Glycerol can be considered a derivative of ethylene glycol where hydroxymethyl (ECL = 4.20 i2) has replaced a hydrogen (ECL N -0.30) in one of the methylenes. The experimental ECL was identical to the calculated, and this agreement allows the assumption that all hydrogens in ethylene glycol have the same incremental ECL and that each of the two carbon atoms has an incremental ECL of +0.7,,. The foregoing results provide the basis for rationalizing the observed ECL of two ethers that could be considered the cyclic and noncyclic dehydroxylated derivatives of di(ethy1ene glycol). Tetrahydrofuran, also named tetramethylene oxide, has an experimental ECL that corresponds to the sum of an internal oxygen, a tetramethylene unit, and a ring closure (or hydrogen termination). The higher volatility of its noncyclic equivalent, ethyl ether, is probably due to the difference in structure between the tetramethylene chain and the two short ethylenes. Furthermore, ethyl ether is exactly equal to a combination of two terminal methyl groups (as in diglyme) with an internal ethylene oxide unit (ECL = 2S0), if this unit is viewed as -CHz-O-CHzas found in the middle of the di(ethy1ene glycol) chain. This view is justified because the conversion of ethylene glycol to di(ethy1ene glycol) is equal, structurally, to the insertion of the symmetrical -CH2-O-CH2unit into the middle of the ethylene glycol
chain, although, mechanistically, 4H2-CH2--Ois inserted at the terminal position. These observations reveal the potentialities of the ECL method in alerting the experimenter to the differences that exist in electronic environments for a structural unit, such as for ethylene in ethylene glycol, ethylene glycol monoethyl ether, and ethyl ether. The calculated and experimentally determined ECL of ethylene glycol and its derivatives are listed in Table 111. Experimental values were constant over the range 230-250 “C. ACKNOWLEDGMENT
Dr. W. H. Tallent contributed helpful suggestions in the preparation of this manuscript.
RECEIVED for review August 19, 1968. Accepted November 15, 1968. Presented at the Great Lakes Regional Meeting of the American Chemical Society, Milwaukee, Wisconsin, June 13-14, 1968. The Northern Regional Research Laboratory is headquarters for the Northern Utilization Research and Development Division, Agricultural Research Service, U S . Department of Agriculture. The mention of firm names or trade products does not imply that they are endorsed or recommended by the Department of Agriculture over other firms or similar products not mentioned.
Identification of Aromatic Ketones in Cigarette Smoke Condensate J. H. Bell, Sue Ireland, and A. W. Spears Research Division, Lorillard Corp., Greensboro, N . C . Because of the complexity of cigarette smoke, extensive fractionation was necessary to isolate and identify minor components. The separation techniques involved solvent partition, column, paper, and gas chromatography. The gas chromatographic system allowed the collection of smoke constituents for subsequent analysis by ultraviolet and infrared spectroscopy and mass spectrometry. From the study of one subfraction which represents 0.08% of the original weight of the condensate, fluoren-9-one, the four methylfluoren-9-ones and seven other alkylated fluoren-9-ones were identified.
