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A comparison of capillary chromatographic techniques for the separation of very large polycyclic aromatic molecules
Anolytico Chimica Acta, 127 (1981) 55-61 Ekevier Scientific Publishing Company, Amsterdam -
Printed in The Netherlands
A COMPARISON OF CAPILLARY CHROMATOGRAPHIC TECHNIQUES FOR THE SEPARATION OF VERY LARGE POLYCYCLIC AROMATIC MOLECULES
YUKIO HIRATA. Chemistry
and MILOS NOVOTNY*
Department,
Indiana
PAUL A. PEADEN and MILTON Chemistry
Department,
Brigham
University, Bloomington,
IN 47405
(U.S.A.)
L. LEE
Young
University.
Provo,
UT 84602
(U.S.A.)
(Received 24th September 1980)
SUMMARY Current advances in the technology of capillary columns for gas chromatography permit extension of the range of polycyclic aromatic compounds which may be eluted. High-performance liquid chromatography (h.p_l.c.) in the non-aqueous, reversed-phase mode shows promise forseparation of even larger molecules: an inquiry into the feasibility of capillary h.p.1.c. in this area has resulted in elution of polycyclic compounds containing up to eleven aromatic rings.
The detrimental health effects caused by polycyclic aromatic compounds (PAC) that are generated in various combustion processes are wellestablished. Certain of these substances are known to be mutagenic and carcinogenic. While various analytical procedures have been developed in numerous labortories for their determination, chromatographic separations play a prominent role in this field [ 11. Mixtures of PAC isolated from various products of combustion (e.g., soot, coal tar, or smoke condensates) are frequently very complex, and the same is true for materials derived from petroleum or coal. When highefficiency gas chromatographic columns are employed, it is not unusual to observe 1OW200 compounds in such mixtures [2--41 in the range of 2-ring to 6-ring PAC structures. Capillary gas chromatography (g-c.) and high-performance liquid chromatography (h.p.1.c.) have been developed over the years as successful methods for PAC separation. Both methods hold advantages and disadvantages of their own. As has been established in previous studies [2--41, relatively short thin-film glass capillary columns (yielding typically 30 OOO80 000 theoretical plates for such separations) can clute PAC containing up to six rings within the usual temperature range of modem g.c. The typical “Present address: School Toyohashi. Japan.
of Materials
0003-2670/8l~0000+30001902.50
Science, Toyohashi
8 1981 Eisevier
Scientific
University of Technology,
Publishing
Company
56
upper temperature limits for these analyses lie between 240 and 260°C. Not only does capillary g-c. have the advantage of superior resolution (often necessary to distinguish various toxicologically important PAC isomers), but its combination with mass spectrometry provides a powerful idcntification tool. Very precise measurements of retention data [5, 61 are of additional benefit in structural assignments. High-performance liquid chromatography, usually in the non-polarstationary phase mode, is a relatively easy separation technique for PAC from the operator’s point of view. In conventional modem h.p.l.c., typical plate numbers arc about an order of magnitude lower than in capillary g-c. Correspondingly, lower component resolution is obtained. This lack of resolution can often be overcome by the use of selective detectors [ 7-91, such as the variable-wavelength absorbance or spectrofluorimetric monitors. Thus, reliable quantitative measurements of selected PAC are feasible with h.p.1.c. methods in spite of the obvious complexity of certain mixtures. The compound range in typical determinations overlaps with that indicated for capillary g.c. above. The shortcomings of h.p.1.c. are seen primarily in the lack of ancillary techniques for “on-line” structure elucidation of PAC, as well as insufficiently standardized column technology [lo] to measure retention date reliably. Somewhat different considerations apply for PAC larger than 6-ring structures. Because of volatility constraints, liquid chromatography becomes preferable. Although recent advances in the preparation of more thermally stable capillary columns [ll--141 can extend the range of separated compounds somewhat, the scope of this method for heavier PAC appears limited. This study examines the roles of both methods using the extracts of carbon black and coal tar as examples. Temperature limitations of the state-of-the-art glass capillary columns, including those with advanced surface treatments [ll] and chemically bonded polymers [ 12, 131, are demonstrated here. The recently developed capillary h.p.1.c. [ 15-171 is offered as an alternative for the separation of large PAC. although this method at present has limitations of its own. However, separations of up to ll-ring structures are shown to be feasible using non-aqueous, reversed-phase capillary h.pl.c. EXPERJMENTAL
AND
RESULTS
Sample prepam tion
The aromatic fraction of coal tar was obtained through a solvent partition scheme as previously described [18]. The carbon black sample used in this study was a gift from Cabot Corporation, Boston, MA. A Soxhlet extraction with methylene chloride was carried out as previously described by Lee and Hites [ 191. Extract aliquots w&& concentrated through solvent evaporation to the volumes appropriate for chromatographic separations.
