117
V O L U M E 2 3 , NO. 1, J A N U A R Y 1 9 5 1 Tahle 11. W e i g h t of
Sample Gtarn
0 8956 0 0603 0.3121 0.2204 0,0983 0,1556
Determination of Superoxide Oxygen in Sodium Superoxide-Sodium Peroxide Mixtures Oxygen Liberated on Total Decomposition cc./g.
(S. T P.) 170
Siinerowde
Calcd. 6%
Superoxide Ovygena Liberated
Superoxide Obsd.
cc :g.
%
16.1
30.9 15.2 30.7 15.1 171 18.G 37.4 18.4 35.9 17.7 36.0 27.6 191 28 .i 58.5 28.8 26 0 111 5G.0 0.1617 234 0.1647 113 55.8 0,0691 273 80.2 159 78.1 0,0397 161 79.4 0 . 12,53 206 94.3 188 92.6 0.1178 192 94.5 " Piire uhite sodium ueroxide, supplied by .\line Safety Appliances Co., Pittsburgh, Pa., liberated no oxygen when treated with acetic acid-dlethyl phthalate solution. Game material treated with water a t O n yielded 7.7 and 12.0 cc. of oxygen (S.T.P.) per gram i n two experiinents.
ivlit~w the denominatcr is tlir t1icoretir;d quaiit,ity of oxygt'il avnilablc: per gram of pure sodium superoxide upon conversion to iwroside. The data of T:ihle I1 s h o ~t h a t i n the rase of sodium superoxide-sodium peroxide mixtures 1 1 1 t h vitlurs obtained for superoxide roiltent agrer rc:tsonably n.c.11 n-ith the theoretical, when one considers the extreme viisiit iyit). of superoxides to moisture. It is j)articularl,v striking that pure sodium peroxide liberates no oxygen upon treatment with acetic acid-diethyl phthalate mist,ures. This again emphasizes the fact that there is little, if any,
release of oxygen by the secondary decomposition of hydrogen peroxide in solution, and demonstrates t'hat the method employed is applicable to mixtures containing superoxide ion. Samples of sodium superoxide which on total decomposition with ferric chloride-hydrochloric acid catalyst solution released 296 cc. of oxygen (S.T.P.) per gram, liberated 203 and 245 re. of oxygen (S.T.P.) per gram Lvhen treated with water a t 0". Since theoretical release of superoxide oxygen for these samples is 192 cc. (S.T.P.) per gram, these dat,a emphasize the unreliabi1it.y of the latter treat,ment as a method for the determination of superoxide oxygen. Hoxever, George ( I ) has reported that treatment of potassium superoxide with water at room teinperatue causes release of the theoret'ical quantity of oxygen required for conversion to peroxide. The authors are unztble t o sc.count for the discrepancy between his results nith potasGum superoxide anti theirs with the sodium compound. ACKNOWLEDGRIENr
The authors are indebted t o the Office of Saval Research for a grant which has made this aiid continuing investigations possible. LITERATCRE CITED (1) Gcoi.ge, Discnssio~zsFaraday SOC.,KO.2, 196 (1917). ( 2 ) Holt a n d Sims, J . Chem. Soc., 65, 433 (1894). ( 3 ) Kaaartiovskii et al., D o k l a d y Aknd. S a u k , S.S.S.R., 64, 6 9 (1919). f4) Kram and Parmenter, J . Am. Chem. Soc., 56,2385 (1934). (5) S t e y h a n o u , Ph.D. thesis, Cniversity of Kansas, 1949. 16) S t e p h a n o u , Schechter, Argersinger, a n d Kleinherg, J . rim. Chem. SOC.,71,1819 (1949).
(7) Whaley and Kleinberg, Ibid., in press. RECEIVED M a y 19, 1950.
Determination of Hydroxy Compounds in Amine Mixtures SIDNEY SIGGIA AND IRENE R . KERVENSKI General Aniline a n d F i l m Corp., Euston, P a .
