milk contaminants-\veri: greater than lo3. Three other nuclides, l3aIM, and La1@,are possible short-lived milk contaminants immediately following nuclear teats. The D.F ' s for I I 3 l and La140 are high because of the combined discriminations in the katch resin exchange preparation of mil complex fract o m but often fail\ to give pure individual cornponent-. For example, bcr d(a)anthraceiic and chrysene are not ne11 resolved nor are benzo(a)pyrene, bens?(e)pyrene, and benzo(k)-fluoranthene. Further separations can sometimes be achieved by partition chromatography on payer or by gas-liquid chromatography. The mAHAiwN
latter methods are usually limited to the use of rather small quantities. The use of solvent extraction for the purification of polycyclic aromatic compounds has been reported by several workers. Steidle (14) utilized nitromethane to extract this type of compound from pentane. More recently, nitromethane has been used for the extraction of polycyclic aromatic hydrocarbons from cyclohexane solutions of cigarette smoke condensates (9, 11). Other solvents evaluated by Haenni, Howard, and Joe (10) are acetonitrile, dimethylformamide, and methylsulfoxide, which were applied t o the removal of polycyclic aromatic compounds from heptane solutions of paraffin wax. These solvents are useful for the separation of unsubstituted polycyclic aromatic hydrocarbons as a class but do not selcctivcly separate individual comJ)ounds. Golumbic (8) qtudied several solvent pairs to obtain a system suitable for the separation of individual polycyclic aromatic compounds by countercurrent distribution. These solvent systems included cyclohexane: 80% ethanol, cyclohexane: 98% acetic acid, benzene:
80% acetic acid, iso-octane: Si% ethanol and n-heptane: aniline. H e concluded that the first of these n a s the most selective for iiomeric methylnaphthalenes. Relatively little separation was observed for unsubstituted polycyclic hydrocarbons. In our approach to this problem, we felt that the use of a complexing agent selective for these compounds would be desirable. The solubilization of polycyclic aromatic hydrocarbons in water by purines had been demonstrated by Brock, Druckrey, and Hamper1 (4). Certain hydrocarbons of this elas$, including benzo(a)pyrene, were solubilized t o a greater extent than were others. Weil-Malherbe carried out extensive studies with several purines (16). H e found that tetramethyluric acid was the most effective of the several purines which he tested for dubilization of I)cnzo(a)pyrene. Wanlcss ( I 6 ) utilized aqueouq caffeine solutions to prepare a polycyclic aromatic concentrate from high boiling petroleum products. Solubilities of several carcinogenic aromatic amines were shown by Seish ( I S ) to be greatly increased by the addition of caffeine or tetramethyluric acid. SirniVOL. 35, NO. 13, DECEMBER 1963
a
2071
lar findings for the carcinogenic dibenzocarbazoles and dibenzacridines were reA C D E ported by Booth and Boyland (IAl). though the purine, tetramethyluric acid, markedly increased the solubility of polycyclic aromatic compounds in water, the amounts of these materials dissolved were still too small to be of practical use in a countercurrent extraction operation. We therefore utilized an aqueous 90% methanol solution containing tetramethyluric acid for the lower phase to give increased solubility for the polycyclic compounds. Cyclohexane was used for the upper phase. By the use of this solvent pair it has been pos01 I I I I I I I I I I sible by countercurrent distribution to 110 120 130 140 150 160 170 180 190 200 achieve separations of several comTUBE NUMBER pounds which are difficult to separate by other means. Figure 1. Countercurrent distribution of several polycyclic aromatic compounds between cyclohexane and 0.83% tetramethyluric acid in methanol:water (9:1 EXPERIMENTAL
PREPARATION OF TETRAMETHYLURIC ACID. 8-Chlorocaffeine was prepared in 97% Materials and Apparatus.
