SPECTRUM 215 GACKGROUND MAX ABS INT 1'30' MASS RCNGL 10 - 280
loo
1
i
11
I
50
k
170
I
270 1%
260
250
M/E SPECTRUM 219 BACKGROUND MAX A B S IN1 ' 6 3 3 MASS RANGE 30 - 280
1
u9
I
lW1
119
-3 I
50
205 L Id0
223
L 150 M/E
260
50
250
Figure 5. Comparison on the display terminal of spectrum numbers 215 and 219 identified as methylpalmitate and phthalate, respective-' ly, and with the background number one subtracted. The spectrum numbers correspond to the numbers on the chromatograms in Figure 4.
which were identified as methylpalmitate and phthalate. By using a special command, the differences of peak intensities for these spectra are shown on the display which extracts the most characteristic peaks of each spectrum as shown in Figure 6. The mass spectrum of scan number 175 (peak No. 2) is, as suspected, similar to phenazone. Scan number 330 is identified as dextropropoxyphene (peak No. 4). Other compounds such as caffeine and several methyl esters of fatty acids were also identified. Although the hard copy unit provides an easy way to reproduce the mass spectra shown on the display, the resolution obtained with the electrostatic plotter is of much higher quality. The results from studied samples show that fast scanning magnetic mass spectrometers in combination with computers can be used to advantage in capillary column work. With the described system, the measurement of the ion intensities can be set to optimal accuracy by choosing a suitable bunching factor for each scan speed. In analysis of
100
150
200
250
b, /E
Figure 6. The difference between the two spectra presented in Fig-
ure 5, plotted on the display terminal organic compounds by mass fragmentography, this method can probably notably increase the precision of the ion intensity measurements. Another advantage with the system is that it is mainly operated using software technique and is much more flexible compared to systems which use the push-button hardware technique.
LITERATURE CITED (1) R. A. Hites and K. Biernann, Anal. Chem., 40, 1217 (1968). (2) 6.Hedfjall, P.-A Jansson, Y. MArde, R. Ryhage, and S. Wikstrom, J. Sci. instrum., 2, 1031 (1969). (3) C. C. Sweeley, B. D. Ray, W. I. Wood, J. F. Holland, and M. I. Krichevsky, Anal. Chem., 42, 1505 (1970). (4) W. E. Reynolds, B. A. Bacon, J. C. Bridges, T. C. Coburn, B. Halpern, J. Lederberg, E. C. Levinthal, E. Steed, and R. B. Tucker, Anal. Chem., 42, 1122 (1970). (5) B. Hedfjall, A Akerlind, and R. Ryhage, "Advances in Mass Spectrometry," Vol. 6, 1973. (6) R . Ryhage, Quart.Rev. Biophys., 6, 311 (1973). (7) P. M. J. van den Berg and P. H. Cox, Chromatographia,5, 301 (1972).
RECEIVEDfor review October 25, 1974. Accepted December 3, 1974. The work described was made possible by grants from the Knut and Alice Wallenberg Foundation and from the Swedish Board for Technical Development.
Use of a Nematic Liquid Crystal for Gas-Liquid Chromatographic Separation of Polyaromatic Hydrocarbons George M. Janini,' Kevin Johnston, and Walter L. Zielinski, Jr.2 NCI Frederick Cancer Research Center, Frederick, Md. 2 170 1
Gas-llquld chromatography in the nematic region of N,N'bls(p-methoxybenzylldene)-a,&'-bi-p-toluidine has shown base-line separations for geometric isomers of 3-5 ring p0lyaromatic hydrocarbons (PAH). This application is appropriate for 2-6 ring PAH compounds. The unique selectivity of this liquid phase, based upon differences in the molecular length-to-breadth ratio of solute geometric isomers, has enPresent address, University of Tripoli, Tripoli, Libya.
* Author to whom correspondence should be addressed. 670
ANALYTICAL CHEMISTRY, VOL. 47, NO. 4, APRIL 1975
able- the complete GLC separation of mixtures here fore not possible. Retention ratios for benzo[a]pyrene/benro[ elpyrene and chrysene/benz[ a]anthracene/triphenylene mixtures at 260 OC were 1.6/1.0 and 1.0/1.4/1.0, respectively. Temperature programming from 185-265 ' C resulted in the separation of sixteen 3-5 ring PAH compounds. The results of this study should be of direct significance to programs in carcinogenesis research and air pollution monitoring.
