Anal. Chem. 1980, 52, 736-740
736
assistance of John Potter concerning the design and construction of the test atmosphere system. (6) (7) (8)
LITERATURE CITED ( I ) Horiguchi, M. J . Osaka City Med. Cent. 1960, 9 ,5257-93. (2) Sandage, C. "Tolerance Criteria for Continuous Inhalation Exposure to Toxic Material - 11. Effects on Animals of 90day Exposure to H,S, Methyl Mercaptan, Indole, and a Mixture of H,S, Methyl Mercaptan, Indole and Skatole", Report #ASD-TR-61-519 (11); Biomedical Laboratory, Aerospace Medical Laboratory, Aeronautical Systems Division, Air Force Systems Command, United States Air Force: Wright-Patterson Air Force Base, Ohio, 1961; 30 pp. (3) Shults, W. T.; Fountain, E. N., Lynch, E. C. J . Am. Med. Assoc. 1970, 211. 2153-54. (4) "Threshold Limit Values for Chemical Substances and Physical Agents in the Workroom Environment"; American Conference of Governmental Industrial Hygienists: Cincinnati, Ohio, 1978; p 21 (5) "Criteria for Recommended Standard ....Occupational Exposure to nAlkane Mono Thiols, Cyclohexanethiol, and Benzenethiol"; U S . De-
(9) (10) (1 1)
partment of Health, Education, and Welfare, Public Health Service, Center for Disease Control, National Institute for OccuDational Safety and Health: Cincinnati, Ohio, 1978; p 3 Cave, G. C. 8. Tappi1963, 4 6 , 15-20. Charlton, J.; Sarteur. R.; Sharkey, J. M. OilGas J . 1975, 73, 96-100. Moore, H.; Helwig, H. S.; Graul, R. J. Am. Ind. Hyg. ASSOC.J . 1960, 21. 466-70. Fekistein, M.; Balestrieri, S.;Levaggi, D. A., J . Air. Poliut. ControlAssoc. 1965, 15, 215-17. Okita, T. Atmos. Environ. 1970, 4 , 93-102. Kendall, W. G.; Stuart, A. "the Advanced Theory of Statistics", Vol. 1; Charles Griffen and Co., Ltd: London, 1963; p 54.
RECEIVED for review October 29, 1979. Accepted January 17, 1980. This work was supported in part by the Grossman fund, by USPHS Grants 5 TO1 OH00020-12, and 08SlRR05441A18, and by the Northern California Occupational Health Center.
Capillary Gas Chromatography with Ultraviolet Spectrometric Detection M. Novotny" and F. J. Schwende Chemistry Department, Indiana University, Bloomington, Indiana 47405
M. J. Hartigan' and J. E. Purcell Analytical Department, The Perkin-Elmer Corporation, Norwalk, Connecticut 06068
A gas-phase UV absorption detector with a 50-pL cell volume and variable-wavelength capabllity is ideally suited for the selective detection of aromatic solutes emerging from widebore capillary columns. Capillary columns with smaller diameters can also be used but with certain sensitivity sacrifice. The descrlbed applications of capillary GC/UV-detection are UV-absorbing compounds in gasoline, volatiles of physiological fluids, and polycyclic aromatic mixtures. Utilization of this detector can be extended to chromophore-tagged molecules, as demonstrated with benzyl esters of fatty acids. The described UV detector has good sensitivity and response linearity.
