atures were analyzed. The particle size of the samples varied from 0.4 to 2.1 pm as the heating temperature increased. However, the points of the inflection of the (1- (1 - x)1/3)-t curves were clear and the BazTiO4 amounts could be determined. Figure 3 shows plots of kinetic data for the BazTiO4 formation. Although the amounts of BazTi04 formed are less, the change of the BazTi04 formation as a function of temperature is the same as the results obtained by Kubo et al. (I).
cussions. We also thank Kikuo Wakino, Director of Research of Murata Manufacturing Co., Ltd., for his encouragement and permission to publish this paper.
ACKNOWLEDGMENT
RECEIVEDfor review January 2, 1975. Accepted April 9,
The authors are grateful to Shigero Ikeda for helpful dis-
LITERATURE CITED (1) T. Kubo. M. Kato, and T. F u j i , J. Chem. Soc. Jpn., hd. Chem. Sect., 70, 647 11967). (2) R. E.'Hamikon, Can. Ceram. Soc. J., 37, LXll(1968). (3) H. U. Anderson, J. Am. Ceram. Soc., 56, 605 (1973).
1975.
Carbon Black Adsorbates: Separation and Identification of a Carcinogen and Some Oxygenated Polyaromatics Avram Gold Harvard School of Public Health, 6 6 5 Huntington Avenue, Boston, MA 0 2 1 15
The industrial importance of carbon black and consequent occupational exposure have for some time sparked interest in characterizing its extractable organic constituents (1-6). A recent epidemiological survey of the rubber tire industry (7) indicating excess stomach cancer among workers with high exposure to carbon black further accentuates the importance of analytical studies on this substance.
,
EXPERIMENTAL The carbon black sample, 900 g of oil furnace black, was extracted for 100 hr with CHzClZ. Evaporation of the solvent on a rotary evaporator a t room temperature yielded 371 mg of brown tar. The total extract was redissolved in benzene and extracted with 0.5N NaOH and 1N HC1. The benzene layer yielded a 312-mg neutral fraction. The neutral extract was further separated by a Rosen fractionation (8) into fractions containing saturated hydrocarbons (17 mg), polycyclic aromatic hydrocarbons (183 mg), and polar compounds (26 mg). The polycyclic aromatic hydrocarbon fraction was separated by isothermal preparative gas chromatography a t 270 "C with Nz as carrier gas a t a flow rate of 150 ml/min. The column was 25% SE-52 (phenyl silicone) on Chromosorb W, Y 4 - h . X 15-ft. The chromatogram is shown in Figure 1. Fractions were collected in Utubes containing a small amount of porous glass and cooled in a Dry Ice-ethanol bath. The collected compounds were desorbed by stirring in CHzC12. T h e polar fraction of the Rosen separation was chromatographed by reverse phase high pressure liquid chromatography (HPLC) on a Perkin-Elmer octadecyl Sil-X-I column (4.6-mm X 1-m) with 3096 H20-70% methanol eluant a t 50 O C and a flow rate of 0.6 ml/min. The chromatogram is given in Figure 2.
DISCUSSION AND RESULTS This study expands the scope of previous analytical work on carbon black and airborne particulate extracts with a systematic effort to identify components in the neutral polar fraction of carbon black extract as well as the constituents of the polycyclic aromatic hydrocarbon (PAH) fraction. Several problems were encountered in analyzing both the aromatic and polar extracts. Although analytical schemes for polycyclic aromatic hydrocarbons have been worked out and the compounds themselves extensively characterized,
Fiaure
1. GC
trace of
Dolvcvclic aromatic hvdrocarhnn fractinn
IO
I
0
IO
20
30 40
50 60 Time (mln)
Figure 2. HPLC trace of neutral polar fraction
there are still aromatic hydrocarbons in environmental samples whose structures have not been elucidated (9). One such compound, cyclopenta[cd]pyrene, occurred in this study and the method employed to deduce its structure should be of general utility for polycyclic aromatic hydrocarbons. ANALYTICALCHEMISTRY, VOL. 47, NO. 8, JULY 1975
