404
ANALYTICAL CHEMISTRY
or reducing indigo carmine, such as nitrates, chlorates, nitrites, iron salts, and sulfites, will interfere with the method unless previously removed (1). Calibrating the reaction by constructing a linear curve from the net absorbances at 0 and 100% saturation as described here provides a simple and rapid method adequate for most purposes; the slight change in blank absorbance between reduced and oxidized samples introduces an error of less than 1%. The practical difficulties involved in preparing a graduated series of water samples of known oxygen content, protected from the air and in sufficient quantity for determination by the Winkler method, would seem to make this method less advisable for routine use. LITERATURE CITED
(1) Am. Pub. Health Assoc., New York, “Standard Nethods for the Examination of Water and Sewage,” 9th ed., 1946.
(2) Efimoff, W. W., Biochem. Z., 155, 371 (1925). (3) Fox, H. XI., and Wingfield, C. A,, J . Ezptl. Bid., 15, 437 (1938). (4) Kirk, P. L., “Quantitative Ultramicroanalysis,” ii’ew York, John Wiley & Sons, 1950. (5) Ramon, D. S., Limnological Soc. Am., University of Michigan, Ann Arbor, LIich., Spec. Pub., 15, 1944. (6) Ricker, W. E., Ecology, 15, 348 (1934). (7) Roughton, F. J. W., and Scholander, P. F., J . Biol. Chem., 148, 541 (1943). (8) Snell, F. D., and Snell, C. T., “Colorimetric Methods of Analysis,” p. 137, S e w York, D. Van Nostrand Co., 1945. (9) Thompson, T. G., and Miller, R. C., Ind. Eng. Chem., 20, 774
(1928). (10) Treadwell, F. P., and Hall, TV. T., “Analytical Chemistry,” 9th ed., Vol. 11, p. 700, Xew York, John TViley & Sons, 1951. ( 1 1) Welch, P. S., “Limnological Methods,” p. 366, Philadelphia, Blakiston Co., 1948. (12) Winkler, L. W., Ber., 21, 2843 (1888). RECEIVED for revicw June l:, 1953. Accepted October 10, 1953.
Determination of Phenanthrene in Coal-Tar Products LEENDERT BLOM
and
WILHELMUS J. VRANKEN
Staatsmijnen in Limburg, Central Laboratory, Geleen, The Netherlands
E
XISTIKG methods for the gravimetric determination of phenanthrene via phenanthrenequinone give unreliable results, chiefly because of the shortcomings of the procedures recommended for oxidation of phenanthrene to the quinone with iodic acid. The determination of phenanthrene in coal-tar products by chemical methods has not been studied extensively. Williams (9) suggested a method based on the ovidation of phenanthrene. The phenanthrenequinone produced is precipitated as toluphenanthrazine in an acetic acid medium with 3.4-tolylenediamineJ and the precipitate is filtered, dried, and weighed. X correction of about 15%, found by experiments, is added, being the weight of the toluphenanthrazine in solution. According to Williams, the oxidation is quantitative. KhmelevskiI and Postovskii (6) reported an improvement of Williams’ method. The oxidation procedure is essentially the same; however, the resulting quinone is dissolved in a saturated sodium bisulfite solution, forming an addition compound. After filtration the quinone is precipitated with ammonia and potassium permanganate, filtered, dried, and weighed. S o solubility correction need be made. These authors also state that the oxidation of phenanthrene to form phenanthrenequinone is quantitative. KhmelevskiI and Levin (4)suggested a second improvementj precipitating the quinone from the bisulfite solution with an aqueous solution of o-phenylenediamine dihydrochloride. The difference from Williams’ method is the bisulfite step, which renders the solubility correction superfluous. I t is remarkable that these authors introduce a correction factor (1.136) due to the incomplete oxidation to form phenanthrenequinone, which they ascribe to iodination. Essentially the same method was reported by KhmelevskiI and Postovskii (5), who also used the correction factor mentioned above. Pavolini ( 7 ) used chromium trioxide in glacial acetic acid as an oxidant, and estimated the resulting phenanthrenequinone gravimetrically as the cobalt complex of the monooxime. DEVELOPMENT O F METHODS
Melting points were taken on the proposed standard apparatus developed by W. M. Smit for the Stichting Centraal Instituut voor Physisch-Chemische Constanten in the Netherlands. Materials. The phenanthrenequinone had a melting point of 209.2’ C. An estimation with 2,4-dinitrophenylhydrazine showed 99.1% phenanthrenequinone.
