B E
I
5
0
10
15
20
MINUTES
Figure 3. A. B.
Separation of ammonia, methanol, and methylamines Trimethylamine Ammonia
C.
D. E.
Column: 9-ft. 15% THEED-5% same as Figure 1
Dimethylamine Methylamine
LITERATURE CITED
Methanol
TEP on Chromororb W:
Amine Separation. The separation of the methyl-, ethyl- and n-propylamines was studied using six liquid phases and combinations of these liquid phases. Initial exploratory work was carried out using diglycerol, polyglycerol, Carbowax 400, Carbowax 1540, T H E E D , and T E P t o separate the methyl amines and ammonia. Of the six liquid phases only T H E E D showed promise of separating the four compounds, but it did not give adequate deactivation of the support. TEP, which showed good properties as a tail reducer in the deactivation study, showed incomplete
column temp.:
60’ C.;
10- component mixture was not satisfactory. However, increasing the concentration of liquid phase to 57, THEED-15% T E P (maintaining the T H E E D / T E P ratio of 1 to 3) showed more promising separation. This is illustrated in Figure 2. For a mixture of only the methyl amines and ammonia, a somewhat better beparation than that obtained in Figure 2 can be achieved with the THEED/TEP ratio of 3 to 1. Using 6% THEED-2% TEP, some tailing of the ammonia peaks TVMS found, but by increasing the concentration of liquid phase t o 15% THEED-5% TEP the tailing of ammonia m s eliminated. This separation, with that of methanol, is shown in Figure 3.
other conditions
separation of the methylamines and ammonia. To study the separation of the nine amines and ammonia, five columns were prepared with varying amounts of T H E E D and TEP. These columns are listed below in Table I, and the retention time data are tabulated in Table 11. From the data in Table 11, one can choose the optimum composition of liquid phase for the separation of the 10 components. The retention time data shows that 2% THEED-6Y0 TEP provides the best separations of all 10 compounds. The actual separation obtained with this column of the
(11 Burks. R. E. .Tr.. Baker. E. B.. Clark. ‘ P., Esslinger, J.; Lace{., J. ’C. Jr.; J. Agr. Food Chem. 7, 778 (1959). (2) Decora, A. W., Dinneen, G. U., “Gas Chromatography,” H. J. Xoebels, R. F. Wall, and N. -Brenner, eds., p. 33, Academic Press, S e w Tork, 1961. (3) Fisher Scientific Co., Pittsburgh, pa., Technical Data Bulletin No. 152, Teflon as a Chromatographic Support,” May 1961. (4) Hughes, R. B., J . Sci. Food Agr. 10, 431 (1959). ( 5 ) James, A. T., Martin, A. J. P., Smith, G. H., Biochem. J . 52, 235 (1952). (6) James, A4. T., Ibid., p. 242. ( 7 ) Ring, R. D., Riley, F. W., “GasLiquid Chromatographic Analysis of Amine Mixtures,” Sixth Detroit Bnachem. Conference, Detroit, Mich., October 1958. (5) Smith, E. D., Radford, R. D., AXAL. CHEV.33, 1161 (1961). RECEIVEI)for review August 6, 1962. Accepted December 10, 1962.
Gas Chromatography of the Reduction Products
of Chlorinated Organic Pesticides HERMAN F. BECKMAN and PETER BERKENKOTTER Pesticide Residue Research laborafory, University o f California, Davis, Calif.
b A technique for the analysis of halogenated hydrocarbons with particular reference to pesticides, using a combination of gas chromatography and sodium-liquid anhydrous ammonia reduction, includes gas chromatographic isolation of the compound followed b y a reduction to liberate the halogen. Both steps measure the halogenated hydrocarbon present. A third and final step for further identification and quantitative measurement of the compound involves gas chromatographic analysis of the organic residue left after reduction. The organic phase is taken from the aqueous system into a solvent and condensed in an evap242
ANALYTICAL CHEMISTRY
orative concentrator. The residue i s then reintroduced into the gas chromatograph. The peak or peaks obtained are characteristic of the parent compound. The procedure gives three measures of a compound: its initial gas chromatography, measurement of the halogen liberated, and gas chromatography of the dehalogenated residue.