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Table I. Retention Times for Compounds Characterized in the Polycyclic Aromatic and Neutral Polar Fractions of Carbon Black Extract Retention t i m e , Peak
W
0.E
0
z a
m
min
U
Polycyclic aromatic hydrocarbons ( G C )
1. Naphthalene 2. Acenaphthalene 3 Phenanthrene 4 Fluor anthene 5 Pyrene 6 Benzo[ghi]fluoranthene 7 C yclopenta[ cdlpyr ene
SOLVENT: phexane
1.75 4.25 8.25 15.25 18.25 32 .OO 35.50
0.4
cyclopenta
a
[cd]
pyrene
\ 0.2
Neutral polar compounds (HPLC)
9 Anhydride 10 Phenalenone 11 4-H-cyclopenta[def]phenanthren-4-one 12. 6- H-benzo[cd]pyren-6-one
5 .O 17.6 29.4 52.8
ANALYTICAL CHEMISTRY, VOL. 47,
NO. 8 , JULY
1
1
I
I
1
250
275
3cO
325
350
I
375
I
400
X (nm)
Compounds 1-6 (Table I) of the polycyclic aromatic hydrocarbon fraction were identified by UV, low resolution mass spectrometry, and peak enhancement. Although resolution of phenanthrene (Table I, compound 3; Figure 1, peak 3) and anthracene would not be achieved on the column used in the gas chromatographic separation, the presence of anthracene in peak 3 was ruled out by a comparison of the UV spectrum of compound 3 with that of an authentic sample of phenanthrene. Compound 7 could not be identified through its UV or low resolution mass spectra, which are given in Figure 3. By high resolution mass spectrometry, this compound had the molecular formula Cp,Hlo (mass required, 226.0782;found, 226.0783). A literature search yielded a structurally uncharacterized carcinogen previously isolated from carbon blacks ( 2 , 3 ) that appeared to be identical by UV and melting point (165') to 7. Possible structural isomers derived from the empirical formula consist of combinations of fused five- and six- or five-, six- and seven-membered rings, not all of which are aromatic. The compound (-3 mg) was hydrogenated over Pd in 95% ethanol under the assumption that the UV spectrum of the remaining aromatic system and the Hz uptake would give a clue to the structure of the original carbon skeleton. Compound 7 took up one equivalent of Hz to yield compound 8 after purification by liquid-solid chromatography over alumina and recrystallization from hexane. Compound 8 formed pale yellow plates having a melting point of 131 OC and had a molecular weight of 228. The UV and low resolution mass spectrum of 8 are shown in Figure 4.The UV is similar to that of pyrene and the data confirm that the hydrogenation product is identical to 3,4-dihydrocyclopenta[cd]pyrene(IO), recently synthesized in an unambiguous manner (11).The dihydrocyclopentapyrene could only have resulted from the uptake of one equivalent of Hz by cyclopenta[cd]pyrene which is, therefore, the structure assigned to compound 7.The technique of correlating UV spectra of partially hydrogenated unknown polycyclic aromatic hydrocarbons with those of known aromatic systems should prove helpful in structure determinations by greatly restricting the number of possible structures for the unknowns. In analyzing the neutral polar fraction, it was necessary first, to work out a method of separation and, second, to establish a method of identification for compounds for which no extensive or specific literature exists as it does for polycyclic aromatic hydrocarbons. High pressure liquid chromatography was decided upon as the method of separation. Porous reverse phase packing was used in order to take ad1470
I
225
1975
z
60
9
40
4 +L + 20
I
W
LL
IC0 I25
Figure 3. UV
I50 175 263 2 5 250 m /e
and mass spectrum of compound 7
ISOLVENT: n-hexane
unr
0.E
y
z a
0.E
m
I
3,4 dihydrocyclopento [cd] pyrene
3
$ 0.4
0. i
2
X (nm)
L 225 2 b m/e
Figure 4. UV
and mass spectrum of compound 8
SOLVENT: n-hexane
0
0.6
benzo [cd] pyren-6-one
r: 2 0 c I
I-
102
125
150
175
XO
250
A
'5
225
rn/e
Figure 5.