57
Capillary gas chromatogmphy All g.c. runs were obtained with a Perkin-Elmer Model 990 gas chromatograph with the injector and the flame ionization detector connections modified for the use of glass capillary columus. A 15 m (0.27 mm i.d.) glass capillary was hydrochloric acid solution at 110°C Cl1 1, then statically with SE52 phenylmethylsilicone gum tories, State College, PA) to have 0.25 pm average
was conditioned
at 350°C
fust leached with a dilute silylated [20] and coated (Applied
Science
Labora-
film thickness. The column for 100 h prior to its use in chromatography. The
carbon black extract was injected at room temperature, and the column was subsequently programmed at 10% min-’ from 40 to 110°C and at 2’C mm-’
from 110 to 350°C. Figure 1 shows the chromatogram obtained, indicating only a slight elevation in baseline between 310 to 350°C. The structures of representative components were assigned, based on their mass spectra (Hewlett-Packard Model 5982A combined gas chromatograph/mass spectrometer) and retention of standard compounds. A complete identification of all sample components was not carried out. The last three structures on the chromatogram are only representative of the recorded “peak clusters”; the numbers placed below the structures represent the molecular weights. A 40 m (0.3 mm i.d.) glass capillary column was prepared with 0.3-tirn bonded film of a methylpolysiloxane according to the technology described by Blomberg and WInnman [ 12, 133 and washed extensively .with a series of organic solvents. The column was subsequently conditioned at 330°C for several hours. Figure 2 shows a comparison of coal tar and carbon black extract samples chromatographed under identical conditions (programmed
from 70 to 320°C
--i
at 2°C min-l).
Good baseline stability
at high temperatures
.Ji :
_.‘.
‘.
.‘. -_
I
Fig. 1. High-temperature separation of the polycyclic aromatic components of carbon black obtained with 15 m (0.27 mm i.d.) glass capillary column coated withSE-52 silicone gum. Carrier gas, helium.
58
a
b
Fig. 2. Chromatrograms of (a) aromatic fraction of cod tar, and (b) carbon black extract, obtained with 40 m (0.3 mm i.d.) glass capillary column provided with a film of bonded
polysiloxane phase. Carrier gas, helium. is demonstrated. was employed
Again, a combined gas chromatograph/mass spectrometer to ascertain the range of separation (see Fig. 2 b).
Capillary high-performance
liquid chromatography
The equipment used for capillary h.p.1.c. was recently described [17]. The column used was a 100 m (70 pm i-d.) thick-walled glass microcapillary packed with 30-pm alumina particles [ 151. The stationary phase (an octadecylsilyl bonded phase) was generated inside the column via an in situ bonding technique [ 161. The inlet pressure was set at 300 atm, while the flow rate was approximately 1 ~1 mm-‘. The stepwise gradient elution technique [ 171 was employed. The separated components were detected with a miniaturized spectrofluorimetric detector [ 171, with a cell volume of 0.1 ~1. A full-scale recorder deflection corresponds to approximately 1-ng amounts. Figure 3 shows the separation of the heavy components of the carbon black extract obtained during a 50-h run. Peaks from the selected parts of this chromatogram were trapped at the column exit and subjected to directprobe mass spectrometry and spectrofluorimetric measurement as previously described [21]. Although most identifications are tentative because of the lack of suitable standards for the high molecular weights, the presence of at least 1 l-ring structures was suggested.
