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Anal. Chem. 1988, 60, 173-174
Evaluation of a Nitrosyl-Specific Gas-Phase Chemiluminescent Detector with High-Performance Liquid Chromatography Albert Robbat, Jr.,* Nicholas P. Corso, and Tyng-Yun Liu Chemistry Department, Tufts University, Medford, Massachusetts 02155
A gas-phase chemiluminescent detector (CD) used In conjunction with high-perfonnancellquld chromatography (HPLC) has been evaluated. The detector responded llnearly from 1000 ng to the detection iimlt for the nitrated polycyclic aromatic hydrocarbons tested. The relative molar response with respect to 2-nitronaphthalene was wlthln 13%. HPLC with reversed-phase solvents under isocratlc and gradlent elution conditions Is shown to be compatible with the CD.
Table I. Comparison of Linear Response and Relative Molar Response of Nitro-PAH
compd'
mol fract
slope
inter
corr
1,3-dnn 1-nn 2-nn 2-nf 9-na
0.422 0.266 0.266 0.218 0.206 0.186
0.505 0.408 0.326 0.255 0.206 0.198
-8.34 -3.86 -0.03
0.999 0.999 0.999 0.999 0.999 0.999
1-np
Of primary importance to analytical chemists is the ability to identify analytes in complex sample matrices. Often the analytes of interest are present at trace levels, with detection and quantification complicated by high levels of interfering components. Notwithstanding the continuing development of high-resolution gas and high-performance liquid chromatographic columns, the development of chromatographic detectors that are selective, sensitive and specific for a given analyte provides unmatched advantages over exhaustive sample cleanup procedures and universal detectors. Chemical interactions which produce chemiluminescent optical responses are highly specific since relatively few compounds undergo chemiluminescent reactions. During the past decade, increasing attention has focused on developing chromatographic chemiluminescent optical detection systems (I, 2). Chemical analyses and, as important, sample cleanup prescreening methods based on chemiluminescent detection may be accomplished more easily since only sample components of interest will produce a signal response. In particular, chemiluminescent detectors based on the nitrosyl radical/ ozone reaction have been used for the determination of nitrogen-containing organic compounds. Previously, we reported on the design, response, and analytical application of a gas chromatographic chemiluminescent detector (GC/CD) for the analysis of nitrated polycyclic aromatic hydrocarbons (nitro-PAH) (3). The GC/CD responded linearly between 0.050 and lo00 ng. The CD detection limit for most nitro-PAH was 50 pg at a S / N of 3. The detector responded on a mole of NOz per mole of compound basis for each nitro-PAH tested (within 8%). The GC/CD has been used to identify and quantify a number of nitro-PAH present in environmentally complex samples, for example, in organic fractions of diesel exhaust particulate matter and waste crankcase oil. T o date, high-performance liquid chromatography with gas-phase chemiluminescent detectors (HPLC/CD) have not been widely used. Reports have appeared in the literature for the determination of N-nitroso and other nitrogen containing organic compounds by HPLC/CD based on the nitrosyl radical/ozone chemiluminescent reaction (4-8). Results indicate that the presence of the mobile phase in the NO'/03 reaction chamber dramatically reduces the CD signal response as compared to GC/CD. For example, when reversed-phase HPLC solvents are employed, the CD signal response is about 5 orders of magnitude less sensitive than our GC/CD. In the present investigation, the application of our gas-phase chemiluminescent detector with isocratic and gradient elution
-4.29
-2.88 -5.12
slope/ mol fract
RMRb
1.20 1.53 1.23
0.98
1.17
0.95 0.81 0.87
1.00 1.07
1.24 1.00
'Compounds are 1,3-dnn = 1,3-dinitronaphthalene,1-nn = 1nitronaphthalene, 2-nn = 2-nitronaphthalene, 2-nf = 2-nitrofluorene, 9-na = 9-nitroanthracene and 1-np = 1-nitropyrene. bThe RMR (relative molar response) is based on the ratio of the slope and the mole fraction for each compound relative to the ratio of 2-nitronavhthalene. high-performance liquid chromatography has been evaluated. The linear dynamic range, the detection limit, and the applicability of reversed-phase solvents are discussed.
