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Anal. Chem. 1981, 53, 946-951
Flame Emission Detection in Microcolumn Liquid Chromatography V. L. McGuffln and Milos Novotny" Department of Chemistry, Indiana University, Bloomington, Indiana 47405
A flame photornetrlc detector has been developed and characterized for use with mlcrocaplllary column hlgh-performance llquid chromatography. The total column effluent was asplrated dlrectly into a cool, hydrogen-nitrogen-air diffusion flame, and phosphorus emlsslon was selectively monitored by uslng an Interference fllter and photomultlpller tube. Moblle phases, containing up to 50% of selected organlc solvents, have been utlllzed wfth no resulting loss In sensitivity. The detection lhlt obtafned wlfh thls system was 7 X lo-'' g/s phosphorus.at the maxlmum of the Gaussian peak.
Recent communications regarding both the theory and application of miniaturized HPLC systems (1-10) have demonstrated their potential in modern separation science. Such systems are capable of providing very high efficiencies, while utilizing only small quantities of sample of solvent. Several technological approaches have been investigated, including open tubular capillaries (443, packed capillaries (7-9), and packed microbore columns (10, 11). Although the level of technical development differs for the various column types, the potential for high resolution and the availability of different column selectivites have been generally demonstrated. There are significant differences between the microcapillary columns, pioneered by Novotny and Ishii et al. (4-9), and the microbore columns, developed by Scott and Kucera (10, 11). The former have typical inner diameters of 30-70 pm and flow rates of 1 bL/min, while the latter have diameters and flow rates which are intermediate between capillary and conventional HPLC columns (1mm id., 50 bL/min). The instrumental and technological requirements for microbore columns are not as stringent as for capillary columns. The very low flow rates encountered in capillary HPLC have considerable influence upon the design of suitable detection devices. The miniaturization of flow cells, while less desirable technologically, has resulted in improved performance for concentration-sensitive detectors, as demonstrated by Scott and Kucera (11). Miniaturized detectors have already been reported for UV (8),fluorescence (9), and electrochemical (12) measurements. There are additional detection and ancillary techniques which are particularly well-suited to the reduced flow rates typically used in capillary HPLC. Direct coupling of the microcolumn with a mass spectrometer or Fourier transform IR spectrometer appears feasible; similarly, moving-band and flame-based detectors show considerable promise. In this paper, a modified flame photometric detector is described, in which the total microcolumn effluent is introduced into a flame, and the characteristic optical emission of certain solutes is measured. The principle of flame photometric detection, originally described by Brody and Chaney (13),has been widely used in gas chromatography for the selective detection of sulfur and phosphorus compounds. More recently, flame photometric detection (FPD) has been employed, with limited
success, to monitor HPLC effluents (14-16). Kirkland and co-workers (14) described a flame emission detector for HPLC which included a right-angle pneumatic nebulizer and modified burner assembly. This detector utilized approximately 25% of the column effluent, and, under favorable conditions, was capable of sensing 2 X g/mL g/mL sulfur. Organic solvents, even phosphorus and 2 x at very low concentrations,severely quenched emission of the chemiluminescentHPO and S2species. Metal ions and many buffers also showed significant chemical interference; thus, applications of this detector were limited to aqueous systems. More recently, Chester described an FPD for liquid chromatography in which the usual gas inlet configuration is reversed (15,16);air is used to nebulize the sample in a hydrogen atmosphere. Quenching effects of many organic solvents, metal ions, and buffers were considerably reduced in the inverted-flame FPD. The observations of West and c+workers (17-19) have been of primary importance to this investigation: they demonstrated that a hydrogen-nitrogen diffusion flame may be used for the sensitive and selective determination of phosphorus and sulfur in aqueous media. The chemiluminescent HPO and S2species are formed most effectively at low flame temperatures (300-400 "C) and in a highly reducing atmosphere. A novel flame photometric detector is reported in this paper which utilizes a total consumption burner. The microcolumn effluent is concentrically nebulized and aspirated directly into a cool, hydrogen-rich diffusion flame. For this application, the total consumption burner offers certain distinct advantages over the right-angle pneumatic nebulizer utilized by Kirkland et al. (14). First, the total column effluent is introduced into the flame; this provides for more efficient use of the small samples and low flow rates associated with microcapillary column HPLC. This also ensures that a representative fraction of the effluent reaches the flame, thus no preconcentration of volatile solutes or solvents occurs, as it might with separate, heated nebulizer chambers. Another advantage is the much lower dead volume of this design; the end of the capillary column may be simply inserted into the flame. The disadvantages of the total consumption burner include the decreased nebulization efficiency (larger droplets) and the dependence of droplet size on solvent viscosity and surface tension.