!CiGi’--I -
I
.20
2
I
I
3 1
, 30
4 1 40
I
I 50
HOURS
Fig. 3. Separation of thecarbon black heavy aromatic components by h.p.l.c. obtained with 100 m (70 rm i-d.) glass capillary column containing 30 rm irregular alumina/C,,-bonded phase. Stepwisc gradient: (1) 100% acetonitrile; (2) 80% acetonitril~20% dichloroethane; (3) 50% acetonitril-50% dichloroethane; (4) 100% dichloroethane. DISCUSSION
The extraction of various PAC-containing materials with powerful organic solvents may result in an appreciable yield of extractables, but frequently only a small percentage of this material can be separated by g-c. For example, about 73% of solvent-refined coal (SRC) was found extractable [4] into methylene chloride, of which amount 18% and 21% correspond, respectively, to the aliphatic and the polyaromatic fractions. However, gas chromatography could cover only some 26% of the polyaromatic fraction and 11% injected aliphatic fraction, corresponding to 1 and 0.395, respectively, of the original SRC sample. At present, the remaining non-volatile compounds can neither be separated effectively nor identified in this and similar materials. Evidence exists for some very large PAC in numerous mixtures (e.g., certain petroleum fractions [22] ), but again an effective methodology for their complete separation and identification has not been developed yet. The toxicological and chemical significance of most PAC above 6-ring structures is unknown because of the unavailability of standard compounds and the lack of structural characterization. Recent advances in the technology of glass [ 11-131 and fused-silica [ 141 capillary columns make it feasible to extend the temperature limits of capillary g-c. As shown by Figs. 1 and 2, these improvements extend the
60
range for PAC up to eight rings. However,
temperatures
in excess of 350°C
are judged to be the present technological
limit of this method. Recently developed polysiloxane bonded phases [12, 131 seem to offer an interesting approach to the preparation of stable columns for hightemperature g-c. work. As this technology develops further, columns with different selectivities may become available. Figure 2 also exemplifies how different combustion products may vary in terms of their PAC composition: coal tar (a by-product of the coking processes) has only a few major components at the high-molecular-weight end of the chromatogram, while the carbon black extract primarily consists of much larger molecules. The peak asymmetry of the lateeluting components can be attributed to their limited solubihty in the methylpolysiloxane stationary pha*. Given the temperature limitations of gc., liquid-chromatographic techniques become the only viable chromatographic alternative for sample components with more than seven or eight condensed aromatic rings. However, the problem of sample complexity becomes enormous (as occurrence of isomers increases with molecular weight) so that conventional h.p.1.c. resolution is insufficient. While capillary h.p.1.c. has the potential (15, 231 for improved resolution, Fig. 3 demonstrates that much improvement is needed. Apart from the current instrumental limitations 115, 171 of smallvolume technology, long separation times are a major difficulty. Identification of the separated PAC components is a major task at present. Although their mass and fluorescence spectra may provide useful information, practically no reference compounds are available for this compound range. Once the techniques of combined liquid chromatography-mass spectrometry are adequately developed, capillary h.p.1.c. will have a distinct advantage with its very low flow rates (typically, 1 ~1 min-‘) for “on-line” investigations of the separated PAC. Meanwhile, repeated trapping of capillary fractions is tedious at best. Because of the limited solubility of large polycyclic molecules, the selection of the column phase system used in this work deserves further mention. The non-aqueous reversed-phase system is generally compatible with both solubility and retention of the large PAC molecules. It should be noted that the polarity of the octadecyl packing appears less than that of any of the solvents used. Severe tailing of the later components in Fig. 3 could be caused by solubility problems, insufficient column deactivation, or a combination of both. This work was supported by Grant No. 24349 from the National Institute of General Medical Sciences, U.S. Public Health Service. REFERENCES 1 M. L. Lee, M. Novotny Compounds, Academic 2 M. L. Lee, M. Novotny
and K. D. Bartle, Analytical Chemistry of Polycyclic Press, New York, 1981 (in press). and K. D. Bnrtle, Anal. Chem., 48 (1976) 405.