EXPERIMENTAL SECTION HPLC grade acetonitrile (Fisher Scientific, Medford, MA) was used as received. Water was purified with the Milli-Q Water Purification System (Millipore, Milford MA). All solvents were fiitered through a 0.45-pm Nylon-66 filter (Rainin, Woburn, MA) and ultrasonically vacuum degassed prior to use. Nitro-PAH have been identified elsewhere and were used without further purification (9). Standard solutions were prepared by dissolving the appropriate amount of nitro-PAH in acetonitrile and then performing serial dilutions. The solvent delivery system consisted of a Constametric I11 pump, a Constametric I pump, and Gradient Master (LDC/Milton Roy, Riviera Beach, FL). A Rheodyne Model 7410 sample injection valve with an internal 2-pL sample loop was used for sample introduction onto the HPLC column. An ODS solvent miser, 25 cm X 2.1 mm, 5-pm particle size reversed-phase column (Alltech Associates, Deerfield, IL) was used. The dynamic range was determined by using acetonitrile as the mobile phase at a flow rate of 0.2 mL/min. The effect of the mobile phase composition on the detection limit was determined by using acetonitrile and water at a flow rate of 0.2 mL/min. Linear gradient elution experiments were performed from 45/55 to 75/25 acetonitrile/water in 60 min at flow rates of 0.5 mL/min. The chemiluminescence detector has been described previously (3). The HPLC column was connected to a 7-cm length of stainless steel tubing. The tubing was extended 5 cm into the quartz tubing (34 cm) housed in a pyrolysis oven at 900 "C. A glass solvent trap (100 mL) located at the exit port of the oven and before the CD system was maintained at -75 "C with a dry icelacetone bath. RESULTS AND DISCUSSION Isocratic HPLC/CD experiments were performed with acetonitrile as the mobile phase at a flow rate of 0.2 mL/min. The dynamic range of the CD for nitro-PAH was evaluated from 1000 ng to the detection limit. The CD responded linearly over the entire concentration range. Table I summarizes the CD response for the nitro-PAH studied. The detection limit for the mononitro-PAH investigated was about 30 ng while that for 1,3-dinitronaphthalene was 10 ng a t a S I N of
0003-2700/88/0360-0173$01.50/00 1988 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 2, JANUARY 15, 1988 3
Table 11. Linear Response and Detection Limits for I-Nitronaphthalene at Different Mobile-Phase Compositions of Acetonitrile and Water detection % CH3CH
slope
inter
corr
100 90 80
0.408
-3.86 -13.20 -7.69 -10.80 -6.59 -5.30
0.999 0.999 0.999 0.995 0.996 0.997
70 60 50
0.163 0.090 0.061
0.044 0.036
limit, ng injected 30
30 60
70 125 250
3. In theory and as reported previously (3),the gas-phase chemiluminescent detector is molar responsive, i.e., the CD responds only to the moles of nitro groups per mole of nitro-PAH. Thus, for example, the detector achieves lower detection limits per compound injected for lower molecular weight mononitrated-PAH than higher molecular weight mononitrated-PAH. The HPLC/CD system responded similarly. This is evidenced by the decreasing slope with increasing molecular weight of mononitrated-PAH. Also shown in the table is the relative molar response for each compound relative to 2-nitronaphthalene. The experimental results are in agreement with theory within about 13%. Isocratic experiments were performed with varying acetonitrile/water mobile phase composition to determine solvent effects on CD signal response. At a given concentration of 1-nitronaphthalene the signal detected a t 50/50 acetonitrile/water was about 10 times less than that observed when the mobile phase was acetonitrile. The detection limit of the CD became worse as the percent water in the mobile phase increased. For example, the detection limit for 1-nitronaphthalene increased from 30 ng injected a t 100% acetonitrile to 250 ng injected at 50/50 acetonitrile/water. Although the CD detection limit varied with percent acetonitrile, as shown in Table 11, the CD responded linearly for all isocratic acetonitrile/water compositions examined. These findings indicate that at any given isocratic experimental condition, concentration calibration curves are not needed for each nitro-PAH measured. The separation of 10 nitro-PAH by reversed-phase linear gradient HPLC/CD is shown in