EXPERIMENTAL SECTION Flame Photometric Detector. The stability of this detector depends upon the carefully regulated flow of high-purity gases. The flow rates of all gases were controlled by single-stage regulators at the cylinder and calibrated flow meters (Brooks, Model R-2-15-D-CO). Type 4A molecular sieves were used to remove trace impurities from the air and nitrogen cylinders. The miniaturized FPD burner base and flame housing were designed and constructed in-house and are shown schematically in Figure 1. Hydrogen, the fuel gas, and nitrogen, a noncombustible nebulizing gas, are premixed in a baffle. The burner base features a fritted disk to allow uniform air diffusion to the hydrogen-rich flame. Stainless steel flame jets were constructed with inner diameters of 0.65, 0.76, 0.89, and 1.02 mm.
0003-2700/81/0353-0948$01.25/00 1981 American Chemical Society
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FLAME PHDTOMETRIC DETECTOR
- IGNITION
COIL
‘IPLIER
AMMETER
FIBER O P T I C
I
CAPILLARY COLUnN
11-11 REC0RDER
LOU PASS FILTER
Flgure 2. Schematic diagram of the chromatographicsystem, flame
photometric detector, and ancillary electronic equipment.
FUEL INLETS
Flgure 1. Schematic diagram of the FPD burner base and flame
housing. A glass capillary (50 ,um i.d., 0.6 mm o.d., and 10 cm length) is inserted through the baffle, burner base and flame jet and extends approximately 1mm above the top1 of the jet. The other end of this capillary is connected to the microcapillary HPLC column with shrinkable IPTFE tubing. The total column effluent is nebulized by the concentric flow of gases over the capillary orifice. An aluminum flame housing was constructed which allows unrestricted flow of exhaust fumes but doles not permit light to enter. The exit tube is short to prevent condensation on the walls of the housing, and the relatively closed design m i n i i e s the effect of drafts on flame stability. An ignition coil and thermocouple are included in the housing. A fiber optics probe (IEdmund Scientific Co., Barrington, NJ) is fixed in the wall of the housing and views the cooler central region of the diffusion flame. An interference filter (530 nm, fwhm 14 nm) is placed at the other end of the fiber optics. This combination of optical devices transmits approximately 25% of the incident light to a photomultiplier tube (Hmamatsu Corp., Model R-372), which is biased in the usual manner. The PMT is supplied with a maximum of 1200 V from a Keithley high-voltage power supply (Model 244). The electrical signal from the PMR is amplified by using a picoammeter (RCA, Model WV-511A) and filtered to remove high-frequencynoise components. The low pass filter is an active noninverting second-order filter, which allows selection of time constank from 1to 30 s. The output is then displayed on a strip chart recorder (Shimadzu Seisakusho Ltd., Model R101). The complete analytical system is shown schematically in Figure 2. Liquid Chromatograph. The chromatographic system utilized in this investigation is similar to that described by Tsuda and Novotny (6, 7). A Varian syringe pump (Model 8500) was chosed to minimize fluctuations in pressure and flow rate, which were expected to have significant effects of the response of the mass-sensitive flame photometric detector. The capillary columns are prepared by packing standard-bore Pyrex or soft glass capillary tubing (0.5 mm, i.d.; 5 mm, 0.d.) with dry silica or alumina partrcles of uniform size. The packed tubing is then drawn out to an appropriate diameter (-70 pm, i.d.) using a commercial glass drawing machine (Hupe and Busch, Groetzingen, West Germany). The packed capillary is washed with dry hexane for several hours at approximately 5 jLL/min. A solution of the bonding agent
is then introduced using four of the miniature reservoirs (150 pL) described by Hirata and Novotny (9). The first and third chambers are fiied with a dilute solution (3-4%) of an appropriate silane in dry toluene. The third chamber may alternatively contain a solution of a smaller silane, such as methyltrimethoxysilane, to “cap” any remaining silanol groups. The second and fourth chambers are filled with dry toluene to wash unreacted silane from the column. The solutions are pumped through the column at a reduced flow rate (- 1pL/min) and at an elevated temperature (60 “C). The column is then washed with dry hexane for 2 days, after which the polarity of the mobile phase is gradually increased. Samples are injected directly onto the packed capillary column using the technique described by Hirata and Novotny (9). Reagents. The solid supports most commonly used in the preparation of packed microcapillary columns were LiChrosorb Si-100 and LiChrosorb Alox T (30 pm), obtained from E. Merck Reagents (Darmstadt, West Germany). The