of the sodium salt, as shown by the fact that p-aminobenzoic acid treated with various amounts of sodium hydrovide (see Table 11) gave the same maxima and identical absorption spectra. This observation appears to be a t variance with those of Doub and Vandenbelt ( 5 ) and of Kumler and Strait (IO), who indicate that there is a shift in the maximum from 284 to 266 mM due to 0.1 S sodium hydroxide (5, or 1 S sodium hydrovide (16) rather than the solvent. Furthermore, these authors indicate that the extinction coefficient of the sodium salt should be higher, whereas in this study it appears essentially the same as that of p-aminobenzoic acid in viater. The difference may be due to the small amount of alcohol used by these authors to facilitate solution (5). However, when paminobenzoic acid was dissolved in methyl alcohol up to 7910 of NA PAiA IN MICROGRAMS PER M L the final volume, the maximum and extinction coefficient xere the Figure 6. Relation between Absorption at same as in water. The sodium salt when dissolved in isopropyl Maximum (266 mp) and Sodium p-Aminobenzoate Concentration in Distilled Water alcohol (in which it is only slightly soluble) showed a maximum from 268 to 270 mp. It is possible therefore that the above 1.0-om. light absorption path workers (5, 10) obtained this shift in alcoholic solution rather than in T+ ater. The sodium salt obeys Beer's law in water, as shown in Figure 6. Procedure I. An accurately weighed sample is transferred to a Thus the quantitative usefulness of these conditions is indicated volumetric flask with 99% isopropanol. Dilutions are further again. made in volumetric flasks so that a 1-ml,aliquot of the final solution will contain from 0.0002 to 0.00025 gram of p-aminobenzoic acid. The extinction coefficients of both p-aminobenzoic acid and its One milliliter of this stock solution is further diluted to 100 ml. sodium salt are of a sufficiently high magnitude to permit the deand is spectrophotometrically assayed a t a wave length of 288 termination of l to 2 micrograms per milliliter of solution, with a mp. This step is also carried out using 2-, 3-, 4-, and 5-ml. high degree of precision (optical density being about 0.1 to 0.2 for aliquots of the stock solution which are diluted to 100 ml. These solutions are also spectrophotometrically assayed and the 1 to 2 micrograms per ml.). density readings obtained are plotted against the milliliters taken The high degree of precision that may be obtained when the from the stock solution. This curve when plotted is linear. ultraviolet absorption maxima are employed in isopropyl alcohol The sample to be assayed is treated in a similar manner to the for p-aminobenzoic acid and in water for the sodium salt is shown standard; the dilutions were calculated t o show a reading approximately in the center of the plotted curve. The densitv in Table I . Here both the spectrophotometric and titrimetric
-'
r
ANALYTICAL CHEMISTRY
922 reading obtained, when plotted against the standard curve, gives the per cent purity of the p-aminobenzoic acid. Procedure 11. Sodium p-aminobenzoate may be assayed in a similar manner, except that the diluent used is distilled water and spectrophotometric determinations are made a t wave length 266 mp. ACKNOWLEDGMENT
The authors wish to express their thanks to A. E. Sobel of the Brooklyn Jewish Hospital for his helpful comments in the preparation of this manuscript. LITERATURE CITED
(1) Ansbacher, S., Science, 93,164 (1941). (2) Ansbacher, S., Vitamins and Hormones, 2,215 (1944). (3) Bell, P. H., and Roblin, R. O., Jr., J . A m . Chem. SOC.,64, 2905 (1942).
(4) Bjerrum, N., Z. physik. Chrm., 104, 1 6 4 (1923).
(5) Doub, L., and Vandenbelt, J. M., J . A m . Chem. Soc., 69, 2714 (1947). (6) Dry, T. J., Butt, N. R., Schei5ey, C. H., and Dunnette, Margaret, Proc. Staf Meetings Mayo Clinic, 21, 497 (1946). (7) Elvehjem, C. h.,A m . Scientist, 32,25 (1944). (8) Havinga, E., and Veldstra, H., Rec. trav. chim., 66, 257 (1947, (9) Kumler, W. D., J . A m . Chem. Soc., 68, 1188 (1946). (10) Kumler, W. D., and Strait, L. A., Zbid., 65, 2349 (1943). (11) Park, C. R., and Wood, W. B., Jr., Bull. Johns Hopkins H O W . , 70, 19 (1942). (12) Strauss, E., Lowell, F. C., and Finland, M., J . Clin. Invmtigation, 20, 159 (1941). (13) Tierney, N. A,, J . A m . Med. Assoc., 131, 280 (1946). (14) U.S. Pharmacopoela,1st Bound Supplement,p. 85, 1943. (15) Wood, W. B., Jr., J . Ezpt. Med., 75, 369 (1942). (16) Wyss, Oiville, Proc. SOC.Eaptl. Bwl. Med., 48, 122 (1941). RECEIVED February 6, 1948. Presented before the Division of Sgriculturd and Food Chemistry at the 113th Meeting of the AXERICAN CEIEMICAL SOCIETY, Chicago, Ill.
