Separation and gas chromatographic determination of normal


Separation and gas chromatographic determination of normal hydrocarbons in tobacco leaves. Roy L. Johnston, and Louis A. Jones. Anal. Chem. , 1968, 40...
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Separation and Gas Chromatographic Determination of Normal Hydrocarbons in Tobacco Leaves Roy L. Johnston and Louis A. Jones’ Department of Chemistry, North Carolina State University, Raleigh, N.C. 27607

THEPOLYCYCLIC COMPOUNDS present in tobacco and tobacco smoke have received considerable attention because of their carcinogenicity in experimental animals (I, 2). Because aliphatic hydrocarbons are the major constituent of the hydrocarbon fraction and, under pyrolytic conditions, will produce polycyclic hydrocarbons (3), methods of separating these compounds from others present in the neutral fraction have been developed. Several workers have employed chromatography on alumina (4-6), followed by removal of the olefinic compounds by either recrystallization from acetone (5) or bromination followed by chromatography on alumina (6). Separation of the normal hydrocarbons from branched isomers has been accomplished by adduction with urea (4) or complexation with Linde 5A molecular sieves ( 5 , 6). Although no quantitative data were given for the urea adduction, decomposition of the urea clathrate followed by mass spectrometry and gas chromatography indicated a homologous series c12-c33, the Calhydrocarbon predominating (4). When molecular sieves were used, the concentrations of the normal hydrocarbons were determined by temperature-programmed gas chromatography (TPGC) before and after complexation, and the efficiency of the alumina chromatography was studied by incorporating isotopically labeled hydrocarbons as internal standards (6). After complexation and removal of the molecular sieves, the filtrate was analyzed by TPGC and mass spectrometry. The normal hydrocarbons were displaced from the molecular sieves and analyzed similarly, and a homologous series C26-C33was found for normal hydrocarbons, CBI being present in the highest concentration. Quantitative data were also presented for iso- and anteiso-hydrocarbons, 2-methyl and 3-methyl branching, respectively(5). During the course of determining those compounds isolated from tobacco by a low temperature water entrainment vacuum distillation (7), it was necessary to develop a procedure for the separation and identification of the hydrocarbon fraction. The criterion was an analytical scheme which would minimize the possibilities of isomerization, rearrangements, condensations, etc. of the other nonhydrocarbon compounds present in the sample. The present communication describes the analysis developed and the quantitative and qualitative applications of the method. 1

To whom inquiries should be sent.

~~

(1) A. I. Kosack, J. S. Swinehart, and D. Taber, J. Natl. Cancer inst., 17,375 (1956). (2) E. L. Wynder and D. Hoffman, “Tobacco and Tobacco Srhoke,” Academic Press, New York, N. Y., 1967, Chapter 7. (3) 3. Lam, AcfuPafhol.Microbiol. Scund., 39,207 (1956). (4) W. Carruthers and A. W. Johnstone, Nature, 184, 1131 (1959). See also R. L. Stedman, Chem. Reu., 68, 153 (1968) for other leading references. (5) J. D. Mold, R. K. Stevens, R. E. Means, and J. M. Ruth, Biochem., 2,605 (1963). (6) . , A. W. Spears, C. W. Lassiter, and J. H. Bell, J. Gas Chromatog., i(4), 34 (i963). (7) L. A. Jones and J. A. Weybrew, Tobacco Sci., 192(1962).