A coal-tar product chiefly composed of anthracene, carbazole, and phenanthrene was dissolved in benzene and successively extracted with 72 and 90% sulfuric acid to remove carbazole. The solution was concentrated, most of the anthracene crystallizing out. After filtration, crude phenanthrene was obtained by evaporation. This was treated three times with maleic anhydride (to remove anthracene), followed by a threefold recrystallization from ethyl alcohol and drying in vacuo over calcium chloride. The phenanthrene obtained in this way had a melting point of 99.0 “C. The ultraviolet spectrum of the sample is shown in Figure 1 and is in excellent agreement with the spectrum shown in the compilation of ultraviolet spectral data of the American Petroleum Institute ( 1 ) except in the case of curve C, where the authors used a concentration that was ten times larger to obtain nearly the same curve. They believe this discrepancy is due to a printer’s error. Determination of Phenanthrenequinone. A small number of preliminary experiments showed the method of KhmelevskiI and Levin (4)to be the most promising, with regard to speed and accuracy. This method is based on the reaction of phenanthrenequinone, dissolved in a saturated solution of sodium bisulfite, with o-phenylenediamine dihydrochloride to form phenanthrophenazine (I), which is very insoluble in the reaction medium.
I The original method of Khmelevskiy and Levin fied as described below:
( 4 ) was modi-
The acetic acid solution of the uinone was cooled before the sodium bisulfite solution was ad?ed. The results were made higher and more reproducible in this way. The o-phenylenediamine dihydrochloride was added as an aqueous solution instead of in the solid form. The precipitated “azine” was dried a t a temperature of 120’ C. instead of 105’ C. I n this way much time is saved. Twenty-four hours of heating a t 120’ C. did not alter the weight of a 400-mg.
V O L U M E 2 6 , NO. 2, F E B R U A R Y 1 9 5 4
405
sample of azine, which proves the stability of the compound a t this temperature. In many experiments the precipitation of the azine was not entirely quantitative, part remaining in colloidal solution. Therefore the filtrate was always reheated and filtered again. However, in many cases, addition of trichloroacetic acid made the first precipitation quantitative, so that no second filtration was needed. With this modified method the following results were obtained, which are in excellent agreement with those obtained by precipitation with 2,4-dinitrophenylhydrazine. Quinone Used, hlg.
304.8 305.0 303.8 305.5 252.6
Volume of Azine Precipitation, hI1. 340 150 150 340 280
Azine Found,
Quinone Recovered,
31g.
%
404.7 405,3 403.1 405.3 337.8
99.1
Still better results were obtained in a more dilute acetic acid medium, as is demonstrated in Table I. In these experiments heating was continued for 10 minutes after iodine evolution had stopped (iodine sublimes completely into the condenser). At decreasing acetic acid concentration the phenanthrenequinone production increases to a maximum of 94% a t a concentration of 30% acetic acid in water. Below this concentration difficulties arose, due to the very small solubility of the phenanthrene in the reaction medium.
99.1
99.1 99.0 99.3
Oxidation of Phenanthrene to Phenanthrenequinone. In studying the osidation, the amount of phenanthrenequinone obtained was always estimated by the method described above (no use being made of first modification). Attempts to oxidize phenanthrene with chromium trioxide in glacial acetic acid according to the method of Pavolini ( 7 ) failed entirely, the yield of quinone being only 30 to 40%. With this oxidant the chance of overoxidation is very great; therefore this method was not extensively studied.
+WAVE
and 10 ml. of water was first used. About ten experiments showed an average yield of 85% of the quinone, for oxidation times of 0.5 and 2.5 hours. I n all these experiments the iodine production stopped after 0.5 hour, which was an indication that the reaction had finished.