Among the many selective detectors used today in chromatographic analysis, various optical spectroscopic detectors play a n important role. These include fixed-wavelength monitors as well as more recently introduced scanning devices a n d imaging detectors. T h e information sought with such instruments may range from simple quantitative analyses of selected components in complex sample matrices up to an acquisition of entire optical spectra during a chromatographic run. While optical detectors have been very common in liquid chromatography, only a few studies have dealt with gas-phase optical spectroscopic detectors to date. With the technological advances of Fourier-transform IR spectroscopy during the past several years ( I ) ,new identification possibilities are becoming attractive in the fields of both gas and high-performance liquid chromatography. In addition, Hausdorff (2)has demonstrated t h a t a simple selective IR monitor can be a useful device in GC peak identifications. When a nondispersive IR instrument is manually tuned to measure absorbance in a given spectral 'Present address: D e l t a Associates, Inc., M i l p i t a s , Calif. 95035. 0003-2700/80/0352-0736$01 .OO/O
region, various sample components can be selectively traced in complex mixtures. Although structural information potentially obtained with UV absorption instruments is considerably less distinct, somewhat similar considerations are applicable. Uses of heated gas-phase detection cells for both fluorescence (3-7) and UV measurements (8, 9) have been investigated. Kaye evaluated the basic instrumental variables involved in UV detection for packed columns, including the possibilities of repetitively scanning a narrow spectral range in 20-s intervals (8). While the detector sensitivity was better than for a typical thermal conductivity cell, the figures are about 2-3 orders of magnitude different from the measurements obtained in this report. Merritt e t al. (9) developed their UV gas-phase monitor as an integral part of a process stream analyzer. The selectivity of their detector was found advantageous for a petrochemical problem, as aromatics could be selectively traced in the presence of other hydrocarbons. In this study, a standard variable-wavelength UV-visible detector for HPLC was modified to permit an effective coupling of such a detector to GC capillary columns. A 50-pL detector cell permitted an easy coupling with high-efficiency glass capillary columns, while its sensitivity was about three orders of magnitude greater than the previously described devices of this type (8, 9). T h e limit of detection for naphthalene was determined and compared to typical flame-ionization detection data under similar chromatographic conditions. Several applications of capillary GC with this detector are demonstrated. These applications attest to unique capabilities of the detector in typical high-resolution GC analyses. These include detection and/or quantitation of aromatic compounds in complex sample matrices such as gasoline or the concentrate of volatiles in human urine. While wide-bore (0.7-mm i.d.) capillary columns are ideal to use with this detector, the problems of excessive cell volume @ 1980 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 52, NO. 4, APRIL 1980
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Table I. Sensitivity Data for Naphthalene at 212 nm, % T Mode amount injected, ng 5 3 2 1
0.5 0.2
1
signal, cm 46.96 32.60 25.46 12.40 7.28 2.29
noise, peakpeak, cm 1.68 1.40 1.40
1.29 1.21 1.34
SIN
27.95 23.29 18.19 9.61 6.02 1.71
maximum concn. in peak, nghL 13.0 8.57 5.71 2.86 1.30 0.44
b 2 Flgure 1. Schematic of the gas-phase, small volume UV cells. Q =
quartz window. Dimensions, in millimeters, are approximate can also be overcome for conventional (0.25-mm i.d.) glass capillary columns through the use of a purge gas. An example is shown that involves chromatography of polycyclic aromatic hydrocarbons (PAH). Finally, it will be demonstrated that applications of the UV gas-phase detector can be extended to classes of compounds which are easy to “tag”with a UV chromophore. Benzyl esters of carboxylic acids with a medium chain length were prepared t h a t chromatograph easily on glass capillary columns.