In recent years, polycyclic aromatic hydrocarbons (PAH) have been extensively studied, principally because of the exhibited carcinogenic (1, 2 ) and mutagenic ( 3 ) properties of members of this class of compounds. The possible adverse effect of these chemicals on human health is a matter of international concern. Continuing efforts have been made to elucidate the role of the various components and impurities in PAH in order to assist in the development of preventive measures and to provide a basis for assessing hazards. PAH have been detected in such diverse sources as soot, carbon black, coal-tar, pitch, mineral shale, crude oils, rubber tires, etc. More significant is the observed occurrence of PAH in environmental situations of concern to public health (e.g., tobacco smoke, smoked food, and automobile exhaust). Accurate and reliable methods for the separation and quantitative determination of these compounds in occupational, environmental, and biological samples have long been a target of many researchers. Several methods have been published and extensively reviewed by Sawicki ( 4 ) , Schaad ( 5 ) , and Hutzinger et al. (6). These methods include: column chromatography ( 7 ) , thin layer and paper chromatography ( 6 ) ,high pressure liquid chromatography ( 8 ) , UV spectroscopy (9) and many other less versatile techniques (6). However, the separation of many of the PAH positional isomers has been found difficult, if not impossible. Furthermore, these methods do not, in general, provide satisfactory analytical accuracy because of the inherent resolution limitations. It has been long considered that gas-liquid chromatography is superior to alternate techniques and more appropriate for the separation and quantitative analysis of organic compounds, provided that a suitable column is identified. GLC has played an important role in the analysis of PAH (5, 10-15). Applications of capillary GLC have been reported in the analysis of PAH and related compounds in cigarette smoke (16, I T ) , air and automobile exhaust (18, 19). Although the GLC technique has been used with varying degrees of success, in which columns for specific narrow ranges of PAH compounds have been developed (10-19), no liquid phase has been reported which wholly meets the specifications of the UlCC/IARC Joint Working Group (20, 21 ). This Group has specified that an acceptable method should be capable of separating a t least benz[a ]anthracene, benzo[a]pyrene, benzo[e]pyrene, benzo[ghi]perylene, pyrene, benzo[k Ifluoranthene, and coronene. One packed column ( 1 4 ) and another capillary column ( 1 6 ) achieved partial success; however, a mixture of anthracene and phenanthrene, a mixture of benz[a ]anthracene, triphenylene, and chrysene, or a mixture of. benz[a]pyrene, benz[e]pyrene, and perylene and benzo[k Ifluoranthene, has not been adequately separated on these or other GLC columns. It has been recognized, on the other hand, that excellent separations of rigid molecules can be achieved using nematic liquid crystals as liquid phases, which exhibit an ordered molecular arrangement within a defined temperature interval (22-25 ). This work reports base-line separations for a wide range of PAH compounds, including the socalled "benzpyrene fraction," and describes the chromatographic behavior of some PAH positional isomer pairs on such a liquid crystal stationary phase over a temperature range of 185-265 "C.
