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Anal. Chem. 1987, 59, 334-339
Chlorine-Selective Detector for Microcolumn Liquid Chromatography S t a f f a n Folestad,* Bjorn Josefsson, a n d P e t e r Marstorp
Department of Analytical and Marine Chemistry, Chalmers University of Technology, University of Goteborg, S-412 96 Goteborg, Sweden
A novel design of a chlorlnegelectlveflamebased detector adapted to mlcrocokmn reversebphaseliquid chromatography is described. The total column effluent (20-70 pUmln) Is Introduced via a thermospray Interface Into a heated oven system. Chlorinated compwds are converted, after pyrolysls In a hydrogen gas stream, to Indium( I ) chloride, whkh Is subsequently exclted In a cool hydrogen dlffuslon flame to emlt at 360 nm. The detection h i t Is 9 pg/s ( S I N = 3) for l,l,2-trlchloroethane in water. The Influence on lndlum CMOrkle and background emlsdon was lnvestlgated, exploring the range of compatlble solvents and buffers added to the mobile phase. Three selected organlc sdvents were tested and they were found to reduce the senslthrlty by 1 order of magnitude. A linear response from 5 ng up to 70 ng for 1,1,2-trlcMoroethane In 15% methanol/water was observed. Nonvdatlle compounds wch as chlorlnated uraclls, guanine, and guanosine could be detected selectively. For these compounds, 80-90% of the chlorlne content was converted, compared with equivalent amounts of chlorlne Injected as hydrogen chloride. Pharmaceuticals exempllfled with chloramphenicol sodhnn succlnates could also be determlned 88lectively In complex mixtures. The detector has practlcalty no dead volume.
There is a great need for a chlorine-selective detector in liquid chromatography (LC), since a large number of chlorinated nonvolatile and high-molecular-weight compounds are not suitable for chromatography analysis. Various attempts have been made to modify existing halogen-sensitive gas chromatography (GC) detectors for use with LC. In this respect electron-capture detection has been used on-line with nebulizer (I), moving wire (2),and/or furnace systems (3). The more applicable reversed-phase LC has been coupled to an electron-capture detector via an on-line extraction module inserted between the separation column and the detector. In that system, toluene or hexane was used to ektract halogenated organics from the water phase ( 4 ) . The commercial Hall electrolytic conductivity GC detector has also been modified for use with LC. The column eluent was introduced together with hydrogen make-up gas into a heated quartz furnace of a Hall detector (5). The choice of LC solvent was limited to methanol and methanol-water mixtures, since with other organic solvents extensive carbon deposits accumulated in the detector furnace. Still, these detectors possess limitations either in handling nonvolatile compounds or in compatibility with reversed-phase LC. Furthermore, the specificity for chlorine is limited. Flame photometric detection (FPD) seems to be a promising technique (6). These detectors have a small dead volume because the column effluent can be directly introduced into the flame. Furthermore, the detector can be selective for certain elements, and the mobile phase interaction may be controlled. The principle of FPD is that molecules which contain heteroatoms such as phosphorus, sulfur, or halogens, when burned
in a cool hydrogen-rich flame, produce chemiluminescent species (halogen with added indium) that emit light at characteristic wavelengths. Thus FPD can be utilized as an element-selective detector in LC. Flame photometric wlective detection of chlorine using the indium(1) chloride emission band at 360 nm was proposed by Gilbert (7). The detection principle has been demonstrated in combination with GC and shown to give good sensitivity, selectivity, and a large linear dynamic range (8-12). The principle was utilized by Gutsche et al. (13)in combination with LC, with a moving chain interface to eliminate the organic solvents. The drawback is that a significant amount of band dispersion is introduced. Folestad and Josefsson (14)aspirated the LC effluent directly into a primary flame and used a platinum catalyst for efficient conversion of the organic chlorine compounds into hydrogen chloride for selective indium chloride detection. When the column effluent is directly introduced into the flame, the organic solvents severely reduced the emission from InCl so that only small