multifrequency phase-modulation fluorescence lifetime determinations on-the-fly in hplc

Multifrequency Phase-Modulation Fluorescence Lifetime Determinations On-the-Fly in HPLC

W . T Y L E R C O B B a n d L I N D A B . M c G O W N *

Department of Chemistry, P. M. Gross Chemical Laboratory, Duke University, Durham, North Carolina 27706

The first use of a multifrequency, phase-modulation spectrofluorometer for fluorescence lifetime determinations on-the-fly in HPLC is described. Direct, simultaneous measurements of fluorescence intensity, phase-shift, and demodulation are made at one-second intervals for polycylic aromatic hydrocarbons as they are eluted from the chromatograph. Fluo- rescence lifetime is calculated from both the phase-shift and the demodulation; the two independent values can be used to indicate the absence or presence of more than one component at any point in the chromatogram. Special considerations regarding data correction and calibration for phase-modulation lifetime determinations under continuous-flow conditions are discussed, along with effects of flow rate and mobile phase composition. Index Headings: Fluorescence; Luminescence; Chromatographic detection; Spectroscopic techniques; Time-resolved spectroscopy.

INTRODUCTION

Fluorescence decay of an excited-state population is a time-dependent process that provides a fundamental level of kinetic selectivity in chemical analysis. The mean lifetime of the excited state, or fluorescence lifetime, is a characteristic of the fluorescent compound, and lifetime measurements can be used for component identification as well as for the resolution of individual fluorescence contributions in multicomponent systems. 1 In HPLC detection, fluorescence lifetime can be used to identify components in chromatographic peaks 2,a and to resolve the contributions of individual components in overlapping peaks. 4 In this paper, we describe the first use of a multifrequency, phase-modulation spectrofluorometer for on- line fluorescence lifetime determinations under continuous-flow conditions, and application of the technique to the detection of polycyclic aromatic hydrocarbon (PAH) compounds as they are eluted from an HPLC system. Batch and stopped-flow simulations were used in combination with continuous-flow studies to identify instrumental and experimental factors associated with the frequency-domain lifetime measurements on-the-fly in HPLC. Effects of mobile phase composition and flow rate were studied under continuous-flow conditions.

THEORY

The basis of phase-modulation fluorescence lifetime determinations is the use of a continuous (nonpulsed) excitation beam that consists of a high-frequency (ac) component superimposed on a steady-state (dc) component, s The fluorescence response is modulated at the same frequency, but is phase-shifted and demodulated

Received 24 April 1989. * Author to whom correspondence should be sent.

as a function of the fluorescence lifetime of the sample. In both batch and flow experiments, the frequency-domain approach of phase-modulation fluorescence enables us to simultaneously measure fluorescence intensity, phase angle, and modulation, because all of this infor- mation is obtained from the same emission response function. In the continuous-flow experiments described here, these simultaneous measurements were made at one-second intervals, resulting in many measurements across each chromatographic peak.

There are several features of fluorescence lifetime measurements that are of particular importance for HPLC detection. First of all, fluorescence lifetime is generally independent of concentration; therefore, the lifetime for a single-component peak should attain a certain value as soon as the fluorescence intensity is detected and should remain constant over the entire peak. Second, phase-modulation lifetime determinations provide two independent lifetime values: rp, calculated from the phase- shift, and rm. calculated from the demodulation. For single-component peaks, rm, has the same constant value as rp over the entire peak. For overlapping peaks, on the other hand, rm will be greater than r~ in the regions that are comprised of more than one fluorescent component. Phase-modulation fluorescence lifetime detection can therefore be used to indicate the presence of more than one fluorescent component at any point along the chromatogram.

EXPERIMENTAL

The PAHs (99 %, Analabs) and solvents (HPLC-grade, Burdick and Jackson) were used without further puri- fication. Stock solutions (10 #M) of the PAHs were prepared in 100% acetonitrile by dissolution of the solid PAH, followed by sonication for 30 min. Standard solutions and mixtures were prepared by dilution of the stock solutions. Mobile phases and PAH solutions were all passed through 0.45-#m filters before introduction into the HPLC.

