Anal. Chem. 1990, 62, 186-189
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On-Line Fluorescence Lifetime Detection for Chromatographic Peak Resolution W. Tyler Cobb and Linda B. McGown* Department of Chemistry, P. M. Gross Chemical Laboratory, Duke University, Durham, North Carolina 27706
Frequencydomaln fluorescence llfetkne measurements, made on-the-fly durlng hlgh-performance liquid chromatography, were used to resolve overlapplng chromatographlc peaks of polycyclic aromatlc hydrocarbons. twotomponent heterogenelty analysls of the multifrequency Hfethne data provlded fractional lntenslty contrlbutknsfor the individual components, whlch were multlplled by the total fluorescence lntenslty to yield the peaks of each component. Excellent results were obtalned when the fluorescence lifetlmes of the components were fixed In the heterogeneity analysls to values obtalned from the on-the-fly lifetlme chromatograms. Posltlve errors In the peak lntensltk were observed when the llfethnes were allowed to float In the heterogeneity analysls; the source of the error has not yet been identified but may be due to systematlc bias In the heterogenelty analysls software.
The lifetime T can be calculated from both the phase-shift and the demodulation, to give lifetimes T~ and T,, respectively. For a homogeneous system of a single lifetime component, T~ = T,. If the signal is heterogeneous, i.e., due to more than one ground-state component, then T, > T ~ .Determinations of T~ and T, a t multiple modulation frequencies can be used to resolve the heterogeneous signals, yielding the lifetime and fractional intensity contribution of each component. Chromatographic peak resolution is accomplished by performing heterogeneity analysis at discrete points along the chromatogram. At each point, the fractional intensity of each component is calculated by heterogeneity analysis and multiplied by the total intensity at that point to find the intensity of the component. The peak of the component is constructed from the calculated intensities of the component along the chromatogram.
INTRODUCTION
The PAHs (9970, Analabs) and solvents (HPLC grade, Burdick and Jackson) were used as received for solution preparation. Solvents used as mobile phases were further purified by vacuum filtration. Micromolar solutions of individual PAHs were prepared in acetonitrile, and PAH mixtures were prepared directly from these solutions. Reversed-phase chromatography was performed with a dualpump HPLC system (Waters) with a 10 X 0.3 cm glass cartridge column assembly, including Vydac 201P packing and a C-18 guard column, and fixed UV absorption detection at 254 nm. The PAHs were injected manually and eluted isocratically with a flow rate of 0.3 mL/min. The &component mixture was eluted with a 100% acetonitrile mobile phase; an 87% solution of acetonitrile in water was used for elution of the 11-component mixture. The eluted PAHs passed from the absorbance detector into the 48000s phase-modulation spectrofluorometer (SLM Instruments, Inc.), for on-the-fly,simultaneous detection of fluorescence intensity and lifetime. The instrumental configuration for these studies includes a 450-W xenon arc lamp, excitation monochromator, electroopticmodulator compartment,a 20-pL black quartz, "low-fluorescence"flow cell (Hellma) in a thermostated sample chamber, emission filters, PMT detectors, and an IBM PC-AT for data acquistion and analysis. Lifetime calculations and heterogeneity analysis were performed with software provided with the instrument. A reference fluorophore, 9-anthracenecarbonitrile (9AC, T = 11.31 ns in acetonitrile), was used to calibrate the phase and modulation of the excitation, as has been previously descried (7). The instrument heterogeneity analysis software provides a nonlinear least-squares (NLLS) fit to multifrequency data. The fluorescence lifetimes of the PAH compounds used in the two mixtures are shown in Table I. The lifetimes were determined on-line, under the same conditions that were used for the heterogeneity analysis experiments. For both mixtures, phase and modulation data were collected on-line, at 1-5 intervals, at five modulation frequencies: 4, 10, 15, 25, and 35 MHz for the 6component mixture, and 5, 10, 15, 25, and 40 MHz for the 11component mixture. The 6-component mixture was excited at 360 nm, and emission was observed through a combination of a 399-nm longpass filter and a 600-nm shortpass filter. For the 11-component mixture, excitation was at 330 nm, and emission was observed through a combination of a 345-nm longpass filter and a 600-nm shortpass filter. Under these wavelength conditions,
EXPERIMENTAL SECTION Recent studies have explored fluorescence lifetime detection on-the-fly in high-performance liquid chromatography (HPLC), using both time-domain (1-5) and frequency-domain (6, 7)techniques. Analytical applications have included lifetime determinations for peak identification (5-7) and time-delay measurements for improvement of signal-to-noise ratios (1-3). Time-domain techniques have not yet been described for the resolution of overlapping chromatographic peaks. Preliminary studies of peak resolution in the frequency-domain, using on-the-fly measurements of phase shift and demodulation, have been described for a three-frequency instrument (7). In this paper, we describe the first use of on-the-fly lifetime determinations with a multifrequency fluorometer for chromatographic peak resolution in HPLC. Heterogeneity analysis of the multifrequency data with a nonlinear least-squares fit provides the fluorescence lifetimes and fractional intensity contributions of each of the overlapping components, at 1-s intervals along the chromatographic peak. Alternatively, the lifetimes can be fixed to values found from the lifetime chromatograms for the individual components in nonoverlapping peak regions, leaving only the fractional intensities to be calculated. In either case, the peaks of the individual components are reconstructed by multiplying the fractional intensity by the steady-state intensity a t each point. Two mixtures of polycyclic aromatic hydrocarbon (PAH) compounds were studied: a 6-component mixture and an 11component mixture. The chromatographic conditions were intentionally adjusted to give poor chromatographic resolution, in order to test the heterogeneity analysis resolution for a model system of PAHs with extensive peak overlap.