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ANALYTICAL CHEMISTRY

EXPERIMENTAL All solvents were purified by standard procedures, checked for purity by GC, and stored in the dark until used. Hydrocarbons were obtained from Distillation Product Industries, Rochester, N. Y.; Columbia Organic Chemical Co., Columbia, S. C.; Lachat, Chemicals, Inc., Chicago, Ill.; and Humphrey Wilkinson Inc., North Haven, Conn. They were dodecane through tetracosane, heptacosane, octacosane, and dotriacontane (C1z-C24,Cn, Czs, c 3 2 , respectively). All samples were purified when necessary by fractional distillation or clathrate formation with urea (&a infra) and checked for purity by TPGC. Silicic acid (Mallinkrodt Chemicals) was sieved through a 115-mesh screen (Taylor scale), retaining the silicic acid less than 115-mesh. It was washed with distilled water, acetone, and ether; dried in an oven at 140 “C under vacuum for 24 hours; and stored in an airtight bottle. An F & M Model 300 gas chromatograph equipped with a Model 1609 hydrogen flame ionization detector was used in this study. Columns prepared were 2-feet x 0.25-inch copper tubing containing the following column substrates and supports: (1) 1 % Apiezon L on Diatoport S 60-80 mesh, (2) 5 z silicone rubber on Chromosorb W-AW 30-60 mesh, (3) 5% XE-60 on Diatoport S 60-80 mesh. Injection port was maintained at 230 “C, detector at 210 “C, and all runs were programmed from 75-275 “C at 9”/min with a helium flow rate of 75 ml/min at a pressure of 25 psi. RESULTS AND DISCUSSION

The unknown sample of interest was obtained by the chemical separation of 7.300 grams of tobacco “aroma” sample obtained by a low temperature-pressure water entrainment distillation of 125 pounds of Hicks tobacco. The volatiles were trapped in Dry Ice-acetone cooled condensers; the solutions were then thawed, saturated with salt, and extracted with ether. Following separation into acidic, basic, and neutral fractions (7), the neutral fraction containing alcohols, esters, branched and straight chain hydrocarbons, olefins, and carbonyl compounds was investigated. It was decided to attempt separation of the total hydrocarbon sample from the oxygenated material by liquid-solid chromatography since TPGC indicated the complete sample was too complex to be meaningful (7). Although other workers had used alumina (4-6), the numerous reports of secondary reactions, such as dehydrohalogenations, double bond migration, and condensations (8-10) prompted us to investigate the possibility of using a low temperature silicic acid column with pentane as an elutant. A similar technique has been reported for the separation of terpenes from essential oils (11). (8) E. Lederer and M. Lederer, “Chromatography,” Elsevier,

New York, 1957,pp 61-65. (9) H. G. Cassidy, “Adsorption and Chromatography,” Interscience, New York, 1951, pp 255-258. (10) R.L. Stedman, A. P. Swain, and N. J. Rusaniwskyj, Tobacco Sci., l(1962). (11) J. G. Kirchner and J. M. Miller, Ind. Eng. Chem., 44, 318 (1952) and references therein.

A 900-mm x 10-mm i.d. glass column jacketed by two 400mm x 70-mm condensers was packed with 25 grams of activated silicic acid and the column maintained at 0 ”C with circulating ice water to minimize reactions between solute and adsorbent and permit the use of pentane and ether as elutants. Chromatography of known straight chain hydrocarbons under the above conditions indicated the hydrocarbons were eluted in the first 200 to 300 ml of pentane elutant. The 5.230 grams of unknown neutral compounds were placed on a similar column and the process repeated, each 100-ml fraction being monitored by infrared spectrometry and TPGC. The gas chromatograms of all fractions were similar and the infrared spectra were identical to those reported for neophytadiene (12). The total weight of the combined hydrocarbon fractions was 3.200 grams. Oxygenated compounds were removed from the column with ether and methanol and accounted for 38.8 (2.030 grams) of the neutral fraction. Separation of the unsaturated hydrocarbons from the saturated compounds by recrystallization from acetone proved to be inefficient and attention was directed to the technique of urea adduction (13) for the separation of straight chain hydrocarbons from the mixture. Molecular sieves were not considered because they have been reported to cause isomerization of olefins (14) and, because of the presence of neophytadiene, could lead to spurious results. Although urea adduction has been known for some time (13), little work has been done on the quantitative aspects of the method. One report states that the yield of adduct can be maximized when the ratio of urea to alkane is 20: 1. Data were given for the recovery of hydrocarbons CI1and CI6and the gravimetric yields were 71 and 88 %, respectively. Using a 12:3 ratio of urea to alkane and concentrations of 5, 10, and 30% of the CI4alkane in methanol, recoveries of the hydrocarbon were 80, 91, and 96%, respectively (15). However, no further data were given for recoveries of gross mixtures. To determine the quantitative nature of the adduction, a standard solution was prepared by dissolving 0.3 mmole of each hydrocarbon (c12-c24, CZ1,Czs,CZ2)in 10 ml of hexane. The urea solution was prepared by saturating methanol with urea at room temperature. Adduction was accomplished by adding 25 ml of urea solution to 1 ml of the standard and shaking for 1 hour. After being filtered, the white solid was air-dried and the hydrocarbons were recovered by decomposing the adduct in water and then extracting with pentane. The pentane was evaporated and the recovered hydrocarbons were weighed. Triplicate analyses showed that the hydrocarbons could be recovered in an average yield of 92.5 i 5.0x. In addition to the gravimetric procedure, recovery was also investigated by TPGC. Another solution was made c p containing 0.3 mmole of hydrocarbons C12C2*in 10-ml hexane solution. To prepare the standard solution for each of the triplicate analyses performed, 1 ml of the hexane solution was diluted to 5 ml with pentane. Using the same procedure for adduction and, following the evaporation of the pentane, the hydrocarbons were made up to 5 ml with pentane. For each triplicate analyses, four 5O-pl samples were analyzed and the areas calculated by width at half height times height. The results are shown in Table I, and as reasonable agreement was obtained between the gravimetric and