silanes used in this investigation include octyltriethoxysilane,odadecyltriethoxysie, and methyltrimethoxysilane, purchased from Petrarch Systems, Inc. (Levittown, PA). Organophosphoruspesticide standards were “qualitativegrade” and were purchased from the Anspec Co., Inc. (Ann Arbor, MI). Cygon (0,O-dimethyl S-(Nmethylcarbamoylmethy1)phosphorothioate, 98% purity), DDVP (2,2-dichlorovinylphosphate, 93% purity), malathion (0,O-dimethyl S-(dicarbethoxyethy1)dithiophosphate, 95% purity), and guthion (0,O-dimethyl S-(~-OXO1,2,3-benzotriazin-3(4H)-ylmethyl)phosphorothioate,99% purity) were utilized in this investigation. The phosphinyl derivatives (20-22) of deoxycorticosterone, estradiol, cyclohexylamine, and phloroglucinol were obtained through the courtesy of Karl Jacob, Ludwig-MaximiliansUniversitat Munchen, West Germany. Trimethyl phosphate, a model solute used to characterize the miniaturized FPD, was obtained from the Aldrich Chemical Co. (Milwaukee, WI). All organic solvents utilized in this investigation were Fisher Spectranalyzed grade; water was deionized, distilled, and finally redistilled from alkaline permanganate. Ancillary Equipment. A Jasco Uvidec 100-11 variablewavelength UV detector, modified with a 0.07-pL flow cell, was used for comparison of response and dead volume with the miniaturized FPD. A Cary 14UV-visible scanning spectrometer was used to obtain transmittance spectra of interference filters.
RESULTS AND DISCUSSION Optimization. The most critical factors influencing the proper operation of the miniaturized FPD are the nebulization efficiency and rate of solvent evaporation in the flame. The nebulization efficiencyof the total consumption burner depends upon the rate of nebulizing gas flow, the ratio of diameters of the capillary and flame jet, and the viscosity, surface tension, and flow rate of the HPLC effluent. The rate of desolvation depends upon the flame temperature, the combustibility and heat capacity of the solvent, the initial droplet size, and the residence time of the droplet in the flame.
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Thus, there are several compromises which must be considered. First, the nebulizing gas flow should be high enough to form very small droplets in a uniform manner, yet low enough to allow long residence time for complete desolvation in the miniaturized flame. Second, the flame temperature should be high enough for fast evaporation of the solvent, yet low enough to favor formation of the chemiluminescentHPO species. Finally, it is advantageous to decrease the viscosity and surface tension of the effluent by adding surfactants or organics to the mobile phase; however, these additives may retard performance of the chromatographic system and increase background emission of the flame. In preliminary investigations, both stainless steel and glass capillaries of various inner and outer diameters were used to introduce the HPLC effluent into the flame. Glass capillaries were found to be superior, presumably due to reduced adhesion and surface wettability. The best nebulization was achieved with a capillary of inner diameter 50 pm and outer diameter 0.6 mm and a flame jet of diameter 0.76 mm. The height of the capillary is adjusted while the flame is lit and solvent is aspirating. When the capillary is properly positioned, typically 1 mm above the top of the flame jet, a high-frequency sputter is audible. The diffusion flame is inherently less stable than one in which the fuel and oxidant are premixed. For this reason, a quartz or inert gas sheath is frequently used to improve flame stability. Quartz tubes of various dimensions were utilized to shield the flame; however, no significant improvement in performance was observed with the miniaturized FPD. The voltage applied to the PMT was varied from 600 to 1100 V. The response of the FPD to a standard injection of trimethyl phosphate was optimum and relatively constant from 750 to 850 V. The FPD response was nominally optimized by independently varying the gas flow rates to obtain the maximum signal-to-noise ratio. The flow rates are limited, at one extreme, by the inability to sustain a flame when water is aspirated, and, at the other extreme, by high background emission and flame temperatures. The flow rate of hydrogen was varied between 25 and 75 mL/min, and the optimum was found at 55 mL/min. Similarly, nitrogen was varied between 50 and 110 mL/min and air between 50 and 100 mL/min, with maxima at 90 and 75 mL/min, respectively. Thus, the optimum fuel-to-oxidant ratio was approximately 3.7, which is comparable with that of the GC flame photometric detector (13). The use of surfactants has been suggested to minimize surface tension and capillary effects at the point of nebulization (23). Both ionic (sodium dodecyl