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Anal. Chem. 1987, 59. 1733-1736
in 3 for convenience and the NOz groups have been replaced with CHO groups because NOP parameters for MM2 do not yet exist. This substitution is of little consequence and the charge transfer effect is successfully modeled (vida infra). Energies were obtained by sweeping through b' = 0-180' and 4 = 0-360' in 20' increments. The minima in the range 8 = 80-160° and 4 = 20-100' were then reinvestigated with a finer grid and with a larger number of analyte orientations at each 04 grid point. Both contour maps are strikingly similar but difference plots (not shown here) highlight the differential binding of R vs. S on the CSP. As seen in Figure 2 there are two broad and shallow binding regions about the CSP favored by the analyte. One region corresponds to front-side association of analyte while the other involves rear-side association. The shallowness around each minimum assures a large number of diastereomeric structures exist; each of which may contribute to the overall retention of the analyte. The energy difference between the two global minima is 0.3426 kcal mol-', favoring retention of the S analyte. The R enantiomer experimentally has the shorter retention time; hence for this example theory and experiment agree. The small difference in binding energy was anticipated and is consistent with a = 1.33. These minima correspond to CSP and analyte rigidly locked in their preferred conformations. Preliminary work suggests that when all degrees of freedom are relaxed so that CSP can alter its conformation to better accommodate analyte and the analyte can alter its shape to better bind to CSP that the difference in binding energy is somewhat larger but still favoring retention of the S analyte. A chiral recognition model for analyte binding to CSP 1 exists (2). We have not located structures corresponding to those expected from such a model. While we are not yet in a position to confirm nor deny the validity of the established mechanism, snapshots of analyte orientations taken from near the global minima in Figure 2 reveal a consistent pattern of binding . The most highly retained S analyte is found to maintain structures like the one shown in Figure 3 while the R analyte binds like that depicted in Figure 4. In both diastereomeric complexes we find a r acid-r base interaction between the aryl rings (the existing model has this feature) and a hydrogen bond between CSP and analyte (the existing model also has this feature). The difference between binding of R and S analytes involves the direction of analyte as it docks with the CSP and how it utilizes the hydroxyl group. For the R enantiomer the hydroxyl serves as a hydrogen bond donor coordinating with the amide carbonyl oxygen on the CSP. For the S enantiomer, in contrast, the analyte is rotated 180' and the analyte serves as a hydrogen-bond acceptor. In this
orientation the N-H of the CSP amide is directed toward the lone pair of electrons on the oxygen of the analyte's hydroxyl group. In both instances the phenyl group on the CSP serves only as a steric barrier to keep analytes from strongly binding to the rearside. In this communication we have demonstrated a novel approach to the problem of predicting which of two optical analytes is the most retained on a chiral column. We have provided insight about where on the CSP aryl alcohol 2 prefers to bind, and we have presented an alternative mechanism by which enantioselection may take place. Our results are preliminary; they are not meant to credit nor discredit the existing chiral recognition model. In this communication we simply demonstrate that modern computation along with molecular graphics can furnish information heretofore not available and we can provide insight to problems that are important in separation science. ACKNOWLEDGMENT We thank Bill Pirkle for his hospitality and stimulating discussions. LITERATURE C I T E D (1) Souter, Rex W. Chromatographic Separation of Stereoisomers ; CRC Press: Boca Raton, FL, 1985. (2) Pirkle, W. H.; House, D. W.; Finn, J. M. J Chromatogr. 1980, 792, 143-1 58. (3) Lipkowitz, K. E.; Landwer, J. M.; Darden, T. Anal. Chem. 1986, 58, 1611-1617. (4) Lipkowitz, K. 8.; Malik, D. J.; Darden, T. Tetrahedron Lett. 1986, 27(16), 1759-1762. (5) Lipkowitz, K. B.; Demeter, D. A.; Parish, C. A ; Landwer, J. M.; Darden, T. J . Comput. Chem., in press. (6) Allinger, N. L. J . Am. Chem. SOC. 1977, 9 9 , 8127 (7) Burkert, U.; Aliinger, N. L. Molecular Mechanics; American Chemical Soclety, Monograph 177; American Chemical Society: Washington, DC, 1982. (8) Clark, 1.A Handbook of Computational Chemistry: A Practical Guide to Chemical Structure and Energy Calculations ; Wiley-Interscience: New York, 1985.
