1546
Anal. Chem. 1985, 57, 1546-1552
w IOV-IOV
T
T
02nA
'inn
1
2
additional dead volume. The series equipotential mode does not change the rules for selectivity; however, since the phenomenon is based on mass transport rather than electrochemistry, the improvement in signal to noise is obtained for all solutes detected at or below the applied potential. Both modes are useful for compensating base line shifts during mobile phase gradients or temperature changes. The difference mode of detection is simple to implement and can significantly improve both detection limits and selectivity. Registry No. Gentisic acid, 490-79-9; vanillic acid, 121-34-6; caffeic acid, 331-39-5; coumaric acid, 25429-38-3; ferulic acid, 1135-24-6;sinapic acid, 530-59-6.
LITERATURE CITED
minutes
Difference mode detection with depletion (equipotential)using the series configuration. Chromatographic conditions and peak Mentities are given in Figure 2. Figure 9.
those compounds with similar redox potentials from a mixture compounds with varying redox potentials. Series equipotential operation improves detection limits by taking advantage of the difference in flux to the two electrodes. In essence, the downstream working electrode acts as a reference signal for drift and may be incorporated into the cell without incurring
(1) Shoup, Ronald E., Ed. "Bibliography of Recent Reports on Electrochemical Detection"; BAS Press: West Lafayette, IN, 1982. (2) Lunte, S. M.; Kissinger, P. T. J. Chromatogr. 1084, 377,579. (3) Allison, L. A.; Shoup, R. E. Anal. Chem. 1083, 55,8. (4) Mayer, G. A.; Shoup, R. E. J . Chromatogr. 1083, 255,533. (5) Roston, D. A.; Shoup, R. E.;Kissinger, P. T. Anal. Chem. 1982, 54, 1417A. (6) Roston, D. A.; Kissinger, P. T. Anal. Chem. 1982, 54,429. (7) Blank, L. J . Chmmafogr. 1976, 777,35. (8) MacCrehan, W. A.; Durst, R. A. Anal. Chem. 1981, 53, 1700. (9) Lunte, C. E.; Kissinger, P. T. Anal. Chem. 1983, 54, 1458. (IO) Weber, S. G.; Purdy, W. C. Anal. Chem. 1982, 54, 1757. (11) Goto, M.; Zou, G.; Ishii, D. J. Chromatogr. 1983, 275,271. (12) Shoup, R. E. 1982 LCEC Symposium, Indianapolis, IN; BAS Press: West Lafayette, IN, 1982; Abstract 15.
RECEIVED for review March 15, 1985. Accepted April 1, 1985.
Miniature Fluorometric Photodiode Array Detection System for Capillary Chromatography Jennifer C. Gluckman, Dennis C. Shelly,' and Milos V. Novotny* Department of Chemistry, Indiana University, Bloomington, Indiana 47405
A hlgh-sensltlvlty multlchannel fluorescence detector wlth small volume has been developed for use In caplllary column llquld and supercrllcal lluld chromatography. By use of an Intenstfled linear photodlode array to monltor fluorescence emlsslon, on-llne spectral Information can be obtalned over a linear range of loa, wlth mlnlmum detectable quantltles of less than 2 X lo-'' g. Sample chromatograms are presented for standard polycycllc aromatlc hydrocarbons and for the high-efflclency Separation of hlgh-molecular-weight neutral arornatlcs extracted from a fossll fuel materlal. I n addition, the use of spectral subtraction to resolve coelutlng species is demonstrated.