sulfate and tetramethylammonium chloride) and nonionic (Tween 80) surfactants were investigatedin concentrationsranging from 0.01 to 0.1 M. Although a slight improvement in nebulization was observed, the surfactants increased the noise level too greatly to be of any practical value, Organic solvents have also been investigated for this purpose with promising results. Effect of Organic Solvents on Emission. The effect of selected organic solvents on phosphorus emission was studied by using trimethyl phosphate as a model solute. Methanol, ethanol, acetone, and acetonitrile were added to the aqueous mobile phase in concentrations ranging from 0 to 50%. Contrary to the results obtained by Kirkland and co-workers (14), no decrease in HPO emission was observed for moderate concentrations of acetone and alcohols. Furthermore, small increases in detector response (1-to 3-fold) have been observed at certain concentrationsof these solvents. As shown in Figure 3, a 2-fold signal enhancement was obtained when 10% methanol was added to the aqueous mobile phase. This effect was observed for solutions containing up to 50% methanol;
MASS PHOSPHORUS
(no)
Figure 3. Calibration curves for the flame photometric detector, as 10% a function of mobile phase composition: (0)pure water, (0) aqueous methanol, (A)25% aqueous methanol.
however, the degree of enhancement decreases with increasing concentrationof organic solvent. Similar results were obtained for solutions of ethanol and acetone. In contrast, acetonitrile greatly increased background noise and severely quenched phosphorus emission, even at very low concentrations (1-5%). Similar observations were made by Chester (15),in studies of solvent effects on the normal and inverted-flame FPD. The observed signal enhancement in the presence of acetone and alcohols may be due to changes in the physical properties of the HPLC effluent, such as surface tension, viscosity, volatility, and combustibility, which result in improved nebulization and desolvation. The enhancement might also be attributed to an increase in flame temperature in the presence of combustibleorganic solvents. To determine whether flame temperature influences detector response, a standard solution of trimethyl phosphate was injected onto the microcolumn and eluted with pure water. Through a second capillary, solutions of aqueous methanol were introduced into the flame. Under these conditions, phosphorus emission was completely suppressed, which implies that changes in flame temperature do not contribute to the observed enhancement effect. Presumably then, alteration in the physical properties of the effluent is primarily responsible for the enhancement of phosphorus emission in the presence of acetone or alcohols. The gain in emission intensity cannot be simply related to a single specific property of the organiewater mixture; instead it may depend, in a complicated and subtle manner, on the combined influence of many factors (24). The enhanced response does not indicate that quenching of the type described by Kirkland et al. (14) and West et al. (17)does not occur in the miniaturized FPD. It does suggest that vast improvement in other areas, primarily nebulization and desolvation, overshadows any decrease due to chemical quenching which may or may not occur in the flame. Preliminary reports on the gas chromatographic FPD described the tendency of the fuel-rich flame to extinguish whenever a large quantity of an organic compound eluted (25). This problem, commonly referred to as “solvent flameout”, has not been observed with the miniaturized LC FPD, even at flow rates up to 20 pL/min. Furthermore, there have been no problems with soot formation in the flame when moderate concentrations of organic solutions are continually aspirated. It should be noted, however, that the introduction of an organic solvent changes the fuel-to-oxidant ratio of the flame, and consequently the response of the detector also changes. For this reason, gradient elution is not recommended when highly accurate quantitative results are required, unless the method of standard additions is employed. Detection Limits and Linear Dynamic Range. The specification of detection limits in chromatographyhas always been a controversial subject, yet the comparison of detector performance depends upon the unambiguous determination of sensitivity. For minimization of this ambiguity, a statis-
ANALYTICAL CHEMISTRY, VOL. 53, NO. 7, JUNE 1981
H
I
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w
a
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15
I0
5
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Flgure 4. FPD res@rxleo as a function of flow rate: column, 10 m silica microcolumn; mobile phase, 10% aqueous mcsthanol; solute, trlmethyl phosphate, 50 ng of phosphorus.