z

(12) A. Rodgman, J. Org. Chem., 24,1916 (1959). (13) F. Bengen and W. Schlenk, Jr., Experientia, 5,200 (1949). (14) J. G . OConner and M. S. Morris, ANAL. CFEM.,32, 701 (1960). (15) W. J. Zimmerschied, R. A. Dinerstein, A. W. Weitkamp, and R. F. Marschner, Ind. Eng. Chem.. 42,1300 (1950).

Table I. Hydrocarbon Recovery from Urea Adducts na

Standardb

Adductc

12 13 14 15 16 17 18 19 20 21 22 23 24

3.27 f 0.06 3.01 f 0.11 3.23 f 0.05 3.75 f 0.11 3.67 f 0.08 3.58 f 0.25 4.27 f 0.24 4.11 f 0.21 4.00 f 0.13 4.24 f 0.11 4.04 f 0.08 3.75 f 0.04 4.34 f 0.24

1.62 i= 0.23 2.34 f 0.08 2.77 f 0.19 3.31 zk 0.24 3.34 f 0.18 3.38 f 0.31 3.85 f 0.20 3.74 & 0.26 3.32 f 0.17 3.66 & 0.23 3.50 f 0.15 3.32 f 0.11 3.92 f 0.33

0

n of C,H2,+2.

c

Area f mean deviation.

Recovery, % 49.54 77.23 85.23 88.03 91.01 94.41 89.74 91 .OO 83 .OO 86.32 86.63 88.53 90.32

* Area f mean deviation.

Table 11. Comparison of Calculated Equations

Columnsa

Sample

XE-60 os. AL

Standard Unknown Standard Unknown Standard Unknown

SR us. AL

xE-601;~. SR

Equationb

rc

y = 1.27~ 0.87 0.999 y = 1.26~ - 1.16 0.999 y = 1.00~ - 0.68 0.999 y = 1.09~ 0.01 0.990 y = y =

1 . 1 5~ 0.17 0.999 1 . 1 3~ 0.02 0.999

Sd

0.22 0.13 0.04 0.08 0.23 0.13

.Columns: XE-60 = 52 XE-60 on Diatoport S; AL = 1 % Apiezon L on Diatoport S; SR = 5% silicone rubber on Chromosorb W-AW.