T
HE sodium-liquid anhydrous ammonia reduction technique ( I ) has been used as a means of releasing organically bound halogen for analysis. The chlorinated hydrocarbon pesticides as a group are considered t o be repre-
sentative of compounds with organically bound halogen that are generally resistant t o dehalogenation. The reduction technique would then apply to other organohalogen compounds and the subsequent analytical methods described are also generally applicable. Krzeminski and Landmann (Z), reporting on an application of the reduction procedure at the Michigan State University Conference on Pesticide Residues in the spring of 1961, described a method for the determination of chlorinated hydrocarbon residues in animal fats. The actual chloride ion detection was by potentiometric means ; however, amperometric
organohalogen compound, which can be transferred n-ith a small amount of ethyl ether to a beaker for reduction with sodium and liquid anhydrous ammonia. Since this involves a very small amount of pesticide, only small amounts of reagents are neceqsary for the reaction. This yields low blanks for halogen detection. If the organic phase left after reduction is quantitatirely removed from the reaction mixture, by a solvent such as ethyl ether or pentane, it can be concentrated by vacuum or a ivarni water bath to yield the organic residue, which may be injected into the gas chromatograph for further analysis. The data thus obtained serve for further identification or reaffirmation as well as quantitative measurement. The possibility of applying this identification process to pesticide residue analysis may be adapted t o suit the specific crop or insecticide under study.
1
ALDRIN
2 0
0
I kD METHOXYCHLOR
L
I
2
0
4
a
6
10
Tme, Mlnutas 115
137
Figure 1 .
181 M3 225 Co Tmwmlure Increase
159
12
249
14
16
269
29
Retention times of aldrin, TDE, DDT, and methoxychlor
Column, 2 feet, 20% Dow 1 1 on 40- 60-mesh Chromosorb P, program rate 1 1 '/min., helium flow 50 cc./min.
and coulometric procedures are also highly sensitive and accurate. Gas chromatography of pesticides is now rather well established and in many cases highly sensitive methods are available for the analysis of pesticides. Gas chromatography has also been combined with other means (all types of spectrophotometry as well as microcoulometry) for final detection of the material sought. The gas chromatograph may serve for both cleanup and detection. This report concerns a technique combining soclium-liquid anhydrous ammonia reduction with gas chroniatographic analysis of the hydrocarbon residue recovered after reduction. Methods of determining the halogen liberated by this reduction procedure
MATERIALS AND EQUIPMENT
Gas Chromatographs. F and M Model 500 with temperature procramming.. Wilkins iierosranh Model A-90-P. Columns. For F and 11 instrument. Two-foot. '/*-inch 0.d.. stainless steel column packed with '20% Dow 11 silicone oil on 40- 60-mesh Chromosorb P. Two-foot, l/r-inch o.d., stainless steel column packed with 20y0 SE-30 silicone rubber on 30- 6O-nlesh Chro-gum mosorb W. Six-foot, l/d-inch o.d., stainless steel column nacked with 20% Ucon nolar on 30- 60-mesh Chromosorb P. For Aerograph instrument. Thirtyfoot, '/e-inch o.d., copper column packed with 30% SE-30 silicone gum rubber on 30- 60-mesh Chromosorb W. Six-foot, l/c-inch o.d., stainless steel column packed with 20% Doiv 710 silicone oil on 30- 60-mesh Chromosorb TiT. Both instruments were equipped with thermal conductivity detectors and operated a t 175 ma. In column operations involving collection of samples, a collector of a U-tube design was used.
-
are !vel1 established, and need not be discussed. When a pesticide is extracted from a crop, and the extract is concentrated and injected into a gas chromatograph, a response is obtained when sufficient pesticide is present to be vithin the sensitivity of the instrument detector. Preliminary cleanup of the original extract improves sensitivity and specificity. In a considerable number of cases, such as fats, oils, milk, or plants containing much oil or way, the preliminary cleanup is essential. The pesticide or other organohalogen may be quantitatively trapped in a collection device attached t o the effluent stream of gas a t the time the detector responds to the chemical. At this point one has a rather pure fraction of the
u
U
n
4
K 2
0 8
10
Figure 2.
