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Anal. Chem. 1086, 58, 1717-1721
Elimination of Quenching Effects in Luminescence Spectrometry by Phase Resolution d. N. Demas,*l-sWesley M. Jones,' and R. A. Keller' Los Alamos National Laboratory, Los Alamos, New Mexico 87545,and Chemistry Department, University of Virginia, Charlottesville, Virginia 22901
We descrlbe two methods of reducing quenching effects In analytical luminescence measurements. These methods are a form of tlme resolutlon based on the amplitudes and the phase shifts of modulated slgnals. I n method 1 the slgnal Is moddated at a high frequency where we show that the M a l Is essentially Independent of sample quenchlng. Method 2 simultaneously measures the modulated amplitude and the phase shift between the emlsslon and the excitation; using these data, we correct the modulated amplitude to the unquenched value. Method 1 Is not as accurate as method 2 but lends itself to use wlth automated Instruments where the sample composition may change and rapld real-time measurements are deslred. Method 2 ls more accurate but requires more calculations and careful control of experlmental conditions. Experlmental resuits are presented on the reduction of errors in the quenching of uranyl by chloride. Our methods are compared with other approaches for ellmlnatlng quenchlng errors.
Luminescence spectrometry is a pervasive analytical tool (I).Its popularity rests, in part, on exceptionally high sensitivity and selectivity. Luminescence methods are, however, subject to errors caused by quenchers in the analyzed sample. These quenchers reduce the luminescence signal and, if not accounted for, produce erroneously low concentration estimates. Quencher problems are especially severe in complex media such as biological, mineralogical, or environmental samples where the nature and amounts of the quenchers are unknown and not easily controlled. There have been several approaches to the elimination of luminescence quenching effects: (1)dilution, (2) standard addition (2),and (3)back extrapolation of luminescence decays or correction for the degree of quenching (3-5). In the dilution approach the sample is diluted until the quencher concentration is too low to affect the luminescence intensity. In the standard addition method a known concentration of the luminescent species is added, and the emission intensity is remeasured; since the luminescence of the standard is quenched to the same extent as the unknown, the concentration of the unknown can be readily inferred. In the decay imethod the sample is excited with a short-duration light pulse, and the decay curve is extrapolated back to the flash. The extrapolated signal is directly related to the concentration of the luminescent species and is independent of quenching (3, 4). Alternatively, the observed intensity is corrected for quenching from the measured lifetime (5). All three methods have weaknesses and merits. All methods fail if quenching is too large; sample purification before *To whom correspondenceshould be addressed at the University of Virginia. * Los Alamos National Laboratory. *University of Virginia. Work done in part while at Los Alamos National Laboratory on a Sesquicentennial Fellowship from the University of Virginia. 0003-2700/88/0358-1717$01.50/0
analysis may then be necessary. Dilution is simple, but requires extra sample handling and is only suitable if the concentrations of the luminescent species are high enough so that dilution does not reduce the signal below the analytically useful range. Finally, without a priori knowledge of the degree of quenching, the degree of dilution must be established experimentally: Standard addition does not depend on knowledge of the extent of quenching. However, the sample handling is relatively labor-intensive, and the method does not readily lend itself to on-line processing. The decay methods are elegant, avoid additional sample handling, and inherently eliminate errors from sample scatter and sufficiently short-lived luminescence (3-5). However, the instrumentation is elaborate and expensive, and the high computational requirements generally preclude real-time analysis. We describe two new methods of reducing or eliminating quenching effects in luminescence spectrometry. Both approaches are related to phase-resolved spectroscopy (PRS) (6,7), an increasingly important analytical tool (8,9). Our instrumentation is simpler than that of pulsed methods, and we avoid the extra sample handling of the dilution and standard addition methods. We describe results based on quenching of the UOzz+luminescence.
