atures were analyzed. The particle size of the samples var- ied from 0.4 to 2.1 pm as the heating temperature in- creased. However, the points of the inflection of the (1 - (1 - x)1/3)-t curves were clear and the BazTiO4 amounts

cussions. We also thank Kikuo Wakino, Director of Re- search of Murata Manufacturing Co., Ltd., for his encour- agement and permission to publish this paper.

could be determined. Figure 3 shows plots of kinetic data for the BazTiO4 formation. Although the amounts of

LITERATURE CITED (1) T. Kubo. M. Kato, and T. F u j i , J. Chem. Soc. Jpn., hd. Chem. Sect., 70, BazTi04 formed are less, the change of the BazTi04 forma- 647 11967).

tion as a function of temperature is the same as the results obtained by Kubo et al. (I) .

(2) R. E.'Hamikon, Can. Ceram. Soc. J., 37, LXll(1968). (3) H. U. Anderson, J. Am. Ceram. Soc., 56, 605 (1973).

RECEIVED for review January 2, 1975. Accepted April 9, 1975.

ACKNOWLEDGMENT The authors are grateful to Shigero Ikeda for helpful dis-

Carbon Black Adsorbates: Separation and Identification of a Carcinogen and Some Oxygenated Polyaromatics

Avram Gold

Harvard School of Public Health, 665 Huntington Avenue, Boston, MA 021 15

The industrial importance of carbon black and conse- quent occupational exposure have for some time sparked interest in characterizing its extractable organic constitu- ents (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 accent- uates the importance of analytical studies on this sub- stance.

EXPERIMENTAL The carbon black sample, 900 g of oil furnace black, was extract-

ed 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 at 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, Y4-h. X 15-ft. The chromatogram is shown in Figure 1. Fractions were collected in U- tubes containing a small amount of porous glass and cooled in a Dry Ice-ethanol bath. The collected compounds were desorbed by stirring in CHzC12.

The polar fraction of the Rosen separation was chromato- graphed 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 constit- uents of the polycyclic aromatic hydrocarbon (PAH) frac- tion.

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 struc- ture should be of general utility for polycyclic aromatic hy- drocarbons.

ANALYTICALCHEMISTRY, VOL. 47, NO. 8, JULY 1975 1469

Table I. Retention Times for Compounds Characterized in the Polycyclic Aromatic and Neutral Polar Fractions of Carbon Black Extract

Retention t ime,

Peak min

Polycyclic aromatic hydrocarbons (GC)

1. Naphthalene 1.75 2. 3 4 5 6 7

9 10 11

Acenaphthalene Phenanthrene Fluor anthene Pyrene Benzo[ghi]fluoranthene C yclopenta[ cdlpyr ene

Anhydride Phenalenone 4-H-cyclopenta[def]phenanthren-4-one

Neutral polar compounds (HPLC)

4.25 8.25 15.25 18.25 32 .OO 35.50

5 .O 17.6 29.4

12. 6- H-benzo[cd]pyren-6-one 52.8

Compounds 1-6 (Table I) of the polycyclic aromatic hy- drocarbon fraction were identified by UV, low resolution mass spectrometry, and peak enhancement. Although reso- lution of phenanthrene (Table I, compound 3; Figure 1, peak 3) and anthracene would not be achieved on the col- umn used in the gas chromatographic separation, the pres- ence of anthracene in peak 3 was ruled out by a comparison of the UV spectrum of compound 3 with that of an authen- tic 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 un- characterized carcinogen previously isolated from carbon blacks (2 ,3 ) that appeared to be identical by UV and melt- ing 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 assump- tion that the UV spectrum of the remaining aromatic sys- tem 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 recrystalli- zation 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 dihydro- cyclopentapyrene 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 tech- nique 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 possi- ble structures for the unknowns.

In analyzing the neutral polar fraction, it was necessary first, to work out a method of separation and, second, to es- tablish a method of identification for compounds for which no extensive or specific literature exists as it does for poly- cyclic aromatic hydrocarbons. High pressure liquid chro- matography was decided upon as the method of separation. Porous reverse phase packing was used in order to take ad-

0.E W 0 z U a m

0.4 a

0.2

SOLVENT: phexane

\ cyclopenta [cd] pyrene

I 1 1 I I 1 I I 225 250 275 3cO 325 350 375 400

X (nm)

z 60 9 40

4 20 I W +L LL

IC0 I25 I50 175 263 2 + 5 250

m /e

Figure 3. UV and mass spectrum of compound 7

0. E

y 0.E z a m

$ 0.4 3

0. i

2

ISOLVENT: n-hexane u n r

I 3,4 dihydrocyclopento [cd] pyrene

X (nm)

L 225 2 b m /e

Figure 4. UV and mass spectrum of compound 8

1470 ANALYTICAL CHEMISTRY, VOL. 47, NO. 8 , JULY 1975

I- r: 2 0 c I 102 125 150 175 X O 250

rn/e

Figure 5. Mass spectrum of compound 9

'5

225 2% 275 300 325 350 375 400 425 X(nrn)