tically derived definition is herein proposed. The minimum detectable quantity is defined as the absolute amount (grams or moles) of solute whic:h produces a signal-to-noise (rms) ratio of
where t is the Students: t value at a specified confidence level for n measurements of the signal-to-noise ratio (26). This definition requires that the time constant of the detector and ancillary electronic equipment be small with respect to the full width at half-maximum (fwhm) of the eluted peak. Note that six to eight separate determinations are required to specify the minimum dletectable quantity at a signal-to-noise (rms) ratio of 2, at the 99.5 to 99.9% confidence level. The detection limit is then defined as the concentration (g/mL or mol/L, for concentration-sensitive detectors) or mass flux (g/s or mol/s, for mass-sensitive detectors) at the maximum of the Gaussian peak produced by the minimum detectable quantity. This may be calculated from the fwhm of the peak [2.3540 (27)],according to the method described by Diebold and Zare (28). The detection limits and linear range for the miniaturized LC FPD were determined by using trinnethyl phosphate as a model solute under nonretained conditions. The minimum detectable quantity was 2.0 f 0.1 ng of phosphorus, and the mass flux at the Gauseiian maximum was 71 f 3 pg/s phosphorus. These values were obtained at a signal-to-noise(rms) ratio of 5 and at the 99.5% confidence level. Despite the enhancement of signal response in the presence of selected organic solvents, no improvement in detection limit was observed, due to the increased background emission of the flame. The linear range of this detector was determined for mobile phases containing pure water, 10% aqueous methanol, and 25% aqueous methanol. The results summarized in Figure 3 indicate that response is linear for all tlhree solvent systems from the detection limit to 100 ng of plhosphorus. Flow-Rate Studies. & a mass-sensitive detector, the FPD is expected to show signd intensity which is linearly dependent on flow rate. With trimethyl phosphate as a model solute, the response of the FPD was determined at flow rates ranging from 0.5 to 20 pL/min. As shown in Figure 4, the detector behaves ideally at flow rates greater than 7 pL/min; however, at lower flow rates, the response is relatively constant and much higher than predicted theoretically. Similar results were reported by Manahan et al. (29), in their investigation of mobile phase effects on an atomic absorption detector for HPLC. They attributed the improved performance to higher nebulization efficiency when the effluent flow rate was less than the natural uptake rate of the nebulizer (“starving the burner”). If this is the case with the miniaturized LC FPD, it fortuitously occurs at the flow rates typically used for analysis (-1 pL/min). These results also imply that small
949
changes in flow rate, such as pump pulsations, should have little effect on signal intensity. Dead Volume Studies. Several recent theoretical studies of the potential application of packed and open tubular capillary columns have emphasized the necessity for low volume detectors before the high efficiencies of such columns may be fully utilized (2, 3). There are three major sources of band broadening in the LC FPD: dead volume within the capillary and connecting tubes, residence time of the solute in the flame, and the combined time constant of the electronic equipment. A modified commercial UV detector, with cell volume 0.07 pL, was used to estimate the contribution of the FPD capillary and connecting tubes to the extra-column band broadening. The capillary and connections were installed in series between the column and UV detector. These devices contributed less than 5% to the fwhm of a nonretained peak. This source of band broadening may be eliminated by introducing the effluent from the capillary column directly into the flame. The residence time of a droplet in the flame is dependent upon the droplet diameter, as well as other factors; thus, a wide range of diameters produced by the nebulizer can lead to broadening of the chromatographic peak. Since droplet diameter is a function of effluent viscosity and surface tension, it is important that solutes do not appreciably influence these parameters. The addition of a small amount of an organic solvent to the mobile phase may moderate the solute effect, thus decreasing the residence time spread of the droplets. A slight decrease in bandwidth with increasing methanol concentration has been observed, consistent with both the improved nebulization efficiency and decreased residence time spread. The absolute magnitude of the residence time and residence time spread is not known; however, it is expected that contributions to the bandwidth from this source will be small in comparison with other sources of peak dispersion. Some tailing of chromatographic peaks has been observed due to adhesion of less volatile solutes on the tip of the capillary. The greatest contribution to band broadening in the miniaturized LC FPD is the combined time constant of the ancillary electronic devices. Although the amplifier and recorder possess measurable time constants, under most circumstances the low pass filter will determine the system response. For this reason, it is imperative to select a cutoff frequency which provides adequate filtering of the signal, yet does not appreciably increase the solute bandwidth. In general, the time constant should be no more than 32% of the time standard deviation of a nonretained peak. If the void volume and efficiency of the column are known, the maximum permissible time constant may be calculated (30);however, empirical methods may also be utilized. A low pass filter with time constants selectable from 1to 30 s has proven effective with packed capillary columns. Several chromatograms, obtained independently with the modified UV detector and the miniaturized FPD, were compared to assess their relative contributions to solute bandwidth. The FPD and ancillary electronics contributed significantly less to the band dispersion than the UV device; however, the absolute dead volume of this detector is not known. Applications. Numerous efforts have been made to utilize flame ionization or thermionic detectors in HPLC with a transport device to facilitate solvent evaporation; however, these investigations have had limited success (31). At the same time, conventional flame spectrometric devices, designed to handle bulk samples, are not always compatible with HPLC conditions. The emerging microcolumn technology is likely to provide certain solutions to these problems. While the
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Flgure 5. Chromatogram of organophosphorus pesticides: column, 10 m octylsilane microcolumn; mobile phase, 42% aqueous methanol; solutes, (1) solvent and phosphorus-containing impurity, (2) cygon, (3) DDVP, (4) phosphorus-containing impurltles, (5) malathion, (6)guthion.