IN RECENT YEARS a number of papers have appeared dealing with the fractionation of cigarette smoke condensate into acidic, phenolic, basic, and neutral fractions. The ether soluble neutral fraction is a large and complex fraction and has received considerable attention. While the number of investigations dealing with the separation of the neutral fraction are too numerous to cite, bibliographies can be found in recent publications by Wynder and Hoffmann ( I ) and Stedman (2). The neutral fraction is usually separated by column chromatography into a series of subfractions of increasing polarity, and from this point the separation scheme is designed for the isolation and determination of either a single compound or a particular group of compounds. The intention of this work was to reproducibly fractionate the smoke condensate to the extent that even the minor smoke constituents -___
(1) E. L. Wynder and D. Hoffmann, “Tobacco and Tobacco
Smoke,” Academic Press, Inc., London and New York, 1967. (2) R. L. Stedman, Chern. Rev., 68,153 (1968). 310
ANALYTICAL CHEMISTRY
could be isolated in sufficient quantities to obtain reliable identification, and therefore more completely characterize the composition of cigarette smoke. EXPERIMENTAL
Smoking and Collecting of Condensate. Nonfilter cigarettes of 85 mm length were smoked on an automatic smoking machine with a capacity of 100 cigarettes, 20 of which were smoked simultaneously. The cigarettes were smoked to an approximate 20-mm butt with a puff frequency of 3 puffs/min and a puff duration of 2 sec. The smoke was drawn into a tubular shaped trap which was maintained at room temperature and then passed into a series of three 2-liter flasks submerged in a dry ice-methanol slurry. After the smoking of each 12,000 cigarettes, the condensate was collected from the traps. Because of the great number of cigarettes being smoked, a rather large volume of water was also condensed. By warming the collection vessels with hot water, the condensate could be poured from the flasks and collected without the use of organic solvents. The initial separation of the condensate followed the general procedure of extracting an ethereal solution of the condensate with HzSOa and NaOH solutions. The ether portion was dried over Na2SO4and the ether evaporated at a low temperature under reduced pressure. Approximately 90 grams of the neutral fraction thus obtained was chromatographed on a 135-cm X 6.5-cm column containing 1400 grams of florisil (60/100 mesh) which had previously been washed with methanol and reactivated at 165 “C for 3 hours. Fractionation of Condensate. As seen in the fractionation scheme (Figure l), the column was eluted with increasingly polar solvents. The NPfraction, which is similar to fraction
NA FRACTION (o.s%of whole tar)
NEUTRAL FRACTION
I Florisil column
I
1
7
HEXANE
V
1
HEXANE-BENZENE (8:l)
1
BENZENE-ETHER (4:l)
METHANOL
Bright blue fluorescent band collected and preclpltated
PARAFFINS
V N2 FRACTION I Partitioned between HEXANE
NITROMETHANE NA FRACTION
I CARBON DISULFIDE
I 1
'I
HEXANE NAlH Fraction
ACETONITRILE NAIA Fraction (0.08%whole tar)
I
+
Alumlna column
Florisll column
J V 1 HEXANE-BENZENE (4:l)
HEXANE NT FRACTION
HEXANE
NAiN Fractlon
1 1 1
v
HEXANE-BENZENE(4:l) NA2 METHANOL NA3
HEXANE NA1
NITROMETHANE
hexane & nitromethane
I
I
v I v v 1
METHANOL-H~O(B:l) NA1M Fractlon
_ I
V
Florisi1,coiumn
NE FRACTION
Figure 1. Fractionation scheme of the neutral fraction of cigarette smoke condensate
B reported by Wynder and Hoffmann (3), was eluted with hexane-benzene (8:l) but was actually defined by the observation of the fluorescence of the eluate when exposed to an ultraviolet lamp. Collection was begun at the elution of a bright blue fluorescent band and continued until the fluorescence became very pale blue and the eluate was colorless. Approximately 1 liter/lO grams of neutral fraction was necessary to elute the entire band. The solvent was flash evaporated at approximately 40 "C under reduced pressure, and the residue redissolved in 15 grams of acetone per gram of residue. The acetone solution was refrigerated for 16 hours at 0 "Cand the paraffinic precipitate removed by filtration. After removal of the acetone at reduced pressure, the residue was dissolved in 5 ml of hexane per gram of material and extracted 5 times with nitromethane (NA fraction). The amount of each nitromethane extraction was the hexane