THEORY The theory of phase-modulation fluorescence lifetime determinations and heterogeneity analysis has been described elsewhere (8). Briefly, the sample is excited with intensity modulated light, resulting in a similarly modulated fluorescence signal that is phase-shifted and demodulated as a function of the fluorescence lifetime of the emitting species.
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 2, JANUARY 15, 1990
I I
Table I. Abbreviations, Peak Numbers and Fluorescence Lifetimes of the PAH Compounds abbreviation
PAH anthrecene fluoranthene benz[a]anthracene chrysene benzo[e]pyrene benzo [b]fluoranthene benzo[k]fluoranthene benzo[a]pyrene dibenzo[a,h]anthracene benzo [ghi]perylene indeno(l,2,3-cd)pyrene
peak n 0 . O 6-comp 11-comp 1
BaA BeP BbF BkF BaP DBA BgP IP
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Figure 1. Fluorescence lifetime chromatogram of the 6-component mixture at 10 MHz. Solid line is steady-state (dc) intensity, squares , triangles are are T ~ and numbered in Table I.
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Peak numbers correspond to PAHs as
background from scattered light and Raman scattering are negligible.
RESULTS AND DISCUSSION 6-Component Mixture. Figure 1 shows the fluorescence lifetime chromatogram for the 6-component mixture a t 10 MHz. In regions where only one fluorescent component is present, T~ = T, = constant (where the constant is the lifetime of the component), as expected. For regions where the chromatographic peaks overlap, T , > T~ and the lifetimes change continuously across the region of overlap, reflecting the changing fractional intensity contributions of the two different components. For example, the extensively overlapping BgP and IP peaks show the homogeneous BgP lifetime of 20.3 ns a t the beginning of the peaks, followed by the heterogeneous lifetime region in which T, > T~ and the lifetimes are decreasing, converging on the shorter lifetime of 6.9 ns for IP in the homogeneous IP region at the end of the peaks. In this case, as well as in the overlapping peaks of BbF and BkF, and BkF and BaP, the lifetime chromatogram is able to indicate (1)the presence of more than one component ( T, > T ~ and ) (2) the lifetimes of both components in the overlapping peaks. I t is important that the chromatogram in Figure 1 also shows fluorescence intensity, which is simply the dc, or unmodulated component, of the emission signal. It is an advantage of the frequency domain approach that the phase, modulation, and steady-state intensity information is all obtained simultaneously, on-the-fly, from the same emission signal. The NLLS heterogeneity analysis of the five-frequency data set was performed point-by-point for the 6-component mixture. The fractional intensity contributions calculated by
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Figure 2. Heterogeneity analysis results for the 6-component mixture. Solid lines are peaks for the same amount of the each PAH as in the mixture, injected separately (Le.,the “true values”). Squares and triangles represent the peaks calculated from the two-component fiis. (a)Lifetimes of the two components allowed to float. (b) Lifetimes fixed to values found from Figure 1.