microflame emission detector described in this report has been used specifically for the detection of organophosphorus compounds, this detector is believed to be the predecessor of other useful flame-based devices. Many synthetic industrial products and some naturally occurring substances contain phosphorus or sulfur; thus, it would be advantageous to employ a selective device for their detection and quantitation in complex matrices. To illustrate the potential of the flame photometric detector, we chromatographed a mixture of four organophosphorus pesticides on a short, octylsilane capillary column using 42 % aqueous methanol as the mobile phase (Figure 5). The injected quantities of cygon, DDVP, malathion, and guthion were 81, 280,230, and 200 ng of phosphorus, respectively; hence, the response of this detector is not entirely independent of solute structure. While universal detection is not currently practicable in HPLC, the range of detectable substances can frequently be extended by suitable chemical derivatization techniques. This approach is exemplified by the incorporation of chromophores into molecules, which subsequently become detectable using UV or spectrofluorimetric devices. Similarly, molecules containing certain polar functional groups may be derivatized to incorporate phosphorus, and may be subsequently detected with the flame emission detector. Jacob and co-workers (20-22) have derivatized the less polar hydroxysteroids, amines, and aromatic hydroxy compounds using dimethylthiophosphinyl chloride, followed by gas chromatographic analysis with thermionic detection (alkali flame ionization detector). Since such derivatives were reported to be stable toward hydrolysis, the more polar classes of compounds, which were difficult or impossible to analyze by gas chromatography, might be resolved by using reversed-phase HPLC. The application of microcolumn HPLC technology and the miniaturized FPD has been investigated in this area, and some preliminary results are presented here. A simple model mixture, containing derivatives of an amine and an aromatic hydroxy compound (approximately 200 ng each), was chromatographed under reversed-phaseconditions on an octadecylsilane microcolumn. This chromatogram (Figure6) demonstrates that such derivatives are indeed stable in aqueous solution and that this general approach should be feasible for other solutes. Our initial studies with hydroxysteroids have been disappointing: while derivatives are quite easily formed, they are too hydrophobic for conventional aqueous reversed-phase chromatography. The derivatives of deoxycorticosterone and estradiol were insoluble in the mobile phases required for optimum operation of the miniaturized
Figure 6. Separation of dimethylthiophosphinatederivatives: column, 12 m octadecylsilane microcolumn; mobile phase, 38 % aqueous methanol; solutes, (1) solvent, (2) cyclohexylamine derivative, (3)
phloroglucinol derivative.
FPD ( 0 4 0 % aqueous methanol). Investigations of other polar classes of compounds and other derivatization procedures are currently under way.
