Anal. Chem. 1995, 67, 1371-1376
Limits of Detection and Resolution for On-the-Fly Fluorescence Lifetime Detection in HPLC Maria Brak Smalley and Linda B. McGown* Department of Chemistry, P.M. Gross Chemical Laboratory, Duke Univemity, Box 90346, Durham, North Carolina 27708-0346
The limits of lifetime detection and resolution in on-thefly fluorescence lifetime detectionin HPLC were evaluated for simple, binary systems of polycyclic aromatic hydrocarbons. It was found that peak heterogeneity due to coelution was clearly indicated for two compounds with fluorescence lifetime ratios as small as 1.2, and the individual peaks could be recovered using predetennined values for the lifetimes of the compounds. The meaning of detection limits in lifetime measurementswas explored and definitionswere proposed. Mts of lifetime detection were determined to be 6 and 0.3 pmol for benzo[blfluoranthene and benzo[k Ifluoranthene, respectively, Continuous, on-the-fly fluorescence lifetime detection in highperformance liquid chromatography (HPLC)has recently been described.’ The detector is a commercially available, frequency domain fluorescence lifetime instrument (MHn that employs multiharmonic Faurier transform technology to acquire phase and modulation data at multiple modulation frequencies in a single measurement. In this approach, both fluorescence lifetime and fluorescence intensity are obtained at intervals of milliseconds or greater during a single chromatographic run. Indication of chromatographic peak heterogeneity and resolution of the individual, component peaks through lifetime detection were demonstrated for binary systems of benzo[klfluoranthene and benzo[blfluoranthene. In contrast to other approaches to resolution of overlapping chromatographic peaks, MHF lifetime detection does not require any degree of chromatographic resolution across the overlapping peak region, nor does it require any spectral resolution. It provides on-thefly lifetime determination, indication of peak overlap, and resolution of coeluting peaks, all from a single chromatographic run, without any a priori assumptions regarding chromatographic peak shape or the identity, properties, or spectral characteristics of the eluting compounds. Moreover, lifetime detection provides hghly sensitive indication of minor components and matrix effects. This paper investigates limits of lifetime detection, limits of lifetime resolution, and limits of resolution of overlapping peaks that are associated with MHF lifetime detection in HPLC. Traditional approaches to determining detection limits cannot be directly applied to fluorescence lifetime detection since fluorescence lifetime is concentration independent and traditional “blank measurements” cannot be made. Therefore, other means of expressing these limits were defined and explored. Binary (1) SmaUey, M. B.;Shaver, J. M.; McGown, L. B. Anal. Chem. 1993,65,34663472. 0003-2700/95/0367-1371$9.00/0 Q 1995 American Chemical Society
systems of polycyclic aromatic hydrocarbon (€‘AH) compounds were used here as in the previous work. THEORY AND BACKGROUND Limit of Detection. The subject of detection limits has been discussed by numerous a~thors.2-l~ There is general agreement that such limits should be based on statistical concepts and should only be used to describe a complete analytical procedure, although there is a divergence of opinions as to how the limits should be dehed. The following discussion is based on the most commonly recommended and accepted definitions. Three considerations must be addressed before determining detection limits. First, any nonanalyte contributions to the measured signal must be identified and quantified. Generally, such measurements are made using a field blank, which ideally contains every component of a specific sample except the analyte of interest.3 Second, a calibration curve is constructed that describes the relationship between analyte concentration and detector response. Third, the analyst must accept the possibility of incorrect results, which include both false positives (type I) and false negatives (type ID. Serious consequences may arise if the confidence level at which the experimental results are reported is allowed to be too large. It is therefore important to lower the probability of obtaining such errors to a reasonable level, realizing that a probability of 0 for either error is unreasonable and generally impo~sible.~,~ With regard to analyte detection, the analyst must make both a priori and a posteriori decisions. In determining detection limits, the analyst must estimate, a priori, the minimum concentration that may be expected to yield a signal S sufficientlylarge to be detected and then decide a posteriori, given an observed signal (2) Ingle, J. D.; Crouch, S. R SpectrochemicalAnalpk; Prentice-Hall: Englewood
Cliffs, NJ, 1988; pp 172-176. (3)Kurtz, D.A;et al. In Detection in Analytical Chemistty: Importance, Theoy, and Practice; Currie, L.A, Ed.; ACS Symposium Series 361;ACS: Washington, DC, 1988; Chapter 16. (4)Cume, L.A In Detection in Analytical Chemistty: Importance, Theory, and Practice; Cume, L. A,Ed.: ACS Symposium Series 361;American Chemical Society: Washington, DC, 1988 Chapter 1. (5)Boumans,P. W. J. M. Spectrochim. Acta B 1978,33B, 625-634. (6)Currie, L.A. Anal. Chem. 1968,40, 586-593. (7)Oppenheimer, L.; Capizzi, T. P.; Weppelman, R M.; Mehta, H. Anal. Chem. 1983,55,638-643. (8) Borman, S.A Anal. Chem. 1986,58,986A (9)Hecht, H.G.Mathematics in Chemistty: An Introduction to Modem Methods; Prentice Hall: Englewood Cliffs, NJ, 1990; pp 293-296. (10)IUPAC. Spectrochim. Acta 8 1978,338, 242-245. (11) Caulcutt, R; Boddy, R Statistics for Analytical Chemists; Chapman and Hall: New York, 1983;Chapter 4. (12)Hecht, H. G.Mathematics in Chemistty: An Introduction to Modem Methods: Prentice Hall: Englewood Cliffs, NJ, 1990; Chapter 4. (13)Ferrces, R;Egea, M. R Anal. Chim. Acta 1994,287,119-144.