* Least squares equation where y = retention time in minutes of first named column and x = retention time in minutes of second named column. c r = correlation coefficient. d s = standard deviation of x from calculated line.

z),

TPGC analysis (average 84.7 the procedure was considered satisfactory. The 3.200 grams of hydrocarbon fraction were adducted in a like manner, except the hydrocarbon-urea mixture was shaken for 22 hours. Following pentane extraction, chromatography on silicic acid served to assure no carry-over of impurities in the extraction procedure and evaporation of the pentane yielded 0.035 gram of normal hydrocarbons. This was dissolved in 0.5 ml of heptane and stored in a vial capped with a rubber septum. TPGC, using columns and conditions previously described, indicated that the sample was composed of primarily a homologous series of straight chain hydrocarbons (Figure 1). Identification of the n-paraffins was made by the method of Lewis, Patton, and Kaye (16) and consisted of plotting the retention times of a known series of hydrocarbons obtained on three different columns (under the same operating conditions) us. one another. The straight lines thus obtained were calculated by the method of least squares (17). The same (16) J. S. Lewis, H. W. Patton, and W. I. Kaye, ANAL.CHEM., 28, 1370(1956). (17) E. J. Williams, “Regression Analysis,” Wiley, New York, 1959, Chapter 6. VOL. 40, NO. 1 1 , SEPTEMBER 1968

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I2 W

K

E

z:

5

m 1I i

r_

i-

I/

/I

I

TIME

IN MINUTES

Figure 2. Chromatogram of normal hydrocarbons from tobacco gum on a 2-foot X 1/4-inchcolumn of 1 Apiezon L on Diatoport S

x

TIME IN MINUTES

Figure 1. Chromatograms of normal hydrocarbons on a 2-foot x column of 1 Apiezon L on Diatoport S

x

Top. Standard hydrocarbon mixture Bottom. From tobacco aroma

procedure was employed for the unknowns and the results are shown in Table 11. It should be noted that intercepts of the equations differ from standard to unknown by less than 0.3 second in two of the three cases. Excellent agreement was obtained for the slopes of the equations as well as high correlation coefficients and small standard deviations. These results, in addition to the similarity in retention times (Figure l),lead to the con-

Table 111. Areas and Per Cent Composition for CnHzn+2 1 Apiezon L 5 Silicone rubber Average

z

7 3

z

n Area. C0mp.b Areaa Comp.b C0mp.c 0.54 0.51 0 . 0 7 k 0.03 0.57 15 0.80f0.05 4.18 7 . 1 0 k 0 . 1 0 4.17 6 . 5 5 k 0 . 0 4 4.19 16 10.15 1 6 . 9 0 f 0 . 0 5 9.94 17 16.18k0.36 10.37 17.30 18J 27.55 k 0.31 17.70 28.75 f 0.08 16.91 7.61 1 2 . 0 0 f 0 0 . 1 5 7.06 19 1 2 . 7 2 f 0 . 2 6 8.15 3.87 6 . 7 0 f 0 . 1 8 3.94 20 5 . 9 2 f 0 . 0 6 3.79 14.85 25.95 f 0.04 15.26 21d 22.52 f 0.20 14.44 5.44 8 . 1 8 f 0 . 2 4 4.81 22 9 . 4 5 k 0 . 1 4 6.06 2.94 4.48320.09 2.63 23 5 . 0 5 f 0 . 0 4 3.24 2.72 4 . 3 5 f 0 . 1 0 2.56 24 4.50k0.02 2.88 6.01 9.82f0.09 5.77 25 9 . 7 5 f 0 . 0 8 6.25 2.84 4 . 8 8 f 0 . 1 4 2.87 4 . 3 8 f 0 . 0 9 2.81 26 5.48 9 . 4 0 f 0 . 2 5 5.53 21 8 . 4 5 f 0 . 0 4 5.42 2.64 5 . 6 8 f 0 . 1 1 3.34 28 3 . 0 2 f 0 . 1 4 1.94 2.90 5 . 6 0 f 0 . 1 4 3.29 29 3.90f0.05 2.50 2.29 4 . 3 5 f 0 . 0 8 2.56 30 3 . 1 5 ~ l ~ 0 . 0 52.02 4.80 8 . 5 2 k 0 . 1 4 5.01 31 7.15=t00.11 4.58 2.05 3 . 7 0 k 0 . 0 8 2.18 32 3 . 0 0 f 0 . 0 8 1.92 1.46 2 . 7 5 f 0 . 0 6 1.62 33 2.02+0.02 1.29 a Average of four determinations 32 mean deviation. Calculated from normalized areas. Average of two compositions. d Plus isomer.