Microcolumn liquid chromatography (LC) and capillary supercritical fluid chromatography (SFC) are becoming increasingly well-recognized as the principal separation methods for the analysis of complex nonvolatile or thermally labile mixtures. The analytical advantages of both methods have recently been reviewed (1,2). As the large resolving powers of slurry-packed capillary columns in microcolumn LC ( 3 , 4 ) and open-tubular columns in SFC (5-7) fully demonstrate *Presentaddress: Department of Chemistry and Chemical Engineering, Stevens Institute of Technology, Hoboken, NJ 07030. 0003-2700/85/0357-1546$01.50/0
their potential impact on biochemical, environmental, and technologically important separations (8-12), these highly efficient separations must be matched by equally effective detection and ancillary identification methods. Detection volumes on the order of nanoliters or below are required for most work in microcolumn chromatography. Among the several detector types which have been successfully miniaturized (13,14), fluorescence detectors are particularly attractive because of their inherent high sensitivity and selectivity. Indeed, by using lasers as excitation sources, impressively low detection limits have been achieved (15, 16). In addition, on-line optical methods, combined with microcolumn LC, can complement recent LC/mass spectrometric techniques (17), providing detailed, multiparameter, structural information despite the small detection volumes. Optical multichannel imaging spectroscopy (18) has recently undergone many technological improvements (19)which have allowed both the silicon vidicon (20,21) and the linear photodiode array (PDA) (19,22-25) to be successfully employed as detectors in conventional scale LC. While vidicon tubes have traditionally been used for fluorometric detection, the more recent linear photodiode arrays, or their intensified counterparts, are rapidly gaining in popularity as UV-visible absorbance detectors. It is interesting, however, that although fluorescence-based detection is currently one of the most sensitive and inherently selective methods available in both liquid and supercritical fluid chromatography, linear photo@ 1985 Amerlcan Chemlcal Society
ANALYTICAL CHEMISTRY, VOL. 57,
diode arrays have not yet been tested in this capacity. In addition, the ability of these imaging detectors to monitor any single emission wavelength at the same time as the total fluorescence signal can simplify many complex chromatograms, and the on-line spectral information that they provide can be combined with information derived from other sources, such as mass spectrometric, nuclear magnetic resonance, or Fourier transform infrared measurements for the identification of many unknown substances contained in complex mixtures. On the basis of promising results which have been obtained with conventional-scale PDA absorbance detectors (23,24) and the demonstrated ability of the arrays to make sensitive static fluorescence measurements (26, 27), we decided to construct a miniaturized fluorescence imaging detector for capillary column chromatography. T o date, there has been very little effort toward the utilization of miniaturized optical multichannel imaging detectors for high-resolution chromatography. Thus far, the only reported work is that of Takeuchi and Ishii (25),who detected constituents from a commercial cold medicine using a PDA absorbance detector with a 50-nL detection volume. Lee and co-workers (28) have taken an alternative approach in the construction of an “on-the-fly” fluorescence scanning detector, which has been utilized in capillary column SFC. Two important limitations of photodiode array devices, which may account for their scarcity in microcolumn separations, are their lower sensitivity and higher noise as compared to photomultiplier tubes. These characteristics often necessitate both long integration times and spectral averaging, leading to large detection time constants incompatible with the high efficiencies of capillary column chromatography. Recent developments in array technology have, however, led to rapid scanning intensified PDA devices, which are more thermally stable and have higher detection efficienciesthan did previous versions. These improvements, in conjunction with both the increased mass sensitivity of the microcolumns in concentration-sensitive detectors (29) and the increased sensitivity of fluorometric detection, should permit high sensitivities and low noise levels to be achieved in multichannel fluorescence imaging detection coupled with high-resolution separations. Although a variety of samples may readily benefit from multiparameter analyses, technologically important fossil fuel materials contain many large polyaromatic compounds, whose structural similarity and many isomeric forms make their identification virtually impossible using a single analytical technique. In addition, the molecular fluorescence of polycyclic aromatic compounds has been extensively discussed in the literature (30-36) and is known to exhibit a relatively high degree of detail, which is frequently indicative of molecular structure. For example, increasing ring conjugation, alkyl and phenyl substitution, and the inclusion of heteroatoms into the ring all produce distinct spectral changes (30, 32, 33). The detection system described in this study incorporates either broad-band or single-wavelength-laser excitation together with an intensified photodiode array and a miniaturized fiber optic flow cell. In addition to evaluating its analytical performance, we have applied the system to LC analyses of some technologically and environmentally important mixtures of polycyclic aromatic compounds. Although only liquid phase microcolumn separations are demonstrated, the system is equally compatible with capillary SFC.
EXPERIMENTAL SECTION ChromatographicSystem. Separations were performed on 200 pm, i.d., slurry-packed capillary columns, prepared as previously described (4). A S h m d z u Model LC-SA pump (Shimadzu Corp., Kyoto, Japan) was employed in the constant-pressure mode to provide pulsation-freesolvent flow at pressures up to 7250 psi. Sample injections were made with a low-volume internal loop valve having a 0.1 p L volume (Model CI4W.2, Valco Instruments Co.,
NO. 8, JULY 1985
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P
Figure 1. Schematic dlagram of the photodiode array fluorescence detector: A, lamp power supply; B, lamp; C, current-to-voltage converter/amplifler; D, focusing lens; E, double monochromator; F, mlcrobench optical system; 0, collimating lens; H, focuslng lens; I,flow cell; J, photodiode; K, fiber optic; L, fiber optic coupler; M, emission spectrograph; N, intensified, 512 channel diode array; 0, computer Interface; P, computer; Q, source monitor AID.