Aromatic
61 3 M. L. Lee, M. Novotny and K. D. Bartle, Anal. Chem., 48 (1976) 1566. 4 R. V. Schultz, J. W. Jorgenson, M. P. Maskarinec, M. Novotny and L. J. Todd, Fuel, 58 (1979) 783. 5 M. L. Lee, D. L. Vassibrros, C. M. white and M. Novotny, Anal. Chem., 52 (1979) 766_ 6 M. Novotny, R. Kump, F. Merii and L. J. Todd, Anal. Chem., 52 (1980) 401. 7 H. Boden, J. Chromatogr. Sci., 14 (1976) 391. 8 D. C. Hunt, P. J. Wild and N. T. Crosby, J. Chromatogr., 130 (1977) 320. 9 K. Ogan, E. Katzand W. Slavin, Anal. Chem., 51(1979) 1315. 10 K. Ogan and E. Katz, J. Chrornatogr., 188 (1980) 115. 11 M. L. Lee, D. L. Vassilaros, L. V. Phillips, D. M. Hercules, H. Azumaya, J. W. Jorgenson, M. P. Msskarinee and M. Novotny, Anal. L&t., 12(A2) (1979) 191. 12 L. Blomberg and T. Wannman, J. Chromatogr., 168 (1979) 81. 13 L. Blomberg and T. Wtinnman, J. Chromatogr., 186 (1979) 159. 14 R. Dandeneau, P. Bente, T. Rooney and R. Hiskes, Am. Lab., 11 (9) (1979) 61. 61. 15 T. Tsuda and M. Novotny, Anal. Chem., 50 (1978) 271. 16 Y. Hi&a, M. Novotny, T. Tsuda and D. Ishii, Anal. Chem.. Sl(l979) 1807. 17 Y. Hi&a and M. Novotny. J. Chromatogv., 186 (1979) 521. 18 M. Novotny, M. L. Lee and K. D. BartIe, J. Chromatogr. Sci., 12 (1974) 606. 19 M. L. Le and R. Hites, Anal. Chem., 48 (1976) 1890. 20 M. Novotny and K. Tesarik, Cbromatographia, 1(1968) 332. 21 P. A. Peaden, M. L. Lee, Y. Hirata and M. Novotny, Anal. Chem., 52 (1980) 2268. 22 K. H. Altgelt nad T. H. Couw, Adv. Chromntogr., 13 (1975) 71. 23 J. H. Knox and M. T. Gilbert, J. Chrometogr., 186 (1979) 405.
Printed in The Netherlands
A COMPARISON OF CAPILLARY CHROMATOGRAPHIC TECHNIQUES FOR THE SEPARATION OF VERY LARGE POLYCYCLIC AROMATIC MOLECULES
YUKIO HIRATA. Chemistry
and MILOS NOVOTNY*
Department,
Indiana
PAUL A. PEADEN and MILTON Chemistry
Department,
Brigham
University, Bloomington,
IN 47405
(U.S.A.)
L. LEE
Young
University.
Provo,
UT 84602
(U.S.A.)
(Received 24th September 1980)
SUMMARY Current advances in the technology of capillary columns for gas chromatography permit extension of the range of polycyclic aromatic compounds which may be eluted. High-performance liquid chromatography (h.p_l.c.) in the non-aqueous, reversed-phase mode shows promise forseparation of even larger molecules: an inquiry into the feasibility of capillary h.p.1.c. in this area has resulted in elution of polycyclic compounds containing up to eleven aromatic rings.
The detrimental health effects caused by polycyclic aromatic compounds (PAC) that are generated in various combustion processes are wellestablished. Certain of these substances are known to be mutagenic and carcinogenic. While various analytical procedures have been developed in numerous labortories for their determination, chromatographic separations play a prominent role in this field [ 11. Mixtures of PAC isolated from various products of combustion (e.g., soot, coal tar, or smoke condensates) are frequently very complex, and the same is true for materials derived from petroleum or coal. When highefficiency gas chromatographic columns are employed, it is not unusual to observe 1OW200 compounds in such mixtures [2--41 in the range of 2-ring to 6-ring PAC structures. Capillary gas chromatography (g-c.) and high-performance liquid chromatography (h.p.1.c.) have been developed over the years as successful methods for PAC separation. Both methods hold advantages and disadvantages of their own. As has been established in previous studies [2--41, relatively short thin-film glass capillary columns (yielding typically 30 OOO80 000 theoretical plates for such separations) can clute PAC containing up to six rings within the usual temperature range of modem g.c. The typical “Present address: School Toyohashi. Japan.