EXPERIMENTAL Chemicals. The nematic liquid crystal employed as the liquid phase, N,N'-bis(p-methoxybenzylidene)-a,cu'-bi-p-toluidine, was obtained from Eastman Kodak Co. and found to be in excess of 99% pure, as determined by differential scanning calorimetry. T h e transition temperatures, 181 "C (solid-nematic) and 320 "C
J L I
0
I
I
5
10
Time, minutes
Figure 1. Separation of 1-methylnaphthalene and 2-methylnaphth-
alene Column: 6-R X 0.25-in 0.d. Supelco glass. Packing: 20% (w/w). Conditions: Oven, 165 "C; injector, 200 "C: detector, 200 O C ; hydrogen flow 5 ml/rnin; carrier flow rate, 5 rnl/rnin. Concentration: About 0.05pg of each
(nematic-isotropic), observed upon heating, were in excellent agreement with the manufacturer's published data. The observed heats of transition, 43.5 f 0.8 kJ/mole for the solid-nematic and 7 . 5 f 0.8 kJ/mole for the nematic-isotropic transition have not been previously reported. The methyl naphthalenes were obtained from Chem Service (West Chester, Pa). All other PAH compounds used in this study were obtained from sources identified under Acknowledgment. Glass-distilled chloroform (used for the preparation of the packing material) and benzene (used for the preparation of the solutions) were purchased from Burdick & Jackson (Muskegon, Mich.). Apparatus and Procedure. A 1440 Varian gas chromatograph equipped with a flame ionization detector and a linear temperature programmer was used. The chromatograms were recorded on a 1-mV f.s. strip chart recorder, using an electrometer setting of 16 X lo-" A f.s. Nitrogen carrier gas, hydrogen and air flow rates were measured with a soap bubble flowmeter. The air flow rate was maintained a t 300 ml/min, while the hydrogen flow was usually kept a t about 5 ml/min lower than the carrier gas flow rate. Sample injection volumes of 1 pl were delivered using a Hamilton 701N 10-pl syringe. The packing material (2.5-20 wt % on 100/120 mesh HP Chromosorb W) was prepared by the solvent slurry method, fluidized drying with nitrogen, followed by resieving to 100--120 mesh. The columns were conditioned overnight a t 265 "C prior to use. The glass columns were visually inspected daily, and no deterioration or darkening of the packing was observed. One column was emptied after two-weeks use and the liquid phase retrieved in chloroform. Its UV spectrum was identical to that prior to use in GLC analysis (absorbance maxima a t 285 f 2 nm and 328 f 2 nm). Chromatography on a 2-ft X 0.125-in. 0.d. column containing 2.5% (w/w) packing, operated a t 300 O C for 72 hours evidenced some deterioration (loss in resolution) and loss of liquid phase (about 10% estimated from decreased retention volumes). In columns programmed to 265 "C bleeding was substantially diminished to insignificant and acceptable levels. The number of theoretical plates and the retention volume of anthracene on a 6-ft X 0.25-in. 0.d. column containing 2.5% (w/w) packing were reproducible to within 3% over a period of three weeks of continuous operation. The DSC analyses were performed on a Perkin-Elmer DSC-2, and the UV spectra were run on a Cary 17 by E. Barr of the Frederick Cancer Research Center. A N A L Y T I C A L CHEMISTRY, VOL. 47, NO. 4 , APRIL 1975
671
I
io
20 Time, minutes
Figure 3. Chromatogram of selected tetracyclic aromatic hydrocar-
-L____I___L_-0 5 10 Time, minutes
bons
Figure 2. Chromatogram of selected tricyclic aromatic hydrocarbons. Column: 6-ft X 0.25-in. 0.d. Supelco glass. Packing: 2.5% (wlw). Conditions: Oven, 210 OC; injector, 250 OC; detector, 250 O C ; flow rate, 18 ml/min. Concentration: About 0.05 pg of each component
RESULTS AND DISCUSSION The selection of a nematic liquid crystal as a potential liquid phase for the gas-liquid chromatographic separation of PAH compounds was based upon two particular considerations. First, the structures PAH geometric isomers in a given class (same molecular weight) differ in their lengthto-breadth ratio (differences in rod-like character). Second, nematic liquid crystals have been well known as unique liquid phases for GLC separations of positional isomers of rigid molecules (22, 26, 2 7 ) . Roughly, the more rod-like a solute molecule is, the more readily it can spatially approach, and hence more strongly interact with, a nematic solvent. The specific liquid crystal chosen (N,N'-bis(pmethoxybenzy1idene)-oc,a'-bi-p-toluidine), is unusual in its thermal stability and its selectivity for rigid molecules over a surprisingly wide temperature range. Such properties should make it uniquely appropriate for the separation of PAH compounds, their analogs, and derivatives. Furthermore, its well-defined chemical structure and its inherent capabilities for participation in 7r-type charge transfer interactions should render it useful for the evaluation of such operative forces in solutions. Either other nematic liquid crystals are too volatile for this purpose, (22, 23, 2 6 ) or other polymeric liquid phases previously employed for PAH separations are of poorly defined composition (12, 14, 16, 28). The separation of 1-methyl naphthalene from 2-methyl naphthalene is shown in Figure 1. Based upon boiling point differences alone, the a-isomer should elute last, yet the reverse is observed here. This is due to the greater length-tobreadth ratio for the @-isomer,prolonging its retention by 672
*
ANALYTICAL CHEMISTRY, VOL. 47,
1
30
NO. 4.
