c‘dllU/~~tiC*~t
Chil~IkCJ
/iCttl.
77 ( 1975)
Publishing
309-S
A.‘!Elscvicr
Scientific
SHORT
COMMUNICATlON
309
11
Company.
Amsterdam
D. B. WALTERS.
W. J. CHAMBERLAIN
(Received
5th Dcccmbcr
Rcsetrrc~lr
in The Ncthcrhnds
smoke condensate
Cis and rrwns fatty acids in cigarcttc
Riclrctrtl U. R~r.well AgrvMrwal of A fpicirltrtr~. Arli~~rrs. Grorqict
- Printed
;lnd 0. T. CHORTYK Corrcr.
Agrl~rtlrwwl
Rescw~~cl~ Stwicc,
U~rircrl
Srures
Dcqwrrrwrrr
30604 ( U.S. A.)
1974)
A rapid method for determining the cis-ZIWIS composition of unsaturated fatty acids of cigarette smoke with gas chromatography and the new stationary phase. Silar-1OC (ref. 1) is described. Such lipid analyses have been performed by sophisticated techniques2 or by gas chromatographic methods necessitating timcconsuming formation of derivatives3. Various fatty acids have been found in cigarette smoke condensate ranging from the saturated C2 to Cz6 compounds including odd and even numbered carbon studiesLl*s have indicated the isomers j-.‘. Previous lengths as well as branched-chain presence of several unsaturated fatty acids. most abundantly palmitoleic (Ci;). oleic (C:;). linoleic (C:;). and linolenic (Cfi). all of which have been reported as the cis isomer. No work has been done on the tr*ans isomers present in smoke condensate. The cis-trms composition of unsaturated fatty acid ester mixtures is of interest for several reasons: (a) nutritional studies indicate that the t~rtr~s isomer may not be nutritionally equivalent to the naturally occurring cis isomer”; (b) animal studies indicate that blood-clotting time is altered when W~IIISisomers are substituted for cis isomers’ I: (c) subtle molecular structural changes often account for major flavor changes’ 2; and (d) the difference between the cls and rr’ctns acids could be a result of chemical changes which may produce important biological differences.
A dual-column Varian Model 2800 gas chromatograph equipped with flame ionization detectors wns used. Flow rates of 10. 30. and 300 ml min- l were used for helium. hydrogen, and air, respectively. Stainless steel columns. l/8 in. x 10 ft. packed with loo/, Silar-1 OC on loo-120 mesh Gas-Chrom Q. were used isothermally at 170 “C. with injector and detector temperatures of 250 “C. A Varian Model 25A recorder was used at a chart speed of 0.25 in. min - *. Peak areas were determined with a Varian Model 485 integrator. Percent compositions were normalized and reported relative to percent total volatiles. Individual peaks were identified by comparative retention times and co-chromatography with knowns. Retention time was measured relative to methyl palmitate.
310
SHORT
COMMUNICATION
:* /; / .“,!
10
TIME.
I,WUTES
co
0 60
-..-..60
100
120 TIME.
,J ,!
> ,
140 MINUTES
r
,‘. ,
5
160
Fig. 1. Chromatogram of cstcrificd cigarctlc smoke condcnsntc fraction F-59. (a) 1 methyl palmitotc: 2 methyl palmitokxtc: 3 methyl palmitclaidatc: 4 methyl olcntc: 5 methyl linolcutc: 6 methyl linoIclaidutc. (b) Continuation of Chromutogram ut diffcrcnt uttcnuation.