yield from caffeine by treatment with chlorine in chloroform solution according t o the directions of Fischer and Reese (6). The chlorocaffeine (map. 191" C.) mas converted to 8-methoxycaffeine in 86% yield by refluxing in methanol with sodium methoxide ( 5 ) . Rearrangement to tetramethyluric acid was accomplished by heating 8-methoxycaffeine (m.p. 177-8" C.) for 1 hour at 200-30" C. ( I ) . The product crystallized from boiling water upon cooling (m.p. 228.5-9" C.). A yield of 60% was obtained for the synthesis from caffeine. Tetramethyluric acid can be obtained in two crystalline forms, stout heavy blocks or long, fine needles. The former is the more stable and can be obtained upon heating the needles (12). Calculated for C9HI2N4O3: C, 48.21; H, 5.36. Found: C, 48.15; H, 5.17. Ultraviolet absorption maxima a t 240 and 294 mfi with log Emol3.80 and 4.04 (H2O) were consistent with the spectrum reported by Fromherz and Hartmann (7). COMPOUNDS USED. Samp!es of dibenzofuran, acridine, fluoren-9-one, dibenzothiophene, caffeine, and indole were obtained from Distillation Products Industries, (Rochester, N. Y.). Benz (c)acridine, benz(a)acridjne, benzo(k)fluoranthene, and benzo(e)pyrene were obtained from Aldrich Chemical Co., Inc. (ACilwaukee, 'A7is.). Anthanthrene, I 1 H-benzo(a)fluorene and dibenzo(n,Z)pyrene were obtained from L. Light and Co., Ltd. (Colnbrook, Bucks., England). Chrysene, benz(a)anthracene, benzo (a)pyrenc, and pyrene were obtained from Rutgerswerke A.G. (Frankfurt, West Germany). Carbazole was obtained from Matheson, Coleman 6. Bell (East Rutherford, N. J.). Perylene and benzo(g,h,i)perylene were obtained from Bios Laboratories, Inc. ( K e w York, II'. Y . ) . IIIZBenzo(b)fluorene was obtained from Edcan Laboratories (South Norwalk, Conn.). Fluorene, phenanthrene, dibenz(a,h)anthracene, anthracene, and fluoranthene were obtained from com2072
ANALYTICAL CHEMISTRY
v./v., A. Benzo(g,h,i)perylene B. Benzo(e)pyrene C. Benzo(a)pyrene D. Chrysene E. Benz(a)anfhracene
mercial sources and were further purified by repeated column chromatography on alumina. SOLVENTSYSTEM FOR COUNTERCURRENT DISTRIBUTIONS.Nine grams of tetramethyluric acid was dissolved in one liter of 9 : l (v./v.) methano1:water. This solution was equilibrated with 1 liter of cyclohexane to give 1080 ml. of lower phase and 920 nil. of upper phase. Only a negligibly small portion of the tetramethyluric acid was present in the upper phase. It was calculated that the lower phase contained 0.83% (w./v.). Solvent pairs containing other conrentrations of tetramethyluric acid were prepared similarly. COUNTERCURRENT DISTRIBUTION APPARATUS. .4 100-tube glass Craig countercurrent distribution apparatus (H. 0. Post Scientitic Instrument Co., Maspeth, N. Y.) mas used for the separation. Each tube contained 10 ml. of each solvent phase. Procedure for Countercurrent Distribution. Three hundred transfers were performed in the 100-tube Craig countercurrent distribution apparatus by recycling after the 99th transfer. Ten milliliters of each phase of the solvent pair was used in each tube. A mixture of 4.9 mg. of chrysene, benz(a)anthracene, benzo(a)pyrene, benzo(e)pyrene, and benzo(g,h,i)per3rleiie mas introduced into tube 0 9s the sample to be separated. After the final Iransfer, 5.0-ml. aliquots of upper phase were nithdrawn from every third tube for analysis. Each withdrawn aliquot was washed with 5.0 ml. of water and the ultra\ iolct absorption spectrum rvzs determined. The concentrations of the materials present were calculated from the absorLance a t an appropriate maximum. The measured amounts of these materials are plotted as a function of the distribution tube number in Figure 1.