percentages of an organic solvent could be tolerated. Furthermore, a gradient of combustible organic solvents changes the temperature and fuel-to-oxidant ratio of the flame. Consequently, the response of the detector also changes. Large mass-flows can disrupt the delicate balance of chemical and physical processes in the flame. The introduction of organic solvents, metal ions, and buffers even at low concentrations has been reported to cause severe chemical and spectral interference with flame-based detectors for conventional LC (14-16).Novotny and co-workers (17,18) have shown that miniaturized LC systems, using packed capillary columns with an inner diameter (i.d.) of 0.2 mm or less, are well suited for flame-based detection of sulfur and phosphorus. The reduced volumetric flow rates, 0.5-20 kL/min, allow the direct introduction of column effluents containing organic solvents into the detector. In addition, flame-based detectors are mass-sensitive devices so that their sensitivity will generally not be reduced with further miniaturization, as is the case with optical detectors. A novel chlorine-selective detector is presented, compatible with micro LC flow rates from 10 to 100 pL/min and based on the selective indium chloride emission at 360 nm. The total microcolumn effluent is introduced into an oven system on-line with a cool hydrogen diffusion flame. Chlorinated compounds are pyrolyzed in a hydrogen atmosphere at temperatures up to 1000 OC. The performance of the detector is characterized, and its potential as a versatile detector for reversed-phase micro LC is shown. EXPERIMENTAL SECTION Detector. The chlorine-selective flame photometric detector is shown schematically in Figure 1. A fused silica capillary, with an inner diameter of 50 p m or 100 pm, or a stainless steel capillary, 120 pm i.d., transports the total LC column effluent into the detector system. Two configurations of the capillary inlet to the detector have been used. In the first (A in Figure 11, the capillary is embedded in a 4 cm long quartz wool plug, located in a quartz tube with 2.6 mm i.d. and 13-15 cm length. A second configuration (B in Figure 1)consists of a fused silica capillary inserted through
0003-2700/87/0359-0334$01.50/00 1987 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 59, NO. 2, JANUARY 1987
335
Indium region
Quartz tubes 0
A
AI
,Quartz
wool
ii
Detector oven
! ~I !lI
Nitrogen inlet Hydrogen inlet Capillary
Flgure 1. Schematic view of the detector unit.
a piece of stainless steel capillary. A 2-cm length of the tip of the stainless steel capillary is heated electrically, thus heating the inner fused silica capillary which in turn produces a jet of vapor. Silica particles coated with indium (see below) are placed between two small plugs of quartz wool and positioned 6 cm above the outlet of the LC connection. The length of the indium region is 12 mm and terminates 6 mm from the analytical flame. The column effluent is transported by hydrogen gas through the inner reactor tube. This tube is surrounded by an outer quartz tube, 5 mm i.d. and a length of 10-12 cm, which leads nitrogen gas (300 mL/min) to the flame. The flame tip cap was made of stainless steel. The oven system was homemade from a Kanthal wire, Alkrothal, 2 X 0.2 mm, 3.36 Q/m, and 35-40 cm length. The wire was coiled in 15 rounds making the length of the heating zone 4.5 cm. The wire was insulated with a needled blanket of ceramic fiber, 1 2 mm thick, enclosed by a glass fiber cloth in a brass cylinder, The oven power supply was normally set to give a power of 65 W. A Pt/Pt,Rh thermocouple was used for measuring temperatures. The oven system was mounted on a rack and pinion arrangement for vertical adjustment purposes. The flame was positioned in the middle between a concave spherical reflector (focal length f = 25 mm, 60-mm diameter) and a biconvex synthetic fused silica lens (f = 50 mm, 50-mm diameter) all in an aluminum housing which prevents external light from entering. The distance between the reflector and lens is 100 mm. An interference filter, Zeiss UV 360, fwhm 8 nm, having a transmission of 33% at 360 nm is placed just behind the lens. A second biconvex lens, (f = 150 mm, 50-mm diameter) is positioned 200 mm from the primary lens. A spot at the flame with a radius of 7 mm is then viewed by the photomultiplier tube (PMT), EM1 9558 QB. The PMT was normally supplied with 800 V from an EM1 high-voltage power supply (Model