Kenny B. Lipkowitz* David A. Demeter Carol A. Parish Department of Chemistry Indiana-Purdue University Indianapolis, Indiana 46223 Thomas Darden Laboratory of Molecular Biophysics National Institute of Environmental Health Science P.O. Box 12233 Research Triangle Park, North Carolina 27709 RECEIVEDfor review February 2,1987. Accepted April 1,1987. This work was funded by the donors of the Petroleum Research Fund, administered by the American Chemical Society.
Pulsed-Laser Photothermal Refraction Detection in Capillary Liquid Chromatography Sir: The absence of detectors that are versatile and sensitive and have extremely low volumes has impeded the development and utilization of capillary liquid chromatography (LC) separation techniques in chemical analysis (1,2).Commercially available spectrophotometric (absorbance-based) detectors are used extensively in analytical-scale LC. However, the majority of these detectors are not compatible with LC due to the large volumes (ca. 10 fiL) of their flow cells. The volumes of solute bands eluted by capillary LC columns range from low nanoliters for true open capillary columns to high nanoliters for 0003-2700/87/0359-1733$01.50/0
the slurry-packed capillary columns employed in this work (3). While the flow cells of some commercial spectrophotometric detectors have been modified to accomodate certain capillary columns ( 4 , 5 ) ,these modified detectors generally suffer from poor sensitivity (due to short optical path lengths), relatively large amounts of stray light, and poor light throughput. The intense, highly collimated outputs of lasers can be focused to produce large photon densities, in extremely lowvolume flow cells, without large amounts of stray radiation. 0 1987 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 13, JULY 1, 1987
This often provides for sensitive fluorescence detection in capillary LC (6), which, whenever possible, should be performed “on-column” (7,8).The effective flow-cell volume of a laser beam focused to a 10-pm spot at the end of a 20-pm-id. capillary column is a mere 3 pL. Unfortunately fluorometry is not a particularly versatile technique, due to the small percentage of analytes that possess appreciable fluorescence quantum efficiencies. Methods based on the measurement of absorbance are much more versatile but, when applied to capillary LC detection, exhibit the aforementioned problems. Recently, attention has been given to developing laser-based thermal optical detection techniques for LC (8-13). These techniques provide for sensitive absorbance-based measurements by relying for their signals on temperature increases in a sample, generated by the nonradiative dissipation of absorbed laser radiation. The techniques that have been used most in LC are thermal lens (8-10) and photothermal refraction (11)and deflection (12) detection. Signal generation with these techniques is critically dependent on the spatial properties of the laser beams that are employed. When a laser beam with a Gaussian spatial distribution of light intensity is passed through an absorbing sample, Gaussian temperature and refractive index gradients are created in the sample. With the thermal lens technique a probe laser beam is focused through the sample collinear with an absorbed laser beam. This probe beam will experience a lensing effect, which can be monitored in the far field with a pinhole and a photodetector. The photothermal refraction technique differs in that the absorbed and probe beams are perpendicularly intersected in the sample and the optical element experienced by the probe beam is that of a cylindrical, rather than spherical, lens. Because the volume probed within the sample is the volume of the intersected beams, the photothermal refraction technique is constrained to measuring absorbances in very small volumes. Photothermal beam deflection involves monitoring the deflection (as opposed to the divergence) of the probe laser beam. With this technique the absorbed and probe beams typically intersect within the sample a t an angle that is less than that of the perpendicular offset used in photothermal refraction, although in one report a crossed beam configuration has been successfully employed for photothermal deflection (14). Previous applications of these techniques to LC detection have involved the use of continuous wave (CW) lasers. These lasers offer excellent beam properties and high average powers, but they do not readily offer access to the analytically important ultraviolet (UV) portion of the electromagnetic spectrum. A pulsed, pumped-dye laser, using the appropriate dyes and harmonic generation optics, offers a convenient means to access any wavelength in the UV for detection purposes. The uses of pulsed-laser thermal lens detection for flowing liquid samples (13) and pulsed-laser photothermal deflection for gas chromatography detection (25) have been reported. In this report we discuss our use of pulsed-laser photothermal refraction detection in capillary LC. Separations are performed with slurry-packed capillary columns. The technique is illustrated for the separation of nitrated pyrene isomers in a synthetic coal fly ash extract. CW laser, photothermal refraction detection has provided for the sensitive measurement of dabsyl-amino acids (dabsyl = (dimethylamino)azobenzenesulfonyl), following separation with a microbore LC column (11). The work presented herein differs in that we (1)employ for detection an excimer pumped-dye laser tuned to the near-UV absorbance maximum of the underivatized analyte of interest and (2) employ for the separation true capillary LC using slurry-packed capillary columns. Eliminating the need to derivatize the sample in order to match the wavelength of the laser, we do not have to be concerned with whether derivatization was complete and also
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do not have to resolve the analytes of interest from unreacted and hydrolyzed derivatizing reagent, which may be present in the sample. The capillary columns provide excellent efficiency in this application and afford the possibility of injecting extremely small sample volumes.