Houston, TX). A 10-8 injection period was employed in order to obtain reproducible plug injections. A miniaturized continuous-gradient device (37)was utilized for separations requiring solvent programming. Intensified Photodiode Array Detector. A schematic diagram of the detector is given in Figure l. A 200-W mercury-xenon arc lamp (Hanovia Model 901B-1), contained in a multipurpose housing (Model LH 150, Kratos Analytical Instruments, Ramsey, NJ) and powered by a regulated, low ripple power supply (Model LPS 251HR, Ki-atm Instruments), provided an intense and stable, broad-band excitation source. An adjustable reflector mounted behind the light source and a UV-transmitting fused-silicacondensing lens cf/1.5) maximized the total output power, while forced air cooling and feedback regulation within the power supply minimized power fluctuations due to arc wander. Fluctuations which did occur were monitored and compensated through the ratio circuitry described below. The focused output of the Hg-Xe lamp passed through a 0.2-m double grating monochromator optimized for the UV region (240 nm blaze) and having an f/4 aperture with adjustable slits to provide bandwidths from 1 to Kratos Instruments). The entire lamp 20 nm (Model GM-200-3, housing/monochromator unit was rigidly mounted on a kinematic mount, constructed in-house, to allow precise alignment of the arc image with the excitation optical train. Due to the necessarily small detection volumes, it was important both to minimize the size of the arc image (which measured 1.4 X 3.0 mm at the exit of the monochromator when a 20 nm bandwidth was employed) and to maintain careful control of its alignment with respect to the flow cell. The image reduction was accomplished by means of an f/4 fused-silica collimating lens (Oriel Corp., Stamford, CT) followed by an f / l fused-silicacondensing/focusing lens (Physitec Corp., Norfolk, MA), reducing the arc size to 0.7 X 1.5 mm at the detection cell. Both the excitation and emission optics were rigidly mounted on an optical microbench (Physitec Corp.) to facilitate precise and reproducible alignment. In addition, the excitation focusing lens was coupled with a precision micrometer (Physitec Corp.) to allow for changes in focal length with changing excitation wavelength. A second lens (f/l Oriel Corp.), placed beyond the flow cell, focused excitation light passing through and around the cell onto a UVenhanced photodiode (Type BD/PSB, United Detector Technology, Culver City, CA). The photodiode allowed fluctuations in lamp intensity to be monitored and provided a proportional signal, which was converted to a voltage and then digitized. This digital value was subsequently ratioed with the fluorescence data from the diode array detector in order to compensate for changes in excitation power. The fluorescence signal could be transferred to the emission spectrograph via two alternativepathways. The first was designed
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ANALYTICAL CHEMISTRY, VOL. 57, NO. 8, JULY 1985
t
-
.05mm, I , D. Capillary
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Figure 2. Cross-sectional view of the flow ceii body, showing expanded detail of the cell interior.