THEORY If a sample is excited with a sinusoidally varying source, the sample emission will vary sinusoidally. The emission will, however, be phase shifted from the excitation, and the degree of modulation of the emission will be lower than the degree of modulation of the excitation source (6-12).These differences arise from the inability of the excited-state concentration to follow the excitation. The phase shift, 6, and the degree or percentage of emission modulation, m, are
6 = tan-' m = M/[1
(27rf7)
+(2~fr)~]~/~
(la) Ob)
where f is the modulation frequency, T is the excited-state lifetime, and M is the degree of excitation modulation. If the excitation is not sinusoidal, the wave form is decomposed into its Fourier components and a series of equations similar to eq 1is derived for the fundamental and the harmonics. Since it is possible to discriminate instrumentally against the harmonics, we consider only the fundamental in subsequent discussions. Equations l a and 1b are the basis of the well-known phase-shift method for measuring excited-state 7's (7-1I). Either 6 or m can be used to evaluate T from eq l a and l b , respectively. Our methods for eliminating quenching effects require that quenching arises from diffusional (dynamic) quenching and not from ground-state associational (static) quenching (1,lO). Under these conditions 7 and total emission intensity are related by
1/10 = 0 I988 American Chemical Society
7/70
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 8, JULY 1986
reference. The two outputs are denoted by the in-phase, P, and quadrature, Q, signals, respectively. The vector sum of the P and Q signals is S. The phase shift, 6, relative to the reference and S is are by
S = (P2+ Q 2 ) l l z 6 = tan-' ( Q / P )
&2.
0.4
i 6
i i 3
S is the modulated amplitude of eq 3. Commercial dualchannel LI amplifiers generally directly calculate S and 6.
91'0
A (2ilfT,)
Figure 1. Variation of normalized signal as a function of A (=2AfT0), and the extent of quenching, 0,indicated by each curve.
where I is the total luminescence intensity (i.e., at very low f s or dc), and the subscript 0 denotes the unquenched value. We now describe the two approaches for eliminating quenching errors. Method 1. We observed that there is an interesting opposing effect between T and the percentage of signal modulation (see p 52 of ref 10). Increased quenching decreases 7. However, as the 7 decreases, m increases. Thus, while quenching decreases the total emission yield, at least some of the lost intensity is regained in the detected modulated amplitude because of the higher percentage modulation. As we will show, operation a t high-modulation f s can largely eliminate the detrimental effects of even extensive quenching. The observed modulated emission signal as a function of f and the extent of dynamic quenching is given by
+ A2]1/2 S = SoA(1 + A2)/[(1/0)2 + A2]{'/2
(3b)
A = 2i?f70
(3c)
@ =
(34
S = So/[(1/@)2
7/70
(4b)
(34
where S is the observed modulated amplitude and So is the modulated amplitude for an unquenched sample at very low f s . Sof is the unquenched signal at f. 0 is the extent of quenching, which is also the low-frequency emission intensity of a quenched sample relative to that of an unquenched one. Note that at very low f s, S / S , = I/Io(eq 2 and 3a). Figure 1shows S/Sovs. the dimensionless normalized frequency, A, for several values of Clearly, a t very low f (A = 0), S decreases most with changes in the extent of quenching. As f increases, however, the differences between the curves for different 0's become progressively smaller, and all the curves approach each other. Thus, by modulation of the signal a t high enough frequencies, the measured signal becomes almost independent of the extent of quenching. Method 2. This approach takes advantage of the fact that, with a dual-channel lock-in (LI) amplifier, one can simultaneously determine the modulated amplitude, S, and the phase shift, 6, of the emission relative to the excitation. Once S and 6 (eq la) are known, the unquenched modulated intensity at zero modulation frequency, So, is calculated from eq 3a. This approach does require a determination of T~ in the analysis medium but this is easily done with a reference. Once T~ is obtained, it is generally unnecessary to remeasure it for every experiment. This method is similar to, but simpler than, one of the pulsed methods (5). Methods 1 and 2 depend on being able to measure the ac signal amplitude unaffected by noise or variations in the phase angle. Dual-channel LI amplifiem provide this capability. A dual-channel LI amplifier can be simply described as containing two normal LI amplifiers. One amplifier processes the signal that is in-phase with the reference, and the other amplifier processes the signal relative to a 90° phase-shifted