PM28B). An electrometer, Keithley 610C, was used for amplifying the electric signal from the PMT. The high frequency noise components were removed with a Spectrum 1021A low-pass filter before the signal was monitored on a Perkin-Elmer strip chart recorder, P E 561. The overall time constant was measured to be 0.25 s when operating without the low-pass filter. The complete analytical system is schematically presented in Figure 2. Chromatographic System. A Varian 8500 syringe pump was modified for the purpose of delivering pulse-free flow rates in the range 5-250 pL/min. The driving frequency to the stepping motor was divided by a factor of 10 or 100, facilitated by installing a frequency divider circuit in the pump control electronic system. Injections were made with a Valco, air-actuated, 0.2-pL internal loop valve and also, during optimization procedures, with a Valco external loop value equipped with a 200-pL or a 3.5-pL loop. The microcolumns were constructed from 1/16 in. 0.d. stainless steel
tubing, 250 X 1mm, and slurry packed at 900 atm with methanol as the driving solvent and methyl iodide or methanol/glycerol (75/25) as the slurry solvent for Nucleosil OH 7 pm and Spherisorb ODS2 5-pm packing material, respectively. Chemicals. The indium-coated silica particles were prepared from kieselguhr, supplied by Merck for GC, particle size 0.2-0.3 mm. Sedimentation in water was carried out several times to remove fines. Indium(II1) nitrate, purchased from Merck, was dissolved in water and the large granulates of the kieselguhr were poured into this solution. The water was evaporated from the slurry and the dried particles were heated until both the conversion to the red-brown indium(II1) oxide and the removal of nitrous gases were complete. Subsequently, a quartz tube was filled with the particles and heated while purging with hydrogen gas. A final sedimentation of the coated particles was carried out prior to use. The coated particles contained approximately 1.3 mg of indium per mg of kieselguhr. All organic solvents utilized in this investigation were Rathburn HPLC grade and the water was double distilled. The other chemicals used were of reagent grade. 5-Chlorouracil, 4-chlorouracil, 6-chloroguanine, and 6-chloroguanosine were purchased from Sigma and chloramphenicol sodium succinate from Parke-Davis & Co., Pontypool, Gwent, Great Britan. Ancillary Equipment. A Jasco Uvidec 100-111 variablewavelength UV detector was modified to make it possible to draw a fused silica capillary through the cell compartment. After removal of the fused silica capillary polyimide coating on the part in the light path, this arrangement was used to monitor effluents in the connecting tube between the microcolumn and the flame detector. Transmission spectra of interference filters were obtained on a Hitachi 100-60 UV spectrophotometer. Procedures. During optimization flow-rate studies and the study of the effect of organic solvents on emission, 200-pL injections were made. Thereby, both the response of chlorinated compounds and chemical interferences, causing deviations from the expected ideal plug profile, could be monitored. Water was used as mobile phase throughout the gas-flow-rate investigations and 15% MeOH in water was used when varying the mobile phase flow rate.
RESULTS AND DISCUSSION A successful combination of a flame-based detector with a liquid chromatograph relies on handling the transport of a solute from the liquid effluent t o the analytical flame. In order to utilize the principle of the specific emission of indium chloride, the chlorinated solute must first be decomposed and subsequently converted to indium chloride. Dual flame systems have been the common design of these flame pho-
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 2, JANUARY 1987
Table 1. Conversion Efficiency, Signal, Noise, and Signal Reduction due to Organic Solvents for Different Reactor Tube Inner Diameters Compared under Similar Conditions conversion efficiency reactor tube i.d., mm compared to HCl, 9i
noise"
b
C
b
C
1 3.3 12.3
1 3.4 4.4
1 3.2 6
1
2.8 10.7
1 0.9 3.6
90 97 95
1.4 2.6 4.9
signal"
cross-sectional area ratioa
Normalized to the 1.4 mm i.d. tube. Solute, 1,1,2-trichloroethane. *Mobile phase: 100% water. 'Mobile phase: 10% acetonitrile in water. /LENSES\
FL?ME
MICRO COLUMN
LOWPASS
MIRROR
U INJECTOR
I
RECORDER
1
PUMP
Figure 2. Schematic diagram of the chlorine-selective detector coupled to a micro LC system.