Colorimetric Determination of Certain Alpha, BetaUnsaturated Aldehydes RICHARD B. WEARN, WILLIAM M. MURRAY, JR., MATHILDE P. RAMSEY,
AND
NELLADEANE CHANDLER
Southern Research Institute, Birmingham, Ala. m-Phenylenediamine dihydrochlorideis a specific color reagent under the proper conditions for a,& unsaturated aldehydes and ketones in addition to a few other highly reactive aldehydes. Advantage has been taken of this specificity in developing successful proceduresfor the quantitative determination of cinnamaldehyde, crotonaldehyde, and furfural without interference from such common impurities as acetaldehyde and benzaldehyde.
D
L'RIXG an investigation in this laboratory, it became necessary to develop a method for determining cinnamal-
dehyde in the presence of benzaldehyde and acetaldehyde, which are the usual accompanying impurities. S o method was available from the literature, as procedures based on the use of such reagents as hydroxylamine, fuchsin-sulfurous acid, sodium bisulfite, semicarbazide, thiosemicarbazide, and semioxamazide showed little or no selectivity in their reactions. Feigl (31 reveals that the colored Schiff base derived from o-dianisidine and cinnamaldehyde is detectable in extremely small concentrations, whereas the corresponding benzaldehyde and acetaldehyde reactions are much less sensitive. However, preliminary investigation showed that the color was unstable and not suitable for adoption as a quantitative method. Fellenberger (4)reported a method for the determination of cinnamaldehyde based on a color reaction with sulfuric acid and isobutyl alcohol, but it was not investigated because the color tone was said to vary with dilution. A note on the A.O.A.C. colorimetric method (1) for the determination of citral as originally reported by Hiltner ( 5 ) revealed that ethyl alcohol could be used as solvent without purification, as the usual impurity, acetaldehyde, did not interfere under the conditions of the determination. This method is based upon Schiff base formation between rn-phenylenediamine dihydrochloride and citral, giving a highly sensitive yellow to orange coloration in 80% aqueous alcohol containing oxalic acid. The inclusion of the oxalic acid in the reagent to obtain more uniform colors in the presence of easily oxidizable terpenes and the use of a blue 420 mp filter in conjunction with a photoelectric colorimeter to compensate for dyes present in some lemon and orange extracts have been discussed ( 2 , 6). During the course of this investigation, an abstract of a recent paper by Wachsmuth and Lenaers (8)was noted, which described
a colorimetric method for the determination of cinnamaldehyde in cinnamon essence and extracts based upon a reaction with pphenylenediamine in acetic acid solution. Hiltner (6, 6) had reported earlier that reproducibility of results with this amine in the determination of citral had been difficult, and that mphenylenediamine was a superior reagent. Because of the reported specificity of this reagent for citral in the presence of acetaldehyde, it was decided to investigate its use for the determination of cinnamaldehyde. In a preliminary evaluation of the selectivity of the reagent solution as prepared by the A.O.A.C. directions, a large number of aldehydes and ketones in concentrations approximately 10 to 20 times that required to produce a strong color with citral were tested. When no color developed a t room temperature, the solution was heated a t 60' to 65' C. in a water bath for 15 minutes (Table I). It is apparent that the sensitivity of the reaction varies directly with the general reactivity of the aldehydes and ketones, Strong positive reaction was given by a,@-unsaturated aldehydes and ketones and by highly reactive aromatic aldehydes such as vanillin. Ordinary aliphatic and simple aromatir aldehydes as well as ordinary ketones (including methyl and methyl aryl ketones) did not react even on heating. The only borderline cases were Zethylhexaldehyde, cyclamal, and isobutyraldehyde, in which the formyl group is apparently more reactive on account of its attachment to a secondary carbon atom A postulation that the reagent is not specific for citral and possibly applies generally to a,@-unsaturated aldehydes has just appeared in a paper by Price and Dickman (7), on a study of the cyclization of citral and citronellal. Their prediction is in good agreement with the work presented here. These results indicate the possibility of adapting the reaction to the colorimetric determination of a number of relatively re active aldehydes and ketones without interference from the