12
14 me,Mlnutes
1'6
ia
30-60-mesh
5
15
10
20
Figure 3.
Toxaphene response curves
Column, 30 feet, 3070 SE-30 gum rubber on 2 7 5 ' C., helium flow 70 cc./min.
Chromosorb
temp.
I
25 T i m , Minutes
20
30
Strobane response curves
Column, 2 feet, 20% SE-30 gum rubber on 30- 60-mesh Chromosorb W, helium 5 5 cc./min., program rate 1 1 '/min., Initial temp.
50' C. VOL. 35,
NO.
2, FEBRUARY 1 9 6 3
243
Time , Minutes
TIME, MINUTES
Figure 4. Response curve for d e chlorination product of DDT or TDE
Figure 5. Response curves for dechlorination products of technical methoxychlor
Column, 6 feet, 20% Dow 710 silicone oil on 30- 60-mesh Chromosorb W, temp. 200' C., helium flow 55 cc./min.
Column, 6 feet 20% Dow 710 silicone oil on 30- 60-mesh Chromosorb W, temp. 2 5 6 ' C., helium flow 5 5 cc./min.
The collector was held in a Dewar flask containing a dry iceacetone mixture and one open end was fitted with a female 7/16 standard-taper joint to ensure a tight seal to the exit port of the chromatographic column. Ammonia Tank. The conditions of operation and the assembly for removal of liquid anhydrous ammonia have been described ( I ) . Standards. All the chlorinated compounds-DDT, aldrin, T D E , and methoxychlor-were originally of high purity and were recrystallized until a single peak on the gas chromatograph was obtained. This ensured a purity above 997,. The
TIME .MINUTES
Figure 6. Response curves for d e chlorination products of aldrin Column, 2 feet 20% SE-30 silicone gum rubber on 30- 60-mesh Chromosorb W, helium flow 5 5 cc./min., program rate 1 1 "/ min., initial temp. 100' C.
244
ANALYTICAL CHEMISTRY
standards of toxaphene and Strobane were used as received from the manufacturer, showing only a chlorine content guarantee. RESULTS AND DISCUSSION
Any halogenated organic compound may be reduced by the liquid anhydrous ammonia-sodium technique. For compounds that will chromatograph or will yield a chromatographable product, further analyses may be used t o identify products or to estimate the quantities present. The pesticides for this work were chosen because of their general resistance t o complete dechlorination as we11 as the identity of their reduction products. The data presented are designed to show that several pesticides may be separated by gas chromatography, and that when these materials are dechlorinated, their organic residue may be rechromatographed for further identification. Figures 1 to 10 show these data as well as some infrared analyses of the reduction products. Figures I , 2, and 3 give gas chroniatographic data for the pesticides themselves. Figure 1 Phoxs the conditions and retention times for aldrin, TDE, DDT, and methosychlor. As little as 10 pg. of these compounds mav be detected, Figures 2 and 3 show the same data for toxaphene and Strobane. S o t e the extremes in temperature required for the separation of the components of Strobane. While toxaphene is said to be chlorinated camphene, it would seem t o be a mixture of many chlorinated compounds. These compounds, which are probably closely related and of about the same chlorine content, have similar retention times. Strohane is said to be a mixture of
chlorinated terpenes, mainly camphene and &-pinene. Under the temperature conditions used, the retention times for the examples shown are characteristic of the particular compound. Since toxaphene and Strobane are such complex mixtures, no specific retention time can be used for them, although the nature of the gas chroniatogram might indicate their presence. To detect the presence
I
p _p-o
1 W
v,
z
0 a v,
w
(t:
0
2
4
6
8
T I M E , MINUTES
Figure 7. Response curves for dechlorination products from Strobane Column, 6 feet, 20% Ucon polor on 30- 6 0 mesh Chromosorb W, initial ?emp. 100; C., helium flow 55 cc./min., progrom rate 1 1 C./ min.
