silica gel columns by the sulfuric acid saves time and removes the risk of losing corticosteroids during air-drying of the eluent from the column. CS values reported b y Sweat are higher than by this method: 4.3 as compared to 2.0 y per 100 ml. of plasma. T h e more active silica gel enables removal of some non-CS fluorescing sub-tances, previously reported as CS. This may explain why CS values reported by Sweat (13-26) are higher than h y the present method, where 1.8% ethyl :ilcohol i q used instead of 0.0 and 0.5% in chloroform. This is in accordance with Takeda's critical evaluation of S w a t ' s method (26). ACKNOWLEDGMENT
The columns used in the chromatography of the steroids were made by Charles H. Jones, Instrumentation Division> Walter Reed Army Institute of Research. The authors wish to thank Karl Pfister of Merck & Co., Iiic., Rahn-ay, X. J., for generously providing the CS, 17-OH-CS, and other corticosteroids used in this investigation.
LITERATURE CITED
(1) Ayres, P. J., Garrod, O., Simpson, S. A,, Tait, J. F., Biochem. J . 65, 639 (1957). (2) Ayres, P. J., Simpson, S. A., Tait, J . F., Ibid., 65, 647 (1957). (3) Bayliss, R. I. S.,Steinbeck, A. W., Zbid., 54, 470 (1953). (4) Bondy, P. K.. Altrock, J. R., J . CEin. Invest. 32, 703 (1953). ( 5 ) Bondy, P. K., Upton, G. V., Proc. Soc. Erptl. B i d . M e d . 94, 585 (1957). f6) Bush. I. E.. Biochem. J . 50. 370 (1951)' (7) Chen, C., Wheeler, J., Tewell, H. E., Jr., J . Lab. Clin. Jfed. 42, 749 (1953). (8) Corcoran, -4.C., Page, I. H., Zbid., 33, 1326 (1948). (9) Gold, J. J., J . Clin. Endocrinol. and Metabolism 17, 296 (1957). (10) Goldzieher, J. IT-.,Bodenchuk, J. %.I., Kolan, P., h s a ~ . Cmst. 26, 853 (1954 j. (11) Harnood, C. T., >fason, J. W., .I. Clin. Endociinol. and Zfelabolism 16. 790 (1956). (12) Lev&, B., J . Clz'n. Pathol. 10, 148 (1957 ). (13) PIIader, W,J., Buck, R. R., A N ~ L . CHEM.24,666 (1952). (14) Morris, C. J. 0. R., Williams, D. C., Biochem J . 54, 470 (1953). (15) Selson, D. H., Samuels, L. T., J . Clin. Enrlocrinol. and Metabolism 12, 519 (1952). \
,
(16) Paschkis, K. E., Cantarow, A. F.,
Walkling, A. A., Boyle, D., Endocrinology 47, 338 (1950). (17) Peterson, R. E., J . Biol. Chem 225, 25 (1957). (18) Peterson, R. E., Karrer, Aurora, Guerra, L., ~ A L CHEM. . 29, 144 (1957). (19) Porter, C. C., Silber, R. H., J . Biol. Chem. 185, 201 (1950). (20) Reichstein, T., Shoppee, C. W., Vitamins and Hormones 1, 345 (1953). (21) Silber, R. H., Busch, R. D., J. Clan. Endocrinol. and Metabolism 15, 970 (1955). (22) Silber, R. H., Porter, C. C., J . Biol. Chem. 210, 923 (1955). Sweat. M. L.. h A L . CHEV. 26.
s.
(25 102, 341 (19i3). (28) ITTeichselbaum,
H.
1'. J,:,,
JIargraf,
olism 15, 970 (1955). (29) Vheeler, J., Freeman, S., Chen, C., J . Lab. Clin. N e d . 42, 758 (1953). (30) Wintersteiner, O., Pffiffner, J. J., J . B i d . Chem. 116, 291 (1936).
RECEIVED for review June 6, Accepted ilpril 25, 1958.
1957.
Determination of p-Cresol in Industrial Waste Waters GEORGE R. TALLON and ROBERT D. HEPNER Mellon Institute, Pittsburgh, Pa.
,*
.