for use without a fiber optic probe and, in the present work, was employed in preliminary alignment procedures. It consisted of an f/4 fused-silica collimating lens (Oriel Corp.) followed by an f/1.5 focusing lens (Oriel Corp.), which imaged the emission signal onto the entrance slit of the spectrograph. For data acquisition, interference from scattered or stray light was minimized by transmitting the emitted fluorescence via a 20-cm segment of single fiber optic probe (P/NWF00200, Maxlight Fiber Optic Division, Raychem Corp. of Arizona, Phoenix, AZ), which was inserted into the base of the flow cell as illustrated in Figure 2. This fiber terminated at the entrance slit of the spectrograph, where it was secured by a fiber-optic coupler, designed in-house and shown in Figure 3. It should be noted that, since the core diameter of the fiber optic was 200 pm, this served as the effective entrance slit width of the spectrograph when entrance slits of larger value were employed. The remaining components of the PDA detector were the emission spectrograph, the intensified diode array, and the computer system. The emission spectrograph (f/3.85, Model TN 1149-9, Tracor Northern, Middleton, WI), also kinematically mounted, contained a 150 groove/mm holographic grating, which dispersed the fluorescence signal onto the face of a 512-element photodiode array coupled to a proximity-focused microchannel plate intensifier (Model TN6123, Tracor Northern). The output from the array (Peltier-cooledto -3 "C and purged with nitrogen) was converted to a digital signal by the detector controller/interface (Model TN-6200, Tracor Northern) and transferred under direct memory access (DMA) to a dedicated IBM personal computer (IBM Corp., Boca Raton, FL). The rapid data transfer rate allowed detection time constants to be determined solely by the acquisition parameters. In this study, signal integration times of 200-300 ms/scan provided adequate sensitivity with an average of 8 scans/spectrum yielding low background noise levels. This resulted in total detection time constants of 1.6-2.5 s. Since slurry-packed capillary columns generally exhibit chromatographic peaks with typical widths of 30 s or more, little band-broadening was introduced by the detector. Software developed in-house provided real-time viewing of the totalfluorescence chromatogram and of the fluorescence intensity at two preselected wavelengths, while displaying the spectra as they were acquired. The data were written directly to floppy disk, thus facilitating postrun examination using more detailed processing programs. Chromatograms and spectra were plotted on a Hewlett-Packard 7470A plotter (Hewlett-Packard, San Diego, CA). Detection Cell. The fiber-optic flow cell (see Figure 2) utilized in the present study was similar in design to that described for
Black Surf-
Flgure 3. Schematic diagram of the flber optic coupler: (A) fiber coupling with emission spectrograph; (B) expanded detail showing interior of coupler and coupler plate.
the laser fluorescence detector (16). The relatively large size of the arc image did, however, necessitate placing the column outlet at a greater distance from the fiber-optic probe than previously described. In the PDA system, the spacing was on the order of 3 mm, yielding a detection volume of 150 nL. The cell was mounted on a precision x-y translator to facilitate centering at the focal point of the arc image. Reagents. The linearity and sensitivity of the detector were measured using anthracene (Mallinckrodt, Inc., Paris, KY) in a mobile phase of 10% water in acetonitrile and 1,3,6,8-tetraphenylpyrene (Chem Service, West Chester, PA) in a 100% acetonitrile mobile phase, respectively. Fluoranthene was obtained from City Chemical Corp. (New York, NY), while rubrene and decacyclene were purchased from Aldrich (Milwaukee, WI). These standard compounds, while all natively fluorescent, are known to exhibit a range of fluorescencequantum efficiencies. The fossil fuel sample, derived from SRC2 fuel oil blend in a 5.75/1 ratio of middle-to-heavydistillate (Pittsburgh and Midway Coal Mining Co., DuPont, WA, code no. 1701), was obtained from the Fossil Fuel Research Matrix Corp. repository administered by the Oak Ridge National Laboratory. The sample analyzed was the neutral fraction of this fuel, prepared as previously described (38). All solvents were HPLC grade (Fisher Scientific) and passed through a 0.2-pm filter, while water was purified through a cation, anion, and activated charcoal system (ContinentalWater Systems, Inc., El Paso, TX). Mass Spectrometry, Mass spectra were obtained from collected peaks using direct probe insertion to a Hewlett-Packard 5982A dodecapole mass spectrometer (Hewlett-Packard Corp., Palo Alto, CA) in conjunction with an INCOS 2300 data system (Finnigan MAT, Sunnyvale, CA).
RESULTS AND DISCUSSION The ability to obtain complete on-line fluorescence spectra at high sensitivity and without sacrifice to chromatographic efficiency is of extreme utility in a multiparameter approach to the characterization and identification of unknown compounds contained in complex mixtures. The intensified PDA detector for capillary column chromatography provided this information, allowing spectral distinctions to be made between mass spectrometrically identical compounds and facilitating
ANALYTICAL CHEMISTRY, VOL. 57, NO. 8,JULY 1985
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A 1
1=400nm
LOG A M T . INJECTED
Flgure 4. Linearity of detector response to 0.1-pL injections of increasing amounts of anthracene in a mobile phase of 10% water in acetonitrile (k' = 1.4).