Analytical Chemistty, Vol. 67, No. 8, April 15, 1995 1371
S, whether a real signal has been detected.5~~ The smallest “real (3) signal” that is detectable and discernible from measurements of the field blank is termed the critical level (Lc)or criterion of detection and is determined by the maximum acceptable value for the type I significance criterion, a,and the standard deviation where s is the standard deviation of ‘c&s and n is the number of of the net signal (uo),when the true value (limiting mean), ,US, is points used to determine robs and s. The value of fdcis compared equal to 0. Mathematically,for a large number of meas~rements~>&~ to a critical t value, tmt,at the desired significance level. If tcdc
tdt,then there is a significant difference between the two, meaning that robs is inaccurately detected and is therefore reported as not detected.”Jz Although this is an acceptable way to compare an experimental value with a “known” value, and is analogous to the standard treatments of detection limits, the presence of a large standard deviation will result in a small tealcand lead to the acceptance of the two values as being the same under questionable circumstances.11J2 In order to minimize such acceptances in lifetime detection, we add a second criterion: in order to accept Tobs and rpEas being indistinguishable, Tob$S must be 1 3 . This is similar, but not identical in theory, to a required signal-bnoise (S/N) ratio of 2 3 that is often used to define instrumental detection limits. Therefore, to say that a given analyte in the chromatogram has been idenaed through lifetime detection, the t test and “ S / N criteria must both be met. On-the-FlyIifetime Measurements. The theory of phasemodulation fluorescence lifetime measurements has been well described.14 Briefly, the sample is excited with an excitation beam of a given wavelength that is intensity modulated at a given frequency. This excitation produces a fluorescence emission signal that is intensity modulated at the same fmquency but phase shifted and demodulated relative to the exciting light. The observed phase shift and demodulation are functions of the fluorescence decay characteristics of the sample and the applied modulation frequency and may be used, independently, to calculate the fluorescence lifetime of the sample. If the two independently calculated lifetimes are equal, then a homogeneous system containing a single lifetime component is indicated. If the lifetime calculated from demodulation is greater than the lifetime calculated from the phase shift, then ground state heterogeneity (Le., the presence of more than one fluorescence lifetime component) is indicated. In this case, the lifetime and fractional intensity contribution of each of the components can be determined by NLLS analysis of the phase and modulation data collected at multiple modulation frequencies. This approach was used in the initial on-the-fly lifetime detection work,15-18 which required multiple sample injections to obtain the multifrequency information, one modulation frequency at a time. The introduction of MHF technology greatly improved the capabilities of on-the-fly lifetime detection. Since multifrequency phase and modulation information are acquired from a single chromatographic injection, both data acquisition and data analysis are simphied. Thorough discussions and characterization of data (14) Lakowicz, J. R Principles of Fluorescence Spectroscopu; Plenum: New York, 1983. (15) Cobb, W.T.; McGown, L B. Anal. Chem. 1990, 62,186-189. (16) Cobb. W.T.;Nithipatikom, K; McGown, L. B. In Progym in Analytical Luminescence; Eastwood, D., Cline Love, L. J., Eds.; Asru Srp 1009; ASTM: Philadelphia, PA, 1988, pp 12-25. (17) Cobb, W.T.;McGown, L B. Appl. Spectrosc. 1987, 41, 1275-1279. (18) Cobb, W.T.;McGown, L. B. Appl. Spectrosc. 1989,43, 1363-1367.