EXPERIMENTAL SECTION Apparatus. A block diagram of the equipment used in this work is shown in Figure 1. The photothermal refraction detector consisted of a Lambda Physik FL 2000 dye laser pumped by a Lambda Physik EMG 50 excimer laser. The excimer uses a Xe/HCl gas fill to pump the 2-(4-biphenylyl)-6-phenylbemxazole (PBBO) dye (Lambda Physik LC 4000) at 308 nm. The dye laser, which produces the absorbed beam, was tuned to 400.0 nm. The energy of the laser pulses was 30 pJ and a repetition rate of 23 Hz was employed. The probe laser was a Spectra-Physics Model 145-01helium-neon laser. The absorbed laser beam was focused with a 25-mm-focal-length (f) quartz biconvex lens. The probe laser beam was focused with a 22-mm-f lens. The intensity of the probe beam at beam center was monitored with a Clairex Model CL707HM photoconductive cell placed 67 cm from the flow cell. An Ithaco, Model 391A lock-in amplifier, referenced to the synchronous output of the excimer laser, was employed to monitor and amplify the photocell signal. A sensitivity of 300 p V and time constant of 4 s were typically used. The lock-in output was monitored with a Cole-Parmer Model 8376-20 recorder. The entire system was rigidly mounted on a pneumatically stabilized optical table (Modern Optics), which served to minimize vibration induced contributions to the background. Chromatography. The slurry-packed microcolumns were constructed and packed following the procedure of Gluckman and co-workers (3). The columns were 200-pm-i.d. fused silica (Scientific Glass Engineering, Austin, TX) and were packed with 5-pm CPS-Hypersil particles (Shandon Southern Products, Ltd., Cheshire, England). The particles were slurry packed at 5000 psi with a Haskel Model 17502 pneumatic amplifier pump employing acetone as the slurry and packing solvent. Porous Teflon frits, 20 p m thick, were employed as exit frits and were held in place by a 1 cm length of 50-pm4.d. fused-silica capillary, which was secured with epoxy into the end of the column. Detection was performed in a short length of 200-pm-i.d. fused silica from which the polyimide coating had been removed. This short length of tubing was attached to the 50-pm-i.d. fused silica at the terminus of the column by means of epoxy. Injection was performed as described in ref 3. The injection volume was approximately 20 nL. The mobile phase, 95/5 n-hexanel2-propano1,was pumped by the aforementioned Haskel pump, generally at 1000 psi. Reagents. All solvents used for separations were Mallinckrodt Chrom AR grade. 1-Nitropyrene and dinitropyrene isomers were received from the NCI Chemical Carcinogen Reference Standard Depository, a function of the Division of Cancer Etiology, NCI, NIH, Bethesda, MD. The sample analyzed was an extract of coal fly ash that had been coated with pyrene and subsequently ex-
ANALYTICAL CHEMISTRY, VOL. 59, NO. 13, JULY 1, 1987 I 1201
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posed to an atmosphere of NO2 (16). The fly ash was then extracted in a Soxhlet apparatus with toluene, concentrated, and injected onto the column.