and the organic residue is isolated, they are again ready for gas chromatography. The results of this chromatographic step are shown in Figures 4 t o 8. The sample should contain a t least 10 pg. of the dechlorinated compound for thermal conductivity detection. The column conditions were similar for the chromatography of the reduction products of D D T and T D E . The retention time for each was similar, which was not unexpected because of the similarity of the parent compounds. K h e n the reduction products of T D E and D D T were chromatographed, the fractions u ere collected and rechromatographed. The second pass through the chromatograph showed that the products are not affected by the conditions of chromatography and that the retention times are reproduced. Figure 5 shows the results of chromatography of the reduction products of methoxychlor. The major peak is due t o the p,p'-isomer. Figure 6 shons the reduction products obtained from aldrin. Rechroniatography indicated the same retention time with no decomposition due to chromatography. Figures 7 , 8, and 9 deal with the reduction products of Strobane and toxaphene and five known terpenes. Strobane and toxaphene are both shown with 11 peaks, which superimpose in terms of retention time but not concentration. Figure 9 s h o w the retention times for several terpenes under the same conditions as Figures 7 and 8.
W v)
z
0 P
v)
W
a
T I M E , MINUTES
Figure 8. Response curves of dechlorination products from toxaphene Column, 6 feet, 20% Ucon polor on 30- 60mesh Chromosorb W, initial temp. 100' C., helium flow 5 5 cc./min, program rote 1 1 "/min.
of such a mixture of compounds one must have at least 50 pg. of either material for injection into the gas chromatograph. *4fter these materials are taken through the dechlorination procedure
m
W
xmz W
m
T I M E , MINUTES
Figure 9. Chromatographic separation response curves for terpenesa-pinene, camphene, $-pinene, dlimonene, and p-cymene Column, 6 feet, 20% Ucon polar on 30- 60-mesh Chromosorb W, initio1 temp. 100' C., helium flow 55 cc./min., program rate 1 1 ' C./min.
The retention times correspond for both compounds and all five terpenes. The two trace components shown with the standard terpenes are unknown, but
SPECTRUM NO
-
67
DATE-_SEPLB,
-
OPERATOR
___ SAMPLE
h
- ' v&cU&D!~T f r o c l h f u m GI C
ORIGIN -._IBBaBBLaBy_ PURITY -
9 9 '1,
+
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CONCENTRATION
W
n
0 . 5 %
REFERENCE--
z 0
~
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340-
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52
L I T PROGRAM -A__-
-
ATTENUATOR SPEED
4 K
3-
GAIN
t-
CHART SPEED A
20 -
SCALE ORDINATE I)(L ABSCISSA REMARKS-
0:
a
I
I
I
I
5
6
7
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Figure 10.
I
'9
I
1
I
I
I
1
10
II
12
13
14
15
L E N G T H , MICRONS
Infrared spectra for reduction product of DDT and TDE VOL 35, NO. 2, FEBRUARY 1 9 6 3
245
have the same retention times and temperatures of elution as two of the components in the reduction products of both toxaphene and Strobane. High blank levels would be encountered in pesticide residue work in the analysis of carrots or citrus, which have high contents of naturally occurring terpenes. This in part may be avoided in the initial chromatography and cleanup of the extract. One advantage gained by using the sodium-liquid anhydrous ammonia procedure for the reduction of the chlorinated hydrocarbon pesticides is thelorv temperature reaction that takes place and its speed. These factors avoid side reactions and possible polymer formation of the organic residue left after dechlorination. Thus, the use of sodium-liquid anhy-
drous ammonia as a reducing medium for liberating both the halogen and organic fraction for further analysis has distinct possibilities and certain advantages not obtainable with other reduction procedures. Investigations of the identity of the reduction products of DDT and T D E have shown that 1,l-diphenylethane is the product formed. Gas chromatography of the reduction products showed identical retention times and infrared spectra the same as for known 1,l-diphenylethane (see Figure 10). The yield appeared quantitative in that no other observable compounds could be detected. Methoxychlor was shown to yield 1,l-dianisylethane [l,l-bis(4methoxypheny1)ethanel by the procedures used for D D T and TDE. The retention times of the several
known terpenes shown correspond exactly to those found for the reduction products of toxaphene and Strobane. This is an indication of the composition of the starting materials used in the original preparation of these products. Reduction of aldrin indicates the presence of several products Ivith one predominant. The reduction is quantitative on the basis of chlorine content, but the identity of the organic residues has not been determined. LITERATURE CITED
(1) Beckman, H. F., Ibert, E. R., ddams,
B. B.. Skovlin. I>. 0.. J . Aor. Food Chem.’6, 104 (1958). ‘ (2) Krxeminski, L. F., Landmann, W. A., Ibid., 11, 81 (1963).