tometric detectors (7-12). The column effluent is burnt in a primary hydrogen diffusion flame and compounds that contain chlorine are decomposed, followed by conversion to indium chloride in a region between the primary and a secondary cool hydrogen diffusion flame. The secondary flame, where the chemiluminescence appears, is supplied with the excess of hydrogen gas from the primary flame. When nonvolatile compounds are present in the column effluent, it is important that the temperature in the primary flame is kept sufficiently high in order to achieve efficient vaporization and decomposition of organochlorine compounds. There is a need for careful control of the temperatures and the chemical environment during the stages of decomposition, conversion to indium chloride, and, finally, emission measurements. A dual flame construction was initially miniaturized in the development of a detector suitable for microcolumn liquid chromatography. However, problems associated with difficulties in efficient evaporation with 20-100 pL/min mobilephase flow rates resulted in a low sensitivity. Additionally, it was difficult to maintain a stable flame and to control the temperatures in the different stages. Detector Design and Temperature Control. On the basis of experiences with dual flames, an alternative design with a reaction chamber in an oven system is presented. In this device the decomposition of high-molecular-weight nonvolatile organohalogens could efficiently be carried out at high and precisely controlled temperatures. The presented detector works with temperatures up to lo00 "C in the center of the oven, Figure 1. The alternative of a lower temperature and an added catalyst was not successful since deactivation occurred. This effect has been observed earlier when chlorinated organics were catalytically decomposed (19). Different inlet capillaries made from stainless steel or fused silica were inserted into the oven in the region where the temperature can be varied between 100 and 500 "C. For the codiguration A, Figure 1, where the capillary is embedded in untreated quartz wool, the temperature must exceed 300 "C for efficient evaporation. However, for nonvolatile compounds, adsorption occurred onto the quartz wool. With configuration B this adsorption was eliminated. The evaporation temperature with the electrically heated capillary tip was separately controlled. The system was operated with the
temperature set at 250 "C. The performance of this interface is similar to LC-MS thermospray interfaces (201, although it is not operated under vacuum conditions. With eluents containing phosphoric acid, the 120-pm-i.d. steel capillary was rapidly clogged, a consequence of the active surface of stainless steel at elevated temperatures. The surface of the fused silica capillary was inert toward phosphoruscontaining buffers. Therefore, a fused silica capillary was used, inserted into the heated steel capillary. However, during long run operation, carbonaceous deposits caused clogging even of the fused silica capillary. To prevent this, it was regularly replaced after 2-4 days. In addition, this fused silica capillary is the only connecting tube between the column and the oven system, and consequently, the detector has practically no dead volume. The metallic indium should be kept in the liquid state a t a temperature within the range 200-420 "C in order to obtain optimal sensitivity. It is also essential that the indium surface area should be as large as possible. Metallic indium can be suspended on a wire mesh (9, IO), in a steel cup ( I I ) , in a heated reservoir (12) or coated on copper-beryllium coils (21). In order to obtain as large an area of indium as possible, coating of silica particles has been used in this work. The indium-coated silica particles were positioned in the inner reactor tube where the temperature was in the range 250-400 "C. When a freshly prepared indium-coated particle plug had been installed, the system had to be conditioned for a t least 1h. Initially there is a high background which decreases and then stabilizes. The dominating species in the background emission at 360 nm is Inz and a drifting base line occurs until the flux of Inz becomes stable. The lifetime of the indium layer depends on the conditions under which the system has been operated but is typically a t least 1month. The use of high concentrations of organic solvents (50-100% methanol) caused carbonaceous deposits on the indium-coated particles. The system could be reconditioned by passing oxygen through the heated inner reactor tube followed by hydrogen. During the optimization three different