The standard Gibbs and 4-amino.antipyrine methods do not measure p cresol. Control of a recovery process -for chemical process waste waters .which contain p-cresol and lesser amounts of other phenols requires a .reliable procedure for the determination of p-cresol. A method is described using Gibbs reagent to couple with Snterfering phenols to form indophenols. p-Cresol, separated from the nonvolatile indophenols b y steam distillation, is measured b y a diazo colorimetric method. Analyses of prepared mixtures showed that 98.5% or more o f the p-cresol was recovered. Replicate tests on chemical process waste waters indicated good precision for the method. Exploratory tests indicated +he procedures may b e useful for de.terminations of p-cresol, and certain .other p-substituted phenols, in effluents from tar and by-product coke plants.
T
wo colorimetric methods, the Gibbs and the Paminoantipyrine method, w e generally used for the determination of phenols in industrial waste waters . ( I , 2 ) . Neither measures pcresol and
certain other para-substituted phenols. I n niarij- phenolic effluents, the parasubstituted phenols are present in minor proportions compared to the other phenols, and their omission may not be important. Certain chemical process waste waters may contain 8 large proportion of p-cresol and only minor aniouiits of other phenols. Control of dephenolizing and treatment processes for such waste streams requires a reliable procedure for the analysis of p-cresol. This procedure can be of value in aiding industry in its efforts to abate stream pollution. d wide variety of phenolic compounds. including p-cresol, may be determined by nitrosophenol ( I , 5 ) , infrared (9), and ultrariolet ( 7 ) methods. Recently published statistical data (6) on five different methods for the dctermination of phenolic compounds showed that results from the nitrosophenol method were inaccurate, and subject to other disadvantages and sources of error. Xhile the infrared and ultraviolet absorption methods have certain advantages for analyses of phenolic compounds, such equipment is generally not available for waste con-
trol purposes, especially in the laboratories of smaller plants. The proposed method for the determination of p-cresol in phenolic waste waters utilizes the Gibbs reaction of phenols to separate para-substituted phenols. The mixture of phenolic conipounds is treated with Gibbs reagent (2,6 - dibromoquinone chloroimide) ; characteristic blue-colored indophenols are formed, but p-cresol and similar para-substituted phenols do not react. The unreacted para-substituted phenols may then be separated by steam distillation from the nonvolatile indophenols. .4 diazo colorimetric procedure is applied to measure the p-cresol and the para-wbstituted phenols in the distillate. APPARATUS
Beckman Model G p H meter. Shaw (8) steam distillation apparatus. Bausch & Lomb, Spectronic-20 colorimeter, with 1-inch cells. SOURCES AND PREPARATION OF PHENOLIC COMPOUNDS
Phenol, Mallinckrodt, analytical reagent. VOL. 30, NO. 9, SEPTEMBER 1958
1521
o-Cresol, Koppers Co., Inc. Approximate impurities as determined by infrared:
'u I/
Buffer
1
?; I
S H . NH,
c1 Phenol
p-Hydrazinobenzenesulfonic acid
2,B-Dibromoquinone chloroimide (Gibbs reagent)
I1
0
2,6-Dibromoindophenol (blue dye) OH
0
1. Oxidation
I
I1
2. Coupling with p-Cresol OH
so3-
I
I
I
I
0
I
SH,OH
I
+
\
I
CHd
Br-O-Br
\/
.Illialine
+
Buffer
no reaction
CH3
+K=X
1;
OH
N
I
I
C1
p-Cresol I
Phenol, o-cresol, m-cresol, and other similar phenols react to form the blue indophenol dyes. Coupling with Gibbs reagent occurs at the open para position of such phenols. When the para position is blocked by an alkyl group as in p-cresol, 2,4-xylenol, 3,4-sylenol, and similar para-substituted phenols, no coupling occurs. Since a n overnight reaction with Gibbs reagent is required to couple with phenolic compounds which would interfere, the time required for a pcresol determination is about 18 to 24 hours. However, actual working time is less than 4 hours. Data summarized in Table I emphasize the necessity of screening out the phenols which form indophenols. Phenol, o-cresol, and m-cresol in the distillate would lead to a n erroneous high value for the p-cresol content of a sample. I n the early investigations, distillates prepared for p-cresol determinations were tested qualitatively for free phenols by the Gibbs method. The development of any trace of blue color in the distillate indicates free phenols which would interfere in the p-cresol analysis. However, if the prescribed procedures are used, interfcrence from other phenols can be avoided. Gibbs buffer (pH 9.4), employed in the usual Gibbs reaction for phenols, is not used. Tests showed t h a t Gibbs buffer slowly decomposed Gibbs reagent during the coloring reaction and caused an interference. Steam-distillable compounds, possibly of the quinone type, were formed which bleached the color produced by the reaction of p-cresol and DPHBSA reagent, and yielded low results. Interference from decomposition products of Gibbs reagent was avoided when sodium bicar-