Figure 1;solvent ramp 45/55 to 75/25 acetonitrile/water over 60 min. The flow rate was 0.5 mL/min. Plots of chromatographic peak area versus analyte injected yielded linear dynamic ranges for these compounds between 70 and 1000 ng. The correlation coefficient in all cases was 0.998. For determination of many nitro-PAH in complex samples, gradient programmed HPLC yields the best separation. The results of this experiment indicate that the CD detection limit was only 2 times less sensitive than isocratic experiments performed in the absence of water. However, it is at least 15 times better than similar nitrosyl/ozone detectors reported in the literature for HPLC (4, 6). Previous attempts to use a NO'/03 CD in conjunction with solvent programming were unsuccessful due to gas-dynamic problems (10). Other researchers have indicated that the use of polar solvents, organic as well as aqueous, have resulted in NO'/03 detector instability, Le., excessively high background noise (7,11).In contrast, as can be seen in Figure 1, base line stability is achieved even at high mobile-phase water content, Le., 50% H 2 0 , with our HPLC/CD system. For optimum signal-to-noise the HPLC eluent was aspirated into the quartz pyrolysis chamber. The day-to-day reproducibility of the CD for both isocratic and gradient experiments over a 4-month period was about 5 % . It appears that the HPLC/CD detection response is flow rate dependent. For example, in the gradient experiments
c
I
1
20
I
0
10
I
I
I
50
I
55
60
30
I
40
T I ME,minutes 45
% CH3CN
I
65
Flgure 1. Chromatographic profile of a solution of 10 nitro-PAH obtained by using reversedphase gradient elution with CD. Labeled peaks are (1) 8-nitroquinoRne, (2) 1,5-dinitronaphthalene, (3) l-nitronaphthalene, (4) 2-nitronaphthalene, (5) 2-nitrobiphenyl, (6) l-nitro-2methylnaphthalene, (7) 4-nitrobiphenyl, (8) 2-nitrofluorene, (9) 9-nitroanthracene, and (10) 1-nitropyrene.
1-nitronaphthalene elutes a t a solvent composition of 51/49 acetonitrqe/water with CD detection limit of 125 ng, this result is in contrast to the isocratic experiment where the detection limit was 250 ng at a similar solvent composition. OBrien and co-workers (12) have shown that the NO'/03 reaction is sensitive to changes in sample pressure and flow rate. Pressure variations in the reaction chamber are more likely to occur with HPLC/CD than with GC/CD due to mobile-phase transition from liquid to gaseous state. Moreover, since the solvent composition is constantly changing during gradient elution, pressure changes within the CD reaction chamber are to be expected. Currently, work is in progress to optimize the HPLC/CD with respect to reaction chamber variables such as pressure, temperature, solvent flow rate, and reactant composition for improved detection of nitrated organic compounds. The use of HPLC/CD as a screening tool to obtain organic fractions of nitrogen containing compounds from complex sample matrices should prove applicable. A forthcoming publication will describe nitro-PAH HPLC isocratic and gradient retention characteristics on an octadecylsilane stationary phase.
ACKNOWLEDGMENT The authors thank LDC/Milton Roy for providing the HPLC equipment. LITERATURE CITED Hutte, S. R.; Sievers, R. E.;Birks, J. W. J. Chromatogr. Sci. 1988, 2 4 , 499-505. Barth, H. G.; Barber, W. E.; Lochmuller, C. H.; Majors, R. E.; Regnier, F. E. Anal. Chem. 1988, 58, 211R-250R. Robbat, A., Jr.; Corso, N. P.; Doherty, P. J.; Wolf, M. H. Anal. Chem. 1988, 58, 2078-2084. Baker. J. K.:Ma. C. IARC Sci. Publ. 1978. 19. 19-32. Fan, T. Y.; Kr&I. S.:Ross, R. D.; Wolf, M.'H.; Fine, D. H. IARC Sci. Publ. 1978, 19. 3-15. Fine, D. H.; Huffman, F.; Rounbehler, D. P.; Belcher, N. M. IARC Sci. Publ. 1978. 14. 43-50. Massey, R.'C.; Key, P.E.;McWeeny, D. J.; Knowles, M. E. IARC Sci. Publ. 1984, 57, 131-136. Sen, N. P.; Seaman, S. IARC Sci. Publ. 1984, 5 7 , 137-143. Whne, C. M.; Robbat, A., Jr.; Hoes, R. M. Chromatographia 1983, 17, 605-6 12. Oettinger, P. E.;Huffman. F.; Fine, D. H.; Lieb, D. Anal. Lett. 1975, 8(6), 41 1-414. Kubacki, S. J.; Havery, D. C.; Fazio, T. IARC Sci. fubl. 1984, 5 7 , 145-1 55. Mehrabzadeh, A. A,; O'Brien, R. J.; Hard, T. M. Anal. Chem. 1983, 55, 1660-1665.
RECEIVED for review June 22,1987. Resubmitted August 26, 1987. Accepted September 22, 1987.