EXPERIMENTAL Chromatographic System a n d Measurements. A PerkinElmer Sigma 3 gas chromatograph was modified with an injection port designed for precolumn sampling procedures (10, 111, and a Porter Instrument Co. VCD 1000 flow controller to control the low carrier gas flow-rates with the wide bore glass capillary columns. Wide-bore glass capillary columns (0.7-mm i d . ) were prepared with different lengths and stationary phases, using the “whisker” surface modification (12)and the mercury-plug dynamic coating procedure ( 1 3 ) . The outlets of capillary columns were connected either to the standard flame ionization detector (FID) or the Perkin-Elmer GC-55 variable-wavelength monitor. When using a 0.25-mm i.d., glass capillary column coated with SE-52 (14),preheated helium purge gas (6 mL/min) had to be applied in a mixing tee to overcome the detector dead-volume problems. A 50-pL detection cell was constructed as a compromise between the detector sensitivity and the necessity to preserve chromatographic resolution. Constructional details of the gasphase UV cell are shown in Figure 1. The cell material is stainless steel. For sensitivity determinations, naphthalene stock solution was prepared (500 ng/gL of hexane) and appropriate dilutions were made. The wavelength setting was optimized experimentally and was found to be 212 nm which closely corresponds to the reported value of 210.6 nm (15)for naphthalene in the vapor phase. A 32 m X 0.7 mm i.d. glass capillary column coated with UCON 50 HB2000 was used for these measurements. The limit of detection for naphthalene was determined by plotting the signal-to-noise ratio vs. the total amount of naphthalene in the injection. A concentration value was calculated to account for peak volume, enabling the detection limit to be expressed in a term independent of chromatographic parameters. Applications of Capillary GC a n d UV Detection. The applications of the capillary GC/UV detector were explored with a wide variety of samples. Lead-free gasoline (0.1pL) was directly injected into a 32 m X 0.7 mm i.d. glass capillary column coated with UCON 50 HB2000 and detected by the flame ionization detector and the UV gas-phase detector operated at two different wavelengths. Volatiles from normal human male urine were trapped on a Tenax GC precolumn using techniques developed in this laboratory (10, 16) and chromatography of the volatiles on the same wide-bore UCON column described above was
c
f %
V
Figure 2. Detection of 1 ng of naphthalene (injected in hexane) at 210
nm, demonstrating signal-to-noise ratio at 8 X signal attenuation monitored by the W detector at two different wavelength settings. A conventional (0.25-mm i.d.) glass capillary column coated with SE-52 was coupled to the UV detector and a standard mixture of polynuclear aromatic hydrocarbons containing approximately 30 ng of each compound was traced by the flame ionization and the UV detectors. In order to extend utilization of GC/UV detection, certain chromophores can be introduced into otherwise nonabsorbing molecules through a suitable chemical reaction. As an example, benzyl esters of C8-CI8 fatty acids were prepared and chromatographed on a 40 m X 0.7 mm i.d., glass capillary column coated with FFAF’ (from Supelco, Inc.). Benzyl esters were formed using benzyl bromide in methylene chloride and tri-n-but.yl ammonium hydroxide in a two-phase reaction system (17, 18). A standard solution of c&8 monocarboxylic acids was prepared by weighing the proper amounts of pure acids and diluting the mixture with methylene chloride to give an approximate 0.01 M concentration for each acid. To a 1-mL aliquot of the above standard solution the following reagents were added: (1)3 mL of aqueous 0.3 M tert-n-butyl ammonium hydroxide; and (2) 100 pL of 1 M benzyl bromide in methylene chloride. The reaction was carried out in a screw-capped vial with Teflon liner at room temperature with continuous, vigorous stirring for 60 min. In addition to the standard acids, a sample of coconut oil was subjected to a similar procedure. The coconut oil was saponified in alcoholic KOH (19) and the free acids recovered by ether extraction after acidification and dilution with water. A 20-wL aliquot of the recovered acids was reacted with 200 pL of the benzyl bromide solution and 3 mL of 0.3 M tert-n-butyl ammonium hydroxide. A one-tenth dilution aliquot of the reaction product was chromatographed.
RESULTS Sensitivity. T h e results for the sensitivity determination are shown in Table I and represent mean values from triplicate measurements. T h e detection limit at S / N = 3 is approximately 300 pg, corresponding t o a concentration of about 1 ng/mL. Under identical chromatographic conditions, the detection limit for naphthalene using FID was 20 pg. Figure 2 shows a 1-ng naphthalene peak as well as the nature of detector noise.