APRIL 1975
Column: 6-ft X 0.25-in. 0.d. Supelco glass. Packing: 5 % (w/w). Conditions: Oven, 260 OC; injector 260 OC; detector, 265 OC; flow rate 37 ml/min. Concentration: About 0.1 pg of each component except for naphthacene, which was less, but undetermined
the nematic liquid phase. The same behavior was essentially reported by Kelker and his coworkers (29) and by Chiavari and Pastorelli (231, who studied other substituted naphthalenes. Frycka ( 1 3 ) succeeded in separating phenanthrene and anthracene by GSC using graphitized carbon black, but the column was unsatisfactory for the higher molecular weight compounds such as benz[a ]anthracene and benzpyrenes. Figure 2 shows base-line separation of an anthracene, phenanthrene, and fluorene mixture with comparable resolution. Previous attempts to base-line separate anthracene from phenanthrene by GLC were unsuccessful by SJ3-52 capillary (16),OV-7 (12,14),or Dexsil-300 (28).As expected, anthracene, the more rod-like molecule, was retained longer. While the tetracyclic isomers, fluoranthene and pyrene, have been successfully separated on several columns (12, 14, 16, 28), the separation of benz[a]anthracene, chrysene, and triphenylene has been incomplete. I t is significant that this latter mixture often amounts to about 20% of the total measured PAH in urban airborne pollutants (28).This separation is of utmost importance for the quantitative measurement of benz[a]anthracene, a potent carcinogen. Figure 3 shows complete base-line separation of this mixture. While solute elution roughly correlates to the degree of solute rod-like (length-to-breadth ratio) character, it is possible that solute-solvent charge transfer interactions may contribute somewhat to solute retention. Further study is required to discern the magnitude of such contributions. The quantitative determination of the carcinogen benzo[alpyrene is of special interest to several areas of study (cancer research, pollution monitoring, petroleum fractionation, preparation of smoked food, etc.). The meaningfulness of such measurements requires the complete chroma-
0
I
I
5
10
I
I
1
1
15
20
25
30
Figure 5. Dependence of the separation factor ( a )of some PAH positional isomers on column temperature in N,N‘-bis(p-methoxybenzylidene)-a&-bi-p-toluidine nematic phase
Time, minutes
( A ) t ’ ~Benzo[a]pyrene/ t ’ ~Benzo[e] pyrene. (B) t ’ ~BenZ[a]anthraCene/t’~ Triphenylene. ( C ) t ‘ ~AnthraCene/f’R Phenanthrene. (D)t ’ ~ ChVSenelt’R Benz [alanthracene
Figure 4. Chromatogram of selected pentacyclic aromatic hydrocarbons Column: 4-R X 0.125-in. 0.d. stainless steel. Packing: 2.5% (w/w). Conditions: Oven, 260 ‘C; injector. 260 ‘C; detector, 265 ‘C; flow rate, 40 ml/ min. Concentration: About 0.2 pg of each component
Table I. Gas Chromatographic Retention Data PAHisomen
Column tern perature,
T
OC
Tricyclic compmndsb 190 200 2 10 220 240
Tetracyclic compounds‘
t’R, minutesa
-
Phenanthrene
Ant h r acene
12.9 9.6 7.5 5.6 3.2
17.6 12.9 10.0 7.4 4.1
Triphenylene 235 250 265
Pentacyclic compounds’ 260 270
14.9 10.2 9 .o
BenzlalChrysene anthracene 22.3 14.6 12.3
29.1 18.7 15.6
Benzo[e]pyrene
Benzo[n]pyrene
22.9 18.3
36.6 28.6
a Net retention time: t ’ ~ = [ t (compound) ~ - t~(benzene)]. The tricyclic compounds were run on a 6-ft X 0.25-in. 0.d. column with 5% (w/wi packing at a flow rate of 21 ml/min. The tetracyclic and pentacyclic compounds were run on a 6-ft X 0.25-in. 0.d. column with 2.5% (w,’w) packing at flow rates of 20 ml/min and 57 ml/min, respectively.