reactor tubes with inner diameters of 1.4, 2.6, and 4.9 mm were compared with regard to their performance under similar conditions. Sensitivity, signal, background, and reduction due to organic solvents were compared by using only hydrogen gas. From Table I it can be concluded that, although the signal is proportional to the cross-sectional area for the two smaller tubes, the signal to noise (S/N) ratio is approximately the same. The lower S / N ratio with the large tube may be caused by an insufficient transport of indium into the analytical flame. With reduced signals, due to organic solvents, the S / N ratio differs more between the three reactor tubes, mainly due to the difference in noise. A change in the ratio of the amount of hydrogen gas to water vapor or a change in evaporation conditions are the most likely explanations. In addition, alteration of the ratio of hydrogen gas to indium chloride may also contribute to this behavior. The 2.6-mm-i.d. reactor tube was selected for further investigation on the basis of a compromise between the gas flow rate, the residence time of the solute in the reactor, and the
ANALYTICAL CHEMISTRY, VOL. 59, NO. 2, JANUARY 1987
Table 11. Effects of Organic Solvents on InCl Emission
70
organic solvent methanol
5 20
50 acetonitrile THF
10 30 10
relative emission intensity at different flame positions upper part flame base of the flame 67
58 34 34 25 32
49
45
Solute, 1,1,2-trichloroethane. mobile-phase flow-rate range. Gas Flow Rate Studies. The hydrogen gas flow rate was varied in the range 50-200 mL/min. An increase in the gas flow rate within this range caused a slight decrease in the signal. Additionally, the S / N ratio decreased significantly due to an increase in noise accompanied by an increase in background. A t low hydrogen flow rates ( 0.9989) was obtained from 0.3 and 4.3, respectively, to at least 70 ng of C1. The methanol-water mixture yielded a response reduction of 22 % . Applications. With a UV detector coupled on-line, a separation of some phenolic compounds was performed, as shown in Figure 3. The selectivity of the chlorine detector is demonstrated, whereas only the chlorinated compounds are indicated. The selectivity is further demonstrated in the separation of some therapeutic drugs in Figure 4b,c. Chloramphenicol and its monosuccinate ester where chosen as model chlorine-containing compounds, since they are not suited for direct gas chromatographic separation. Also chloramphenicol and its intravenously administered form, the ester, are of medical importance as antibiotic drugs. The two peaks from the chloramphenicol monosuccinate are due to the two isomers, chloramphenicol 1-succinateand chloramphenicol 3-succinate (25). The other drugs added to the sample are compounds that might be coadministered with chloramphenicol monosuccinate. In the chromatogram from the UV detector the peaks from theophylline and chloramphenicol are overlapped by the peaks from (P-hydroxyethy1)theophyllineand paracetamol. The peak from carbamazepine is also overlapped by the peaks from the two chloramphenicol monosuccinates. These interferences are eliminated in the corresponding
338
ANALYTICAL CHEMISTRY, VOL. 59, NO. 2, JANUARY 1987 1 i
3
Ci-Det
II ’
CI-Det
UV 274 nm
I
I
0
$0 min
10
I
0
I
10
$0 min
$0
Flgure 3. W i t e c t o r selectivity: column, 250 X 1 mm, Nucleosil OH, 7 pm; mobile phase, acetonitriWwater, 20:80; flow rate, 30 pL/min; solutes, (1) phenol, (2) 0-cresol, (3) 2-chlorophenol, and (4) 2,5-dichlorophenol. Gas flow rates were as follows: H2 85 mL/min
Figure 5. Chromatogram of four nonvolatile chlorinated bases: column, 250 X 1 mm, Spherisorb ODS2, 5 pm: mobile phase, methanoVwater, 10:90 with 25 mM acetic acid; flow rate, 50 pL/min; solutes, (1) 5-chlorouracil, (2) 4-chlorouracil, (3) 6-chloroguanine, and (4) 6-
and N, 330 mL/min.
chloroguanosine. Gas flow rates are given
in Figure 3.
with respect to equivalent amounts of chlorine content injected as hydrogen chloride. The chlorine-selective detector offers possibilities to study nonvolatile chlorinated compounds with reversed-phase LC. For optimal performance, flow rates within the range 30-50 wL/min must be used. Thus, the detector i s suited for work with packed columns of about 1mm i.d. For application t o separations where higher concentrations of organic solvents (>50%)as well as where higher sensitivity are necessary, more studies are needed.
7
ACKNOWLEDGMENT We thank Bo Galle for helpful suggestions in construction o f the detector optics and Ben@Kinberger and Goran Ostling for the gift of t h e drugs. Registry No. Cl,, 7782-50-5; In, 7440-74-6; InCl, 13465-10-6.