'
bonate was used as the alkaline buffer. It was made u p by adding sodium carbonate to the sodium hydroxide extract, and adjusting the p H to 8.0 with dilute sulfuric acid. After the distillate, free from interfering phenols, is obtained, any one of several diazo reagents and certain other sensitive colorimetric reagents could be used to measure the p-cresol. Exploratory investigations indicated that a color reagent prepared from ph ydrazinobenxenesulfonic acid was more satisfactory for p-cresol determination than other colorimetric reagents tested. Preliminary purification, often required for other reagents to be diazotized, was found unnecessary for p-hydrazinobenzenesulfonic acid. Tests over a period of more than a year, indicated no decomposition of the solid reagent. Table I. Color Reactions of Some Phenols with DPHBSA Reagent
Phenolic Compounds" Color Absorbance* p-Cresol Pink 100 Phenol Yellow 122 o-Cresol Yellow-orange 201 159 m-Cresol Yellow-orange a 100 y used for each test. 6 Absorbance compared to p-cresol; measured at 495 mp.
However, certain stabilized diazonium salts prepared for the dyeing industry may also be sufficiently stable and convenient for the colorimetric measurement of para-substituted phcnols. The chemistry of the reactions involved in the colorimetric determination of p-cresol with DPHBSA is not definitely known: The probable reactions are:
CH3 (Red dye) ?
Treatment of p-hydraeinobenzenesulfonic acid with nitrous acid probably produces a diazonium dipolar salt (DPHBSA reagent) which couples with p-cresol. A spectral curve of the clear, red solution produced with DPHBSA reagent and 50 y of p-cresol was obtained using the Spectronic-20 spectrophotometer. This curve (Figure 1) shows that the ma\;imum absorbance was obtained at 495 mp. This wave setting was used for the prepof the standard p-cresol reference curve and the measurement of p-cresol in samples. The relationship of absorbance to weight of p-cresol was found t o be linear from 10 to 140 y o Time studies (Table 11) indicate t h a t the red color formed from DPHBSA and p-cresol was a mayimum value within 5 minutes, and was stable for at least 4 hours. KO precaiitions were necessary to avoid fading of the color by light. RESULTS
Replicate tests (Table 111) were conducted on a prepared solution containing 18 p.p.m. of phenol and 2.00 p.p.m. of p-cresol. Results show that the average value of the determinations was 1.97 p.p.m. of p-cresol and was about l.5yo low. Similar tests, also shown in Table 111, were conducted on a solution CORtaining 2 p.p.m. of phenol and 18.0 p.p.m. of p-cresol. The p-cresol found was 17.9 p.p.m. or an error of 0.5601, low. Results of replicate tests on samples VOL. 30, NO. 9, SEPTEMBER 1 9 5 8
1523
of chemical process waste waters alone and fortified with known amounts of p-cresol are presented in Table IV. Calculations of the standard deviations indicate good precision for the determination of p-cresol. However, as pointed out in the tests on prepared p-cresol-phenol solutions, the actual p-cresol values may be slightly higher. J t was of interest to determine whether the analytical procedure for p-cresol might be applied to ohenolic waste waters from tar and by-product coke plants. Because such waste waters would be likely to contain xylcnols including 2,4- and 3,4-xylenols, as well as p-cresol, tests ‘ivere conducted to determine. the color reactions of these xylenols with DPHBSA reagmt. They produced a red color similar to that obtained with p-cresol. On a weight basis, 2 , 4 and 3,4xylenol w r e equivalent to 84 and l O l % , respectively, of p-cresol. Samples of effluents from tar and by-product coke plants were analyzed for phenols by the 4-aminoantipyrine method. The p-cresol method was applied for the determination of the para-substituted phenols. Qualitative tests with Gibbs reagent showed that interfering phenols had been eliminated from the distillates used for the pcresol determinations. The results of the 4-aniinoantipyrine tcsts arid the p-cresol determinations are summarized in Table V. This method for the determination of p-cresol was developed primarily for analysis of chemical process waste waters which contained more than 1.0 p.p,ni. of p-cresol. Appropriate modifications of the procedures may extend the range of the method t o measure as little as 10 p.p.b. of para-substituted phenols.