NLLS were multiplied by the dc intensity at each point, in order to obtain the resolved peaks of the individual components. The peaks resolved by NLLS for each component in the mixture were compared with peaks obtained for indentical amounts of each component, injected separately. The comparison is shown in Figure 2a for the five-frequency data set. The heterogeneity analysis was also performed on various fourand three-frequency subsets of the five-frequency data set. Results for four-frequency data sets were very similar to those for the five-frequency set, whereas the results showed significant degradation when only three frequencies were used. As shown in Figure 2a, the NLLS results for a two-component fit are excellent for the chromatographically resolved fluoranthene, as well as for the peripheral, homogeneous regions of the five partially resolved PAHs. In the heterogeneous, overlapping regions, results are fairly good for BbF, BaP, and BgP, including good estimates of peak height. Results for BkF and I P are poor; in both cases, peak heights are overestimated. Of course, the performance of the heterogeneity analysis is not bad, considering the almost complete overlap of BgP and I P and the overlap of BkF on both sides of its peak (Figure 1). In order to improve the resolution of peaks in the mixtures, we fixed the lifetimes of the components in the heterogeneity analysis to the values found from the lifetime chromatograms. As shown in Figure 1,there is a region at the periphery of each peak where the lifetime information is essentially homogeneous. Excellent results were obtained when the lifetimes were fixed, as shown in Figure 2b. 11-Component Mixture. Our first observation for the 11-component mixture was the presence of a small but significant background signal that had not been observed for the 6-component mixture. The background was eventually
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 2, JANUARY 15, 1990 40
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RELATIVE TIME ( S E C / 3 ) Figure 4. Fluorescencelifetime chromatogram of the 1 1-component mixture at 10 MHz, with an expanded time axis relative to Figure 3: (a) peaks 1 through 4, expanded time axis: (b) peaks 5 through 8, expanded time axis; (c) peaks 9 through 11, expanded time axis. Legend as in Figure 1.
identified as emission from the flow cell when excited at 330 nm, which is required for simultaneous excitation of the 11 PAHs. The background is not excited at the 360-nm wavelength used for the 6-component mixture, which is why the background did not appear in the earlier studies. The background emission is a broad peak from 400 to 600 nm, with a maximum around 530 nm. The excitation spec-
trum has an undetermined maximum below 290 nm in the UV and decreases to zero near and above 350 nm. Similar spectra are obtained for the flow cell containing acetonitrile, water, methanol, or air. For fluorescence intensity detection, the flow cell emission can be “zeroed out” using PMT voltage offsets. Unfortunately, when lifetime measurements are made, the background signal can still contribute to the observed phase and modulation, even though its dc contribution is offset to zero. We attempted to determine the lifetime of the flow cell luminescence but were unable to detect an ac (modulated) signal in the 1-250 MHz range of the instrument. The complete demodulation at these frequencies, along with the large Stoke’s shift of the emission, is indicative of phosphorescence (consistent with the “low-fluorescence”designation by the manufacturer), but this could not be verified within the frequency range of our instrument. Fortunately, the complete demodulation of the flow cell background means that it will not interfere with the PAH lifetime determinations, since it will appear as a constant, unmodulated dc contribution. It is therefore valid to use the P M T offset to eliminate the background signal, and this was done for the chromatograms of the 11-component mixture. The steady-state (dc) fluorescence intensity chromatogram of the mixture is shown in Figure 3. Under these chromatographic conditions, extensive (almost complete) overlap is observed between peaks 1 and 2 (anthracene and fluoranthene) and peaks 5 and 6 (BeP and BbF). Peaks 3 and 4 (BaA and chrysene) are also highly overlapping. The remaining components, corresponding to peaks 7 through 11, are wellresolved. The lifetime chromatogram for the mixture a t 10 MHz is shown in Figure 4, in which the time scale is expanded to emphasize the regions of peak overlap and lifetime heterogeneity. Figure 4 clearly illustrates the ability of frequencydomain measurements to provide accurate lifetimes and to indicate regions of heterogeneity. Only in one case, that of the overlapping peaks 3 and 4 (Figure 4b), are the results misleading. The region clearly contains two peaks, yet the lifetimes appear homogeneous. This is due to the very similar
Anal. Chem. 1990, 62, 189-200
lifetimes of the two components (16 ns for BaA and 13 ns for chrysene), which are not discernibly different at 10 MHz. Heterogeneity analysis was performed on the five-frequency data set. The NLLS-resolved peaks and the peaks obtained for the corresponding, individually injected components, are shown in Figure 5 for the partially resolved peaks 1through 6. Results are shown for NLLS both with floating lifetimes (Figure 5a) and with the lifetimes fixed (Figure 5b). In both cases, results are good for peaks 1 and 2, which have the greatest lifetime difference. Results are systematically high for peaks 5 and 6 with floating lifetimes and are greatly improved when lifetimes are fixed. Peaks 3 and 4, with the very similar lifetimes, are unresolved when lifetimes are allowed to float; some improvement is observed when the lifetimes are fixed.