acquisition by the MHF may be found elsewhere.’Jg Briefly, the exciting light intensity is modulated at the generated harmonics of a designated base frequency, F. The emission contains the same multiple frequencies (base frequency and harmonics), and the signal at each frequency is phase shifted and demodulated as described above. The phase shift and demodulation at each frequency are determined by the crosscorrelation technique,zo in which the harmonics of a second, slightly offset, frequency, F + AF, which is phase locked to the excitation frequency, are used to modulate the gain of the PMT. The frequency information is digitized and Fourier transformed to yield phase and modulation values at each frequency. The value of A F determines the interval at which the signal is sampled (as its reciprocal) and therefore the number of data points collected per unit time. EXPERIMENTAL SECTION
Benzo[a]pyrene, benzo[e]pyrene, benzo[ghi]perylene,benzo[klfluoranthene, and chrysene @UP, BeP, BgP, BkF, and Chy, respectively; each 99%,AccuStandard),benzo[blfluoranthene (BbF 99%,Aldrich), and Santhracenecarbonitrile (9AC; 98%,Lancaster Synthesis) were used as received. Acetonitrile (HPLC grade, Burdick & Jackson) and water (HPLC grade, in-laboratory Modulab type I water puritication system), which were used for sample preparation and as the HPLC mobile phase (80%CH&N in HzO), were puritied further by vacuum filtration as suggested by the HPLC manufacturer. Stock solutions (-5.00 x loe4M) of the individual PAHs were prepared in mobile phase, stored in amber bottles with Teflonlined caps, and refrigerated to prevent degradation. The stock solutions were used to prepare solutions of BbF, BaP, BeP, BgP, and Chy, all at -1.00 x M, and a mixture of BbF (8.03 x M) and BkF (3.21 x M). Eleven additional BbFIBkF mixtures were prepared by serial dilution, to concentrations of 3.92 x and 1.57 x M for BbF and BkF, respectively,for the limit of lifetime detection experiment. Scattering solutions were prepared by addition of kaolin (Sigma) to water. Steady-state and dynamic fluorescence measurements were made using a multiharmonic Fourier transform phasemodulation spectrofluorometer (Model 4850 MHF, SLM Instruments, Inc.) with He/Cd laser excitation at 325 nm (LiCONiXModel 4240NB). For dynamic-state measurements, the intensity of the excitation beam is electrooptically modulated by a Pockels cell. The emission beam was passed through a combination of a 39Snm long-pass filter and a 600-nm short-pass filter (Oriel) and into a PMT (Hamamatsu type R928) for detection. For collection of steady-state emission spectra, a scanning monochromator was used instead of filters for wavelength selection. Reversed-phase chromatography was performed using an HPLC system (Waters) that was interfaced to the MHF. Manually injected samples were passed through a C-18 guard column and a Vydac 201-TP-B-5 packed 10 cm x 0.3 cm glass carhidge analytical column, with a particle size of 5 pm, that is designed specifically for separation of PAHs. Timed injections were used to create varying degrees of component overlap for the lifetime resolution experiment. The PAH solutions and mobile phase were not degassed or deoxygenated. Compounds were isocratically eluted from the analytical column at a flow rate of 0.5 mL/min (19) Mitchell, G.: Swift, IC In Time-Resolved h e r Spectroscopy in Biochemistry II; Lakowicz, J. R, Ed.; SPIE Vol. 1204; SPIE -The International Society for Optical Engineering: Bellingham, WA, 1990; Part 1, pp 270-274. (20) Spencer, R D.; Weber, G. Ann. N.Y. Acad. Sci. 1969,158, 361-376.
Table 1. Lifetime Resolution Experiment: Predicted Lifetimes and Retention Times of PAHs.