of Materials
0003-2670/8l~0000+30001902.50
Science, Toyohashi
8 1981 Eisevier
Scientific
University of Technology,
Publishing
Company
56
upper temperature limits for these analyses lie between 240 and 260°C. Not only does capillary g-c. have the advantage of superior resolution (often necessary to distinguish various toxicologically important PAC isomers), but its combination with mass spectrometry provides a powerful idcntification tool. Very precise measurements of retention data [5, 61 are of additional benefit in structural assignments. High-performance liquid chromatography, usually in the non-polarstationary phase mode, is a relatively easy separation technique for PAC from the operator’s point of view. In conventional modem h.p.l.c., typical plate numbers arc about an order of magnitude lower than in capillary g-c. Correspondingly, lower component resolution is obtained. This lack of resolution can often be overcome by the use of selective detectors [ 7-91, such as the variable-wavelength absorbance or spectrofluorimetric monitors. Thus, reliable quantitative measurements of selected PAC are feasible with h.p.1.c. methods in spite of the obvious complexity of certain mixtures. The compound range in typical determinations overlaps with that indicated for capillary g.c. above. The shortcomings of h.p.1.c. are seen primarily in the lack of ancillary techniques for “on-line” structure elucidation of PAC, as well as insufficiently standardized column technology [lo] to measure retention date reliably. Somewhat different considerations apply for PAC larger than 6-ring structures. Because of volatility constraints, liquid chromatography becomes preferable. Although recent advances in the preparation of more thermally stable capillary columns [ll--141 can extend the range of separated compounds somewhat, the scope of this method for heavier PAC appears limited. This study examines the roles of both methods using the extracts of carbon black and coal tar as examples. Temperature limitations of the state-of-the-art glass capillary columns, including those with advanced surface treatments [ll] and chemically bonded polymers [ 12, 131, are demonstrated here. The recently developed capillary h.p.1.c. [ 15-171 is offered as an alternative for the separation of large PAC. although this method at present has limitations of its own. However, separations of up to ll-ring structures are shown to be feasible using non-aqueous, reversed-phase capillary h.pl.c. EXPERJMENTAL
AND
RESULTS
Sample prepam tion
The aromatic fraction of coal tar was obtained through a solvent partition scheme as previously described [18]. The carbon black sample used in this study was a gift from Cabot Corporation, Boston, MA. A Soxhlet extraction with methylene chloride was carried out as previously described by Lee and Hites [ 191. Extract aliquots w&& concentrated through solvent evaporation to the volumes appropriate for chromatographic separations.
57
Capillary gas chromatogmphy All g.c. runs were obtained with a Perkin-Elmer Model 990 gas chromatograph with the injector and the flame ionization detector connections modified for the use of glass capillary columus. A 15 m (0.27 mm i.d.) glass capillary was hydrochloric acid solution at 110°C Cl1 1, then statically with SE52 phenylmethylsilicone gum tories, State College, PA) to have 0.25 pm average
was conditioned
at 350°C
fust leached with a dilute silylated [20] and coated (Applied
Science
Labora-
film thickness. The column for 100 h prior to its use in chromatography. The
carbon black extract was injected at room temperature, and the column was subsequently programmed at 10% min-’ from 40 to 110°C and at 2’C mm-’
from 110 to 350°C. Figure 1 shows the chromatogram obtained, indicating only a slight elevation in baseline between 310 to 350°C. The structures of representative components were assigned, based on their mass spectra (Hewlett-Packard Model 5982A combined gas chromatograph/mass spectrometer) and retention of standard compounds. A complete identification of all sample components was not carried out. The last three structures on the chromatogram are only representative of the recorded “peak clusters”; the numbers placed below the structures represent the molecular weights. A 40 m (0.3 mm i.d.) glass capillary column was prepared with 0.3-tirn bonded film of a methylpolysiloxane according to the technology described by Blomberg and WInnman [ 12, 133 and washed extensively .with a series of organic solvents. The column was subsequently conditioned at 330°C for several hours. Figure 2 shows a comparison of coal tar and carbon black extract samples chromatographed under identical conditions (programmed
from 70 to 320°C
--i
at 2°C min-l).