250
275
303
325
350
375
400
425
453
Mass spectrum of compound 9
Id0
i25
I50 I75
2bl 225 2kl 275 m /e
Flgure 7. UV
and mass spectrum of compound 12, uc0(CCI4),1656
cm-'
225
2%
275
300
325
350
375
400
X(nrn)
425
T-LL 3 20
W
lx
Kx)
125
150 175 203 225 m /e
Figure 6. UV
and mass spectrum of compound 11, uc0(CCI4), 1725
cm-' vantage of the solubility of the polar fraction in methanol so that the chromatography could be done on a preparative scale. Sufficient amounts of compound were obtained for characterization by UV, IR, mass spectrometry, and, in some cases, by melting point. The empirical formulas of compounds not readily identifiable by these data were determined by high resolution mass spectrometry to facilitate a literature search in Chemical Abstracts. Components of the neutral polar fraction, designated compounds 9-12 (Table I), were initially characterized by UV, IR, and low resolution mass spectrometry. The exact identity of compound 9 could not be determined from these data. The empirical formula of 9, by high resolution mass spectrometry, was established as Cl8H803 (expected for C18H8O3, 272.0473; found, 272.0472). The fragmentation pattern of the low resolution mass spectrum (M.+ - 44, M.+ - 72, Figure 5) and the IR ( V C O (CHC13) 1775, 1735 cm-l) indicate a six- or seven-membered cyclic anhydride
(12). The fragment a t mle 200, presumably C16H8 (M-+ C203), suggests an anhydride of pyrene or fluoranthene dicarboxylic acid, which for pyrene would imply 1 , l O substitution (perhaps arising from oxidation of compound 7) and for fluoranthene, 3,4 or 1 , l O substitution. UV, IR, and low resolution mass spectrometry were sufficient to identify compound 10 as phenalenone. The molecular formulas of ll and 12, determined by high resolution mass spectrometry enabled these compounds to be identified through the literature as 4-H-cyclopenta[deflphenanthren-4-one (13) and 6-H-benzo[cd]pyren-6-one (14), respectively. The UV and low resolution mass spectra for compound 11 are given in Figure 6 and for compound 12, in Figure 7 . The identity of 1 2 was, in addition, confirmed by melting point [252 "C ( 1 5 ) ] . Although the neutral polar fraction of airborne particulate matter, which might be closely related in composition to the neutral polar fraction of carbon black, has been shown to possess considerable carcinogenic potency (16), concerted efforts have not been made to identify the polar compounds either in airborne particulates ( 1 7 ) or carbon black. Screening the neutral polar compounds identified in carbon black for carcinogenic activity might therefore be useful in discovering hitherto unsuspected environmental carcinogens.
ACKNOWLEDGMENT The author thanks 0. Grubner for helpful discussions and the Chemistry Department of Harvard University for the use of the mass spectrometer. LITERATURE CITED (1) H. L. Falk and P. E. Steiner, Cancer Res., 12, 30 (1952). (2) J. Neal and N. M.Trieff, Health Lab. Sci., 9, 32 (1972). (3) A. H. Oazi and C. A. Nau, in press. (4) C.A. Nau, J. Neal and V. A. Steinbridge, AMA Arch. Ind. Health, 17, 21 (1958). (5) C. A. Nau, J. Neal, and V. A. Steinbridge. AMA Arch. hd. Health, 18, 511 (1958). (6)C . A. Nau, J. Neal, and R. N. Cooley, Arch. fnviron. Health, 4, 415 (1961). (7) R. Monson and K. Nakano, Harvard School of Public Health, 665 Huntington Ave.. Boston, MA 021 15. unpublished results. (8)A. A. Rosen and F. M. Middleton, Anal. Chem.. 27, 790 (1955). ANALYTICALCHEMISTRY, VOL. 47, NO. 8, JULY 1975
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(9) R. Long, "Studies on Polycyclic Arom. Hydrocarbons in Flames", €PA R372 020, June 1972. (10) L. Wallca, Environ. Sci. Techno/., 3, 948 (1969). (11) N. P. Buu-Hoi, P. Jacquignon, J. P. Hoeffinger, and C. Jutz, Bull. SOC. Chem. Fr., 2514 (1972). (12) L. J. Bellamy, "The Infra-red Spectra of Complex Molecules", 2nd ed., John Wiley & Sons, New York, 1964, pp 127-129. (13) J. Michl, R . Zahradnik, and P. Hochmann. J. fhys. Chem., 70, 1732 (1966). (14) R. Zahradnik, M. Tichy, and D. H. Reid, Tetrahedron, 24, 3001 (1968). (15) D. H. Reid and W. J. Bonthrone, J. Chem. SOC.5920 (1965).
(16) W. C. Heuper, P. Kotin, E. C. Tabor, W. W. Payne, H. Falk, and E. Sawicki, Arch. fatho/., 74, 89 (1962). (17) L. L. Ciaccio, R . L. Rubino, and J. Flores, Environ. Sci. Techno/., 8, 935 (1974).
RECEIVEDfor review December 2,1974. Accepted March 3, 1975. This work was funded by the B. F. Goodrich Company-United Rubber, Cork, Linoleum and Plastic Workers Joint Occupational Health Program.