tographic resolution of benzo[a]pyrene from benzo[e ]pyrene, as well as from other PAH compounds present in the benzpyrene fraction. Of those methods recently reviewed ( 5 , 6 ) , none have fully achieved this requirement. Figure 4 shows base-line separation of benzo[a Ipyrene from benzo[e Ipyrene, and from other representatives of the benzpyrene fraction. Since benzo[k Ifluoranthene has the largest length-to-breadth ratio possible for benzo-fluoranthene isomers, no benzo-fluoranthene isomer present in real sam-
Figure 6. Chromatogram of a synthetic mixture of sixteen polycyclic hydrocarbons of wide molecular weight range Column: 4-ft X 0.125-in. 0.d. stainless steel. Packing: 2.5% (w/w). Conditions: Initial oven temperature 185 OC; initial hold, 2 min. Program rate, 4 ‘C/min; final temperature, 265 ‘C; injector, 265 ‘C; detector, 265 ‘C; flow rate, 40 ml/min. Concentration ranges from about 0.1 pg of phenanthrene to about 0.2 pg for benzo[gbi]perylene, except for naphthacene, which was less, but undetermined
ples is expected to overlap with the chromatographic peaks generated by perylene or benzo[a Ipyrene. ANALYTiCAL CHEMISTRY, VOL. 47, NO. 4, APRIL 1975
673
It is interesting to note that PAH compounds analyzed on isotropic liquid phases (12,14,16,28) resulted in elution patterns of chrysene < triphenylene and benzo[a]pyrene < benzo[e]pyrene < perylene. As shown in Figures 3 and 4, elution patterns on a nematic liquid crystal phase are, in contrast, consistent with the degree of solute rod-like character for a given set of geometric isomers. Figure 5 demonstrates the temperature dependence of selected separation factors (a),based upon the retention data and operating conditions given in Table I. While a decreases with increasing column temperature, the rate of decrease varies significantly for different isomeric pairs and is greater, the larger the difference in length-to-breadth ratio between two isomers. In addition, the larger this difference is, the larger is the value of a. The surprising selectivity of the nematic liquid crystal phase employed in this study results in large separation factors for pairs of the most troublesome PAH isomers. This is especially significant since a considerable reduction in the number of theoretical plates is possible for practical separations, and makes the use of conventional laboratory columns of 0.6-1.0 X lo3 platedfoot feasible. The broad utility of this liquid phase is illustrated in the programmed temperature separation of 16 of the most significant 3-5 ring PAH isomers as shown in Figure 6. This separation should be of immediate interest to workers in air pollution measurements of PAH components. An investigation of the thermodynamics of PAH solutes in this liquid crystal solvent is presently under consideration to quantitatively discern the relative contributions of the PAH molecular characteristics of size, shape, and n-type interaction to retention behavior.
ACKNOWLEDGMENT The authors express their appreciation to Delmo P. Enagonio of the National Bureau of Standards, and R. B. Ashworth of the United States Department of Agriculture, Ag-
ricultural Research Center, for their donation of several of the compounds reported in this study.