Good baseline stability
at high temperatures
.Ji :
_.‘.
‘.
.‘. -_
I
Fig. 1. High-temperature separation of the polycyclic aromatic components of carbon black obtained with 15 m (0.27 mm i.d.) glass capillary column coated withSE-52 silicone gum. Carrier gas, helium.
58
a
b
Fig. 2. Chromatrograms of (a) aromatic fraction of cod tar, and (b) carbon black extract, obtained with 40 m (0.3 mm i.d.) glass capillary column provided with a film of bonded
polysiloxane phase. Carrier gas, helium. is demonstrated. was employed
Again, a combined gas chromatograph/mass spectrometer to ascertain the range of separation (see Fig. 2 b).
Capillary high-performance
liquid chromatography
The equipment used for capillary h.p.1.c. was recently described [17]. The column used was a 100 m (70 pm i-d.) thick-walled glass microcapillary packed with 30-pm alumina particles [ 151. The stationary phase (an octadecylsilyl bonded phase) was generated inside the column via an in situ bonding technique [ 161. The inlet pressure was set at 300 atm, while the flow rate was approximately 1 ~1 mm-‘. The stepwise gradient elution technique [ 171 was employed. The separated components were detected with a miniaturized spectrofluorimetric detector [ 171, with a cell volume of 0.1 ~1. A full-scale recorder deflection corresponds to approximately 1-ng amounts. Figure 3 shows the separation of the heavy components of the carbon black extract obtained during a 50-h run. Peaks from the selected parts of this chromatogram were trapped at the column exit and subjected to directprobe mass spectrometry and spectrofluorimetric measurement as previously described [21]. Although most identifications are tentative because of the lack of suitable standards for the high molecular weights, the presence of at least 1 l-ring structures was suggested.
!CiGi’--I -
I
.20
2
I
I
3 1
, 30
4 1 40
I
I 50
HOURS
Fig. 3. Separation of thecarbon black heavy aromatic components by h.p.l.c. obtained with 100 m (70 rm i-d.) glass capillary column containing 30 rm irregular alumina/C,,-bonded phase. Stepwisc gradient: (1) 100% acetonitrile; (2) 80% acetonitril~20% dichloroethane; (3) 50% acetonitril-50% dichloroethane; (4) 100% dichloroethane. DISCUSSION
The extraction of various PAC-containing materials with powerful organic solvents may result in an appreciable yield of extractables, but frequently only a small percentage of this material can be separated by g-c. For example, about 73% of solvent-refined coal (SRC) was found extractable [4] into methylene chloride, of which amount 18% and 21% correspond, respectively, to the aliphatic and the polyaromatic fractions. However, gas chromatography could cover only some 26% of the polyaromatic fraction and 11% injected aliphatic fraction, corresponding to 1 and 0.395, respectively, of the original SRC sample. At present, the remaining non-volatile compounds can neither be separated effectively nor identified in this and similar materials. Evidence exists for some very large PAC in numerous mixtures (e.g., certain petroleum fractions [22] ), but again an effective methodology for their complete separation and identification has not been developed yet. The toxicological and chemical significance of most PAC above 6-ring structures is unknown because of the unavailability of standard compounds and the lack of structural characterization. Recent advances in the technology of glass [ 11-131 and fused-silica [ 141 capillary columns make it feasible to extend the temperature limits of capillary g-c. As shown by Figs. 1 and 2, these improvements extend the
60
range for PAC up to eight rings. However,
temperatures
in excess of 350°C
are judged to be the present technological