CONCLUSIONS A novel detection device for HPLC is described here which requires the employment of microcolumns and the associated miniaturized equipment. The total column effluent is nebulized and aspirated into the flame, yet the system does not suffer from response quenching by organic solvents. The detector can be used with a reasonable range of organic mobile phase concentrations, some buffer systems, such as ammonium formate or borate (14),and even some acids, such as formic acetic, nitric, or hydrochloric, in moderate concentration (18). Thus, sufficient variety is available to design chromatographic systems which will accomplish useful separations. The FPD has high selectivity and fairly good sensitivity. Further improvement of the signal-to-noise ratio is possible by using more efficient signal processing and spectroscopic techniques (32, 33). A further advantage of this device is the very small dead volume. This volume can be made negligible by directly inserting the end of the microcolumn into the flame. Like most flame-based detectors, the FPD has a noisy base line, particularly in the presence of organic solvents. While some signal processing is feasible, relatively large time constants are necessary. This problem might be overcome by the use of a sliding-averagefilter (34);however, a small computer is required to implement this system. This work is currently being extended in two important directions: emission of other elements, such as sulfur compounds and metal chelates; and secondary ionization phenomena, for the selective detection of phosphorus, nitrogen, and halogen compounds. ACKNOWLEDGMENT The dimethylthiophosphinate derivatives of deoxycorticosterone, estradiol, cyclohexylamine, and phloroglucinol were obtained through the courtesy of Karl Jacob (Ludwig-Maximilians-Universitat Munchen, West Germany). Technical assistance was provided by Francesca Perugini, John Dorsett, and Stephen Williamson. LITERATURE CITED (1) Knox, J. H.; Gllbert, M. T. J. Cbromtogr. 1979, 186,405-418. (2) Guiochon, G. J . Cbromatogr. 1979, 185,3-26. (3) Knox, J. H. J . Cbromatogr. Sci. 1980, 78, 453-461. (4) Ishii, D.; Asai, K.; Hibi, K.; Jonokuchi, T.; Nagaya, M. J. Cbromatogr. 1977, 144,157-168. (5) Ishii, D.; Takeuchi, T. J . Cbromatogr. Sci. 1980, 18,462-472. (6) Tsuda, T.; Novotny, M. Anal. Cbem. 1978, 50,632-634.
Anal. Chem. 1981, 5 3 , 951-954 (7) Tsuda, T.; Novotny, M. Anal. Chem. 19711, 50, 271-275. (8) Hirata, Y.; Novotny, M.; Tsuda, T.; Ishil. iD. Anal. Chem. 1979, 51,
1807-1809.
(9) Hirata, Y.; Novotny, M. J. Chromatogr. 1079, 186, 521-528. (10) Scott, R. P. W.; Kucera, P. J. Chromatogr. 1979, 169, 51-72. (11) Scott, R. P. W.; Kucera, P. J. Chromatogr. 1979, 185, 27-41. (12) Hirata, Y.; Lin, P. T.; Novotny, M.; Wightman, R. M. J. Chromatogr. 1980, 181, 287-294. (13) Brody, S. S.; Chaney, J. E. J. Gas Chrorniatogr. 1966, 4 , 42-48. (14) Juiin, B. G.; Vandenborn, H. W.; Kirkiand, J. J. J. Chromatogr. 1975, 112,443-453. (15) Chester, T. L. Anal. Chem. 1980, 52,638-642. (16) Chester. T. L. Anal. Chem. 1980. 52, 1821-1624. (17) Dagnall, R. M.; Thompson, K. C.; West, T S. Analyst(London) 1967, 92,506-512. (18) Dagnaii, R. M.; Thompson, K. C.; West, T S. Analyst (London) 1988, 93. 72-78 _-, - ._ (19) Aldous, K. M.; Dagnali, R. M.; West, T. S. Analyst(London)1970, 95, 417-424. (20) Jacob, K.; Vogt, W.; Knedel, M. Justus Lleblgs Ann. Chem. 1979, 878-a85. (21) Jacob, K.; Falkner, IC.; Vogt, W. J. Chromatogr. 1978, 167, 67-75. (22) Jacob, K.; Maier, E.; Schwertfeger, G.; Vogt, W.; Knedei, M. Biomed Mass Spectrum. 1978, 5,302-311. (23) Dean, J. A. “Flame Photometry”; McGraw-Hill: New York, 1960;p 134.
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(24) Avni, R.; Alkemade, C. Th. J. Mikrochlm. Acta 1960, 460-471. (25) Wenzel, 6. J. Chromatogr. 1979, 17, 687. (26) St. John, P. A.; McCarthy, W. J.; Winefordner, J. D. Anal. Chem. 1987, 39, 1495-1497. (27) Bevington, P. R. “Data Reduction and Error Analysis for the Physical Sciences”; McGraw-Hill: New York, 1969;p 45. (28) Diebold, G. J.; Zare, R. N. Sclence 1977, 196, 1439-1441. (29) Jones, D. R.; Tung, H. C.; Manahan, S. E. Anal. Chem. 1978, 48, 7-10. (30) Scott, R. P. W. “Liquid Chromatography Detectors”; Elsevier: New York, 1977;p 28. (31) Compton, 6. J.; Purdy, W. C. J. Chromatogr. 1979, 169,39-50. (32) Sydor, R. J.; Hieftje, G. M. Anal. Chem. 1978, 48, 535-541. (33) Pao, Y.-H.; Zitter, R. N.; Griffiths, J. E. J. Opt. Soc. Am. 1966, 56, 1133-1135. (34) O’Haver, T. C. Anal. Chem. 1978, 50, 676-679.