Determination of Anionic Surfactants with Azure A and Quaternary Ammonium Salt Lawrence K. Wang and Pedro J. Panzardi Department of Chemicaland EnvironmentalEngineering,Rensselaer Polytechnic Institute, Troy, N Y
The common surfactant portion of synthetic detergents is an anionic surfactant named linear alkylate sulfonate (LAS), shown in Figure 1. Analysis of the residual concentration of LAS in water and wastewater is of importance to environmental chemists and engineers. The Methylene Blue method ( I , 2) and the carbon adsorption method ( I ) are the recommended standard methods for the analysis of LAS or other anionic nonsoap surface-active agents. Both the standard methods involve the use of spectrophotometer, and are extemely time consuming. Recently, Wang ( 3 ) has proposed a modified Methylene Blue method for the determination of LAS as well as other anionic nonsoap surfactants with a spectrophotometer. Wang simplified the number of solvent extractions from seven to two (including the extraction for a blank) and reduced the possible interference caused by chloroform-extractable pollutants. Sufficient experimental data have been obtained ( 4 ) to conclude that the modified Methylene Blue method is more precise than the standard Methylene Blue method. For the environmental water quality control, there is a need to establish a standard titration method for rapid analysis of anionic surfactants in the field. The objective of this paper is to introduce the two-phase titration techniques which can be used for determining LAS and other anionic nonsoap surfactants. The titration techniques would be convenient to be used in the field due to the fact that there is no need for sophisticated instrumentation (not even a colorimeter or simple photometer). In the next section, a general survey of the two-phase titration is presented. In the Experimental section, the authors offer a new two-phase titration involving the use of Azure A and Methyl Orange as primary dye and secondary dye, respectively. Its advantages and limitations are also discussed.
BACKGROUND Two-phase titration using Methylene Blue was initially proposed by Weatherburn in 1950 (5). In his method, the sample containing anionic nonsoap surfactants is first treated with the Methylene Blue reagent and chloroform. After extraction, a blue-colored, chloroform-soluble dyesurfactant complex is formed in the chloroform phase. The treated sample is then titrated with a solution of alkyltrimethylammonium chloride with intermittent vigorous shaking until the end point is reached (i.e., the blue color is completely discharged from the chloroform layer). Weath1472
ANALYTICAL CHEMISTRY, VOL. 47, NO. 8 , JULY 1975
erburn's method has been tried by the authors. It is difficult to detect the end point because the blue color in the top aqueous layer will reflect to the bottom chloroform layer. In 1954, Edwards (6) offered a modified two-phase titration method for use on sewage, to eliminate the interference by proteins, hardness, and soap. Azophloxine dye was substituted for Methylene Blue, hexane for chloroform, and cetyltrimethylammonium bromide for alkyltrimethylammonium chloride. The titration end point is reached when color appears in the hexane layer. A drawback of Edward's method is the necessity of using a centrifuge to facilitate the phase separation. Turney suggested an alkaline Methylene Blue two-phase titration method in 1965 (7). He used Methylene Blue reagent as complexation agent, chloroform as organic solvent, and Hyamine 1622 (diisobutylphenoxyethoxyldimethylbenzylammonium chloride) solution as titrant. The sample containing anionic surfactant is at first treated with the Methylene Blue reagent, chloroform, and 15% sodium hydroxide, then titrated with Hyamine 1622, shaking after each addition. The organic solvent layer is initially blue in color, and changes gradually to red-violet to pink. The aqueous layer, however, becomes colorless at the end point. Hyamine 1622 was later adapted as cationic titrating solution by Reid (8, 9) for his new two-phase titration method using the mixed indicators (dimidium bromide and Disulphine Blue VN). Reid also selected chloroform as organic solvent. The anionic surfactant sample containing all necessary reagents is titrated with Hyamine 1622 solution until the pink is discharged from the organic solvent layer. The end point is reached before the color in the organic solvent layer turns to distinct blue. Wang et al. (10) recently developed an indirect twophase titration method for the identification of LAS in water. Their indirect method uses cetyldimethylbenzyl ammonium chloride (DCBAC) in excess amount to form a complex with the water-soluble anionic surfactant, and uses Methyl Orange (MO) as an indicator (reacts with excess CDBAC) in the presence of chloroform. The color of the CDBAC-MO complex in the chloroform phase is yellow. This water-chloroform two-phase mixture is then titrated with sodium tetraphenylboron (STPB) reagent with intermittent shaking to ensure equilibrium between the chloroform and the aqueous phases. The disappearance of the yellow color in the bottom chloroform layer indicates
