1
3
Figure 6. 1.OO-,d injection of human ultrafiltered serum, sensitivity X 2. Peaks (1) Injection artifact, (2) TMA, (3) DMA, (4 and 5) Unknown. Amine 220 column with NH3 conditioner. Chart speed 1 inch per 2 min
lowing the ultrafiltration procedure was 83-85% for both DMA and TMA. Separation of the methylamines in very small amounts is made difficult by their tendency to tail. Tailing seems to be related to the ability of the compound to hydrogen-bond, with tailing increasing as the ability to hydrogen-bond increases ( I 7). consequently, tailing is much more noticeable with MMA than DMA, probably because of the former having one more hydrogen atom available for hydrogenbonding interactions. Tailing is not a problem with TMA, perhaps because it lacks a free hydrogen for bonding. The elution order of the three aliphatic amines is important in regard to their separation. A column with an elution order of TMA, DMA, MMA would minimize the effects of tailing, but such a column with appropriate sensitivity has not been found. Knights (18) showed that tailing can be reduced by adding a strongly adsorbed material with the carrier. Ilkova (19) noted a substantial decrease in tailing of high molecular weight amines by using ammonia as a carrier. We chose to add a trace of ammonia rather than use it as the carrier because of the problem of fume disposal. The 8-10% increase in detector response with the ammonia conditioner installed we attribute to ammonia binding of acidic sites on the internal surfaces of the column or manifold. The column, when conditioned properly exhibited alAFS most no bleed a t 60 "C a t a sensitivity of 5 X
and thus is excellent for determinations of low concentrations. The tailing of the volatile bases which plagued even James and Martin is essentially eliminated and enables measurements of the amines a t the high picogram levels. The life of the column was almost 1 year with daily use. The following points should be emphasized: 1) scrupulous cleaning of the glass column; 2) protection of the packing from atmospheric 0 2 and COn by installing a filter drier in the carrier gas line containing 4 A, 5 A, and 13X molecular sieves and silica gel in equal amounts; 3) the addition of ammonia to the carrier; and 4) having a clean caustic-treated injector port liner.
ACKNOWLEDGMENT We thank Dan M. Ottenstein of Supelco Inc. for advice and column packings, and Robert J. Mandle and Thomas Wade for the use of the Wang computer. LITERATURE CITED (1) M. L. Simenhoff, M. D. Miine, and A. M. Asatoor, Ciin. Scb, 25, 65 (1965). (2) M. L. Simenhoff, Kidney lnternat., Suppi. No. 3, 314 (1975). (3) T. L. Perry and W. A. Schroeder, J. Chromatogr. 12, 358 (1963). (4) A. M. Asatoor, J. Chromatogr., 4, 144 (1966). (5) K. Biau, Biochem. J., 80, 193 (1961). (6) N. Seiler and M. Weichman, "Progress in Thin Layer Chromatography and Related Methods", Vol. i, A. Neiderwieser and G. Patahl, Ed., AnnArbor Humphrey Sci. Pub., Ann Arbor, Mich., 1970, pp 94-144. (7) J. Spinelli et ai., J. Food Sci., 29, 710 (1964). (8) A. T. James, A. J. P. Martin, and G. Howard Smith, Biochem. J., 52, 238 (1952). (9) Y . L. Sze, M. L. Borke, and D. M. Ottenstein, Anal. Chem., 35, 240 (1963). (10) G. R. Umbreit, R. E. Nygren, and A. J. Testa, J. Chromatogr., 43, 25 (1969). (11) E. H. Gruger, Jr., J. Agric. FoodChem., 20, 781 (1972). (12) C. E. Andre and A. R . Mosier, Anal. Chem., 45, 1971 (1973). (13) A. Miller Ill, et at., J. Agric. FoodChem., 21, 451 (1973). (14) A. DiCorcia, Denes Fritz, and F. Bruner, Anal. Chem., 42, 1500 (1970). (15) A. DiCorcia and R . Samperi, Anal. Chem., 46, 977 (1974). (16) E. D. Smith and A. B. Gasnell. Anal. Chem., 34, 438 (1962). (17) D. M. Ottenstein, J. Chromatogr.. Sci., 11, 136 (1973). (18) H. S. Knights, Anal. Chem., 30, 2030 (1958). (19) E. L. ilkova and E. A. Mistryukov, J. Chromatogr., 54, 425 (1971).