Procedure for Single-Tube Distributions. An amount of the polycyclic compound sufficient to permit accurate absorbance measurements was dissolved in the upper phase of the solvent systems prepared as described above. An aliquot of the upper-phase solution was equilibrated with a measured volume of lower phase by vigorously shaking for about 1 minute. The two phases mere allowed to separate and the upper phase was centrifuged to clarify it. Aliquots of the unextracted and extracted upperphase solutions were then removed, washed once with water, and centrifuged to provide initial and final solutions for ultraviolet absorption measurement. A Perkin-Elmer Model 350 spectrophotometer was utilized for these measurements. Absorption intensities of the two solutions were estimated a t an appropriate maximum using base line corrections. The distribution coefficients were calculated using the equation where A , = absorbance of upper phase after equilibration At = absorbance of upper phase before equilibration Vl = volume of lower phase VZL = volume of upper phase Values obtained for a number of polycyclic aromatic compounds in several solvent pairs are presented in Tables I and 11 RESULTS AND DISCUSSION
An advantage of the countercurrent distribution method for separation of mixtures is that quantitative estimates of components can be made on the basis
of the binomial distribution if determinations of the quantities in two tubes can be made accurately. This permits the estimation of materials which have not been completely resolved. The degree of resolution required will depend on the reliability of the assay method for each component in the presence of the contaminant. The solvent system described offers a method for the quantitative estimation of a number of individual polycyclic compounds in a mixture without resorting to an excessive number of transfers. fi combination of some other fractionatio procedure with the countercurrent extraction will separate most of those paiss which are not well resolved by the countercurrent procedure alone. If it is desired to isolate purified components, additional transfers can be utilized to give the required separation. The distribution coefficients for pyrene and benzo(a)pyrt:ne were essentially constant over a wide range in concentrations (Table 11). This was confirmed by the close rtgreement of the measured amounts t o the theoretical distributions (Figure 1). Single-tube distributions indicated that each polycyclic compound tested was more soluble in aqueous methanol containing tetramethyluric acid (TMU) than in the same solmnt without this complexing agent. The extent of this increased solubility is given by the ratio of the distribution coefiicients in these two systems, K B I K A ,where K A is the distribution coefficient in the solvent system containing TMU and K B is that for the same system without TMU. These values are recorded in Table I. A higher value for this ratio indicates greater solubilization b j the purine. In agreement with the findings of previou9 workers (3), tetramethyluric acid was found to increitse the solubility of polycyclic hydrocarhons without regard to their carcinogenic properties. Although only relatively few compounds were examined in the present study, for those polycyclic aromatic hydrocarbons tested compounds with more fused rings were solubilized by the purine to a greater extent. For compounds with the same number of fused rings the TMU-solubilization m . i greater for the compound with more compact structure. The presence of a partially nonaromatic structure such as is fouiid in fluorene or the benzofluorenes con 'erred decreased solubilization as compared to the fully aromatic structures such as anthracene or benzanthracene. For the limited number of heterocyclic compounds tested, thwe Kith more fused rings were also solubilized by tetramethyluric acid to :t greater extent. The quantitative effects as measured by the ratio K B / K A werc! in the same ranges as were found for the hydrocarbons. Carbazole, with a five-mem-
Table
1.