LITERATURE CITED (1) P. Shubik, Proc. Mat. Acad. Sci. USA, 69, 1052 (1972). (2) H. W. Gerade, "Toxicology and Biochemistry of Aromatic Hydrocarbons," Eisevier, Amsterdam, 1960. E. C. Miller and J. A. Miller, "Chemical Mutagens," A. Hollaender, Ed., Vol. 1. Plenum Press. New York. 1971. D 105. E. Sawicki, Chemist-Analyst, 53, 24, 56, 88 (1964). R. Schaad, Chromatogr. Rev., 13, 61 (1970). 0. Hutzinger, S. Safe, and M. Zander, Analabs Inc. Res. Notes, Vol. 13, No. 3 (1973). E. Sawicki, R. C. Corey, A. E. Dooiey, J. B. Gisclard, J. L. Monkman, R. E. Neligan, and L. A. Ripperton, Health Lab. Sci., 1, 31 (1970). N. F . lves and L. Guiffrida, J. Ass. Offic. Anal. Chem., 55, 757 (1972). E. Clar, Spectrochim. Acta, 4, 116 (1950). V. Cantoti, G. Y . Cartoni, A. Liberti, and A. G. Torri, J. Chromatogr., 17, 60 (1965). L. DeMaio and M. Corn, Anal. Chem., 38, 131 (1966). K . Bhatia. Anal. Chem., 43, 609 (1971). J. Frycka, J. Chromatogr., 65, 341, 432 (1972). D. A. Lane, H. K. Moe, and M. Katz. Anal. Chem., 45, 1776 (1973). A. Zane, J. Chromatogr., 38, 130 (1968). N. Carugno and S. Rossi, J. Gas Chromatogr., 5, 103 (1967). K . Grob. Chem. ind. (London), 248 (1973). G. Grimmer, A. Hildebrandt, and H. Bohnke, Erdoel Kohle. 25, 442, 531 (1972). G. Grimmer, Erdoel Kohle, 25, 339 (1972). UlCC Technical Report Series, Vol. 4 (1970). IARC Internal Techn. Rep., No. 711002 (1971). W. L. Zielinski. Jr., D. H. Freeman, D. E. Martire, and L. C. Chow, Anal. Chem., 42, 176 (1970). G. Chiavari and L. Pastorelli, Chromatographia, 7, 30 (1974). H. 2. Kelker, fresenius' Z.Anal. Chem., 190, 254 (1963). A. 8. Richmond, J. Chromatogr. Sci., 9, 690 (1971). H. Keiker and E. von Schivizhoffen, Advan. Chromatogr. 6, 247 (1968). L. C. Chow and D.E. Martire, J. Phys. Chem., 75, 2005 (1971). R. C. Lao, R. S. Thomas, H. Oja, and L. Dubois, Anal. Chem. 45, 908 (1973). H. Kelker, B. Scheurle, and H. Winterscheidt, Anal. Chin?. Acta, 38, 17 (1967).
RECEIVEDfor review July 29, 1974. Accepted October 30, 1974. This study was sponsored by the National Cancer Institute under Contract No. N01-(20-25423 with Litton Bionetics. Inc.
Gas-Liquid Chromatography System with Flame Ionization, Phosphorus, Sulfur, Nitrogen, and Electron Capture Detectors Operating Simultaneously for Pesticide Residue Analysis H. A. McLeod, A. G. Butterfield, D. Lewls, W. E. J. Phillips, and D. E. Coffin Food Research Laboratories, Health Protection Branch, Health and Welfare Canada, Ottawa, Ontario
A gas chromatograph with one column, a three-way effluent splitter and five dlfferent detectors operating slmuitaneously is described. The column was 42 In. of 5 % OV-17 on Chromosorb W HP. One outlet of the splitter directed approxlmateiy 5 0 % of the effluent to a Coulson electrolytic detector operating in the nitrogen mode; the second directed 45% to a Meipar flame photometric detector (FPD) operating as a trimdetector (flame ionization, sulfur emisslon at 394 nm, and phosphorus emisslon at 526 nm); the remaining 5 % of effluent was monitored by a Nickel-63 electron capture detector (EC 83Ni). The system determines different pesticide compounds such as organochlorine, organosulfur, organophosphorus, carbamate, triazines, and compounds such as piperonyl butoxide from a single injection of sample extract representing 50 mg of sample. Data from analysis of 674
ANALYTICAL CHEMISTRY, VOL. 47, N O . 4 , APRIL 1975
spiked plant and animal tissues are used to Illustrate the application of the system as part of a multiresidue screening procedure.
Multiple detector systems are used with gas-liquid chromatography to improve the selectivity, increase the number of compounds that can be detected, aid in confirming identity, and reduce the time of analysis. Configurations that use gas splitters with combinations of detectors such as flame ionization, electron capture, and the mass spectrometer have been reported for pesticide (1,2)and toxicological analysis (3). Two or three detectors that function simultaneously around one or two flame ionization burners have been developed in the last decade. These are the flame photometric detector (FPD) introduced by Bowman