RECEIVEDfor review April 23, 1975. Resubmitted July 30, 1975. Accepted September 29, 1975. This study was supported by NIAMDD grant 72-2209.
Bound-Monolayer Cation Exchanger for Gas-Liquid Chromatographic Separation of cis and trans Alkenes Paul Magidman, R. A. Barford,
D. H. Saunders, and H. L. Rothbart"
Eastern Regional Research Center, Agricultural Research Service, U S . Depattment of Agriculture, Philadelphia, Pa. 19 1 18
The sulfobenzyl derivative of Porasil C was synthesized, converted from the H+ to the Ag+ form, and used for the first time as a stationary phase in gas chromatography. It showed marked selectivity for alkenes relatlve to alkanes. Retention times of the former were reduced by treatment of the column packlng wlth hexamethyldisllatane and coating with ethylene glycol succlnate. The resultant column packing was useful for the analytical separatlon of trans-2-hexene, cis4-hexene, trans-2-octene, cis-2-octene, trans-5decene, cis-5-decene, trans-3-dodecene, cis-3-dodecene, trans-7-tetradecene, cis-7-tetradecene, trans-9-octadecene, and cis-9-octadecene, and was stable in excess of 10 weeks at temperatures ranging from about 100-180 ' C . 44
*
ANALYTICAL CHEMISTRY, VOL. 48, NO. 1, JANUARY 1976
Silver ion-containing materials have been used to provide specific interactions with r-electron cloud systems in a variety of chromatographic approaches ( I ). Gas chromatographic applications of argentation have been limited to species of low molecular weight, because of the lack of liquid phases which provided adequate solvent power for silver compounds and which also had low volatility and high thermal stability. In gas-solid chromatography, the strong interaction of double bonds with silver ion requires elevated temperatures. For example, the elution of hexenes from columns of Chromosorb W or P impregnated with silver nitrate requires temperatures of 140-220 "C ( 2 ) and elution of these compounds from macroreticular ion exchange res-
2
Table I. Properties of Porasil a n d Some ' Surface-Bound Derivatives m eq uiv/gra rn Bound Moiety ElemenCompound
%C
tal anal.
Porasil C Silica chloridea Benzylsilicaa Sulfobenzylsilicaa Silica chlorideb Benz ylsilicab Sulfobenzylsilicab Methylsilica
...
...
... ...
2.68 1.58
0.32 0.19
...
2.90 2.13
0.85
...
Titration
0.01 (Hf) 0.36(C1-) 0.16'(Hf) 0.41 (Cl-)
0.34 0.25 0.70
0.25'(H+)
...
Surface areac, m2/g
106.0
... 95.9 111.5
...
...
... 88.6
Preparation using tribenzyltin chloride pathway. b Preparation using benzyl m e t h y l ether pathway. C Determination b y argon adsorption procedure. a
ins i n the Ag+ form requires temperatures of 160-190 "C ( 3 ) .In this work, we demonstrate the utility of a bound monolayer cation exchanger in the Ag+ form for the separation of higher alkenes, some of which are potent insect sex attractants (pheromones) ( 4 ) . Related stationary phase materials have been shown to be useful i n liquid and t h i n layer chromatography (5), but this is the first disclosure of their properties i n gas-chromatography.