RESULTS AND DISCUSSION Pulsed-laser photothermal measurements do offer advantages when compared with CW-laser-based methods. The shorter rise time of the signal that results when a pulsed laser is used allows for a shorter signal collection time and reduced errors (15). The use of a pulsed laser for thermal lens measurements in a flowing liquid (14) illustrated that there was no dependence of signal on flow rate while CW-laser-based thermal lens measurements have shown a significant dependence on flow rate (8). The theoretical enhancement offered by pulsed-laser thermal lens measurements compared to CW-laser thermal lens has been reviewed (17). The pulsed method is favored for small samples, rapid analyses, and gas-phase samples. For this application of pulsed-laser photothermal refraction detection the primary advantage is the tunability of the dye laser employed. The slow flow rate encountered when using capillary liquid chromatography columns would probably not have a deleterious effect on either CW or pulsed laser measurements. The ability to tune the source provides the opportunity to detect analytes at a wavelength corresponding to their maximum absorption coefficient. The near-UV wavelength of 400.0 nm used in these experiments was the optimum wavelength for detection of 1-nitropyrene in the mobile phase employed. Measurements a t wavelengths further into the UV require a change of dye or frequency doubling. Frequency doubling was not required in order to perform measurements at 400.0 nm. We employ the crossed-beam photothermal refraction configuration because of its proven ability to make sensitive absorption measurements in small volumes (18) and its ease of alignment relative to thermal lens detection, which employs collinearly aligned laser beams. Once aligned and optimized, the photothermal refraction detector remains aligned from day to day, requiring only occasional “tweaking” of the signal. This is readily achieved by replacing the capillary column flow cell with a flow cell attached to a syringe pump. The syringe pump delivers a test solution to the flow cell at a reproducible flow rate so that the signal level can be set to the same value as previous alignments. The crossed-beam technique makes the process of switching and aligning flow cells fairly easy. Placing the flow cell at the intersection of the two beams is simply a matter of observing the image of the two lasers in the far field as the flow cell is translated into the intersection point. With some practice, the flow cell can be reproducibly placed a t the intersection point of the beams. Nitro-substituted polynuclear aromatic hydrocarbons have received considerable attention in recent years due to their mutagenic and carcinogenicactivity (19-22). Figure 2 contains
1735
a series of chromatograms illustrating the use of a slurrypacked silica column with pulsed-laser photothermal refraction detection to indicate the presence of nitropyrene isomers in the sample previously described. The column employed was 200 bm i.d. X 62 cm packed with 5-pm CPS-Hypersil particles. Isocratic elution with a mobile phase of 9515 n-hexane12propanol was accomplished with a constant inlet pressure of 1000 psi. Chromatograms A and B are separations of the sample. Chromatogram C of the figure is the separation of a standard mixture of (in elution order) 3.0 ng of 1-nitropyrene (1-NP), 0.31 ng of 1,3-dinitropyrene (1,3-DNP), 0.80 ng of 1,6-dinitropyrene (1,6-DNP), and 1.3 ng of l,&dinitropyrene (1,8-DNP). This chromatogram illustrates the separation capability of slurry-packed capillary columns under isocratic elution conditions (analytical scale separations of this mixture typically require gradient conditions). The ability to accomplish this separation under isocratic conditions preserves the thermooptical properties of the mobile phase and, hence, serves to maintain a constant photothermal refraction background signal (23). The small injection volume used (20 nL) for these separations is also advantageous. The complex mixtures resulting from extractions of air particulate matter, soil samples, and other naturally occurring samples often contain many components that are at concentrations below the limit of detection. Concentration of extracts to microliter volumes prior to injection can often enhance the opportunity to detect these trace components. This was the case with the synthetic fly ash sample investigated