volume. The hexane portion was separated into NT and NE fractions by rechromatographing on florisil. The NT fraction was eluted from the column first and was collected until the eluted material showed the presence of carbonyl absorption when subjected to infrared analysis. At this point the remaining residue was eluted from the column as NE fraction. The NA fraction was again chromatographed on florisil (1 gram/80 grams florisil) and eluted initially with hexane. The elution (NA1) was continued until practically no material was found in the eluate. Approximately 1.7 literslgram of sample were required. Elution with hexane-benzene (4 :1) was continued until the concentration of the residue was less than 50 mg/500 ml solvent. About 1.4 liters/gram of sample were adequate for this collection. The remaining residue, NAI, was eluted from the column with methanol. Solvent Partitioning. Preliminary studies of the NA1 fraction allowed us to determine solvent partition coefficients for certain compounds which typify the major classes of compounds present in this fraction. These coefficients were used to establish a solvent extraction scheme with approximations as to what each part contained. The fraction was
180 Fractlons
Figure 2. Fractionation of the NA fraction of cigarette smoke condensate showing the solvent partition scheme partitioned between three different solvent pairs as shown in Figure 2. First, the condensate was dissolved in hexane and methanol-HzO (9 :1). After thorough shaking, the two phases were allowed to separate and the lower portion was transferred to a second separatory funnel. The two phases were partitioned again by shaking with the appropriate solvents. The like solvents were combined and extracted again. This procedure was repeated twice more, and thus each phase was enriched by the backwashing technique. The solvent was evaporated from the hexane portion, and the residue redissolved in equal volumes of carbon disulfide and nitromethane. The same partition procedure was followed. The solvent was removed from the carbon disulfide part, and the residue partitioned between acetonitrile and hexane. The total volume of each solvent used in the extraction scheme was 200 ml or less. The acetonitrile fraction (NAIA) represents approximately 17% of the NA fraction or 0.08% of the original weight of the condensate. Column Chromatography of Acetonitrile Fraction. The acetonitrile fraction was chromatographed on alumina (Merck, neutral) which had been washed with methanol and reactivated at 105 "C for 2 hours. The column dimensions were 68 cm X 5 cm and contained 100 grams of the low activity adsorbent per gram of residue. Elution of the column was initiated with pentane, and the polarity of the solvent was steadily increased by the addition of hexane, benzene, ether, and methanol. An automatic fraction collector, equipped with a 60-ml siphon, was used to arbitrarily collect fractions. On the basis of weight and apparent separation, the more than 800 fractions were recombined to give a final total of 180 fractions. A preliminary examination of some of the 180 fractions by gas chromatography revealed that the majority of the fractions were still too complex to obtain the degree of separation desired for detailed identification of the components. From reported RI values for many polycyclic aromatic hydrocarbons, and from our own experience, reverse phase paper chromatography appeared to be a fast and reproducible means to achieve additional separation. Paper Chromatography. Whatman No. 1 filter paper was acetylated according to the procedure of Spotswood (4). Each fraction requiring additional refinement was streaked
(3) E. L. Wynder and D. Hoffmann, Cancer (Philadelphia) 14, 1306 (1961).
(4) T.M. Spotswood, J . Chrornarogr., 2,90 (1959). VOL. 41, NO. 2, FEBRUARY 1969
311
0
WAVELENGTH (MICRONS)
Fluoren-Pone
8o
3 I
25
Kx-i
fq
I
4 I
3 /
6 I
7
I
8 9 1 0 1 2 I I I I
1520 I
I
40'
.,
II
ll.l.i-
~
-1
2 -Methylfluoren - P o n e
1-Methylfluoren-9-one
I
801
I
Dimethylfluoren-9-one
Bo,
401
I
I.
111
130 1 . :_.I I I I , .-._A .. , - I I , 1 9 , 150 , 160 , 170 , 180
~
,
190
IL-_--ll , 200
h
-
2!0
mie
Figure 3. Mass spectral plots of isolated fluoren-9-ones showing characteristic fragmentation patterns. Acceleration voltage scan, 3200 V to 200 V; magnet current, 5.5 A; volts/stage, 140; ionizing current, 10 PA; ionizing energy, 70 eV; ion source temp, 250 OC.