Distribution Coefficients for Several Polycyclic Aromatic Compounds
Distribution coefficient cyclohexane/ KA Compound Hydrocarbons Anthanthrene Dibenzo(a,Z)pyrene Beneo(g,h,i)perylene Perylene Benzo(e)pyrene Benzo(a)pyrene Benzo(k)fluoranthene Dibenz(a,h)anthracene Pyrene Fluoranthene Chrysene Benz(a)anthracene 11H-Benzo(a)fluorene 11H-Benzo(b)fluorene Phenanthrene Anthracene Fluorene
Number of fused
0.83%TMU 1D. 90% methanol
6 6 6 5 5 5 5 5 4 4
1.22 0.99 1.05 1.36
Heterocycles Benz(c)acridine Benz(a)acridine 9H-Carbazole Dibenzothiophene Dibenzofuran Acridine Fluoren-9-one Indole
ringa
cyclohexane/ KB 90% methanol
KBIKA
1.76 2.44 2.38 2.24 1.96 2.18 2.44 3.42 3.42 2.33 2.82 3.86
5.85 4.75 4.72 4.51 4.42 4.63 5.50 4.70 4.60 3.90 4.11 4.51 5.65 5.55 4.10 4.50 4.30
4.SO 4.80 4.50 3.32 3.25 2.63 2.25 1.98 2.05 1.99
1.46 0.31 0.093 3.16 3.06 0.28 0.61 0.075
2.35 0.48 0.138 4.34 3.88 0.33 0.70 0.090
1.61 1.55 1.43 1.37 1.27 1.18 1.15 1.20
1.36
4
4 4 4
3 3 3 4 4
2
1.88
1.85 1.65 1.62 1.76 1.60
1.11
Table II. Distribution Coefficients for Polycyclic Aromatic Compounds between Cyclohexane and 9:l Methanol : H20 Containing Varying Amounts of Tetramethyluric Acid
Polycyclic compound Benzo(a)pyrene Benao(a)pyrene Benzo(a)pyrene Benzo(a)pyrene Benzo(a)pyrene Pyrene Pyrene Pyrene Pyrene Pyrene
Concentration of polycyclic compound in cyclohexane before extraction, lCcg./ml.
0.0
1.54 14.7 146 2.9 5 .O 1.13 25.8 191 38.8 2.5
4.63
Distribution coefficients %TMU in lower phase 0.46 0.83 1.76 1.78 1.81 2.52
1.22
2.19 2.29 2.29
4.55
ber heterocyclic ring, was solubilized to a greater extent than acridine, with a six-member heterocyclic ring. For the heterocyclics containing the hetero atom in a five-member ring, the order of increased solubilization by the purine was carbazole > dibenzothiophene > dibenzofuran. A marked effect on the distribution coefficients was noted for different concentrations of tetramethyluric acid in the lower phase. This effect was noted by Weil-Malherbe (16) and later confirmed by Boyland and Green (3). These workers measured the solubility of polycyclic aromatic compounds in aqueous solutions containing W e r e n t amounts of purines. They concluded
1.20
2.98
1.75
that the solubilities obeyed the relationship
k = - CP' CEC
where
CP = concentration of purine CEK = concentration of the f
test compound = a constant with values between l and 2, indicative of a reaction involving one to two molecules of purine per complex
For the present study the concentration of the test compound solubilized in VOL 35, NO. 13, DECEMBER 1963
2073
the aqueous methanol by the purine is given by the expression
where ACHCand BCHC are the concentrations of the hydrocarbon in the upper phase for the system containing ThIU or for the system without TMU, respectively. JF7hen the equilibrium concentrations of hydrocarbon in the upper phases of the two systems are the same, Equation 3 may be simplified as shown in Equation 4. This simplification is generally valid throughout the range of concentrations of hydrocarbon studied, since distribution coefficients were essentially constant throughout this range.
The simplified Expression 4 may then be substituted in Equation 2 to give
1 1 (z c)
The slope of the plot of log vs. log CPis a n estimate off. For the two compounds studied, pyrene and benzo(a)pyrene, the values of this slope calculated for consecutive pairs of points were 1.11, 1.18 and 1.12, 1.47. WeilhIalherbe found values for the exponent, f, either near 1 or near 2 suggesting reactions involving either one molecule or two molecules of purine per complex. ACKNOWLEDGMENT
The synthesis of tetramethyluric acid used in these studies was performed by Marvin Crutchfield and T. P. Chen. The authors acknowledge the information provided by Charles Keith regarding his preliminary attempts to use purine complexation for the countercurrent separation of closely-related polycyclic aromatic hydrocarbons. LITERATURE CITED
(1) Biltx, H., Strufe, IC, Ann. 413, 197 (1916).