the deconvolution of chromatographically unresolved peaks through spectral subtraction. Linearity. The linear dynamic range of the system was determined in its role as a detector for high-resolution capiUary chromatography. Anthracene was injected in a mobile phase of 10% water in acetonitrile (capacity factor of 1.4) as the model solute. On the basis of duplicate injections of increasingly concentrated solutions, which were prepared by serial dilution, the signal was found to be linear (correlation coefficient of 0.99) over nearly 3 orders of magnitude, as shown in Figure 4. The lamp power was monitored during data acquisition, and minor variations were corrected for by normalizing all peak-height measurements to correspond to a constant excitation power. Detection Limits. The minimum detectable quantity was determined by using the Student's t test and the procedure described by Hieftje (39) to be an injected amount of 15.5 pg of anthracene in a mobile phase of 10% water in acetonitrile and 11.6 pg of the more highly fluorescent 1,3,6,8-tetraphenylpyrene in 100% acetonitrile. These detection limits corresponded to a concentration of 76.2 pg/L (ppb) anthracene and 57.0 pg/L (ppb) tetraphenylpyrene in the detection cell at the peak maximum and were obtained at a signal-to-RMS noise ratio of 5 (99% confidence level). Chromatographic and Spectral Resolution. In the separation of complex mixtures, it is important to maintain the full resolving power of the slurry-packed capillary column and, therefore, the detector itself can add little extracolumn broadening to the chromatographic system. This has traditionally been a problem for diode array-based detectors, since their lower sensitivities and relatively slow scan speeds have necessitated long detection time constants. The miniaturized, rapid-scan, intensified PDA detector described herein has, however, been able to maintain excellent chromatographic efficiency without sacrifice to detection sensitivity. As is evident in Figure 5A, which presents the separation of a standard mixture of polycyclic aromatic compounds monitored at two selected wavelengths and through the total fluorescence signal, high chromatographic efficiencies (on the order of 150000 theoretical plates for anthracene) were well preserved and no peak distortion was apparent. In addition to chromatographic considerations, an evaluation of the detector's spectral resolution is important to its use for compound characterization and identification. Since many structural differences between similar compounds produce only subtle changes in their respective fluorescence emission spectra, the higher the resolution available, the more useful the detector is as an ancillary identification technique. With a mercury pen ray lamp to provide a well-defined line source, peak widths on the order of 6 nm (full width a t half
*I
I
A = 4 6 5 nm
41
2
1
TOTAL FLUORESCENCE
TINEIMIN)
Flgure 5. Chromatogram of a standard mixture of polycyclic aromatic
hydrocarbons, showing on-line fluorescence spectra obtained for each column, 200 pm, i.d. X 0.8 m length, packed with 5-pm Spherisorb ODs; mobile phase, continuous gradient 100% acetonitrile to 30% ethyl acetate In acetonltrlle (2.0 pL/min); solutes, (1) anthracene (10 ng), (2)fluoranthene (8 ng), (3) rubrene (16 ng), (4) 1,3,6,8-tetraphenyipyrene (1 ng), (5) decacyclene (7 ng); injection volume, 0.1 pL; array exposure time, 0.2 s/scan; 8 scans averaged/spectrum; excitation, 365 nm. maximum) were achieved for all lines measured (360-650 nm). The high spectral quality of the detector is clearly evident in Figure 5B, which presents the emission spectra obtained for each peak in the standard chromatogram. In particular, the high resolution is apparent in the structure of the anthracene spectrum and in the subtle spectral features which are clearly visible in the other spectra,
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ANALYTICAL CHEMISTRY, VOL. 57, NO. 8, JULY 1985
/I TOTAL FLUORESCENCE
L.