2041 3-Methylfluoren-9-one
-I
40
on the- acetylated sheets and ihromatographed using a solvent system of MeOH-Ether-H*O (4 :4:1). The developed chromatograms were examined under ultraviolet light, and the fluorescent bands used as guides for cutting the paper into various segments. By combining the like parts from all chromatograms, the components of the fraction were enriched and allowed more complete analysis. Various spray reagents were also employed in some instances to locate certain compounds. The reagents which have proved most successful were: Ehrlich's reagent (1 % p-dimethylaminobenzaldehyde in 1N HCl), methyl yellow and high intensity ultraviolet radiation, saturated benzene solution of chloranil, and a saturated benzene solution of tetracyanoethylene. Gas Chromatography. The gas chromatographic analyses were carried out on a Perkin-Elmer Model 880 gas chromatograph equipped with a dual column system, dual flame ionization detectors, and linear temperature programming. A modification of the splitters was necessary to prevent eluting compounds from condensing inside the splitter and changing the split ratio. Several substrates, including LiCl, OV-1, OV-17, SE-30, SE-52, and Apiezon L, have been used for the separation of these fractions. The thermal stability of all stationary phases, except OV-17, allowed oven temperatures up to 310 "C without excessive column bleed. All columns were made of 1/8-inchstainless steel and packed with coated Anakrom ABS (80/90 mesh). The gas chromatographic conditions, as well as the column system, were changed whenever the composition of the fractions varied enough to reduce maximum resolution. In most cases a 10-foot, 8 Apiezon L column gave satisfactory resolution. However, if a change in the operating parameters could not effectively separate certain constituents, the unseparated compounds were collected and rechromatographed on an alternate column. The compounds emerging from the gas chromatograph were collected by butting a glass capillary tube against a silicone rubber septum surrounding the exit port. For some samples a 4-mm i.d. looped glass tube with the loop submerged in a dry ice-acetone slurry was employed. In a few extreme cases the effluent was trapped in a specially constructed bubble bottle containing ether. Spectral Analysis. A collected sample was washed from the trap with carbon disulfide, and a KBr pellet prepared. 312
ANALYTICAL CHEMISTRY
i
4-Methylfluoren-9-one
o l ; ; ; : : ; : ; ; ; ; ; ; ; ; ; i ; ; ; : ; ; ; 1ooo 3500 3Mx) 2500 Moo lea, 1600 M o o 1200 loo0 800 FREW ENCY (CM '1
Figure 4. Infrared spectra of the methylfluoren-9-ones isolated from the acetonitrile fraction By carefully placing the sample in the center of the pellet and by using scale expansion, good spectra were obtained from samples smaller than 4 pg. The sample was recovered from the pellet by solvent extraction, and the ultraviolet spectrum obtained from the cyclohexane extract. The volume of the cyclohexane solution was reduced, and the sample was again subjected to the gas chromatograph for the purpose of eliminating any possible contaminates. The compound was collected in a capillary tube as described above, and without further manipulation, the mass spectral data were obtained by using the direct probe system. A Consolidated Electrodynamic Corp. mass spectrometer Model 104 was used for this work. The concentration of isolated compounds made it necessary to use the electronmultiplier detector. For the other operating parameters, refer to Figure 3. RESULTS
In our present study of the acetonitrile fraction (NAlA), we have identified by ultraviolet, infrared, and mass spectral analyses and by GLC retention values fluoren-9-one and a number of alkylated fluoren-9-ones (Table I). Fluoren-9-one was recently identified in cigarette smoke by P. Testa (5). The ultraviolet spectra of the four methylfluoren-9-ones were identical with the methylfluoren-9-onessynthesized by the method of Sprinzak (6) in this laboratory and also match the spectra reported in the literature (7, p i . The infrared spectra ( 5 ) P. Testa, Ann. Direc. Etudes Equipmei,i, SEITA (Seru. 'Exploit. Ind. Tabacs Allumettes) 4, 117 (1966). (6) Y . Sprinzak, J. Amer. Chem. Soc., 80, 5449 (1958). (7) R. A. Friedel and M. Orchin, "Ultraviolet Spectra of Aromatic Compounds," John Wiley and Sons, Inc., New York, N. Y . ,
1951. (8) J. J. Godfroid, BuU. SOC.Chirn. Fr., 11, 2929 (1964).