EXPERIMENTAL Materials. Tribenzyltin chloride was prepared according to the method of Sisido (6) or purchased from Ventron Corporation. Methyllithium was used as a 1.65 N solution as supplied by Ventron Corporation. All other solvents and reagents were ACS grade or purified as described in a prior publication ( 7 ) . Porasil C, 80-100 mesh, Waters Associates, was Soxhlet extracted with constant boiling HC1 (20.2%) until the extractant was colorless (- 48 hr). The silica was washed until neutral on a fritted Buchner funnel, then washed several times more with acetone, followed by hexane. The air-dried silica was heated to 150 "C for 24 hr before use. Most of the hydrocarbon solutes were purchased from Chemical Samples Company. The octenes and tetradecenes were purchased from Pfaltz and Bauer Company, and the octadecenes were synthesized in-house. Stationary phases for gas chromatography (EGS and OV-1) and hexamethyldisilazane were purchased from Applied Science Laboratories, Inc. Syntheses. Sulfobenzyl Porasil C was synthesized using the pathway previously described ( 7 ) .An alternate procedure was also used (8). It consisted of the dropwise addition of 50 mmol of methyllithium (Methyllithium must be handled cautiously. All auxiliary glassware was soaked in absolute ethanol or isopropanol before residual solid residues left by evaporation of ether could react with moisture in air.) solution to 12 mmol of tribenzyltin chloride in 200 ml of dry ethyl ether with continuous stirring. After 4 hr, the solution was cooled in an ice bath and 6 mequiv of silica chloride added. After 5 min, the bath was removed and the reaction allowed to proceed at 23 "C for 20 hr. Several times during the period, the suspension was boiled and quickly cooled to 0 "C to aid in transfer of reagent into the silica pores. The reactions were carried out in a 500-ml round-bottom flask, which was fitted with a condenser, gas inlet tube, stirrer, and dropping funnel that had been dried and flushed with dry argon before the experiment had begun. Dry argon was bled into the reaction flask throughout the procedure. The reaction mixture was acidified and the benzylsilica isolated and analyzed as described previously (7). Gas Chromatography. A Varian Model 1520 gas chromatograph with hydrogen flame ionization detector was used. Columns were constructed of type 304 stainless steel tubing, 61 cm long, 1.6-mm i.d., 3.2-mm 0.d. and packed for a length of 50 cm. The empty portion extended into the injection port to a point just short of the septum. Packing weights were approximately 0.5 g. Detector and injector temperatures were 267 and 244 OC, respectively. Gases were "Zero Grade" except in the cases noted where the helium carrier was of a special high purity supplied by Airco as Grade 6. It contained total impurities of 1 part per million and was par-
ij
6T
a
IT
0
c Y v
c Y a
u
0
-
1
6
I
16
4
30
60
TIME, MINUTES
Figure 1. Chromatograms of (a) straight chain hydrocarbons, (b)cisand trans-2-hexenes, (c) cis- and trans-4-octenes. Column and conditions: silver sulfobenzyl Porasil C; column temperature, 188 "C: sample size, 1 PI of CS2 solution containing -10 mg/ml: helium flow rate, 20 cm3/min; 1.2 X IO-" A full scale (range 0.1, attenuation 4X)
ticularly low in hydrogen (0.2 ppm). Carrier gas flows are reported in cm3/min measured with a Matheson mass flowmeter, Model LF-100. Preparation of Coated GC Packings. One gram of sulfobenzyl Porasil C (0.16 mequiv/gram) was packed into 41 cm X 6.35-mm o.d., 304 stainless steel tubing, and installed in the chromatograph with the column outlet not connected to the detector. A helium flow of 30 ml/min, an injector temperature of 240 "C, and a column oven temperature of 200 "C were used, and then two 100-11charges of hexamethyldisilazane were injected into the column in 30min intervals. The operating conditions were then maintained for approximately 24 hr. The silanized product was removed from the stainless steel tubing and lightly packed into a 40-cm length of 5-mm i.d. glass tubing. Tha following acid treatment was carried out to ensure that all the sulfobenzyl groups were in the H+ form, since some might have been converted to the NH4+ form as a result of formation of NH3 during silanization. The column packing was washed first with aliquots of 0.1 N "03 totaling 50 ml, and then with water until the effluent was acid-free. At this point, the column was wrapped with aluminum foil to avoid reduction of Ag+. A silver nitrate solution, 1 g AgN03 in 50 ml H20, was passed through the column in several aliquots totaling 50 ml. This was followed by a water wash until the effluent was free of Ag+, as demonstrated by tests with KC1 solution. A stream of dry nitrogen was passed through the column until the packing was visibly dry. Final drying, after removal from the column, was carried out in a vacuum oven a t approximately 70 "C for several hours at a pressure of 150-170 mm (absolute) of dry nitrogen. The extent of conversion to the Ag+ form was monitored by combining the effluents from the AgN03 and subsequent water washes, adding concentrated KC1 solution, filtering the AgCl on a Buchner funnel, washing the filter cake thoroughly, and titrating the free Hi' in the filtrate with 0.02 N NaOH. The resulting silver sulfobenzyl Porasil C (AgSP) was coated with stationary phase using CHlClZ as the solvent.