Detection Selectivity. In the analysis of complex mixtures separated by capillary column chromatography, it is often desirable to selectively detect one or more compounds, or compound types, from among a variety of others within a single chromatographic run and without the use of multiple detectors. Indeed, even selective monitoring can allow many substances to be undetected when they coelute with molecules of similar molecular weight or structure. In addition, wellresolved peaks may remain unidentified when suitable ancillary identification techniques are either unavailable or difficult to implement. The PDA fluorescence detector provided flexible detection selectivity and detailed spectral information, which, in combination, can be important in elucidating the composition of complex samples. The fluorescence spectra of the standard compounds shown in Figure 5B illustrate the relationship between molecular structure and spectral detail which forms the basis of this inherent selectivity and which can allow even closely eluting compounds, such as anthracene and fluoranthene, to be distinguished through a judicious choice of emission wavelength. Indeed, the selection or exclusion of a given compound type can often be predicted based on known spectral trends, such as the tendency for larger aromatics like rubrene and decacyclene to fluoresce at longer wavelengths than do smaller molecules. Analysis of Complex Mixtures. Many factors play an important role in the successful separation and analysis of complex samples. In addition to the need for high resolving powers and detection selectivity, spectra must frequently be collected for the duration of a lengthy chromatogram. It is in this respect that adequate data collection and storage
r
systems become important. Figure 6, which presents the high efficiency separation of the large neutral polyaromatics found in a fuel oil blend, illustrates the ability of the PDA system to monitor complex mixtures over an extended period of time. While only the total fluorescence chromatogram is presented, over 2800 spectra were acquired during the 1.5 h separation allowing selected emission wavelengths to be monitored during postrun analysis. To illustrate the utility of fluorescence emission data, which is frequently indicative of compound type, as one component of a multiparameter approach to compound identification,two chromatographically well resolved peaks have been chosen for spectral examination. On the basis of their mass spectra alone, both appear to contain a variety of compact, four-ring structures (molecular weight 202,230,244,258), with a smaller percentage of less compact four-ring (molecular weight 242) and five-ring (molecular weight 252, 266) species, some of which may include a sulfur atom (40). Confronted with the mass spectral similarity of even chromatographically resolved peaks, such as those in Figure 6, and with the coelution of many similar compounds within a single peak, the need for a wide range of information to aid in compound identification becomes obvious. The ability to obtain real-time fluorescence spectra for each eluting peak is one such source of information. On the basis of a unique set of molecular properties, fluorescence profiles frequently differ for compounds which are indistinguishable through other means. For example, catacondensed species, such as anthracene, exhibit highly detailed emission spectra, while pericondensed molecules, such as fluoranthene, fluoresce over a rather broad, featureless region. In this way, additional knowledge is gained and, as
ANALYTICAL CHEMISTRY, VOL. 57, NO. 8, JULY 1985
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Spectrum (823-
Flgure 7. Example of spectral subtraction used to resolve coelutlng compounds from the fuel oil blend in Figure 6: (A) expanded view of chromatographic region of interest; (6)component and residual spectra obtained.
the understanding of structural/spectral relationships grows and libraries of known spectra are accumulated, multichannel fluorescence detectors can potentially acquire a significant role in compound identification. Spectral Isolation. The coelution of many substances within one chromatographic peak is a frequent occurrence in complex chromatograms, such as that of the fuel oil blend. When the migration pattern is such that the concentration of one component falls to nearly zero over the course of the peak, spectral subtraction provides a convenient means to isolate and identify this compound. An example of the spectral resolution of a chromatographically unresolved peak appears in Figure 7 . In this case, the first component of a single, 30 s wide chromatographic peak, which appears in expanded view in Figure 7A, fell in concentration as the elution band passed through the detection cell, leaving the remaining components to form a “shoulder” region. Therefore, by subtracting the spectrum obtained at the height of this shoulder (no. 829) from that obtained at the maximum concentration of the first component (no. 823),a residual spectrum providing qualitative information on the first component could be obtained. (Note that this same approach could equally well have been applied to the analysis of the shoulder on the leading peak edge.) This feature of fluorescence data, which allows spectral deconvolution through subtraction, can be very important in the identification of compounds contained in complex mixtures, where chromatographic resolution is frequently incomplete.
CONCLUSIONS An intensified PDA fluorescence imaging detector has been constructed for capillary column chromatography and has enabled the sensitive, selective detection of many fluorescent compounds contained in complex mixtures. Compatible with both liquid and supercritical phase separations, the instrument
permits rapid acquisition of emission spectra without degradation of chromatographic performance. These features, combined with spectral analysis and deconvolution, will enable fluorescence imaging detection to serve as a valuable component in a multiparameter approach to compound characterization and identification.
ACKNOWLEDGMENT The authors wish to thank Robert Ensman, Ray Sporleder, and Jeff Russ of the Indiana University Department of Chemistry technical support staff for their invaluable assistance in the electronic circuit design, computer interfacing, and software development for this project. We also wish to acknowledge Donald Wiesler for his continued help in mass spectral interpretation. Registry No. Anthracene, 120-12-7; fluoroanthene, 206-44-0; rubrene, 517-51-1; 1,3,6,84etraphenylpyrene,13638-82-9; decacyclene, 191-48-0.