(2) Booth, J., Boyland, E., Riochzni. Riophys. Acta 12, 75 (1953). (3) Boyland, E., Green, B., Brzt. J . Cuncer 16, 347 (1962). (4) Brock, N., Druckrey, H., Haniperl, H., Arch. Ezptl. Puthol. Phamakol. 189, 709 (1938). (5) Fischer, E., Ach, L., Ber. 28, 2480 (1896). (6) Fischer, E., Reese, L., Ann. 221, 336 (1883). ( 7 ) Fromherz, H., Hartmann, A., Ber. 69B, 2420 (1936). (8) Golumbic, C., ANAL. CHEM.22, 579 (1950). (9) Grimmer, G., Beitr. Tabakforsch. 1, (No. 7), 291 (1962). (10) Haenni, E. O., Howard, J. W., Joe, F. L., Jr., J . dssoc. Ogic. Agr. Cheinzsts 45. 67 (19621. ( 11) 'Hoffman; L, D., Wgnder, E. L., ANAL. CHEM.312,295 (1960) (12) John son, T. B., J . .4m. C h e m SOC. 59, 1261 (1937). (13) Neisl1, W. J. P., Rec. Trav. ChznL. 67,361 (1948). 114) Steid le. W.. dissertation. Tubineen UniversitG. German\-. 1953. ' (15) Wanless, G. G., Eby, L. T., Rehner, J., Jr., ANAL.CHEhf. 23, 563 (1951). (16) Weil-Malherbe, H. , Bzochenr. J . 40,351 (1946). RECEIVEDfor review June 19, 1'363. Accepted August 7 , 1963. Y
A General Method for the Chromatographic Separation of Nonionic Surface-Active Agents and Related Materials MILTON J. ROSEN Department of Chemistry, Brooklyn College of the City University o f New York, Brooklyn, N. Y.
b A general chromatographic method has been developed for separating nonionic surface-active agents from each other and from related nonionic materials, using silica gel as adsorbent. The mixture, adsorbed on a silica gel column, is separated by eluting successively with chloroform, 1 :99 (v./v.) ethyl ether-chloroform, 1 : 1 (v./v.) 1 : 1 (v./v.) ethyl ether-chloroform, 1 : 19 (v./v.) acetone-chloroform, methanol-chloroform, 1 :9 (v./v.) methanol-chloroform, and 1 :2 (v./v.) methanol-chloroform. Results obtained with a number of three- and four-component mixtures are described.
C
nonionic surface-active agents commonly contain more than one nonionic substance. These compositions often contain both water-soluble and oil-soluble surfactants to obtain a better balance of properties, and in addition, usually contain other nonionic, nonsurfaceactive materials, such as hydrocarbon OMPOSITIONS CONTAINING
2074
ANALYTICAL CHEMISTRY
oils, fatty esters, glycols, and unreacted raw materials from surfactant manufacture. The removal of ionic surfaceactive agents present in the composition is conveniently accomplished by ion exchange ( I , 7, 8, IO, l a ) , but all the nonionic surfactants, together with any nonionic nonsurfactant material, pass unadsorbed through the ion exchanger. The analysis of this mixture of nonionic surfactants and related materials is usually a difficult task and, in spite of its common occurrence, only a few attempts have been made to systematize the analysis of this type of mixture. A liquid-liquid extraction procedure has been suggested (9) for separating hexane-soluble nonionics from hesaneiiisolubles and chromatographic techniques have been suggested for separating mixtures of mono-, di;, and triglycerides (4-6) and of fatty acid esters of ethylene glycol and polyoxyethylene glycols ( 3 ) . This paper describes a general columnar chromatographic method which has been developed to separate
the components of mixtures containing various types of nonionic surface-active agents and a number of nonionic nonsurfactant materials commonly encountered with them. A column, rather than a thin-layer or paper, chromatographic technique was used because it permitted convenient qualitative analysis of the eluted fractions by refractive index or melting point, and quantitative measurement by weighing on an analytical balance. EXPERIMENTAL
Column. A 50-ml. buret (60-cm. X 1-cm. i.d.) with ungreased stopcock. Procedure. A few milliliters of chloroform were placed in the column, followed by a small plug of glass wool which was tamped in place below the surface of the chloroform with a glass rod, t o remove air bubbles. Ten grams of the silica gel (Davison KO. 922, through 200-mesh) were slurried with 20 t u 30 ml. of chloroform (U.S.P., dried for a few days over anhydrous CaC12) and poured into the column.