Table II. Color Stability Study of Product Formed from DPHBSA and 100 y of p-Cresol
Time 5 min. 30 min. 90 min. 2 . 0 hr. 4 hr. 20 hr. 24 hr.
Table 111.
Test NO.
Absorbance at 495 mp Stored in Exposed darkness to light 0 30 0.30 0 30 0 30 0 30 0 30 0 30 0 30 0.30 0.30 0.29 0.29 0 29 0 29
Determination of p-Cresol in Prepared Solutions
Solution .4. 2.00 P.P.X. p-Cresol and 18 P.P.M. Phenol
1 96 1 99
5
6 7
8
9 10 Av. Error
1.94 1 98 1 94 2 00 1.97 1 5% (lorn)
Solution B. 18.0 P.P.11. p-Cresol and 2 P.P.M. Phenol 11 18.0 17.8 12 17.8 18.1 13 17.8 14 17.6 15 18.1 16 17.8 17 18.0 18 17.6 19 18.1 20 Av. 17.9 Error 0.56% (low)
1524
ANALYTICAL CHEMISTRY
LITERATURE CITED
(1) American Petroleum Institute, Kew York, “Manual on Disposal of Refinery Wastes, Vol. IV, Sampling and Analysis of Waste Water,” 2nd ed., 1957. (2) Am. Public Health ASSOC.,Inc., XewYork, “Standard Methods for the Examination of Water, Sewage, and Industrial Wastes,” 10th ed., 1958.
pStd. Cresol, Dev., P.P.M. P.P.11.
Sature of Sample Chemical process waste water B Sample A 500 p.p.m. p-cresol C Plant waste water D Sample C 100 p.p.m. p-cresol 2 .
+ +
435
4Z3 0
937 90 2
i 5 0 103
190 5 i l 2
Table V. Phenol and p-Cresol Determinations on Industrial Waste Waters
Para-
p-Cresol, P.P.X. -4dded Found
ACKNOWLEDGMENT
The authors wish to express their appreciation t o W. W. Hodge, senior fellow of the Effluents Treatment Fellowship, for his suggestions and assistance in the preparation of this manuscript; also to L. IT. Suniansky and T. G. Patarlis for their technical assistance.
Table IV. Summary of Replicate p-Cresol Determinations
Phenol Substituted (4-;IA4P Phenols (as Method). n-Cresol). P.P.M. P.P.M.”
Kaste Kater Sample Tar Plant Dephenolizer feed 9,400 Deohenolizer iffluent 150 Coke Plant Dephenolizer feed 3 340 Dephenolizer effluent 140 Xmmonia still waste 110 ~
(3) Gibbs, H. D., J .
-4itz.
/
‘
700 i 20 8 01t0.3
295 & 6 6.54Z0.1 3 5 1 0 1
Che?,i. SOC.49,
839-41 (1927). (4) Gibbs, H. D., J . Bzol. Cheitl. 72, 655 (192i).
(5) Lykken, L., Treseder, R. S., Zahn, V., IND.ENG.CHEV.,rls.4~.ED. 18, 103-9 (1916). (6) Mohler, E. F., Jacob, L. N., ANAL. CHEM.29, 1369-74 (1957). (7) Schmauch, L. J., Grubb, H. M.,Zbzd., 26, 308-11 (1954). (8) Shaw, J. -i.,IND.ENG.CHEM.,~ A L . ED. 1, 118-21 (1929). (9) Simard, R. G., Hasegawa, I., Bandaruk, W.,Headington, C. E., ANAL. CHEK 23, 1384-7 (1951).
RECEIVEDfor review Januarv 6, 1958. Accepted May 7 , .1958. Divigons of Industrial and Engineermg Chemistry and Water, Sewage, and Sanitation Chemistry, 132nd Meeting, ACS, Kew York, N. Y., September 1957. Contribution of the Fellowship on Effluents Treatment sustained a t Xellon Institute by Koppers Co., Inc.