Methyl ester standards (Applied Science Laboratories, Inc.) were certified 99 +‘;/, pure. Cigarette smoke condensate (CSC). obtained from commercial cigarettes, F-8. in 8 manner previously was fractionated to yield a weak-acid fraction. described13. F-8 was further fractionated by partitioning it between equal volumes of petroleum ether and 907; methanol-water. The petroleum ether solution was then extracted with sodium hydrogen carbonate. The aqueous layer was adjusted to pH 1.0 with G M hydrochloric acid and extracted with diethyl ether to yield F-59. which represented about O.S”/, of CSC. A portion of F-59 was esterified with Methyl 8 (dimethylformamidc dialkylacetal. Pierce Chemical Co.); 1 ml of Methyl 8 was used per SO mg of F-59. as described elsewhere l 4. Results
anti discussion
Figure 1 shows the resulting chromatograms of esterified fraction F-59. The presence of the following unsaturated methyl esters was established by retention time and co-chromatography with knowns: palmitoleate, palmitelaidate. oleate, linoleate and linolelaidate. This study was primarily concerned with the detection and differentiation of the C:;. CiE, and C:g cis and n’n~s isomers. Also. for the the only standard available for comparison was the cis isomer Ci’B’ compounds. (cis. cis. cis-9.12.1 S-octadecatrienate). This compound was detected only in trace amounts in smoke condensate. Figure l(a) shows baseline separation between methyl linoleate and methyl linolelaidate. Separation was poorer. but adequate for electronic integration. between methyl palmitoleate and palmitelaidate. Figure l(b) shows the remaining portion of the chromatogram, at a different attenuation. beyond the area of interest. Table I gives the experimental percent composition and the cis and UWIS isomers expressed as relative percent of total volatiles from fraction F-59. Relative retention time (R) was measured from the methyl palmitate peak. Similar amounts of palmitoleatc ( J.O7’j/,) and palmitelaidate ( 1.1 8”/0) were detected. However, a much smaller amount of linolelaidate (0.1 S’Y”)compared to linoleate (13.77%) was found, Although 11.14% methyl oleate was detected, the level of methyl elaidate was either too low to be detected or its presence was masked by the oleate.
SHORT
COMMUNICATION
TABLE
I
PERCENT COMPOSITION UNSATURATED FATTY
311
AND ACIDS
RELATIVE RETENTION FOUND IN F-59
Pulmitoleutc. C!; (ci.+9_hcxadcccnoittc) Palmitcluidntc. Cl; (frcr,r.~-Y-hexodcccnoate) Olcutc. ci ; C’iS (cl.+9-octndcccnoiI&) Linoleahz. Cf; cis ( C~S. ~Qis-9.12-oct~tdce;tdicnoi~tc) Linolclaidatc. Cf; rrcllts (rrws. rrum-9. I2-oct;ldcc;tdicnoutc)
TIMES
(R) OF
SOME
I .07
I.21
I.18
1.33
II.14
2.1 I
0.15
2.54
13.77
2.78
ESTERIFIED
I’ Vulucs arc un uvcrupc of duplicetc dctcrminutions. h R-rclntivc to methyl palmitatc (pcok 1 in Fig. I).
Owing to yield variability in the fractionation-extraction procedures. no quantitation of acid contents in the condensate was attempted. These findings illustrate the significance of the new g.c. support. Silar-IOC. to the tobacco chemist and its use to other analytical organic chemists engaged in lipid research. Further work is in progress with this new support for identification of higher molecular weight fatty acids in cigarette smoke condensate.
Area
The authors thank Mr. R. L. Atkins, Technical School for their cooperation.
Mr.
Lamar
Hendrix.
and
the
Athens
REFERENCES I 2 3 4 5 6 7 8 9 10 11 I2
Gas-Cltrunt Newsleftcr. 14 ( 1974) 1. Applied Scicncc Laborutorics, Inc., State Collcgc. Pu.. U.S.A. D. B. Wriltcrs und R. J. Horva. AI&. Clrim Acrtr. 65 ( 1973) 198. E. A. Emkcn, Llpltls. 6 (1971) 686. D. Hoffmann and H. Waziwodzki. Bdrr. Tdwkjiwwlr.. 4 ( 1968) 167. I. Mokhnuchcv. L. G. Scrdyuk and M. Ivunov. C. R. B~tlgtrw. Sci.. 20 (1967) 445. B. L. van Duurcn und A. 1. Kasuk. J. Org. CAPON.. 23 (1958) 473. M. Dymicky and R. L. Stcdman. Tohtrcco SC/.. 12 (1969) 45. R. L. Stcdman. Cl~etn. Rer.. 68 ( 1969) 153. A. P. Swain and R. L. Stcdmun. J. Ass. O_/jlc. rlgr. Clrcrl~.. 4.5 (1961) 536. P. G. Rund and F. W. Quuckcnbush. J. N~crr.. 87 (1965) 489. G. Ruccugliu and 0. S. Privctt, Lipltls. 5 ( 1970) 85. R. Tcrunishi. P. Issenbcrg. J. Hornstein und E. I. Wick. Flaaor Rewcmlr. Principles wul Tdruiqws. M. Dckkcr. New York. 1971. p. 282. I3 A. P. Swain. F. G. Bock, J. E. Cooper. W. J. Chumbcrluin. E. D. Strilngc. L. Lakritz und R. L. Stcdmun, Belrr. Ttrbrrkfor~sch.. 7 ( 1973) I. I4 J. P. Thcnot. E. C. Horning. M. Stufford und M. G. Horning. ,4r1tr/. Letr.. 5 (1972) 217.