.4 method was necessary for determining alcohols in amine mixtures in order to follow the alkylation of amines with alcohols. By acetylating the mixture, the alcohol is converted to the ester and the primary and secondary amines to the corresponding amides. The ester can be quantitatively saponified without significantly affecting the amides. High degrees of accuracy and precision are attainable. It is now possible to determine hydroxy compounds in the presence of amines without significant interference. This was previously impossible chemically except in isolated rases.
0
S E of the most satisfactory methods of determining hydroxy
compounds is acetylation. Primary and secondary amines constitute an interference because they also acetylate (4). \litcliell, Hawkins, and Smith ( 3 ) devised a method for determining hydroxyl groups in the presence of amines by esterification with an acid, using boron trifluoride as a catalyst. They measured the water formed on the esterification via the Karl Fischer reagent. The procedure described below consists of acetylating the mixture quantitatively by the procedure of Ogg, Porter, and Willits (6). The primary and secondary amines acetylate t o the corresponding acetyl compound, and the hydroxy compounds acetylate to the esters. The samples are brought just to neutrality and then an excess of standard alkali is added for saponification. The ester saponifies quantitatively, whereas the acetylated amines are not affected. This technique of acetylation and then saponification was
employed by several analysts ( 1 , 2, 7 ) in the dcterniination of hydroxylated fatty acids. The same grneral approach was found to be applicable for hydroxyl groups in the presence of amines. The primary and secondary amine content can also be determined in the system. Because these compounds acetylate quantitatively, by subtracting the anhydride consumcd by thr hydroxy compound from the total anhydride consumed, a value can be obtained for the primary plus secondary amine content of the samplr If the content of the various amines in the sdmplc is desired, the procedure of Siggia, Hanna, and Kervenski (6) or of Wagner, Brown, and Prters (8) can be used. REAGENTS
Acetylation reagent, 3 parts of pyridine to 1 part of acetic anhydride. Standard alcoholic sodium hydroxide, 0.5 A'. Approximately
ANALYTICAL CHEMISTRY
118 Table I.
Hydroxy Compounds i n Amine Mixtures Alcohol Composition, or Phenol % Found, %
Moles of Primary a n d Secondary Amine per Gram of Sample Calcd. Found
Sample E t h y l alcohol 17.05 16.69 0.00270 0.00270 Naphthylamine 8.74 Ethylnaphthylamine 35.88 Diethylnaphthylamine 38.33 17.85 18.15" 0.00548 0.00521" Phenol 33.47 Aniline Ethylaniline 22.88 Diethylaniline 25.80 11.47 11.59 0.00282 0.00285 Xaphthol Naphthylamine 16.10 Ethylnaphthylamine 29.15 Diethylnaphthylamine 43.28 Butanediol 18.55 18.84 0.00594 0.00587 Cyclohexylamine 31.71 Dicyclohexylamine 49.74 Lauryl alcohol 28.65 28.80 b 48.28 Primary amine (Ciz range) b 23.07 Seoondary amine (CIPrange) b E t h y l alcohol 10.45 10.28 0.00610 0.00611 34.38 Aniline 29.25 Ethylaniline 25.92 Diethylaniline Dark color of solution obscured end point. b These amines are aliphatic amines of C I ~ average chain length, ranging from Ca-Cis. Molecular weights of amine8 used are indefinite, so t h a t molar amounts of amine present in sample could not be calculated.