Table I. List of the Fluoren-9-ones Isolated and Identified in Cigarette Smoke Condensate Showing Ultraviolet Absorption Maxima and Retention Times
Fluorene-9-one 1-Methylfluoren-9-one 2-Methylfluoren-9-one 3-Methylfluoren-9-one 4-Methylfluoren-9-one Dimethylfluoren-9-one Dimethylfluoren-9-one Dimethylfluoren-9-one Dimethylfluoren-9-one Dimethylfluoren-9-one ethyl fluoren-9-one
Methylethylfluoren-9-one a Condition 1-10 feet x * Condition 11-13 feet x Condition 111-10 feet X
Ultraviolet absorption maxima (mp) 14 (in cyclohexane) 256.5 247.5 259.0 250.0 259.5 251 .O 261 .O 251.0 9.5 256.3 248.0 9.9 262.0 253.0 10.4 262.0 253.0 10.8 264.5 259.5 11.4 260.0 251 . O 11.7 262.5 253.0 257.5 251.5 10.7 261.0 252.0 inch stainless steel, 10% OV-17, 170-280 "C at 6 "/min. inch stainless steel, 16% Apiezon L, 2W310 "C at 6 "/min. inch stainless steel, 10% OV-17, 200-300 "C at 6 "/min.
were also identical to the literature spectra (9, IO) and those of the synthesized reference compounds. The infrared spectra of the methylfluoren-9-ones are shown in Figure 4. Figure 3 shows the fragmentation patterns of fluoren-9one, 2-methylfluoren-9-one and a dimethylfluoren-9-one, all isolated from the condensate. Peaks with relative intensities zare omitted, and only the portion of the specof less than 4 trum above m/e = 130 is represented. The graphs are typical of all reported fluoren-9-ones and clearly indicate the parent ions and the loss of m/e = 28 (c=o) or the loss of m/e = 29 (c=o plus a proton in the case of alkyl substitution). The prominent peak at m/e = 165 is apparently typical of all methyl and dimethylfluoren-9-ones. The mass spectrum of fluoren-9-one has been published (II), but no alkylated fluoren-9-ones have been reported. DISCUSSION Although the reported fractionation scheme satisfies the requirements for our investigation, we do not contend that each final subfraction is a complete entity. However, this uniqueness is not necessary for qualitative studies.
(9) J. J. Godfroid, Bull. Soc. Chim. Fr., 11, 2942 (1964). (10) "Coblentz Society Spectra," published by the Coblentz
Society, Inc., Norwalk, Conn., indexed, printed, and distributed by Sadtler Research Laboratories, Philadelphia, Pa. (11) J. H. Beynon and A. E. Williams, Appl. Spectrosc., 14, 156 (1960).
Retention time (min) II*
IIF 6.6
12.5 8.2 14.7 9.8 14.0 14.7 16.6
9.3
The microgram quantities of isolated compounds necessitated the collection of mass spectral data under nonideal instrumental conditions. However, the use of the direct probe system, the electronmultiplier mode, and a rather high volts/stage output produced spectra for isomeric compounds which could be partly interpreted without the spectra of reference compounds. Parent/parent-15 ratios, which have been elucidated in this laboratory for several alkylated aromatic systems (12), were used, in some instances, in conjunction with other spectral data to distinguish between dimethyl and ethyl substitution. To investigate the possibility that the fluoren-9-ones were oxidation products of fluorenes being produced in some step in our separation procedure, we subjected fluorene to repeated column chromatography and to gas chromatographic conditions more severe than those used for the smoke condensate and found no evidence that such a conversion took place. ACKNOWLEDGMENT
The authors express their indebtedness to Dr. Claude I. Lewis for his contributions to this project. We also thank Katherine Osborne, William Hobbs, and Donald Redmond for their fine technical assistance.
RECEIVED for review September 3,1968. Accepted November 6, 1968. (12) C. I. Lewis, Research Division, Lorillard Corp., personal communication, 1967.
VOL, 41, NO. 2, FEBRUARY 1969
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