RESULTS AND DISCUSSION Table I indicates that similar yields of benzylsilicas were obtained regardless of which m e t h o d of benzyllithium preparation was used. It has been postulated ( 7 ) that the lithium methylate produced b y the reaction of Li with benzyl methyl e t h e r reacts with silica chloride and is subseq u e n t l y hydrolyzed, since only about 50% replacement of chloride groups is observed when Davison 62 or Syloid 72 silica chlorides are t r e a t e d with benzyllithium. The extent of benzylation of macroporous silica chloride i n t h i s s t u d y has been increased to more than 80% b y both pathways. F u r t h e r reaction of the surface chlorides m a y be inhibited by steric hindrance of bound benzyl groups, although some m e t h y l groups could also be b o u n d in the current synthesis. To explore this further, methyllithium was reacted with silica chloride. While the error i n % C determination (f0.2%) m a k e s i t impossible to s a y for certain that greater coverage ANALYTICAL CHEMISTRY, VOL. 48, NO. 1, JANUARY 1976
45
8-
CIS 2 - H E X E N E
0
100
50
150
2
I
CORRECTED R E T E N T I O N T I M E , SECONDS
3
RESOLUTION
Figure 2. Effect of hexamethyldisilazane treatment on the retention time of cis-2-hexene and the resolution of a mixture of cis- and trans-2-hexenes on a silver sulfobenzyl Porasil C (0.16 mequiv) column with a helium carrier flow of 20 cm3/min
I
//
5 % 0v-I
Figure 4. Chromatogram of an alkene test mixture in CS2 Concentration, -70 mg/ml. (1) trans-2-hexene, (2) cis-2-hexene, (3) trans2-octene, (4) cis-2-octene, (5)transd-decene. (6)cisddecene, (7) trans-3dodecene, (8) cis3dodecene, (9) trans-7-tetradecene, (10) cis-7-tetradecene, (1 1)frans-9-octadecene.and (12) cis-9-octadecene. Sample size, 2 +I. Column, 12.9% EGS polyester on silanized silver sulfobenzyl Porasil C. Helium (high purity) flow, 30 ml/min. 9 6 X A full scale (range 1, attenuation 32X)
Table 11. Analysis of a Mixture of Alkenes by GLC Known wt, %
101
6
I
/
I
IO NUMBER
I
OF
I
I4 CARBONS
I
I
I8
Figure 3. Variation of log corrected retention time with number of carbons in straight chain alkenes on various silver sulfobenzyl Porasil C column packings at 188 OC. Volumes indicate amount of hex-
amethyldisilazane used in packing preparation. Helium carrier flows, 20-30 cm3/min was obtained, its value and the lower surface area value [by argon adsorption determination (911 indicate that fewer silanol groups remain when the methyl group is bound than when the larger benzyl group is bound. When used as packings for gas chromatography, the two types of preparations of sulfobenzyl Porasil C performed identically. Figure 1 demonstrates that the silver form of sulfobenzyl Porasil C has a greater attraction for alkenes than alkanes, and that cis alkenes interact more strongly with the stationary phase than do their trans isomers. In contrast, hydrogen-form sulfobenzyl Porasil C does not give such selectivity or extent of retention. Retention times for homologues higher than octene on AgSP were too long to be of practical utility even though the column length was no more than 50 cm; thus a number of approaches were investigated in order to lower retention times. The abundance of surface silanol groups has been evaluated (10) as about 8 pmol/m2 which is greater than the amount of sulfobenzyl groups bound to the surface in this study by a factor of 3 to 5. Only about half of the surface silanols are reactive, however (11).To reduce the number of free silanol groups and the extent of interaction between the stationary phase and solutes, the column was treated with hexamethyldisilazane (HMDS). Separations of cis from trans hexenes were then evaluated as depicted in Figure 2. Retention times decreased significantly as did resolution, but after 700 pl of HMDS had been added, resolution was still acceptable. (Resolution, R , was determined by the equation, R = 46
*
ANALYTICAL CHEMISTRY, VOL. 48, NO. 1, JANUARY 1976
trans-2-Hexene 5.20 cis- 2-Hexene 7.64 trans-2-Octene 8.13 cis- 2-Octene 8.10 trans-5-Decene 9.37 cis- 5-Decene 9.49 trans-3-Dodecene 11.15 cis- 3-Dodecene 8.14 trans-7-Tetradecene 8.39 cis- 7-Tetradecene 2.10 trans-9-Octadecene 11.19 cis- 9-Octadecene 11.19 Average of 5 determinations.