LITERATURE CITED (1) Kucera, P., Ed. “Microcolumn High Performance Liquid Chromatography”; Elsevier: Amsterdam, 1984. (2) Novotny, M., Ishil, D.,Eds. “Mlcrocolumn Separatlon Methods”; Elsevier: Amsterdam. 1985. (3) Yang, F. J. Chromaiogr. 1982, 236, 265-277. (4) Gluckman, J.; Hirose, A.; McGuffin, V. L.; Novotny, M. ChromatograDhia 1983. 17. 303-309. (5) NOVOtny, M.; Sprlngston, 5.R.; Peaden, P. A.; Fjeldsted, J. C.;Lee, M. L. Anal. Chem. 1981, 53,407A-414A. (6) Fjeldsted, J. C.; Richter, B. E.; Jackson, W. P.; Lee, M. L. J. Chromatogr. 1983, 279,423-430. (7) Fjeldsted, J. C.; Lee, M. L. Anal. Chem. 1984, 56, 619A-627A. (8) Hirata, Y.; Jinno, K. HRC CC, J . Hlgh Resoluf Chromatogr. Chromafogr. Commun. 1983, 4, 571-572. (9) Novotny, M.; Hirose, A.; Wiesler, D. Anal. Chem. 1984, 56, 1243-1248. IO) Novotny. M.; Karisson, K.-E.; Konlshi, M.; Alasandro, M. J. Chromafogr. 1984, 292, 159-167. 11) Kong, R. C.; Fields, S. M.; Jackson, W. P.; Lee, M. L. J. Chromafogr. 1984, 289, 105-116.
.
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Smith, R. D.; Kallnoski, H. T.; Udseth, H. R.; Wright, B. W. Anal. Chem. 1984, 56, 2476-2480. Hirata, Y.; Novotny, M. J . Chromatogr. 1979, 786, 521-528. Ishii, D.; Takeuchl, T. J . Chromatogr. S d . 1980, 78, 462-472. Guthrie, E. J.; Jorgenson, J. W.; Dluzneski, P. R. J . Chromatogr. Sci. 1984, 22, 171-176. Gluckman, J.; Shelly, D.; Novotny, M. J. Chromatogr. 1984, 377, 443-453. Henion, J. I n "Microcolumn High Performance Liquid Chromatography"; Kucera, P., Ed., Elsevier: Amsterdam, 1984; pp 260-300. Talmi, Y., Ed. "Multichannel Image Detectors"; American Chemical Society: Washington, DC, 1979; ACS Symp. Ser. No. 102. Fell, A. F.; Clark, B. J.; Scott, H. P. J. fharm. Homed. Anal. 1983, 7 , 557-572. Jadamec, J.; Saner, W.; Talml, Y. Anal. Chem. 1977, 4 9 , 1316-1321, Fogarty, M.; Shelly, D.; Warner, I . HRC CC,J . High Resolut. Chromafogr. Chromatogr. Common. 1981, 4 , 581-568. Mllano, M.; Lam, S.; Grushka, E. J . Chromatogr. 1978, 125, 315-326. Jost, K. W.; Crispln. Th.; Halasz, I. Erdol Kohle, Erdgas fefrochem. 1984. 37, 178. Desilets, 0.J.; Kissinger, P. T.; Lytle, F. E.; Horne, M. A.; Ludwiczak, M. S.; Jacko, R. B. Environ. Sci. Techno/. 1984, 18, 386-391. Takeuchi, T.; Ishii, D. HRC CC,J . H/gh Resolut. Chromatogr. Chromatogr. Commun. 1984, 7 , 151-152. Ryan, M. A.; Mliier, R. J.; Ingle, J. D., Jr. Anal. Chem. 1978, 50, 1772-1777. Talmi, Y. Appl. Spectrosc. 1982, 36, 1-18. Fjeldsted, J. C.; Richter, B. E.; Jackson, W. P., Lee, M. L. J . Chromatogr. 1983, 279, 423-430.