Reversed-phase HPLC was performed on a dual-pump system (Waters) with a 10 x 0.3 cm glass cartridge column assembly, including Vydac 201TP packing and a C-18 guard column, and fixed UV detection at 254 nm. Samples (10-20 #L) were injected manually with a sy- ringe. The PAHs were eluted isocratically, with acetonitrile/water mobile phases ranging from 100% to 70% acetonitrile. A four-port valve was used to connect the flow between the UV absorption detector and the phase- modulation spectrofluorometer, in order to perform stopped flow experiments. The valve had no noticeable effects on the chromatographic peaks in the continuous-

Volume 43, Number 8, 1989 0003-7028/89/4308-136352.00/0 APPLIED SPECTROSCOPY 1363 © 1989 Society for Applied Spectroscopy

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FIG. 1. Batch simulation of the edge of a chromatographic peak, for BkF in 100% acetonitri le (r = 7 ns), [ = 23 MHz: (a) calibration at peak maximum of 9AC reference; (b) in tens i ty-matched calibration with 9AC. Legend: (A) r=; (D) %, (--) intensity.

flow experiments and was therefore left in place for all HPLC experiments.

Fluorescence lifetimes were determined with an SLM 48000S multifrequency phase-modulation spectrofluorometer (SLM Instruments, Inc.), in which electro-optic modulation is used to provide essentially continuous frequency selection in the 1-250 MHz range. This is in contrast to previous work, a,4 in which we used an instrument with acousto-optic modulation that was limited to three modulation frequencies. Other features of the cur- rent instrument include a xenon arc lamp source, pho- tomultiplier tube detection, and an IBM PC/AT for on- line data acquisition and analysis. The kinetic or "t ime" mode of the instrument was used for data acquisition under flow conditions; the raw data--including dc intensity, ac amplitude, and[ phase angle--was transferred to a spreadsheet program for data analysis.

Samples were contained in a 4-mL quartz cuvette for batch experiments. A low-fluorescence, 20-#L flow cell made of black quartz was used for flow and stopped-flow experiments. Wavelength selection was accomplished by means of a monochromator, set at 360 nm for excitation, and a combination of a 399-nm long-pass filter and a 600- nm short-pass filter for emission. A reference solution of 9-anthracenecarbonitrile (9AC, 97%, Aldrich) in 100% acetonitrile was used to calibrate the phase angle and modulation for all of the lifetime determinations. The

9AC was measured in the same cuvette (4 mL or flow cuvette) that was used for the sample in each experiment. The lifetime (rr) of the 9AC solution was determined to be 11.31 ns. Fluorescence lifetimes of the PAH compounds were found from the relationships:

1 % = ~ tan (¢, - q~ + tan-~(cor~)) (1)

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RESULTS AND DISCUSSION

Batch Mode Simulations. Benzo(k)fluoranthene (BkF) was incrementally added to 100 % acetonitrile in a quartz cuvette in order to simulate the concentration profile of the front edge of a chromatographic peak. Fluorescence intensity and phase and modulation lifetimes for each of the solutions are shown in Fig. la. Over most of the "peak" edge, rp = rm = 7 ns, as expected for a peak containing BkF (r = 7 ns). However, we found rm to be increasingly greater than rp as the intensity decreases (approaching the front of the peak). Since these measurements were made under batch conditions, flow is not responsible for this effect. Further studies led us to ex- amine the importance of intensity matching between the reference 9AC solution and the sample. All of the lifetimes shown in Fig. la were obtained by calibration with a 9AC solution that had an intensity similar to that of the highest-concentration BkF solution. For our instrument, intensity matching is not important until the BkF intensity gets close to zero, at which point rm and % become increasingly divergent. Figure lb shows the im- provement in lifetime determinations that is obtained when calibration is performed with a 9AC solution that has the same intensity as the sample. Agreement between rm and Tp occurs over the entire concentration range, yielding the correct value of 7 ns. The increase in random fluctuations of the observed lifetimes at the low intensity edge is due to the relative increase in noise associated with the detection of low-level signals.