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clusion that the normal hydrocarbons in the unknown are a homologous series of c16-c33. The area of each peak was determined with a planimeter and the average of four determinations calculated. The per cent composition was determined by the usual normalization techniques and the results are shown in Table 111. The XE-60 column was not used because of apparent conformational changes which occurred, resulting in changes of retention times and peak areas as had been previously reported (la). This probably accounts for the somewhat larger standard deviations found in Table 11. In general, there was reasonable agreement between the data collected on both columns and, of the 19 hydrocarbons analyzed, only CZZand C28 differed by more than 1% absolute. The CIS and Czlhydrocarbons were present in the highest concentration. It should be noted that the method of obtaining the sample-i.e., low temperature water entrainment vacuum distillation (7)-suggests that the hydrocarbons have significant vapor pressures, and the lower molecular weight compounds should comprise the major portion of the sample. Although CISand Csl are contaminated with isomers. the hvdrocarbons from c 1 6 to Czzaccount for 64Z of the sample, a i d , with the exception of CIg and C I ~the , odd-numbered compounds are present in higher concentrations than the even-numbered homologs. Similar hydrocarbon sequences have been noted previously (5). To determine the qualitative utility of the procedure, a sample of the "gum" or resinous material which is found on a tobacco leaf was obtained. The sample was extracted with ether and the ether evaporated. Using the procedure described, except that urea adduction was accomplished in 30 minutes, the TPGC analysis indicated a homologous series of hydrocarbons C19-C35(Figure 2). Hentriacontane, C31, was present in the highest concentration in accord with previous reports (4, 5, 191, and the total time analysis was less than 3 hours. Normalization of the peak areas showed that 99.35x of the hydrocarbon sample was composed of c2&33 and the compounds with an odd number of carbon atoms predominated. In general, the

(18) C. Chen and D. Gaske, ANAL.CHEM., 36,72 (1964). (19) N. Carugno, J. Narl. Cancer Insr. Monograph, No. 9, 171 (1962).

distribution of the hydrocarbons present paralleled the results of other workers (4, 5, 19) but direct comparison cannot be made because of the different types of tobacco used in the sample preparation. It is apparent, however, that the method described can be used effectively as a rapid qualitative technique for the analysis of straight chain hydrocarbons present in complex mixtures.

RECEIVED for review April 12, 1968. Accepted June 13, 1968. Abstracted in part from the M.S. Thesis of R. L. J., North Carolina State University, June 1965. Presented at the Eighteenth Tobacco Chemists' Conference, October 20, 1964, North Carolina State University, Raleigh, N. C. The authors are indebted to the R. J. Reynolds Tobacco Co. for support of this work.

Determination of Lithium by Potentiometric Titration with Fluoride Elizabeth W. Baumann Savannah River Laboratory, E . I . du Pont de Nemours and Co., Aiken, S . C. 29801