RECEIVED for review December 16,1980. Accepted March 23, 1981. This research was supported by the National Institutes of Health, Grant No. PHS R 0 1 GM 24349. Preliminary results were reported at the 31st Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Atlantic City, NJ, 1980.
Identification of Organic Compounds on Diesel Engine Soot Ming-Li Yu and Ronald A. Hites” School of Public and Environmental Affairs and Department of Chemistfy, Indiana University, 400 East Seventh Street, Bloomington, Indiana 47405
Several studies have shown than extracts of soot collected from Ilght-duty dlesel cmgines cause mulatlons In bacteria and mammalian cells both with and wlthouit metabolic activation. To help ldenttfy the speclflc compounds responsible for these biological effects, we studied the detailed Fhemlcal composltlon of one such extract by gas chromatographic mass spectrometry. The iwo most mutagenic fractlons contain alkylated phenanthrenes, fluorenes, fluorenones, and other polycyclic aromatic hydrocarbons, aldehydes, and qulnones. One nitro polycyclic aromatic compound was also identified. The biological Implications of these findings are dlscussed.
The corporate average fuel economy (CAFE) standard and the price of gasoline have induced the American automobile industry to manufacture more automobiles with improved fuel economy. Since diesel engines generally give 25% better fuel economy than gasoline engines ( I ) , many automobile manufacturers are increasing the “dieselized” fraction of their product. By 1990, it is expected that 1 5 2 0 % of the automobile fleet in the United States will be powered by diesel engines (1). This is a mixed blessing. Although the nation may be able to conserve petroleum by this change, it might be sacrificing the health of both its people and its eeviroment. These potential problems are dlue to the high emission of particulates and associated organic compounds from diesel engines. On the average, light-duty diesels produce 0.5 g of particulates/mile ( I ) . Although these particulates are mostly carbon, 10-40% by weight can be extracted witlh organic solvents (2). Unfortunately, this extractable material is highly mutagenic both with and without metabolic activation (3, 4). The chemical composition of the material extractable from diesel particulates is clearly important information. Although
several such chemical studies are now in progress (5-8), very little compositional information on diesel exhaust has been published. In this paper, we will discuss the composition of two subfractions of diesel particulate extractables which have been shown to be especially mutagenic (4).
EXPERIMENTAL SECTION A particulate sample was obtained from a 350 CID, 1978, Oldsmobile diesel engine running on commercial diesel fuel. Hot start, federal test procedure cycles were run, and the exhaust was diluted by about a factor of 10 in a dilution tunnel. The total flow rate in this dilution tunnel was 100-200 m3/h. About 0.1-0.3 m3/h were sampled, and the particulates were collected on a 0.2-pm glass fiber fiiter (Pallflex T60-A20). The filter was extracted with methylene chloride for 1-6h, and the solvent was evaporated on a steam bath with nitrogen purging. The final residue was about 2 g. Small amounts (-50 mg) of the crude extract were separated into seven fractions on a silicic acid column (8 cm X 0.8 cm i.d.). The sequence of the eluants was as follows: hexane (3 mL), 1:l hexane/toluene (3 mL), toluene (3 mL), methylene chloride (3 mL), 2:l methylene chloride/methanol(3 mL), 1:2 methylene chloride/methanol (3 mL), and methanol (3 mL). Chemical analyses have focused on the hexane/toluene and toluene fractions because these have been shown to be the most mutagenic (4). Gas chromatography was performed on a Hewlett-Packard 5730 gas chromatograph equipped with a flame ionization detector. A 15 m X 0.26 mm i.d. glass c a p i l l q c o l coated ~ with SP-2100 methyl silicone stationary phase was used. Gas chromatographic mass spectrometrty was done on a Hewlett-Packard 5982A mass spectrometer interfaced to a 5933A data system. The identification of the GC/MS peaks was based on careful comparison of the observed spectra with published spectra and on fundamental interpretation. GC retention
0003-2700/81/0353-0951$01.25/00 I981 American Chemical Society