Stopped-Flow Simulations. Our next experiments used stopped-flow conditions to measure BkF as it was eluted from the HPLC, in order to evaluate the effects of the flow cell (but not flow conditions) on the lifetime determinations. The stopped-flow experiments used the same instrumental configuration that would be used for real- time chromatographic detection, but the flow from the HPLC was stopped for every measurement in order to eliminate effects due to flow. First, we used a single 9AC reference solution, with an intensity similar to that of the BkF peak maximum, for lifetime calibration. As shown in Fig. 2a, there is excellent agreement between rp and rm across most of the chromatographic peak, and the only deviation occurs at the peak peripheries. Intensity matching helped to alleviate the problem (Fig. 2b). We

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Stopped-flow simulations for BkF eluted by HPLC, 100% acetonitrile mobile phase, f = 23 MHz; a, b, and legend as in Fig. 1.

concluded from this s tudy tha t the flow cell itself intro- duced no new problems or art ifacts in the intensi ty or l ifetime determinat ions.

Flow Experiments. The figures for all of the continuous-flow experiments (Figs. 3-6) represent relative t ime on the X-axis. I t should be noted tha t zero on the relative t ime scale refers to a point just prior to elution of the peak and does not indicate zero re tent ion time. The left- hand Y-axis is scaled for the phase and modulat ion life- t ime determinat ions, and the r ight-hand Y-axis is a relative scale for the intensi ty measurements . The intensi ty scale is consistent for all plots within a given figure.

Figure 3 shows intensi ty and lifetimes, collected under real- t ime flow conditions, for f luoranthene (r = 29 ns) tha t was eluted from the H P L C with a 100 % acetonitri le mobile phase. The results shown in Fig. 3a indicate two separate effects. First, this run used a single 9AC reference instead of intensi ty matching, causing the lifetime divergence at the peak peripheries. Second, the modulation lifetime is not symmetr ic about the chromatographic peak and has a high, positive error at both the f ront and tail edges of the peak. The lat ter effects are observed only under flow conditions and were eventual ly t raced to an ins t rumental artifact: the dc and ac signals used to calculate a given m are actually measured at different times. In fact, we de termined tha t a given ac measurement corresponds to a point tha t is 0.4 s later than the point corresponding to the dc measurement . The actual delay will depend upon the ins t rument av-

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erage mode, and should be de termined for each instru- men t under a given set of ins t rumental conditions.

The results in Fig. 3b were obta ined by intensi ty matching and corrected for the ac delay. The rp and rm values are in excellent agreement with each other and with the correct l ifetime value of 29 ns over almost the entire peak. In tens i ty matching and correction for the ac delay were used for all subsequent runs discussed in this paper.

Effects of Flow Rate and Mobile Phase Composition. Relative to a flow rate of zero, as exemplified by the stopped-flow exper iments (Fig. 2), all of the flow rates used for on-the-fly lifetime determinat ions exhibit increased noise tha t is especially evident at the edges of the peaks. As flow rate through the H P L C is increased, fewer points are collected per peak and the quali ty of the lifetime data is degraded. This is shown for BkF in Fig. 4. The best da ta are acquired at 0.3 mL/min , with excellent agreement between rp and rm at the correct l ifetime of 7 ns over most of the peak. At higher flow rates, systematic divergence of rp and rm occurs on both sides of the peak maximum.

Introduct ion of water into the acetonitrfle mobile phase causes an increase in the re tent ion of BkF, resulting in broader, shorter peaks as the percent water increases. As shown in Fig. 5, best results for the fluorescence lifetime

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1366 Volume 43, Number 8, 1989

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FIG. 6. On-the-fly fluorescence lifetimes determined for a mixture of six PAH compounds, eluted with 100% acetonitrile at 0.3 mL/min., f = 10 MHz. Legend as in Fig. 1. Compounds, in order of elution (true r in parentheses): (1) fluoranthene (29 ns); (2) benzo(b)fluoranthene (25 ns); (3) BkF (7 ns); (4) benzo(a)pyrene (11 ns); (5) benzo(ghi)perylene (15 ns); (6) indeno(1,2,3-cd)pyrene (7 ns).

determinations were obtained with 90% acetonitrile. Above this concentration, accuracy decreases due to the systematic divergence of r~ and rm; and below this concentration, the relative random error increases.