LENGTH IN ANGSTROM UNITS
Figure 1. Absorption Spectrum of Phenanthrene Solvent, iao-octane Cell length, 10.0 mm. Concentration, gram per liter A. 0.01445 B . 0.001445 C. 0.2168
The authors did not succeed in making the oxidation quantitative by the method described by Williams (9) and Khmelevskii (6). In these experiments the oxidation was performed with iodic acid according to Williams' procedure. However, the same results were obtained with the anhydride of iodic acid (iodine pentoxide) used in all other experiments. The results proved that the average yield of quinone was 70%. Prolongation of the reaction time from 2.5 to 4 hours gave 9 % higher results, but the reproducibility left much to be desired. The cause of these low results is to be found in the dark-broan residue observed after solution of the bisulfite-quinone addition compound. This residue seems to consist mainly of 9-oxy-10iodophenanthrene and its acetic acid ester. This agrees with the view of KhmelevskiK (4,s)and Levin. However, experiments proved that the correction factor recommended by these authors was inadequate. An effort was made to suppress the interfering side reactions by adding water, which should have the following effects : Greater solubility of the iodic acid, resulting in a greater speed of the oxidation. Smaller solubility of iodine, reducing the chance of iodination. Instead of 20 ml. of acetic acid a mixture of 20 ml. of acetic acid
Table I.
Influence of Dilution on Oxidation of Phenanthrene
Phenanthrene, 31g.
-4cetic Acid. hI1.
Water, lM1.
250
20
20
1205,
Phenanthrene Found,
%
Big.
Table 11. Influence of Iodic Acid Concentration Phenanthrenr, RIg. 250
Acetic Acid (Initial), M1. 0
Water, hll. 40
hlg. 1000
250
5
35
1000
260 250 250
5 5 2
35 35 35
2000 3000 3000
1206,
Phenanthrene Found,
%
95,4
I:;
95,6 95,6
98,3 98,l
Some phenanthrene sublimed with the iodine. To avoid this, the phenanthrene concentration was kept low by slowly adding 10 ml. of a solution of phenanthrene in acetic acid to the boiling iodic acid solution in diluted acetic acid ( 5 ml. of acetic acid plus 35 ml. of water) or in water (40 ml). In both cases the yield of quinone was 95.60j0, which is about 2% higher than in the foregoing experiments. Finally the influence of iodic acid concentration was studied (Table 11). Although there is but a small difference between the results for 2000 and 3000 mg. of iodine pentoxide, use of about 3000 mg. per 35 ml. of water is recommended in the standard procedure. owing to the consumption of the oxidant by other compounds in the samples analyzed. In all these experiments the quinone formed was converted into the bisulfite addition product by adding the bisulfite solution to the hot solution of the quinone. Several experiments carried out with previous cooling of the quinone solution and 3000 mg. of oxidant gave nearly quantitative results (compare Table 111). This observation, which was made a t the end of this study, is also taken into account in the standard procedure. The melting point of the phenanthrophenazine obtained in these experiments was 223" C. INTERFERIBG SUBSTANCES
Carbazole. It was proved that a carbazole content of the samples corresponding to about one half of the phenanthrene content effected a relative lowering of the results by about 20%. The higher the iodic acid concentration, the smaller the interference. Perhaps it is the red addition compound of carbazole and phenanthrenequinone, mentioned by B d r e n s ($), which plays a role here in stimulating the further oxidation of the
406
ANALYTICAL CHEMISTRY
quinone. Carbazole had to be removed before the oxidation, by using the reaction of carbazole with formaldehyde ( 3 , 8). This gave a very insoluble product, which was filtered off. When this method wm followed, carbazole no longer interfered. Acenaphthene. Oxidation of a 100-mg. sample of acenaphthene and precipitation of the reaction product with o-phenylenediamine gave an insoluble product weighing about 25 mg. and giving rise to erroneous results. Fortunately, it appeared that acenaphthene was also largely precipitated with formaldehyde, though somewhat more slo~vly. In this way the interference could be largely suppressed. Acridine. The presence of acridine gave rise to somewhat high results, b u t for contents beloiv 5970 the interference was negligible. Kaphthalene, anthracene, fluorene, pyridine bases, and phenolic compounds (the latter are precipitated with formaldehyde) did not interfere. The method of analyses which has been adopted for routine work in this laboratory is the outcome of these studies.
the factor 635.7 and dividing this product by the weight of the sample in milligrams.