andyte
“predicted” lifetime (ns)
retention time (min)
BaP BbF BeP BgP ChY
16.3 28.5 19.2 25.9 17.3
5.95 4.12 4.05 8.65 3.06
Determined from injections of standard amounts of the individual components. (I
and routed through a four-port valve to a 20-pL flow cell with an &pL observation volume (Hellma Cells) located in the thermostated sample chamber (20 OC) of the MHF. The four-port valve allowed the acquisition of reference measurements and steadystate spectra in the stopped-flowmode. The fluorescence emission data, from which both intensity and lifetime information are derived, was collected on-the-fly using a Dell 3252) computer. On-theflyfluorescence lifetime detection was performed in the Kinetic Lifetimes mode of the MHF. This mode allows virtually continuous collection of phase and modulation data at as many as 50 modulation frequencies simultaneously. In this work, the first 15 modulation frequencies were used to create a frequency range of 4.1-61.5 MHz, which sufficientlybrackets the optimum frequencies for lifetime detection of the compounds used. Phase and modulation were sampled at 0.096s intervals (AF = 10.417 Hz) during chromatographic peak elution, and the average of 10 samplings was used to determine both lifetime and intensity, resulting in data points at 0.96s intervals along the chromatogram. In this work, only phase data were used in the lifetime analysis. Since the lifetime data are acquired from a single chromatographic run, replicate measurements at each point are not available for calculation of the real (“acquired”) uncertainty associated with the phase values. Therefore, it was necessary to use a constant (“linear”) error to express the uncertainty of each measurement in the NLLS analysis. Goodness of fit for a given model to the data in the NLLS analysis, performed using a commercial software package (Globals Unlimited), is indicated by the reduced x2 value, x2~.21 RESULTS AND DISCUSSION Limits of Heterogeneity Indication and Lifethe Resolu-
tion. The minimum lifetime difference needed to indicate and resolve two overlapping peaks was determined using six pairs of PAH compounds with lifetime ratios ranging from 1.06 to 1.59 Vables 1 and 2). Various degrees of chromatographic peak overlap were achieved for each pair through appropriately timed injections of the individual compounds (Table 2), in order to see whether the amount of peak overlap would affect the lifetime resolution. Figures 1-3 show results for two of the PAH pairs studied. Results for BeP and BaP are shown in Figure 1. From the data shown in Figure lA,predicted lifetimes, rppre, of 19.3 and 16.3 ns were recovered for BeP and BaP, respectively, giving a lifetime ratio of 1.18 for the two compounds. For two different degrees (21) Globals Unlimited: Technical Reference Manual and UserManual; Laboratory of Fluorescence Dynamics, University of Illinois at Urbana-Champaign: Urbana, IL, 1990.
Analytical Chemistry, Vol. 67, No. 8, April 15, 1995
1373
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of peak overlap, the observed lifetimes, robs (Figure lB,D), change across the peaks, indicating the presence of more than one component. It is important to note that the presence of more than one component is clearly indicated even when the peaks are almost entirely overlapping. Because the lifetimes of BeP and BaP are so similar, it was not always possible to accurately recover the predicted lietimes of the individual components. It was possible, however, to recover the intensity peaks of each component (Figure lC,E) by Exing the lifetimes to the predicted lifetimes of the components in a two-component model to obtain the fractional intensity of each component at each point in the chromatogram. The recovered fractional intensity is multiplied by the total intensity at that point in the chromatogram to obtain the intensity due to that component at that point. The intensity peaks can then be used for quantification of each component. Results for BgP and BeP are shown in Figure 2. Predicted lifetimes of 25.9 ns for BgP and 19.3 ns for BeP were recovered from the data shown in Figure ZA, giving a lifetime ratio of 1.35. Results are similar to those for BeP and BaP, with one exception. As shown in Figure 2D, the observed lifetime dips across the peak apex region instead of gradually changing from one lifetime to another. This anomalous result is echoed in the construction of the component peaks (Figure 2E), which are inaccurate in the peak apex region: the contribution from BgP appears to decrease while that of BeP appears to increase. 1374 Analytical Chemistry, Vol. 67, No. 8, April 75, 7995
Table 2. Lifetlme Resolution Experiment: Lifetime Ratlos, lnjectlon Volumes, Injection Amounts, and Injection Delay Times
PAH Paip
5
ratio
inj vol
01L)
inj amt (nmol)
inj delay time (s)
20.00/3.50
0.20/1.75
180 210 240 60 110 115 180 225 255 120 135 170 20b 45 60 90 150 165
BbF/BgP
1.10
BePIBaP
1.18
1.50/70.00
1.00/0.65
BeP/BgP
1.35
2.00/3.50
1.00/1.75
ChylBaP
1.06
6.00/70.00
3.00/0.70
Chy/BeP
1.11
6.00/2.00
3.00,' 1.00
BaPIBgP
1.59
70.00/3.50
0.70/1.75
The compound in boldface type was injected first, followed by the other compound after the indicated delay time. * In this case, Chy was injected first, followed by BeP after a 2@s delay. (I
In order to investigate this anomaly, steady-state emission spectra were collected at points along the eluting peak and
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Figure 2. Limits of heterogeneity indication and lifetime resolution for BePIBgP. (A) Lifetimes of BeP and BgP recovered from raw MHF data collected on the fly. (B) and (D) Observed lifetime across overlapping BeP and BgP peaks. (C) and (E) Individual, lifetime-resolved BeP and BgP peaks recovered from overlapping peaks. The lifetime data were fit to a one-component model for (A), (B), and (D) and to a two-component model, with the lifetimes fixed to the predicted values of BeP and BgP, for (C) and (E).