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ANALYTICAL
CHEMISTRY, VOL. 52, NO. 4,
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A
C
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Q. . . . : . . 2--0 . . , ’
30
,
70
..
tl-oy . . . . IIO
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Figure 3. Chromatographic analyses of a lead-free gasoline sample under different detection conditions. (A) Flame ionization detector, (B)the UV detector adjusted to 220 nm, and (C) the UV detector adjusted to 260 nm
Applications. Chromatograms for the variety of samples previously described are shown in Figures 3 through 6. In the experiment in which a conventional (0.25-mm i.d.) glass capillary column and helium purge (6 mL/min) was used to chromatograph PAH standards, no substantial decrease in performance was observed (Figure 5 ) . However, the detector, as currently constructed, is better suited to wide-bore capillary columns because of their intrinsically high flow-rates and greater sample capacity. Figure 6 shows the chromatograms for the acid standards and the saponified coconut oil recorded at 200 nm. The mean detection limit calculated a t the signal-to-noise ratio equal
to 3, is 0.4 nmol. The difference between the response of the UV detector to naphthalene and the derivatized acids can largely be explained on the basis of their molar absorptivity values: approximately lo5 for naphthalene, but only 5 X lo3 for the benzyl esters.
DISCUSSION Since the optical spectroscopic detectors are basically of a concentration-sensitive detector type, advantages of using capillary columns are obvious. Much of the sensitivity improvement can be directly attributed to the low flow-rates used in this work. While the 50-pL cell has been chosen as a
ANALYTICAL CHEMISTRY, VOL. 52, NO. 4, APRIL 1980
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739
A
I B
Figure 4.
20
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70
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Separation and detection by the UV detector of the volatile constituents of a normal human urine
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(A) 205 nm and (B) 280 n m
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Flgwe 5. Chromatograms of a standard mixture of polycyclic aromatic hydrocarbons: (1) naphthalene, (2) biphenyl, (3) fluorene, (4) phenanthrene, (5) anthracene, (6) 9-methylphenanthrene, (7) fluoranthrene, (8) pyrene, (9) 1-methylpyrene, (10) chrysene and triphenylene, (11) b e n z o [ e ] p y r e n e , (12) b e n z o [ a ] p y r e n e , (13) perylene, and (14) 1,2,3,4dibenz[a,c]anthracene. (A) Flame ionization detector, (B) the UV detector at 200 n m
Figure 6. Chromatogram of benzyl esters of: (A) 1, octanoic; 2, nonanoic; 3, decanoic; 4 , lauric; 5 , palmitic; 6, stearic acids; and (B) saponified coconut oil. UV detector ajusted to 200 n m
compromise between the sensitivity and the cell volume, smaller cells could be constructed. Alternatively (as shown in this work), additional carrier gas can be introduced a t the column end to avoid dead-volume problems with conventional 0.25-mm i.d., capillary columns; the detector response is
“diluted” correspondingly, however. The selectivity of the UV detector can be advantageous in a number of applications. Numerous molecules can be detected a t shorter wavelengths, while primarily aromatics will exhibit selective responses between 25ct300 nm This is well
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ANALYTICAL CHEMISTRY, VOL.
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illustrated in Figure 4. According to our previous experience with the volatile components of human urine, the UV detector a t 205 nm behaves almost like a general-purpose detector. However, fewer components are detectable a t 280 nm from the same sample. Following the concept of “metabolic profiles” (multicomponent biomedical determinations), this detector should find applicability in selectively tracing certain biological aromatic molecules. Its power could be best utilized in a arrangement parallel to the flame ionization detector, in a fashion similar t o the previously described multidetector instruments (20, 21). I n a different group of applications, it is highly desirable to have a UV detector selectively tracing certain molecules, while “ignoring” other interfering substances in a complex sample. In particular, aromatic molecules of both low and high molecular weight are now recognized to be of considerable environmental importance. Yet, such substances are frequently present in complex sample matrices with “less important” compounds. An application of the UV detector in the gasoline sample analysis, as shown in Figure 3, may be quite typical of such a situation. While every organic component within the given volatility range is detected by the flame ionization detector, olefins and aromatics are likely to be responsible for the peaks detected a t 220 nm by the UV detector. Aromatics only should appear a t 260 nm. Insufficient knowledge of the gas-phase spectra is presently a drawback of this type of detection. As such spectra can be accumulated for a great number of compounds by using imaging detectors in future studies, detection devices of the type described here will be used more effectively. In addition t o maximizing response to certain components, selective tuning of the UV detector can also be beneficial in qualitative studies. Specifically, recording peak area a t different wavelengths for the same sample may aid in identification efforts. This has actually been pointed out by Krstulovic et al. (22) for the case of polycyclic aromatic hydrocarbons separated by HPLC; similar considerations are valid for the gas-phase detection. Numerous methods now exist to attach UV chromophores t o various organic compounds. Chemical derivatization procedures have extended considerably the applicability of the UV detectors in HPLC. Wherever sample volatility permits, similar approaches should be examined for GC. Detection
of carboxylic acids as benzyl esters by the UV detector shown in this work is just one example of the many possibilities using suitable reactions. As demonstrated in this paper, successful coupling of capillary GC with UV absorption measuring devices is feasible. In fact, significant sensitivity improvement is realized because of such combination. It is expected t h a t capillary GC/UV detection will be finding an increasing number of applications in selective detection of aromatics in complex mixtures.