3 20 W lx T - L L 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 de- termined 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 (VCO (CHC13) 1775, 1735 cm-l) indicate a six- or seven-membered cyclic anhydride

SOLVENT: n-hexane

0.6

0

benzo [cd] pyren-6-one

A 225 250 275 303 325 350 375 400 425 453

Id0 i25 I50 I75 2bl 225 2kl 275 m /e

Flgure 7. UV and mass spectrum of compound 12, uc0(CCI4), 1656 cm-'

(12). The fragment a t mle 200, presumably C16H8 (M-+ - C203), suggests an anhydride of pyrene or fluoranthene di- carboxylic acid, which for pyrene would imply 1 , l O substi- tution (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 suf- ficient to identify compound 10 as phenalenone. The mo- lecular formulas of ll and 12, determined by high reso- lution 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, con- firmed by melting point [252 "C (15 ) ] .

Although the neutral polar fraction of airborne particu- late 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

(5) C. A. Nau, J. Neal, and V. A. Steinbridge. AMA Arch. hd. Health, 18,

(6) C. A. Nau, J. Neal, and R. N. Cooley, Arch. fnviron. Health, 4, 415

(7) R. Monson and K. Nakano, Harvard School of Public Health, 665 Hun-

(8) A. A. Rosen and F. M. Middleton, Anal. Chem.. 27, 790 (1955).

(1958).

511 (1958).

(1961).

tington Ave.. Boston, MA 021 15. unpublished results.

ANALYTICALCHEMISTRY, VOL. 47, NO. 8, JULY 1975 1471

(9) R. Long, "Studies on Polycyclic Arom. Hydrocarbons in Flames", €PA

(10) L. Wallca, Environ. Sci. Techno/., 3, 948 (1969). (11) N. P. Buu-Hoi, P. Jacquignon, J. P. Hoeffinger, and C. Jutz, Bull. SOC.

(12) L. J. Bellamy, "The Infra-red Spectra of Complex Molecules", 2nd ed.,

(13) J. Michl, R . Zahradnik, and P. Hochmann. J. fhys. Chem., 70, 1732

(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).

R372 020, June 1972.

Chem. Fr., 2514 (1972).

John Wiley & Sons, New York, 1964, pp 127-129.

(1966).

(16) W. C. Heuper, P. Kotin, E. C. Tabor, W. W. Payne, H. Falk, and E. Saw-

(17) L. L. Ciaccio, R . L. Rubino, and J. Flores, Environ. Sci. Techno/., 8, 935 icki, Arch. fatho/., 74, 89 (1962).

(1974).

RECEIVED for review December 2,1974. Accepted March 3, 1975. This work was funded by the B. F. Goodrich Com- pany-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 Chemical and Environmental Engineering, 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 concen- tration 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 spectrophotome- ter, 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 re- duced the possible interference caused by chloroform-ex- tractable 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 tech- niques 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 sec- tion, a general survey of the two-phase titration is present- ed. In the Experimental section, the authors offer a new two-phase titration involving the use of Azure A and Meth- yl 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 dye- surfactant complex is formed in the chloroform phase. The treated sample is then titrated with a solution of alkyltri- methylammonium chloride with intermittent vigorous shaking until the end point is reached (i.e., the blue color is completely discharged from the chloroform layer). Weath-

erburn's method has been tried by the authors. It is diffi- cult 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 titra- tion method for use on sewage, to eliminate the interfer- ence by proteins, hardness, and soap. Azophloxine dye was substituted for Methylene Blue, hexane for chloroform, and cetyltrimethylammonium bromide for alkyltrimeth- ylammonium chloride. The titration end point is reached when color appears in the hexane layer. A drawback of Ed- ward's method is the necessity of using a centrifuge to facil- itate the phase separation.

Turney suggested an alkaline Methylene Blue two-phase titration method in 1965 (7). He used Methylene Blue re- agent as complexation agent, chloroform as organic solvent, and Hyamine 1622 (diisobutylphenoxyethoxyldimethylben- zylammonium chloride) solution as titrant. The sample containing anionic surfactant is at first treated with the Methylene Blue reagent, chloroform, and 15% sodium hy- droxide, 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 so- lution by Reid (8, 9) for his new two-phase titration meth- od using the mixed indicators (dimidium bromide and Di- sulphine Blue VN). Reid also selected chloroform as organ- ic 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 sol- vent layer turns to distinct blue.

Wang et al. (10) recently developed an indirect two- phase titration method for the identification of LAS in water. Their indirect method uses cetyldimethylbenzyl am- monium 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 ex- cess CDBAC) in the presence of chloroform. The color of the CDBAC-MO complex in the chloroform phase is yel- low. This water-chloroform two-phase mixture is then ti- trated 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

1472 ANALYTICAL CHEMISTRY, VOL. 47, NO. 8 , JULY 1975