+
Determine3 wta, %
4.51 7.35 7.72 7.78 10.77 9.93 12.31 8.73 8.49 1.81 10.94 9.46
0
0.39 0.23 0.12 0.11 0.14 0.11
0.20 0.27 0.28 0.29 0.54 0.40
2Atd(wa W b ) , where A ~ R is the difference in retention time between any two peaks a and b , and wa and W b are the base-line widths of the respective peaks.) Retention times for higher alkenes were still prohibitive (Figure 3). Attempts were made to further reduce retention by coating the AgSP with typical liquid phases. A coating of 5% (w/w) OV-1 reduced retention only slightly, although a 5% (w/w) coating of EGS caused a marked reduction, presumably through the interaction of the carbonyl groups with Ag+. Increasing the loading of EGS decreased retention with concomitant reduction of resolution similar to the trend shown in Figure 2. The best compromise was achieved with the use of a column packing which had been treated with 100 pl of HMDS and coated with 12.5% EGS. A separation based upon this column packing is depicted in Figure 4. The rather short column necessitated the use of low temperatures at the start of the elution. The climb in base line over the 160-188 O C temperature range was due to the expected bleed of EGS. The utility of the separation for analytical work may be evaluated from the accuracy and precision reported in Table 11. The determined weight percent is the average of five experiments and is the area percent of the GLC peak corresponding to a particular solute. Empirical data modifiers such as response factors were not used. Stability of the packing is adversely affected by the pres-
c
,
0
L
0
2 20 30 T I M E , 3AYS
40
Flgure 5. GC retention and resolution data for a mixture of cis- and trans-9-octadecenes on a 12.5% EGS polyester on silanized silver sulfobenzyl Porasil C column using 99.9999% helium carrier at a flow of 30 cm3/min. Column temperature, 180 OC
ence of H2 in the carrier gas. Zero Gas grade helium contains about 1 ppm of H2 (12)which reduced the Ag+ to Ago in 10 days of operation at 180 OC under the conditions of chromatography. The packing was removed from the column, extracted with CHzClz and Ago removed with 5 N "03. About 70% of initial capacity of the ionogenic groups remained. In order to determine the effects of H:! in the carrier, Airco No. 6 grade helium was utilized. Each day for 44 days, over a total period of 10 weeks, two samples of a mixture of the cis- and trans-9-octadecenes were injected into the column. Figure 5 summarizes the experiment and depicts an initial decrease in retention time, relative retention, and resolution although an adequate separation was achieved throughout the course of the study. When the column was operated above 180 "C, alkene retention times decreased, but bleeding of the EGS polyester increased rapidly with temperature and loss of resolution was accelerated. When this loss of resolution becomes excessive, the column packing may be removed and regenerated as the Ag+ form. The extent of interaction of solutes with the surface of the supposedly inert support in GLC is often debated. On
the basis of this study, there can be no doubt that the surface does interact as is evidenced by the effect of HMDS upon retention. In addition, the difference in interaction of AgSP with respect to the hydrogen-form support indicates that solutes penetrate the liquid coating and approach near to the surface. The surprising extent of coating penetration was apparent when cis- and trans-9-octadecenes were separated in 30 min a t 188 "C with R = 1.45 on HMDS-treated AgSP at 15% loading of EGS. When a column with 20% EGS was used, a resolution of 0.57 at 180 "C was obtained. Thus, the approach described here has utility for analytical separations, provides a probe for the study of the migration of a solute through a liquid phase, and may be used to measure interactions of volatile solutes with surface-bonded groups.