LITERATURE CITED
r 0
I
I
1
10 20 30 min Figure 4. Chromatogram of therapeutic drugs: column, 250 X 1 mm, Nucleosil OH, 7 pm; mobile phase, methanoVwater, 3:97 with 25 mM acetic acid: flow rate, 30 wL/min; solutes, (1) trimethoprim (410 ng), (2) papaverine (420 ng), (3) (/3-hydroxyethyl)theophylllne (470 ng), (4) chloramphenicol (420 ng). (5) theophylline (430 ng), (6) paracetamole (430 ng). (7) chloramphenicol 3-succinate (1.5 p g including (9)), ( 8 ) carbamazepine (930 ng) and (9) chloramphenicol 1-succinate. Gas flow rates are given in Figure 3. (a) Separation of the non-chlorinecontaining drugs 1-3, 5, 6, and 8. (b and c) The same separation as in (a) but with the chlorine-containing drugs 4, 7, and 9 added. chromatogram f r o m the chlorine detector. This was confirmed i n j e c t i n g t h e non-chlorine-containing compounds separately, F i g u r e 4a. C h l o r i n a t e d f o r m s o f nonvolatile bases, such as 4-chlorouracil, 5-chlorouracil, 6-chloroguanine, and 6-chloroguanosine have also selectively been monitored with the present detector. T h e separation, as shown in F i g u r e 5, was p e r f o r m e d on a reversed-phase m i c r o c o l u m n with a m o b i l e phase of 10% m e t h a n o l in water. The response was between 80 and 90%
by
Nota, G.; Palombari, R. J . Chromatogr. 1971, 62, 153-155. Maggs, R. J. Column 1988, 2(4), 5. Willmot. F. W.; Dolphin, R . J. J . Chromatogr. Sci. 1974, 12, 695-700. Brinkman, U. A. Th.; Maris, F. A. TrAC, Trends Anal. Chem. (Pers. Ed.) 1985, 2(4), 55-59. Shepherd, M. J.; Wallwork, M. A.; Gilbert, J. J . Chromatogr. 1983, 261, 213-222. McGuffin, V. L.; Novotny. M. V. In Microcolumn Separations; Columns, Instrumentation and Ancillary Techniques: Novotny, M. V., Ishii, D., Eds.; Elsevier: Amsterdam 1985; pp 197-217. Gilbert, P. T. Anal. Chem. 1966, 3 8 , 1920-1922. Gutsche, 9.; Herrmann, R.; Rudiger, K. Fresenius’ 2. Anal. Chem. 1088, 24 1 , 54-66. Overfield, C. V.; Winefordner, J. D. J . Chromatogr. Sci. 1970, 8 , 233-242. Bowman, M. C.; Beroza, M. J . Chromatogr. Sci. 1971, 9 , 44-48. Moseman, R. F.; Aue, W. A. J . Chromatogr. 1971, 6 3 , 229-236. Wells. G. Anal. Chem. 1983. 55. 2112-2115. Gutsche, B.; Herrmann. R.; Hohme, M.; Rudiger, K. Spectrochim.Acta 1978, 3 3 , 609-823. Folestad, S . ; Josefsson, B. J . Chromatogr. 1981, 203. 173-178. Julin. B. G.; Vanderborn, H. W.; Kirkland, J. J. J . Chromatogr. 1975, 112. 443-453. Chester, T. L. Anal. Chem. 1980, 52, 638-642. McGuffin. V. L.; Novotny, M. Anal. Chem. 1981, 5 3 , 946-951. Gluckman, J. C.;Novotny, M. V. J . Chromatogr. 1984, 314, 103. Nystrom, M. Ph.D. Thesis, Chalmers University of Technology, Gothenberg, Sweden 1976. Blakely, C. R.; Vestal, M. L. Anal. Chem. 1983, 5 5 , 750-754. Herrmann, R.; Gutsche, 9. Anayst (London) 1969, 9 4 , 1033-1035. Sugiyama. T.; Suzuki, Y.; Takeuchl, T. J . Chromatogr. 1973, 8 0 , 61
Anal. Chem. 1987, 59, 339-343 Chen, H. H. L. W.D. Thesis, University of Iowa, Iowa CW, I A , 1975. Fredriksson, S. A; Cedergren, A. Anal. Chem. 1081, 53, 614-618. (25) Brent, D. A.; Chandrasurin, P.; Ragouzeos, A.; Huribert, 8. S.; Burke, (23)
(24)
J. T. J . Phafm. Scl. 1080, 89. 906.
RECEIVED for review June 18,1986. Accepted September 9,
339
1986. This paper was presented in part a t the 9th InternaLiquid Chromatography, ~ d tional ~~~~~i~~ on column inburgh, July 1985. The work was supported by the National Swedish Environment Protection Board and the Swedish Natural Science Research Council.
High-Efficiency Microcolumn Liquid Chromatography Separation and Spectral Characterization of Nitrogen-Containing Polycyclics from Fossil Fuels Claudio Borra, Donald Wiesler, and Milos Novotny* Department of Chemistry, Indiana University, Bloomington, Indiana 47405
Very large polycycilc compounds, with one or more nitrogen atoms In their structures, were separated from fossil fuel samples and characterized spectroscoplcaily. High chromatographic efficiencies (around 200 000 theoretical plates) of microcolumn liquid chromatography were essential to separate such complex mixtures: within structures of 3-10-rings, almost 170 peaks were resolved and over 600 nitrogen poIycycuCS were spectrally characterized. Most of the structural data were obtained from mass spectroscopy; however, fluorescence emission spectra yielded additional Information concerning the shape of molecules.