(29) Novotny, M. I n "Microcolumn High Performance Liquid Chromatography"; Kucera, P., Ed.; Elsevler: Amsterdam, 1984; pp 194-259. (30) Beriman, I. "Handbook of Fluorescence Spectra of Aromatic Molecules"; Academic Press: New York, 1971. (31) Kropp, J.; Stanley, C. Chem. fhys. Left. 1971, 9, 534-538. (32) McKay, J.; Latham, D. Anal. Chem. 1972, 44, 2132-2137. (33) McKay, J.; Latham, D.Anal. Chem. 1973, 45, 1050-1055. (34) Clar, E.; Schmidt, W. Tetrahedron 1977, 33, 2093-2097. (35) Ciar, E.; Schmidt, W. Tetrahedron 1978, 34, 3219-3224. (38) Clar, E.; Schmidt, W. Tetrahedron 1979, 35, 1027-1032. (37) Karlsson, K.-E.; Novotny, M. HRC CC,J . H/gh Resolut. Chromatogr. Chromatogr. Commun. 1984, 7 , 411-413. (36) Novotny, M.; Konishi, M.; Hirose, A.; Gluckman, J.; Wlesler, D. Fuel, in press. (39) Hleftje, G. Anal. Chem. 1972, 44, 81A-88A. (40) Wiesler, D., Indiana University, Bloomington, Indiana, private communlcatlon, 1984.
RECEIVED for review January 17,1985. Accepted March 25, 1985. Support for this work was provided by Grant No. DOE DE AC02-81 ER 60007 from the U.S. Department of Energy, and Grant No. N14-82-K-0561 from the Office of Naval Research. J. C. Gluckman was the recipient of a full-year graduate fellowship bestowed by the American Chemical Society, Division of Analytical Chemistry, and sponsored by the Perkin-Elmer Corp.
Multiwavelength Detection and Reiterative Least Squares Resolution of Overlapped Liquid Chromatographic Peaks 5.D. Frans,l M. L. McConnell,2and J. M. Harris* Department of Chemistry, University of Utah, Salt Lake City, Utah 84112
A relteratlve least squares procedure Is used to resolve indlvldual component spectra from overlapped ilquid chromatographic peaks detected wlth a rnultlwavelength,photodlode array spectrometer. Rather than seeklng spectral or elutlon regions In the data which are free of overlap, the method makes use of the predlctabie shape of the eluted peaks, while allowing for variation In retention time and peak wldth. Successful spectral resolution of hlghiy overlapped fused peaks containlng up to seven components Is achieved. The exponentlally modHled Gausslan function used to model the elution response Is found to accurately describe of the obgerved peak shape. The effect of the peak shape model on the accuracy of the results Is evaluated; the detection of mlnor overlapped components Is also studied.
The resolution and identification of the components in complex mixtures continue to represent difficult analytical tasks in spite of improvements in instrumentation and methodology. It appears that the increasing complexity of analytical problems will always exceed the current state-ofthe-art in separation and analysis. Furthermore, Davis and Giddings (1) have shown by way of a statistical argument that a chromatogram must be more than 95% vacant in order to provide greater than 90% confidence that a component of interest will appear as an isolated peak. Since chromatograms lPresent address: Spectra-Physics, 3333 N o r t h F i r s t St., San Jose,
CA 95134.
2Present address: T e c Concepts, 985 U n i v e r s i t y Ave., Suite 31,
Los Gatos, CA 95030.
of complex samples are nearly always less vacant than this criterion, the problem of overlapping peaks becomes a nearly universal concern in the practice of analytical separations. To address this concern, it is valuable to recognize that the use of multichannel detectors in chromatography can significantly increase the number of independent informational degrees of freedom in the measurement (2). Several numerical methods have been developed to utilize this additional informing power to resolve overlapped peaks. All of these data analysis methods share the assumption that the spectral pattern of a particular compound will rise and fall in unison as that compound elutes from the column. One approach to utilizing this behavior requires that one or more spectral channels in a region of chromatographic overlap belong only to one compound. The pure spectral channel is identified as the one which exhibits the sharpest peak within the elution period of interest (3-6). The single component chromatogram, thus identified, serves as a template against which the chromatograms at other spectral channels are compared by correlation to extract the spectrum of the component in question. A convenient approach to identifying the unique spectral channels attributable to single components is the factor analysis technique of Lawton and Sylvestre (7),termed self-modeling curve resolution. This method has been successfully applied to the mass spectra of mixtures (8) and presumably would work with GC/MS data as well. An alternative use of the method is to identify those elution times corresponding to the purest, ideally sisgle component spectra. These pure spectra, typically but not necessarily found at the edges of a pair of fused chromatographic peaks (9), are then used to factor individual component chromatograms out of the data matrix. This method has been applied to binary
0003-2700/85/0357-1552$01.50/00 1985 American Chemical Society