limit of this method. Recently developed polysiloxane bonded phases [12, 131 seem to offer an interesting approach to the preparation of stable columns for hightemperature g-c. work. As this technology develops further, columns with different selectivities may become available. Figure 2 also exemplifies how different combustion products may vary in terms of their PAC composition: coal tar (a by-product of the coking processes) has only a few major components at the high-molecular-weight end of the chromatogram, while the carbon black extract primarily consists of much larger molecules. The peak asymmetry of the lateeluting components can be attributed to their limited solubihty in the methylpolysiloxane stationary pha*. Given the temperature limitations of gc., liquid-chromatographic techniques become the only viable chromatographic alternative for sample components with more than seven or eight condensed aromatic rings. However, the problem of sample complexity becomes enormous (as occurrence of isomers increases with molecular weight) so that conventional h.p.1.c. resolution is insufficient. While capillary h.p.1.c. has the potential (15, 231 for improved resolution, Fig. 3 demonstrates that much improvement is needed. Apart from the current instrumental limitations 115, 171 of smallvolume technology, long separation times are a major difficulty. Identification of the separated PAC components is a major task at present. Although their mass and fluorescence spectra may provide useful information, practically no reference compounds are available for this compound range. Once the techniques of combined liquid chromatography-mass spectrometry are adequately developed, capillary h.p.1.c. will have a distinct advantage with its very low flow rates (typically, 1 ~1 min-‘) for “on-line” investigations of the separated PAC. Meanwhile, repeated trapping of capillary fractions is tedious at best. Because of the limited solubility of large polycyclic molecules, the selection of the column phase system used in this work deserves further mention. The non-aqueous reversed-phase system is generally compatible with both solubility and retention of the large PAC molecules. It should be noted that the polarity of the octadecyl packing appears less than that of any of the solvents used. Severe tailing of the later components in Fig. 3 could be caused by solubility problems, insufficient column deactivation, or a combination of both. This work was supported by Grant No. 24349 from the National Institute of General Medical Sciences, U.S. Public Health Service. REFERENCES 1 M. L. Lee, M. Novotny Compounds, Academic 2 M. L. Lee, M. Novotny
and K. D. Bartle, Analytical Chemistry of Polycyclic Press, New York, 1981 (in press). and K. D. Bnrtle, Anal. Chem., 48 (1976) 405.
Aromatic
61 3 M. L. Lee, M. Novotny and K. D. Bartle, Anal. Chem., 48 (1976) 1566. 4 R. V. Schultz, J. W. Jorgenson, M. P. Maskarinec, M. Novotny and L. J. Todd, Fuel, 58 (1979) 783. 5 M. L. Lee, D. L. Vassibrros, C. M. white and M. Novotny, Anal. Chem., 52 (1979) 766_ 6 M. Novotny, R. Kump, F. Merii and L. J. Todd, Anal. Chem., 52 (1980) 401. 7 H. Boden, J. Chromatogr. Sci., 14 (1976) 391. 8 D. C. Hunt, P. J. Wild and N. T. Crosby, J. Chromatogr., 130 (1977) 320. 9 K. Ogan, E. Katzand W. Slavin, Anal. Chem., 51(1979) 1315. 10 K. Ogan and E. Katz, J. Chrornatogr., 188 (1980) 115. 11 M. L. Lee, D. L. Vassilaros, L. V. Phillips, D. M. Hercules, H. Azumaya, J. W. Jorgenson, M. P. Msskarinee and M. Novotny, Anal. L&t., 12(A2) (1979) 191. 12 L. Blomberg and T. Wannman, J. Chromatogr., 168 (1979) 81. 13 L. Blomberg and T. Wtinnman, J. Chromatogr., 186 (1979) 159. 14 R. Dandeneau, P. Bente, T. Rooney and R. Hiskes, Am. Lab., 11 (9) (1979) 61. 61. 15 T. Tsuda and M. Novotny, Anal. Chem., 50 (1978) 271. 16 Y. Hi&a, M. Novotny, T. Tsuda and D. Ishii, Anal. Chem.. Sl(l979) 1807. 17 Y. Hi&a and M. Novotny. J. Chromatogv., 186 (1979) 521. 18 M. Novotny, M. L. Lee and K. D. BartIe, J. Chromatogr. Sci., 12 (1974) 606. 19 M. L. Le and R. Hites, Anal. Chem., 48 (1976) 1890. 20 M. Novotny and K. Tesarik, Cbromatographia, 1(1968) 332. 21 P. A. Peaden, M. L. Lee, Y. Hirata and M. Novotny, Anal. Chem., 52 (1980) 2268. 22 K. H. Altgelt nad T. H. Couw, Adv. Chromntogr., 13 (1975) 71. 23 J. H. Knox and M. T. Gilbert, J. Chrometogr., 186 (1979) 405.