All fossil fuels appear to contain some nitrogen, which is believed to be present almost exclusively in the organic portion of the crude materials. Its levels usually vary from approximately 0.5% in crude petroleum samples to 1-2% in shale oils and coal. Distillation, extraction, and combustion produda contain nitrogen compounds as minor components. Although these compounds have not been investigated as extensively as hydrocarbons and the neutral aromatic components, the compositional information is important for several reasons. Nitrogen polycyclic aromatic compounds (NPAC) are deemed responsible for some problems during the refining process because of their association with color, odor, corrosive power, and the formation of gums and deposits during storage ( 1 , 2 ) . In addition, they deactivate the catalysts during the cracking and re-forming process (3). Numerous basic nitrogen-containing substances are toxic. Several azaarenes and primary aromatic amines have been reported to be tumorigenic in experimental animals,while additional compounds of this type are suspected carcinogens or cocarcinogens (4-6). Furthermore, several neutral nitrogen aromatics, such as dibenzocarbazoles, show carcinogenic activity (7). Capillary gas chromatography (GC) and its ancillary techniques have been, over the last decade, used extensively to investigate various fossil fuel samples. However, the gas-phase analytical methods fail when the compounds to be analyzed are either too large or highly polar. Concurrently, conventional high-performance liquid chromatography (HPLC) lacks the necessary separation efficiency to provide adequate component resolution of the mixtures with increasing molecular weight and the number of possible isomers. TWOprevious reports from this laboratory (8,9)have demonstrated the ability of
microcolumn liquid chromatography (LC) to separate high molecular weight neutral polycyclic molecules of up to ninering structures. The present report describes the techniques that expand the analytical scope for nitrogen polyaromatics. When microcolumns with more than 200 000 theoretical plates were used, nearly 170 NPAC peaks (up to ten-ring structures) were resolved. Moreover, since up to several microgram amounts can be injected into slurry-packed LC capillary columns (10) without serious overloading, the individual peaks were trapped and investigated by mass spectrometry (MS). To further aid the structural characterization of such compounds, the recently developed intensified photodiode array (IPDA) fluorescence detector (11,129 was coupled with the capillary LC columns. On-line fluorescence emission spectra from the NPAC eluted peaks were thus obtained.
EXPERIMENTAL SECTION SRC-I1 fuel oil blend in a 5.75:l ratio of middle-to-heavy distillate (Pittsburgh and Midway Coal Mining Co., DuPont, WA; code no. 1701),COED syncrude oil (FMC Corp., Princeton, NJ; code no. 1106),and crude shale oil (code no. 4601), obtained from the Fossil Fuel Research Matrix Corp. repository (administered by the Oak Ridge National Laboratory), were used in this study. These materials were of a pilot plant origin and should not be considered as representative of products that may eventually be produced on a commercial scale. NPAC fractions were separated from the crude sample according to the procedure of Later et al. (13),by using a column packed with neutral alumina. The adsorbent was activated at 300 "C for 15 h prior to use. To further fractionate the NPAC samples, a silicic acid column was employed. With a slight deviation from the procedure of Later et al., the following fractions and compound types were recovered: (a) fraction S-1, containing secondary nitrogen polycyclic aromatic heterocycles (2OPANH), was eluted with 50 mL of 1/1(v/v) hexane/benzene; (b) fraction S-2, containing amino polyaromatic hydrocarbons (APAH) and tertiary nitrogen polycyclic aromatic heterocycles (3OPANH),was eluted with 40 mL of benzene. Each fraction was evaporated to dryness and then redissolved in an appropriate amount of tetrahydrofuran (THF). HPLC was employed to eliminate low molecular weight substances. A 4.6 mm X 25 cm, 5 pm particles, Techsphere Ultra CI8 column (Phenomenex, Rancho Palos Verdes, CA) was used. The flow rate was set at 1.5 mL/min. Solvent A was THF/acetic acid/triethylamine 100/0.1/0.2 (v/v/v); solvent B was acetonitrile/water/acetic acid/triethylamine 55/45/0.1/0.2 (v/v/v/v). The initial mobile phase contained 0.1% A. Fifteen minutes after
0003-2700/67/0359-0339$01.50/00 1987 American Chemlcal Society