LITERATURE CITED “Transfam Techniques in chemistry”, Griffiths, P. R., Ed.; Plenum Press: New York, 1978. HaUsdWff, H. H. J . ChrOmafogr. 1977, 734, 131-146. BurchfieM. H. P.; Wheeler, R. J.: Bernos, J. B. Anal. Chem. 1871, 4 3 , 1976-1981. Burchfield, H. P.; Oreen, E. E.; Wheeler, R. J.; Bllledeau. S. M. J . Chromatogr. 1974, 99, 697-708. Freed, P. J.; Faulkner, L. R. Anal. Chem. 1972, 4 4 , 1194-1198. Cooney, R. P.; Vo-DInh, T.; Wlnefwdner, J. D. Anal. Chlm. Acta 1877, 89, 9-19. Cooney, R. P.; Winefwdner, J. D. Anal. Chem. 1977. 49, 1057-1060. Kaye, W. Anal. Chem. 1962, 3 4 , 287-293. Merritt, J.; Comendant, F.; Agrams, S. T.; Smith, V. N. An8l. Chem. 1963, 35, 1481-1464. Novotny, M.; Lee, M. L.; Bartle, K. D. Chromafographie 1874, 7 , 333-338. Novotny, M.; Farlow, R. J . Chromatogr. 1875, 703, 1-6. Onuska, F. I.; Comb, M. E.; Bistrlckl, T.; Wiklnson, R. J. J. C h m f c g . 1977, 142, 117-125. Schomburg, G.; Husmann, H.; Weeke, F. J . Chromafogr. 1874, 99, 63-79. Lee, M. L.; Vassllaros, D. L.; Phllllps, L. V.; Hercules, D. M.; Azumaya, H.; Jorgenson, J. W.; Maskarlnec, M. P.; Novotny, M. Anal. Lett. 1879, 12(A2), 191-203. Welgang, 0 . E. J . Chem. Phys. 1980, 33, 892-899. Novotny, M.; McConnell, M. L.; Lee, M. L.; Farlow, R. CM. Chem. 1974, 20, 1105-1110. Gyllenthaal, 0.;Brotell. H.; Hartig. P. J . Chromafogr. 1876, 729, 295-302. Bethge, P. 0.;Llndstrom, K. Anaksf (London) 1974, 9 9 , 137-142. Christie. W. W. “Upid Analysis”; Pergamon Press: Elmsford, N.Y., 1973; Chapter 4, p 85. McConnell. M. L.; Novotny, M. J . Chromafogr. 1975, 112, 559-571. Hrlvnac. M.; Frlschknecht, F.; Cechova, M. Anal. Chem. 1978, 4 8 , 937-940. Krstulovic, A. M.; Rosle, D. M.; Brown, P. R. Anal. Chem. 1976, 48, 1383-1386.
RECEIVED for review September 19,1979.
Accepted January 28,1980. This work was supported by grant number P H S RO1 GM 23668-03 from the National Institute of General Medical Science, U.S. Public Health Service.