CONCLUSIONS This work has demonstrated the resolution of chromatographic peaks by heterogeneity analysis of multifrequency fluorescence lifetime data, collected on-the-fly for HPLCeluted PAH compounds. Excellent results were obtained when lifetimes were fixed in the heterogeneity analysis to values obtained directly from the lifetime chromatograms. Positive
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errors were generally observed when the lifetimes were allowed to float in the heterogeneity analysis. The source of the positive error is not yet known; however, we have observed similar errors for batch heterogeneity analyses of these components. We are currently investigating the possibility of systematic bias in the system software for NLLS heterogeneity analysis.
LITERATURE CITED Richardson, J.; Larson, K.; Haugen, G.; Johnson, D.; Clarkson, J. Anal. Chim. Acta lQ80, 776,407. Imasaka, T.; Ishibashi, K.; Ishibashi, N. Anal. Chlm. Acta lQ82, 742, 1.
Furuta, N.; Otusuki, A. Anal. Chem. lQ83, 55, 2407. Desilets, D.; Kissinger, P.; Lytle, F. Anal. Chem. lQ87, 5 9 , 1830. Kawabata, Y.; S a m , K.; Imasaka, T.; Ishibashi, N. Anal. Chlm. Acta 1988, 208, 255. Cobb, W. T.; McGown, L. E. Appl. Spectrosc. lQ87, 4 1 , 1275. Cobb, W . T.; McGown, L. E. Appl. Spectrosc. IQ89, 43, 1363. Lakowicz, J. R. Principles of Nuorescence Spectroscopy; Plenum Press: New York, 1983.
RECE~VED for review September 1,1989. Accepted November 6,1989. This work was supported by the US. Environmental Protection Agency (Grant R81-2887-01-0).
The Physical Sense of Simulation Models of Liquid Chromatography: Propagation through a Grid or Solution of the Mass Balance Equation? Martin Czok and Georges Guiochon* Department of Chemistry, University of Tennessee, Knoxville, Tennessee 37996-1600, and Division of Analytical Chemistry, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831 -6120
Two distlnctty different approaches have been used to simulate the movement of bands through a chromatographic column. One example of the first approach is the Craig distribution model, which replaces the continuous column wlth a specific number of discrete equiilbratlon processes. Thus it introduces the concept of (theoretical) plates into chromatography, but is not able to explain satisfactorily their significance. The second approach is based on the mass balance equatlon whlch can be integrated numerically over tlme and space to glve the elutlon profile. This caiculatlon can be performed by uslng finlte dlfference methods to extrapolate from the concentration value at a given tlme and position In the column to the next value. I n this paper we discuss the physical meaning of the numerical lntegratlon process followed by the finite difference methods. We show that both approaches are equlvalent and that the band broadening produced by the different methods, due to “numerical diffusion”, can be explained simllarly. Since this effect is sufficiently well-known now, we can introduce a variable amount of addklonai diffuslon and thereby control the overall dispersion.
INTRODUCTION In analytical chromatography the migration of sample zones through the column is described by simple relationships. The peaks are nearly symmetrical, their retention times are directly proportional to the column length, and so the band variances, 0003-2700/90/0362-0 189$02.50/0
the retention times, and the bandwidths are independent of the composition of the sample. Therefore it is relatively easy to calculate the performance of the chromatographic system under various experimental conditions and to choose the optimum ones for the experiment. This remains true only as long as the sample load is low compared to the saturation capacity of the column. For preparative separations, large volumes of very concentrated solutions are injected in order to produce as much purified material as possible in a given time. Consequently, the column is highly overloaded and the peak profiles become unsymmetrical and depend strongly on the amount of material injected. Most noticeably, the migration velocity of the band front changes with the sample size. A better understanding of these phenomena is needed to develop practical optimization procedures for the applications of preparative chromatography. Several simple equations have been proposed that predict the nonlinear behavior of the band of a pure compound in overloaded chromatography (1-5). As far as a generalization of these equations to the case of mixtures of two or more components is possible (6-9),they can be used to find the optimum conditions for a preparative separation (6, 7, 10, 11). Because of the strong mutual interactions that take place between two solutes and the severe deformation of their bands, however, an accurate description of the complete elution profile is necessary to achieve a correct optimization (8-11). For this purpose computer programs have been developed that imitate the migration of the bands down the column (2, 6, 12-19). The simulated chromatograms obtained under 0 1990 American Chemical Society