ACKNOWLEDGMENT The authors thank T. Foglia of our Center for the samples of cis- and trans-9-octadecenes, and M. A. Kaiser, M. O'Brien, and R. L. Grob, Villanova University, for determining the surface areas reported herein.
LITERATURE CITED (1) 0.K. Guha and J. JanAk, J. Chromatogr., 66,325 (1972). (2) J. J. Duffield and L. 8. Rogers, Anal. Chem., 34, 1193 (1962). (3) R. F. Hirsch, H. C. Stober, M. Kowblansky, F. N. Hubner, and A. W. O'Conneii. Anal. Chem., 45, 2101 (1973). (4) J. J. Vollmer and S. A. Gordon, Chemistry, 47, 6 (1974). (5) R. A. Barford, L. T. Olszewski, D. H. Saunders, P. Magidman, and H. L. Rothbart, J. Chromatogr. Sci., 12,555 (1974). (6) K. Sisido, Y. Takeda, and 2 . Kinugawa, J. Am. Chem. SOC., 83, 538 (1961). (7) D. H. Saunders, R. A. Barford, P. Magidman, L. T. Olszewski, and H. L. Rothbart, Anal. Chem., 46, 834 (1974). (8)D. Seyferth, R. Suzukl, C. J. Murphy, and C. R. Sabet, J. Organomet. Chem., 2,431 (1964). (9) M. A. Kaiser, M. O'Brien, and R. L. Grob, Amer. Lab., June-July (1975). (10) K . Unger, Angew. Chem., In?.Ed. Engl., 11, 267 (1972). (11) V. Y . Davydov, L. T. Zhuralev, and A . V. Kieselev, J. Phys. Chem., USSR, 38, 1108 (1964). (12) Private communication from Liquid Carbonic Corp., Dulty's Lane, Burlington, N.J. (1974).
RECEIVEDfor review June 27, 1975. Accepted October 2, 1975. Mention of commercial products does not constitute an endorsement by the United States Department of Agriculture over others of a similar nature not mentioned,
Chromatographic Determination of Vinyl Chloride in Tobacco Smoke Dietrich Hoffmann, * Constantin Patrianakos, and Klaus D. Brunnemann Division of Environmental Carcinogenesis, American Health Foundation, Valhalla, N.Y.
Gio
10595
B. Cor1
Division of Cancer Cause and Prevention, National Cancer Institute, Bethesda,
A chemical-analytical method has been developed for the quantitative determination of vinyl chloride (VC) in tobacco smoke. VC from the mainstream smoke Is trapped on charcoal, extracted, and subsequently converted to 1,2-dibromo-1-chioroethane (De-VC). The latter Is enrlched by column chromatography and determined by gas liquld chromatography using an electron capture detector with a high sensitivity for DB-VC. From the malnstream smoke of a popular 85-mm clgarette without filter tip, we isolated 12.2 ng of VC per cigarette. The VC content in the smoke of
Md. 200 14
some domestic and forelgn cigarettes and little cigars ranged from 5 to 27 ng, and that of-a marljuana cigarette was 5.4 ng. The analytlcai data suggest that the total inorganlc chloride in tobacco is a determining factor for the amount of VC in the smoke. VC may also be released into our respiratory environment during the burning of other chlorine-containing organic matter.
Vinyl chloride (VC) is causally associated with angiosarcoma of the liver in VC workers ( I , 2 ) . Exposure of mice, ANALYTICAL CHEMISTRY, VOL. 48, NO. 1, JANUARY 1976
47