If the sample contains appreciable amounts of acenaphthene, indole, or phenolic substances, . the pretreatment with formaldehyde has to be prolonged to 200 rnl. a t least 1 hour. As a rule the completeness of Figure 3. Apparatus the precipitation of the azine is for Filtration of Acetic Acid Solution indicated by the appearance of a clear yellow or yellow-brown filtrate. Although not proved necessary, addition of trichloroacetic acid is desirable to get complete precipitation. The same result is obtained by leaving the flask overnight on the steam bath before filtration. The buret used must be protected against heat. RESULTS OBTAINED WITH STANDARD PROCEDURE
PROCEDURE
Apparatus.
The apparatus is shown in Figures 2 and 3.
Reagents and Materials. Hydrochloric acid, 12LV (density 1.19). Iodine pentoxide. o-Phenylenediamine dihydrochloride. Sodium bisulfite, fresh1 prepared saturated solution. Formaldehyde, 40% in water. Glacial acetic acid. Trichloroacetic acid. Pumice. Standard Procedure. Veigh approximately 2.5 grams of sample to the marest milligram into a 250-ml. beaker, add 100 ml. of glacial acetic acid, and heat to boiling until the sample is dissolved. Slowly add 3.4 ml. of a 1 to 1 mixture of concentrated hydrochloric acid and a 40% formaldehyde solution to the hot solution while stirring. Heat under reflux for 15 minutes, cool, and filter through a 1G4 sinteredglass crucible under suction direct into a 200-ml. graduate (Figure 3), taking care that the filter remains wet. Wash with glacial acetic acid, dilute with acetic acid to the 200-mI. line, and mix. Transfer part of this solution into a 50-ml. buret (Figure l ) , add approximately 6 grams of iodine pent.oxide to vessel A (Figure l ) , and add 10 ml. of glacial acetic acid, 70 ml. of water, and some pumice. Heat to boiling and add slowly (30 drops a minute) 20.00 ml. of the phenanthrene solution from the buret. Rinse the neck of the vessel with 3 ml. of acetic acid and continue boiling for 10 minutes. Cool, add 45 ml. of saturated sodium biFigure 2. Apparasulfite solution, and shake 2hile tus for Oxidation Of heating to a maximum of 55 C. Phenanthrene Add 125 ml. of water, shake vigorously, and cool. Filter off through a paper filter (S & S 58g3) into a 750-ml. conical flask and wash with water until the filtrate is free from bisulfite. Add 20 ml. of a 4% solution of o-phenplenediamine dihydrochloride. Fit a condenser on the flask, boil for 25 minutes, and heat for 15 minutes on the steam bath: Cool, add 3 grams of trichloroacetic acid, and filter a t the end of 10 minutes through a weighed 1G4 sintered-glass crucible, porosity 5-15 p , and wash with water until the filtrate is free of chloride ions. Dry a t 120" C. to constant weight (-1 hour). P u t the filtrate on the steam bath (at least 1 hour) to see if more precipitate will form. If so, carry out a second filtration. Calculate the &tual percentage (by weight) Of phenanthrene by multiplying the total weight of precipitate in milligrams by
Table I11 demonstrates the results obtained with phenanthrene and with mixtures containing variable amounts of phenanthrene. ACKNOWLEDGMENT
The authors wish to thank H. W. Deinum for his encouragement and interest in the development of this method, J. H. Ottenheym for the purification of some of the test samples used, and H. A. van Vucht for the spectrophotometric measurements.
Table 111. Results Obtained with Standard Procedure
Sample Phenanthrene
Description of Sample Pure, melting point 99.00 C.