The effects of both flow rate and mobile phase composition can be attributed to similar causes. The systematic divergence of rp and rm, which increases with increasing flow rate or decreasing retention, appears to be related to the resulting increase in slope across the peak; this may be due to the difficulty in exactly compensating for the ac delay, which is more critical at higher slopes. The decrease in precision, which occurs with decreasing flow rate or increasing retention, appears to be related to the expansion of low-intensity regions that results from peak broadening. The effects of flow rate and sol- vent composition can be balanced in order to achieve a compromise between chromatographic retention and fluorescence lifetime determination. For example, the chromatogram obtained for the 90% acetonitrile, 0.5 mL/min experiment (Fig. 5c) combines the quality of the fluorescence lifetime data of the 100% acetonitrile, 0.3 mL/ min experiment (Fig. 4a), with the increased chromatographic retention that results from the addition of water to the mobile phase.

On-the-Fly Lifetime Determinations for a Mixture. Fig- ure 6 shows the results for the lifetime determinations for a mixture of six PAH compounds. As in the previous figures, random fluctuations occur in the absence of fluorescence intensity, i.e., between the peaks. As predicted by theory, rm = % = constant in peak regions comprised

of a single component, whereas rm > rp in regions of unresolved peaks. For the former regions, agreement of the calculated lifetimes with the "true" lifetimes (determined by phase-modulation measurements in batch mode for the individual components) is excellent. For unresolved peaks, the calculated lifetimes across the peaks clearly indicate the number of components and the regions of overlap. Even in the highly-overlapping peaks of benzo(ghi)perylene and indeno(1,2,3-cd)pyrene, lifetime measurements accurately show two components with lifetimes of 15 ns and 7 ns. We are currently studying the resolution of overlapping peaks through lifetime heterogeneity analysis, in which fluorescence lifetimes and intensities of each component at each point in the chromatogram are determined by analysis of intensity, phase- shift, and demodulation at that point, measured at several modulation frequencies.

CONCLUSIONS

This work has demonstrated the accurate and precise determination of fluorescence lifetime for PAH compounds on-the-fly, as they are eluted by HPLC. The frequency-domain, phase-modulat ion determinations have the following advantages over time-domain, pulsed- excitation approaches that have been described for fluorescence lifetime determinations on-the-fly in HPLC: (1) lifetime determinations can be made very rapidly (at one-second intervals in our experiments), yielding many measurements across each peak; (2) the phase-modulation lifetimes readily indicate heterogeneity, i.e., the presence of more than one component at a given point along the chromatogram; (3) calculation of actual lifetimes is easily accomplished by means of simple equa- tions, using a reference compound for calibration; (4) fluorescence intensity is directly measured, simultaneously with phase-shift and demodulation, by means of the same excitation beam and detectors.

ACKNOWLEDGMENT

This work was supported by the U.S. Environmental Protection Agency {Grant R81-2887-01-0).

1. L. J. C. Love and L. M. Upton, Anal. Chem. 52, 429 (1980). 2. D. J. Desilets, P. T. Kissinger, and F. E. Lytle, Anal. Chem. 59, 1830

(1987). 3. W. T. Cobb, K. Nithipatikom, and L. B. McGown, in Progress in

Analytical Luminescence, ASTM STP 1009, D. Eastwood and L. J. Cline Love, Eds. (ASTM, Philadelphia, Pennsylvania, 1988), pp. 12-25.

4. W. T. Cobb and L. B. McGown, Appl. Spectrosc. 41, 1275 (1987). 5. R. D. Spencer and G. Weber, Ann. N.Y. Acad. Sci. 158, 361 (1969).

APPLIED SPECTROSCOPY 1367

multifrequency phase-modulation fluorescence lifetime determinations on-the-fly in hplc

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