RECEIVEDfor review July 16, 1962. Accepted December 3, 1962.
Determination of Nornicotine in Tobacco and Smoke by the lJ-Indanedione Spectrophotometric Method Comparison with a n Improved Paper ChromatographicUltraviolet Spectrophotometric Procedure EUGENE GLOCK and MARY
P.
WRIGHT
Department of Research and Development, The American Tobacco Co., Richmond, Va.
b The 1,3-indanedione reaction for the spectrophotometric determination of nornicotine in tobacco and tobacco smoke was investigated. Modification of the reagents has increased the stability of the colored product and the sensitivity of the reaction, permitting determination o f 5 to 100 fig. o f nornicotine per 10 mi. of reaction system. The precision of the determination as applied to tobacco approached 2%. The mean recovery of nornicotine added to tobacco was 99%. The new method is convenient for routine analysis and shows negligible interference from nicotine, myosmine, anabasine, anatabine, and all other pyridine alkaloids studied. The specificity of the 1 ,3-indanedione procedure, as applied to tobacco and smoke, was confirmed b y comparison with an improved paper chromatographic-ultraviolet spectrophotometric procedure. Isolation of nornicotine b y extraction was compared with steam distillation and preliminary paper chromatography in butanolHCI-H20 ( 1 0: 2 : 3 ../..). Chloroform proved to be a superior solvent for the extraction of nornicotine from tobacco. 246
0
ANALYTICAL CHEMISTRY
S
PECIFIC TIONS
NORXICOTINE
DETERMISA-
are required in biochemical and genetic studies as well as in the analysis of commercial tobacco and smoke samples. Recent studies on the alkaloid distribution in tobacco and its relation t o cigarette smoke have demonstrated the importance of nornicotine as a factor in smoke composition and flavor (6, 6, 16). Many analytical procedures for the tobacco alkaloids (1, 2, 9, 14) do not distinguish nornicotine (3-pyridyl-2-pyrrolidine) from other secondary amine alkaloids, particularly anabasine (3-pyridyl-%piperidine) and anatabine (3-pyridyl-2, A4-piperideine), which are of general occurrence in the A7icotiana species and in commercial tobacco varieties. Paper chromatographic procedures using the cyanogen bromide Konig reaction for detection and estimation of nornicotine have lacked the necessary accuracy and precision (10, 18). Background interference has been a difficulty in ultraviolet spectrophotometric determinations as applied to eluates of paper chromatograms (22, 27). A gas chromatographic method has been reported for analysis of tobacco with high levels of nornicotine (19). The column
used does not resolve nornicotine from myosmine, both of vhich are present in tobacco and smoke. The purpose of this nork was to develop convenient quantitative methods for nornicotine m-hich have adequate sensitivity, specificity, and precision for tobacco and smoke samples over the broad range of alkaloid concentrations encountered with experimental and commercial samples. Two different analytical procedures were developed and applied t o tobacco and smoke in a comparative manner. The first of these methods depends on the reaction of nornicotine with 1,a-indanedione (1,3-diketohydrindene) t o form an intensely-colored, purple product. This reaction was suggested for nornicotine analysis by Feinstein and McCabe in 1950 (3, 4 ) . The original method is not satisfactory for tobacco (24). Modification of the procedure has improved the sensitivity of the reaction and the stability of the color produced, permitting spectrophotometric determination of 5 to 100 pg. of nornicotine per 10 ml. of reaction system. The indanedione method is preferred to the isatin methods of Kuhn ( I d , 13) and of Stephens and Weybrew (21) because