Mixture I
Mixture I1
Acridine, Chrysene, Naphthalene, Same as mixture
I
Phenant hreoe
Coal-tar products
Crude phenanthrene
I
I1 111 Iv
v VI VI1 Crude anthracene
I
I1 I11 IV a
PheoSnthrene Found,
Mg.
%
1170 1640 2500 2500 2500 2500
99.7 100.4 99.2 99.1 39.9 40.3
2500 2500 1500 1500 1000 I500 2000 2000
40.4 39.6
Laboratory A A B
B B B
1.0% 1 .O% 4.0%
Same as mixture I, except phenanthrene Technical (contained 3.0% anthracene and 1.0% carbazole)
Mixture I11
Sample Weight,
No measurable amount of precipitate.
2500 2500 600 600 600 600
2500 2500 2500 2500 2500 2500 2500 5500
2000 2000 5000 5000 2500 2500 2500 2500 2500 2500
0
a
93.2 93.2 93.2 93.4 38.8 38.3 44.3 44.5 45.1 44.7 35.9 35.2 34.6
35.3 36.7 36.4 52.1 51.7 8.3 8.3 8.3 8.4 7.4 7.5 6.1 6.6 6.8 6.8
B B B B B
A
A A B B B B B
A B
B B
V O L U M E 26, N O . 2, F E B R U A R Y 1 9 5 4 Thanks are also due to M. K. J. Janssen and J. A. bl. Susser of the Cokeworks Mauritw (Laboratory A ) for their contribution to the evperimental work. LITER 4TURE CITED
407 (4) Khmelevskii, V. I., and Levin, I. S.,Org. Chem. Ind. (U.S.S.R.), 7, 241 (1940). (5 ) Khmelevskii, V. I., and Postovskii, I. Ya., J . A p p l . Chem. (C.S.S.R.I. 17. 463 (1944). (6) Khmelevskii,’ V.’ I., and Postovskii, I. Ya., Org. Chem. Ind. (U.S.S.R.),4, 3G3 (1937). ( 0 Pavolini, T., Industria chimica, 5, 862 (1930). ( 8 ) Pulvermacher, G., and Loeb, W., Ber., 25, 2766 (1892). (9) Williams, A . G., ,J. Am. Chem. Soc., 43, 1511 (1921). -I
(1) .imerican Petroleum Institute, Research Project 44, Serial No. 92 (June 1952). ( 3 ) Behrens, M. H., Ret. trav. chim., 19, 387 (1900); 21, 252 (1502). (3) D u t t , S., J. Chern. Soc. (London),125, 802 (1524).
R E C E I V Efor D revirw October 22, 1952.
.iccepted July 29, 1953.
Nitrosation Method of Determining d-7-Tocopherol B A R B A R A HINDERER POLISTER] School of Medicine, University of California at Los Angeles, Los Angeles, Calif.
I
N COSNECTIOK withx-irradiation experiments(fi), it was desired to determine directly, rapidly, and simply the concentrations of d-y-tocopherol in aqueous emulsions of methyl linoleate tmth before and after irradiation. Of the several frequently emploj-cd methods investigated ( I d j 7-10), the reaction of 8-, y-, and &tocopherols with nitrous acid, first discussed by Scudi and f3uhc (9), appeared least likely to present difficulties in the system under consideration. An ass..ty bawd upon this reaction has been developed by Quaife (8). Alcoholic solutions of 8-, y-, and &tocopherols, because of the presence of an unsubstituted position ortho to the phenolic group, form yellow nitroso derivatives on treatment with nitrous acid. On treatment with alkali the nitrosotocopherols turn red. Measurement of color a t this stage is unsuitable, since the aqueousalcohol solution would be turbid owing to fat which accompanies the tocopherols. Dilution with water and extraction into petroleum ether give dear, stable, yellow qolutions of the nitrosotocaopherols suitable for spectrophotometric determinations. The absorption maxima rpported for nitroso-y-tocopherol are a t 305 :ind 415 mp (8).