in this work. The original 80-mL extraction volume was reduced to 10 WLbefore the chromatograms in Figure 2 could be accomplished. The use of split injection techniques provides minimal band broadening and the greatest flexibility concerning the quantity injected. Unfortunately, when sample sizes are extremely small, as in this case, split injection is not possible. All three chromatograms in Figure 2 were obtained by using the same column and conditions. The tallest peak of chromatogram A was suspected of being 1-NP due to its match in retention time with 1-NP in the standard mixture. Spiking the extract with 1-NP provided an increase in peak height for the suspect peak while the remaining peaks remained the same height (Figure 2B). The presence of 1-NP in this extract has been confirmed by direct inlet probe mass spectrometry (16). The additional peaks in the chromatogram could include different positional isomers of nitrated pyrene. The analytical utility of photothermal refraction detection is dependent on the relationship of signal to concentration. A linear calibration curve was constructed for photothermal refraction signal response to different concentrations of 1-NP in n-hexane, flowing in a 200-pm fused-silica flow cell. The linear dynamic range for the plot extended from 4 X lo* to M with a correlation coefficient of 0.997. The re6X producibility of response for chromatographic detection is illustrated by the detector response to the amount of 1-NP added to the complex mixture. The chromatogram of the standard mixture containing 3.0 ng of 1-NP (Figure 2C) yielded a lock-in amplifier response of 80.0 kV, or a response factor of 26.7 pVlng injected. The separation of the unspiked sample (Figure 2A) yielded a 1-NP peak of 34.5 pV. A spike of 3.9 nglinjection yielded a 1-NP peak of 137 WVafter separation (see Figure 2B). Taking the difference of the detector response for 1-NP for the spiked and unspiked chromatograms yields a response factor of 26.2 WV/ng. This good correlation indicates the possibility of employing standard addition in order to quantify a n a l y k s present in a sample when using this type of detection. The photothermal refraction measurement is independent of path length (23). Rather than attempt to relate these
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 13, JULY 1, 1987
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bance detector that can provide high sensitivity and low detector-related solute band dispersion should aid in the growth of capillary LC. While the sensitivity obtained in this work is slightly better than that for previously reported work with pulsed-laser thermal optical detection for flowing liquid samples, it is only approximately equivalent to that reported for conventional absorbance detectors that had been designed or modified for detection in capillary LC ( 2 4 ) . In order for the large (several orders of magnitude for the laser system employed in this work) theoretical enhancements in sensitivity over normal Beer's law measurements to be realized, several practical problems associated with the use of pulsed lasers must be solved or reduced. Further development of this detection method should include the use of lower wavelength UV excitation, continuous tuning of the dye laser output for maximization of analyte detection, and the combination of the technique with time-resolved fluorescence detection to provide additional analytical information. Improvements in sensitivity should be possible if more durable flow cells that can tolerate higher laser powers are developed. Sheath flow cells may prove useful in this matter (25,26). More sophisticated laser systems, which provide output pointing and intensity stabilities that are superior to the system employed in this work, could also result in significant improvements in sensitivity.
Figure 3. Chromatogram of 1-NP and dinitropyrene isomers standard mixture (higher laser power than that used in Figure 2).