Chil~IkCJ
/iCttl.
77 ( 1975)
Publishing
309-S
A.‘!Elscvicr
Scientific
SHORT
COMMUNICATlON
309
11
Company.
Amsterdam
D. B. WALTERS.
W. J. CHAMBERLAIN
(Received
5th Dcccmbcr
Rcsetrrc~lr
in The Ncthcrhnds
smoke condensate
Cis and rrwns fatty acids in cigarcttc
Riclrctrtl U. R~r.well AgrvMrwal of A fpicirltrtr~. Arli~~rrs. Grorqict
- Printed
;lnd 0. T. CHORTYK Corrcr.
Agrl~rtlrwwl
Rescw~~cl~ Stwicc,
U~rircrl
Srures
Dcqwrrrwrrr
30604 ( U.S. A.)
1974)
A rapid method for determining the cis-ZIWIS composition of unsaturated fatty acids of cigarette smoke with gas chromatography and the new stationary phase. Silar-1OC (ref. 1) is described. Such lipid analyses have been performed by sophisticated techniques2 or by gas chromatographic methods necessitating timcconsuming formation of derivatives3. Various fatty acids have been found in cigarette smoke condensate ranging from the saturated C2 to Cz6 compounds including odd and even numbered carbon studiesLl*s have indicated the isomers j-.‘. Previous lengths as well as branched-chain presence of several unsaturated fatty acids. most abundantly palmitoleic (Ci;). oleic (C:;). linoleic (C:;). and linolenic (Cfi). all of which have been reported as the cis isomer. No work has been done on the tr*ans isomers present in smoke condensate. The cis-trms composition of unsaturated fatty acid ester mixtures is of interest for several reasons: (a) nutritional studies indicate that the t~rtr~s isomer may not be nutritionally equivalent to the naturally occurring cis isomer”; (b) animal studies indicate that blood-clotting time is altered when W~IIISisomers are substituted for cis isomers’ I: (c) subtle molecular structural changes often account for major flavor changes’ 2; and (d) the difference between the cls and rr’ctns acids could be a result of chemical changes which may produce important biological differences.
A dual-column Varian Model 2800 gas chromatograph equipped with flame ionization detectors wns used. Flow rates of 10. 30. and 300 ml min- l were used for helium. hydrogen, and air, respectively. Stainless steel columns. l/8 in. x 10 ft. packed with loo/, Silar-1 OC on loo-120 mesh Gas-Chrom Q. were used isothermally at 170 “C. with injector and detector temperatures of 250 “C. A Varian Model 25A recorder was used at a chart speed of 0.25 in. min - *. Peak areas were determined with a Varian Model 485 integrator. Percent compositions were normalized and reported relative to percent total volatiles. Individual peaks were identified by comparative retention times and co-chromatography with knowns. Retention time was measured relative to methyl palmitate.
310
SHORT
COMMUNICATION
:* /; / .“,!
10
TIME.
I,WUTES
co
0 60
-..-..60
100
120 TIME.
,J ,!
> ,
140 MINUTES
r
,‘. ,
5
160
Fig. 1. Chromatogram of cstcrificd cigarctlc smoke condcnsntc fraction F-59. (a) 1 methyl palmitotc: 2 methyl palmitokxtc: 3 methyl palmitclaidatc: 4 methyl olcntc: 5 methyl linolcutc: 6 methyl linoIclaidutc. (b) Continuation of Chromutogram ut diffcrcnt uttcnuation.