Table I. Relation of d-pTocophero1 Concentration to .ibsorbance of Kitroso-7-tocopherol Solution Total d-y-Toropherol, l l g . / 5 - M l . Sample
Absorbance at 305 mp
Absorb ance at 415 mp
0.60 1.02 1.06 1.20 1.68 1.80 1.94 2.40 2.55 3.00 5.10 6.00
0.65 1.14 1.18 1.30 2.03 2.04 2.36 2.75 2.98 3.71
0.28 0.48 0.50 0.57 0.88 0.88 1.03 1.23 1.35 1.53 2.50 3.11
6.50
7.06
However, attempts by the auther to repeat Quaife’s work were unsuccessful. Both the determinations and the blank for the determinations-i.e., ethyl alcohol containing no tocopherolexhibited such strong absorption bands a t 385, 3 i 0 , 355, 333, 324, and 315 m r that it was impossible to detect the maxima reported for nitroso-ytocopherol. Subtraction of the blank from the determinations did not clarify the situation appreciably because the former’s absorption was not constant, no matter how accurately the reaction period was timed. These interfering absorption bands were thought to be due to the presence of ethyl nitrite, formed by the action of nitrous acid on ethyl alcohol. 1 Present address, General Mills Research Laboratories, Minneapolis, Minn.
This, indeed, XT as found to be the caw, for comparison of the absorption spectra of the blank and the “tocopherol determinations” with that of butyl nitrite (kindly supplied by D. R. Howton, School of Medicine, University of California. a t Los ilngeles) in the same solvent and over the same visible and ultraviolet range disclosed identical absorption maxima. I t was. therefore, necessary to discover an inert medium for the reaction which was both a good solvent for y-tocopherol and yet was completely miscible with the aqueous reagents. Freshly distilled and peroxide-free dimethoxyethane suited these requirements adequately, and the reported absorption maxima for nitroso-ytocopherol were obtained when this d i s t a n c e was used as qolvent for the determinations. dlthough slight colorations developed in both the dimethoxyethane blank and in a dimethoxyethane solution containing only methyl linoleate upon addition of the reagents, in neither case were the colorations extracted by the iso-octane, nor did they affect the ultraviolet or visible spectra of these blanks. I t is assumed that the large excess of reagents added to the dimethoxyethane solutions containing both methyl linoleate and tocopherol is morc than sufficient to react completely with the tocopherol as well as to be partially consumed by the solvent and the ester. Both of the absorption maxima, 305 mp (molar extinction 4.86 X lo3) and 415 mp (molar extinction 2.08 X l O 3 ) , follow Beer’s law over a wide range, as Table I indicates. The results are completely reproducible, with an average error of deviation from the mean of &5%, REAGENTS
Glacial Acetic Acid, C.P. grade. Sodium Nitrite Reagent ( 2 grams of C.P. crystals per 100 ml. of distilled water). The solution should be prepared fresh every few days, then be kept in the refrigerator and warmed to room temperature before use. Potassium Hydroxide Reagent (20 grams of reagent pellets per 100 ml. of distilled water). Anhydrous Sodium Sulfate, reagent grade. 1,2-Dimethoxyethane (Arapahoe Chemirals, Inc., Boulder, Colo.), freshly distilled from a peroxide inhibitor. Iso-octane, Spectro Grade (Phillips Petroleum Co., Chemical Products Division, Bartlesville, Okla.). This solvent was substituted for the Skellysolve H of Quaife’s procedure because its more constant composition and grade of purity was considered preferable for spectrophotometric work. PROCEDURE
All operations should be performed under subdued artificial
light. Five milliliters of a tocopherol solution in redistilled dimethoxy ethane containing between 0.10 and 1.20 mg. per ml. of ytocopherol (the d-7-tocopherol used in these experiments was purchased from Distillation Products Industries, Division of Eastman Kodak Co., Rochester, N. Y . ) is placed in a 50-ml. glass-stoppered graduated cylinder made of red Low Actinic glass. Exactly 0.2 ml. of glacial acetic acid is added and mixed,