measurements to Beer's law absorbances, the measurements at the limit of detection are best expressed as the product of concentration and molar absorptivity (tC). The 1-NP peak in Figure 2C has a Gaussian profile and therefore the concentration a t peak center for the 3 ng injected is calculated to be 2.7 X lo4 M. Assuming a direct proportionality between signal and amount injected, the limit of detection at a signal to noise ratio (S/N) of 3 is EC = 7.4 X cm-'. This value is relatively poor with respect to previously reported values ( I I , 1 8 ) and with good reason. One problem in using a highly focused pulsed laser is the destructive effects of the large power density involved. In order to obtain the maximum signal, it is advantageous to maximize the power of the absorbed laser beam due to its direct relationship to signal (23). Unfortunately, high incident power can destroy flow cells. The average power used to obtain the chromatograms in Figure 2 was 0.6 mW. Figure 3 shows a chromatogram of the synthetic mixture of 1-NP and dinitropyrene isomers with an improved S/N. A 200-pm X 90-cm column was employed with isocratic elution at 1500 psi with a 95/5 hexane/2-propanol mobile phase. The injected quantitites were the same as in Figure 2C. Detection was obtained by using an average power of approximately 1.0 mW, which corresponds to a peak power density of approximately 0.2 GW/cm2. This incident power eventually burned a hole through the thin-wall fused-silica flow cell. Extrapolation of the signal for the 3.0-ng injection of 1-NP for this chromatogram to a S / N of 3 yields tC = 4.6 X cm-', which corresponds to an absorbance of 9 X We have successfully employed heavy-walled 500-pm-i.d. square quartz flow cells and have obtained a limit of detection cm-'. While this value exceeds the value of tC = 2.7 X obtained with pulsed-laser thermal lens detection of flowing liquids (13),it is a factor of 50 less than the extrapolated value of 5 X lo4 cm-' reported by Dovichi and co-workers for detection of dabsyl-glycine when using CW laser excitation (11). Tunability, compatibility with low-volume flow cells, and the potential for high sensitivity characterize the pulsed-laser-based photothermal refraction detector discussed herein. The development of a an ultraviolet-visible (UV-vis) absor-
LITERATURE CITED (1) Knox. J. H.; Gilbert, M. T. J . Chromatogr. 1979, 786,405-418. (2) Scott, R. P. W. J . Chromatogr. Sci. 1980, 78. 49-54. (3) Gluckrnan, J. C.: Hirose, A.: McGuffin, V. L.; Novotny, M. Chromatographia 1983, 17, 303-309. (4) Kok, W. Th.; Brinkrnan, U. A. Th.: Frei, R. W.: Hanekarnp, H. B.; Nooitgedacht. F.: Poppe, H. J . Chromatogr. 1977, 744, 157-163. (5) Yang, F. J. HRC CC, J . High Res. Chromatogr.. Chromatogr. Commun. 1981, 4 , 83-89. (6) Murray, G. M.; Sepaniak, M. J. J . Liq. Chromatogr. 1983, 6 , 931-938. (7) Gvthrie, E. J.; Jorgenson. J. W.: Dluzneski. P. R . J . Chromatogr. Sci. 1984. 22. 171-176. (8) Sepaniak, M. J.; Vargo, J. D.; Kettler, C. N.; Maskarinec, M. P. Anal. Chem. 1984, 56, 1252-1257. (9) Leach, R. A.: Harris, J. M. J . Chromatogr. 1981, 278, 15-19. (10) Buffett, C. E.; Morris, M. D. Anal. Chem. 1982, 54, 1824-1825. (11) Nolan, T. G.: Hart, B. K.; Dovichi, N. J. Anal. Chem. 1985, 57, 2703-2705. (12) Collette, T. W.; Parekh, N. J.: Griffin, J. H.: Carreira, L. A,: Rogers, L. B. Appl. Spectrosc. 1986, 4 0 , 164-169. (13) Nickolaisen, S. L.: Bialkowski, S. E. Anal. Chem. 1986, 58, 215-220. (14) Westel, G. C . : Stotts, S. A. Appl. Phys. Lett. 1983, 4 2 , 931-933. (15) Nickolaisen, S. L.: Bialkowski, S. E. J . Chromatogr. 1986, 366, 127- 133. (16) Yokley, R. A. Ph.D. Dissertation, Chemistry Department, University of Tennessee, 1965. (17) Bialkowski, S. E. Spectroscopy (Springfield, Ores.) 1986, 1 , 26-48. (18) Nolan, T. G.: Weimer, W. A.; Dovichi, N. J. Anal. Chem. 1984, 56, 1704-1707. (19) Wang, Y. Y.; Rappaport, S. M.: Sawyer, R. F. Talcott. R. E.: Wei, E. T. Cancer Lett. (Shannon, Ire/.) 1978, 5 ,39-47. (20) . . Salrneen, I.: Durisin. A. M.: Prater, T. J.: Riley, T.: Schuetzle, D. Mutat. Res. 1982, 104, 17-23. (21) Wei, E. T.: Shu, H. P. Am. J . Public Health 1983, 79, 1085-1088. (22) Rosenkrantz, H. S. Mutat. Res. 1984, 140, 1-6. (23) Dovichi, N. J.; Nolan, T. G.; Weimer, W. A. Anal. Chem. 1984, 56, 1700-1 ~.704. (24) Walbroehl, Y.; Jorgenson, J. W. J . Chromarogr. 1984, 375, 135-143. (25) Zarrin, F.: Dovichl, N. J. A n d . Chem. 1985, 57, 2690-2692. (26) Van Liet, H. P. M.; Poppe, H. J . Chromatogr. 1985, 346, 149-160. ~~~
C. N. Kettler M. J. Sepaniak*
Department of Chemistry University of Tennessee Knoxville, Tennessee 37996-1600
RECEIVEDfor review December 5,1986. Accepted March 30, 1987. This research was sponsored by the Division of Chemical Sciences, Office of Basic Energy Sciences, U S . Department of Energy, under Contract DE-FG05-8613613 with the 7 Jniversity of Tennessee, Knoxville, TN.