Methyl ester standards (Applied Science Laboratories, Inc.) were certified 99 +‘;/, pure. Cigarette smoke condensate (CSC). obtained from commercial cigarettes, F-8. in 8 manner previously was fractionated to yield a weak-acid fraction. described13. F-8 was further fractionated by partitioning it between equal volumes of petroleum ether and 907; methanol-water. The petroleum ether solution was then extracted with sodium hydrogen carbonate. The aqueous layer was adjusted to pH 1.0 with G M hydrochloric acid and extracted with diethyl ether to yield F-59. which represented about O.S”/, of CSC. A portion of F-59 was esterified with Methyl 8 (dimethylformamidc dialkylacetal. Pierce Chemical Co.); 1 ml of Methyl 8 was used per SO mg of F-59. as described elsewhere l 4. Results
anti discussion
Figure 1 shows the resulting chromatograms of esterified fraction F-59. The presence of the following unsaturated methyl esters was established by retention time and co-chromatography with knowns: palmitoleate, palmitelaidate. oleate, linoleate and linolelaidate. This study was primarily concerned with the detection and differentiation of the C:;. CiE, and C:g cis and n’n~s isomers. Also. for the the only standard available for comparison was the cis isomer Ci’B’ compounds. (cis. cis. cis-9.12.1 S-octadecatrienate). This compound was detected only in trace amounts in smoke condensate. Figure l(a) shows baseline separation between methyl linoleate and methyl linolelaidate. Separation was poorer. but adequate for electronic integration. between methyl palmitoleate and palmitelaidate. Figure l(b) shows the remaining portion of the chromatogram, at a different attenuation. beyond the area of interest. Table I gives the experimental percent composition and the cis and UWIS isomers expressed as relative percent of total volatiles from fraction F-59. Relative retention time (R) was measured from the methyl palmitate peak. Similar amounts of palmitoleatc ( J.O7’j/,) and palmitelaidate ( 1.1 8”/0) were detected. However, a much smaller amount of linolelaidate (0.1 S’Y”)compared to linoleate (13.77%) was found, Although 11.14% methyl oleate was detected, the level of methyl elaidate was either too low to be detected or its presence was masked by the oleate.
SHORT
COMMUNICATION
TABLE
I
PERCENT COMPOSITION UNSATURATED FATTY
311
AND ACIDS
RELATIVE RETENTION FOUND IN F-59
Pulmitoleutc. C!; (ci.+9_hcxadcccnoittc) Palmitcluidntc. Cl; (frcr,r.~-Y-hexodcccnoate) Olcutc. ci ; C’iS (cl.+9-octndcccnoiI&) Linoleahz. Cf; cis ( C~S. ~Qis-9.12-oct~tdce;tdicnoi~tc) Linolclaidatc. Cf; rrcllts (rrws. rrum-9. I2-oct;ldcc;tdicnoutc)
TIMES
(R) OF
SOME
I .07
I.21
I.18
1.33
II.14
2.1 I
0.15
2.54
13.77
2.78
ESTERIFIED
I’ Vulucs arc un uvcrupc of duplicetc dctcrminutions. h R-rclntivc to methyl palmitatc (pcok 1 in Fig. I).
Owing to yield variability in the fractionation-extraction procedures. no quantitation of acid contents in the condensate was attempted. These findings illustrate the significance of the new g.c. support. Silar-IOC. to the tobacco chemist and its use to other analytical organic chemists engaged in lipid research. Further work is in progress with this new support for identification of higher molecular weight fatty acids in cigarette smoke condensate.
Area
The authors thank Mr. R. L. Atkins, Technical School for their cooperation.
Mr.
Lamar
Hendrix.
and
the
Athens
REFERENCES I 2 3 4 5 6 7 8 9 10 11 I2
Gas-Cltrunt Newsleftcr. 14 ( 1974) 1. Applied Scicncc Laborutorics, Inc., State Collcgc. Pu.. U.S.A. D. B. Wriltcrs und R. J. Horva. AI&. Clrim Acrtr. 65 ( 1973) 198. E. A. Emkcn, Llpltls. 6 (1971) 686. D. Hoffmann and H. Waziwodzki. Bdrr. Tdwkjiwwlr.. 4 ( 1968) 167. I. Mokhnuchcv. L. G. Scrdyuk and M. Ivunov. C. R. B~tlgtrw. Sci.. 20 (1967) 445. B. L. van Duurcn und A. 1. Kasuk. J. Org. CAPON.. 23 (1958) 473. M. Dymicky and R. L. Stcdman. Tohtrcco SC/.. 12 (1969) 45. R. L. Stcdman. Cl~etn. Rer.. 68 ( 1969) 153. A. P. Swain and R. L. Stcdmun. J. Ass. O_/jlc. rlgr. Clrcrl~.. 4.5 (1961) 536. P. G. Rund and F. W. Quuckcnbush. J. N~crr.. 87 (1965) 489. G. Ruccugliu and 0. S. Privctt, Lipltls. 5 ( 1970) 85. R. Tcrunishi. P. Issenbcrg. J. Hornstein und E. I. Wick. Flaaor Rewcmlr. Principles wul Tdruiqws. M. Dckkcr. New York. 1971. p. 282. I3 A. P. Swain. F. G. Bock, J. E. Cooper. W. J. Chumbcrluin. E. D. Strilngc. L. Lakritz und R. L. Stcdmun, Belrr. Ttrbrrkfor~sch.. 7 ( 1973) I. I4 J. P. Thcnot. E. C. Horning. M. Stufford und M. G. Horning. ,4r1tr/. Letr.. 5 (1972) 217.