compared to emission spectra of the individual components and to a summed spectrum of the two components. As the peak apex is approached during elution, the BgP contribution to the emission spectrum of the mixture decreases and nearly disappears at the peak apex. As elution continues passed the peak apex, the BgP contribution returns to the expected proportion. These results indicate an interaction between BeP and BgP in the peak apex region, where analyte concentrations are the highest, that prevents accurate resolution of the individual component peaks. Indication of this anomaly is an exampIe of the ability of fluorescence lifetime detection to flag anomalies such as component interactions and concentration effects that may not, as in this case, be indicated in the steady-stateintensity chromatogram. Indication of anomalous behavior is essential for accurate interpretation of the intensity data that is used in component quantification. Even if both the intensity chromatogram and NLLS fit to onecomponent models of lifetime data suggest the presence of only a single component under a peak, higher order fitting models should nevertheless be attempted. For example, two-component fits to the BeP/BaP and BePIBgP data above showed the presence of at least two components under what appeared to be homogeneous, singlecomponent peaks in the steady-state intensity chromatograms (Figure 3). In these cases, higher order models did not significantly improve the fits and the presence of two components could therefore be concluded.
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Figure 3. Fluorescence lifetimes recovered from raw MHF data collected on the fly across overlapping peaks: (A) BeP (0)lBaP (A); (B) BeP (0)lBgP ( A ) . The lifetime data were fit to a two-component model. Analytical Chemistty, Vol. 67, No. 8, April 15, 1995
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In summary, fluorescence lifetime detection indicates coeluting compounds, even when their chromatographic peaks are virtually unresolved, for compounds with a lifetime ratio as small as 1.2. The peaks can then be resolved by NLLS analysis of the lifetime data by fixing the lifetimes to predetermined values. Limits of Lifetime Detection. Fluorescence intensity and lifetime were detected on-the-fly for triplicate, 10-pL injections of 12 different concentration mixtures of BbF and BkF. The injection amounts ranged from 0.392 to 803 pmol for BbF and from 0.157 to 321 pmol for BLF. The mean and standard deviation of the lifetime across each peak were determined for all injections using only the phase information from those points that had a steadystate intensity 250% of the maximum peak intensity. Various models were used in the NLLS analysis to find the one that best described the data, based on the consistency of the recovered lifetimes and fractional intensity contributions across the peaks, the x 2 values, ~ the number of iterations required for completion of the NLLS analysis, and the phase residuals. It was determined that the best model has one floating lifetime and one lifetime that is fixed to zero, in order to account for signiicant scattered light contributions as the signal approaches background levels. As a test of the first criterion for lifetime detection and component identification,values for tCdcwere compared to values for tcit (eq 3) at significance levels of a = 0.05 and 0.01 (95%and 99% coddence levels, respectively). As expected, there were some cases in which a significant difference was observed at a = 0.05 but not at a = 0.01. However, each significance level identified the same limit of lifetime detection. After considering all of the criteria described in the discussion of limits of detection
1376 Analytical Chemistty, Vol. 67, No. 8, April 15, 7995
(see Theory and Background), the minimum amounts of BbF and BRF that must be present for fluorescence lifetime detection are 6.3 (1.6 ng) and 0.31 pmol (79 pg), respectively, which correspond to concentrations of 160 and 7.9 ng/mL. It is important to note that although using a = 0.01 reduces the risk of incorrectly concluding that there is a significant difference, it concomitantly increases the risk of accepting significantly different values as being the same.llJ3 The detection limits reported here are instrumental detection limits and were determined under nonoptimiied conditions. If we were to improve chromatographicconditions by keeping the HPLC column at a constant temperature and degassing and deoxygenating the mobile phase, for example, the sensitivity, precision, and selectivity of this technique should be enhanced. Use of a higher power laser and a more optimal wavelength for these compounds could improve detectability in these simple, binary systems. ACKNOWLEWYENT This work was supported by the Office of Exploratory research of the United States Environmental Protection Agency (Grant R817127-01). Received for review November 28, 1994. Accepted January 24, 1995.@ AC9411334 @Abstractpublished in Advance ACS Abstracts, March 1, 1995.