is noted. The buret reading is taken. Then 50 ml. of 0.5 N alcoholic sodium hydroxide are added to the contents by means of a pipet (this need not be done to the blank), the stopper is again moistened with pyridine, and the flask is put back on the steam bath and kept a t incipient boiling for 2 hours. U on cooling, the excess sodium hydroxide is titrated with 0.5 N suyfuric acid. S o blank is necessary on the saponification. C 4 LCU LATIONS
Hydroxyl
M I . of H2S04used for 50 ml. of
alcoholic NaOH minus ml. of H2SOc used for sample = 9
A X NH~SX O ,mol. wt. of compound X 100 Grams of sample X 1000
% hydroxy compound
Primary plus Secondary Amine
MI. of alcoholic NaOH for blank minus ml. of
alcoholic NaOH for sample = B
~ ~ N ~ o-H( )A X . Y H ~ S O-~ ) Grams of sample X 1000 moles per gram of primary plus secondary amine
(BX
DISCUSSlON
Q
85 ml. of saturated aqueous sodium hydroxides added to 5 pounds of C.P. methanol. Indicator, 1 part of O.lm, aqueous cresol red neutralized with sodium hydroxide to 3 parts of thymol blue also neutralized with sodium hydroxide. Standard aqueous 0.5 S acid. PROCEDURE
A weighed sample containing about 0.01 mole of hydroxyl is introduced into a glass-stoppered iodine flask together with 10 ml. of acetic anhydride-pyridine reagent which has been accurately measured with a pipet. If the amount of hydroxyl is small and the amount of primary and secondary amines is large, taking a sample with 0.01 mole of hydroxyl may consume all the reagent without quantitatively acetylating all the hydroxyl groups. If so, either a smaller sample or a larger amount of reagent should be taken. A blank consisting of the reagent alone is run in the same manner M the sample. A glass stopper is well moistened with pyridine and loosely seated. The flask is heated on a steam bath for 45 minutes. Upon cooling, 10 ml. of water are added and the flask is swirled to bring the water in contact with all the reagent. A few drops of mixed indicator are added and the contents are titrated with 0.5 N alcoholic sodium hydroxide till the blue end point of the indicator
Occasionally more than 2-hour saponification may be required for quantitative results. This was not necessary in determining the alcohols that were studied. Reflux condensers were not needed in the saponification step of the above procedure. However, occasion may arise where the ester formed on acetylation may be very volatile and a reflux condenser may be necessary. Several runs were made heating the amides of aniline, N-ethylaniline, and butylamine for the required saponification time, and, in addition, the samples were allowed to stand for 16 hours before titration. The extent of hydrolysis of the amides amounted t o less than 2%. LITERATURE CITED
(1) Benedikt and Ulger, Chem.-Ztg., 18, 468 (1887). (2) Lewkowitsoh, J., J . SOC.Chem. I n d . , 9, 842 (1890). and Smith, D. M., J . Am. Chem. (3) Mitchell, J., Jr., Hawkins, W., SOC.,66,715 (1944). (41 Ibid.. D. 782. (5j O g g , ' k . L.. Porter, W.L., and Willits, C. O., IND.ENG.CHEM., ANAL.ED.,17, 394 (1945). (6) Siggia, S., Hanna, J. G., and Kervenski. I. R., ANAL.CHEM., 22, 1295 (1950). (7) Steiner, Chem.-Ztg., 59, 795 (1935). (8) Wagner, C. D., Brown, R. H., and Peters, E. D., J. Am. Chem. SOC.,69, 2609-14 (1947). RECEIVED M a y 16, 1950.
Determination of Neutral 17-Ketosteroids in Urine WILLIAM T. BEHER AND OLIVER H. GAEBLER Edsel B. Ford I n s t i t u t e for Medical Research, H e n r y Ford Hospital, a n d W a y n e University, Detroit, M i c h .
D
ETERMIKATIOK of steroid substances in human urine involves hydrolysis of their glycuronides or sulfates, followed by extraction of the free steroids, fractionation of the extract, and, finally, estimation by biological, chemical, or physical methods. The separation of estrogens from androgens by utilizing the phenolic character of the former ( 6 ) , development of Girard's reagent for separating ketonic and nonketonic substances (a), and the digitonin precipitation (19) are among the many noteworthy contributions which made possible a definite system of fractionation (16). For the final estimation, the Zimmermann reaction ( 2 0 ) is commonly used. As Holtorff and
Koch (9j recognized, colorimetric determinations of neutral 1Tketosteroids yield higher values than biological assays for androgens, but they give more complete information concerning excretion of steroids which differ both qualitatively and quantitatively in biological activity. The Zimmermann reaction has been modified by varying the concentrations of alcohol, m-dinitrobenzene, and potassium hydroxide employed, as well as the duration of color development. The principal techniques have been reviewed and evaluated by Kathanson and Wilson ( 1 3 ) . Because the ketonic and nonketonic components in urine extracts give very different colors in