THEFEW published volumetric methods for the determination of lithium are either complicated or nonspecific; consequently, none of the suggested procedures have gained general acceptance. For example, one determination based on iodometric titration of the precipitated complex lithium periodate ( I ) distinguishes lithium from the other alkali metals, but requires that all metals except those of the alkali group be removed and that ammonia be removed if more than a few milligrams are present. Lithium determination by titration of lithium acetate as a base in an acetic acid-chloroform solvent (2) cannot distinguish quantitatively between lithium and sodium, but can distinguish lithium from potassium. A method was developed for analysis of concentrated solutions of LiCl and LiNOa that are used in the Tramex process for separation of lanthanides from actinides (3). This method utilizes quantitative precipitation of LiF in alcohol (4) as the basis for a potentiometric titration with the lanthanum fluoride membrane electrode (5)serving as an indicator electrode. Moderate amounts of sodium can be tolerated. Interference from free acid and some metal ions is eliminated by adding N H 4 0 H and (NH4)2S,respectively. EXPERIMENTAL Equipment. An expanded scale pH meter was used with a saturated calomel reference electrode and a specific fluoride ion indicator electrode (Model 94-09, Orion Research Inc., Cambridge, Mass.). A 2-ml microburet with a micrometer control and a precision Teflon (Du Pont) plunger (Cat. No. 7846, Cole-Parmer Instrument and Equipment Co., Chicago, Ill.) was modified by replacing the glass reservoir with one of polyethylene or Teflon, so that the standard NHIF titrant does not contact glass. Reagents. Reagent grade chemicals were used. LiCl was not further purified; maximum limits of major impurities were: K, Na, Ca, sulfate, 0.01 % each; alkalinity as Li2C03, 0.02 %. Standard 0.5M NH4F was prepared by neutralizing HF, previously standardized against NaOH, with N H 4 0 H to pH -8. Procedure. A 100-p1 sample was placed in -30 ml of 95 % ethyl alcohol. If the sample contained free acid, 1 drop of concentrated NH40H was added. If the sample contained interfering metal ions, 250 to 500 p1 of 20% (NH4)*Sreagent solution was added. (1) ANAL.ED., . . IND. ENG.CHEM., . , L. B. Rogers and E. R. Caley,

15, 209 (1943). (2) C. W. Pifer. E. G. Wollish. and M. Schmall. ANAL.CHEM.. 26. 215 (1954). (3) H. J. Groh, C. S. Schlea, J. A. Smith, R. T. Huntoon, and F. H. Springer, Nuclear Applicatiorzs, 1 (4), 327 (1965). (4) E. R. Caley and G. R. Kahle, ANAL.CHEM., 31, 1880 (1959). (5) M. S. Frant and J. W. Ross, Jr., Science, 154, 1553 (1966).

.,

I

,

The stirred solution was titrated potentiometrically with "IF. Near the equivalence point, the potential was measured after each addition of 100 pl. The volume of titrant at the equivalence point was calculated conventionally as the point of inflection in the curve. RESULTS AND DISCUSSION

Accuracy and Precision. The accuracy of the potentiometric titration method was assessed by determining both the lithium and chloride content of the nominal 11M LiCl solution used throughout the investigation and of a dilute LiCl solution. The latter was selected to eliminate errors inherent in pipetting the viscous concentrated solution; aliquots were concentrated by evaporation for the lithium determination. Chloride was determined by potentiometric titration with 0.1M AgNOa, with a silver indicator electrode. Table I shows that the lithium and chloride determinations agreed well. The precision of the method was calculated for each of the two LiCl solutions (Table I). The higher relative standard deviation for the 11M LiCl (0.4% compared with 0.3% for the dilute solution) probably reflects pipetting error, which is also evident in the chloride values.

Table I. Analysis of LiCl Solutions by Potentiometric Titration

Mean concn, M Standard deviation No. of detns Sample size, ml ~~

~

11MLiCl Lif c110.71 10.67 0.047 0.034 6 3 0.100 0.250

Dilute LiCl Li+ c10.08540 0.08549 O.OOO27 0. oooO3 6

5

10

10

~

Table 11. Comparison of Titration and Ion Exchange Methods Li concentration Nominal found, M Ion composition of solution Titration exchange 11M LiCl 10.71 10.80 11M LiCl 10.82 10.94 9M LiCl 9.07 8.96 11MLiC1,0.3MHCl 11. 16a 11.28 1M LiCI, 6.9M LiN03, 0.215M NzH4,HNOs 8.07 7.93 a 1 drop of NH40H added.

VOL 40, NO. 1 1 , SEPTEMBER 1968

Difference +O. 09

+o. 12

-0.11 +o. 12 -0.14

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