EXPERIMENTAL SECTION Materials. Uranyl solutions were prepared by the dissolution of high-purity U308 in concentrated HNO, and final traces of HzOz. Nitrate was eliminated by fuming with a small amount of sulfuric acid. After almost complete removal of the sulfuric acid, the sample was redissolved in H3POe Solutions were made up to a final H3P04concentration of LOO0 M. All measurements were at 80 ppm of U. Chloride (KC1) was used as the quencher. Instrumentation. The phase-resolved luminescence spectrometer was described elsewhere (12).The excitation system was an argon ion laser (458 nm). Typical laser powers before the optical train were 300 mW. The excitation was square wave modulated by an acoustooptic (AO) modulator driven by a variable-frequency generator. Sample excitation was through the bottom of 30-mL beakers used as cuvettes. The detection system was a Spex double monochromator equipped with an uncooled Hammamatsu R928 photomultiplier. All intensity measurements were made at the primary emission peak at 517 nm, which gave the largest luminescence-to-background ratio. Sample backgrounds were negligible at 80 ppm U. A PAR 5206 dual-channel lock-in amplifier was used for signal processing. The reference was the TTL output from the signal generator. This amplifier was particuarly convenient to use as the phase angle and the emission vector amplitudes could be read directly. Further, it had good harmonic rejection. All data acquisition was done manually. Procedures. Titrations were carried out by sequential additions of aliquots of a quencher solution to an initially unquenched UO2+ solution. The quenched solution was identical with the unquenched sample except for the addition of the quencher. This procedure avoided dilution of the UO?+ during the titration. After each addition the solution was thoroughly mixed. The phase angle and vector amplitude measurements were made at 0.1,0.8, 1, 2, 3,4, 5, 6, 8, 10, 15, 20, and 30 kHz. Higher frequencies were set to better than 0.2% with a frequency counter. The determination of T'S from eq l a required phase-angle shifts between the excitation and the emission. Since the oscillator, the modulator, and the amplifier introduce phase shifts, it was necessary to obtain a reference phase shift for a zero-lifetime emitter. This was done after the quenching titration by replacing the UO?+ solution with aqueous rhodamine 6G. The 3-11s T of this dye (12)is essentially zero relative to UO?+'s multimicrosecond T. Because the phase shift of the amplifier varied with sensitivity range and the frequency, phase shifts were measured for all frequencies and ranges used. RESULTS AND DISCUSSION General Comments. Uranyl was selected as the material to be analyzed for several reasons. Analysis of trace amounts of U is important for exploration, process control, environmental monitoring, and for strategic reasons (3). The UOZz+ luminescence is quite long lived (170 ps in 1 M H3P04),which makes luminescence analytical methods subject to interference even at low quencher concentrations (3,4,13-15). When the analyte is derived from mineral or environmental samples, organic species ( 3 ) or trace metals (4, 14) can quench the luminescence. We selected Cl- 88 the quencher because it is a very efficient quencher of the UO$+ emission (13-15). Further, C1--UO2'+ samples were free of long-term thermal or photochemical decomposition and the oscillating photochemical reactions of some other UO? systems. This stability was especially useful
ANALYTICAL CHEMISTRY, VOL. 58, NO. 8, JULY 1986 1. 0
30
.8
10
20 15 8
LC
Table I. Effect of Modulation Frequency on the Elimination of Quenching Effects
'
. .
6
.6
5 4
-
.4
3
'
.2
2 1 , 0. 8 E. 1 -
Ln
1719
f,
Sfcr,mV
method 1
method 2 So u, mV
kHz
( o / S , %)",b
(u/So, %)",e
0.100
3.31 f 2.54 (77) 2.97 f 1.87 (63) 2.82 f 1.65 (59) 2.28 f 0.93 (41) 1.89 f 0.56 (30) 1.60 f 0.36 (23) 1.38 f 0.24 (18) 1.22 f 0.17 (14) 0.965 f 0.091 (9) 0.807 f 0.052 (6) 0.560 f 0.019 (3.4) 0.429 f 0.009 (1.9) 0.289 h 0.002 (0.7)
\
0.800 1.000
I/@ Figure 2. Normalized modulated amplitudes as a function of 110 and modulation frequency (In kHz indicated by each curve). Alternate frequency data points are denoted by +'s or 0's. The solid lines are the theoretical curves calculated from eq 1 and 3, and T~ = 172 ps.
For the experimental points, 0 was taken as the experimentally measured S I S , for f = 100 Hz. given the long period required for a complete multifrequency titration. Quenching Mechanism. Our methods require that all quenching be diffusional. The chloride anion presented a potential problem as it was more likely to quench by static ion pairing than were the cationic quenchers encountered in some UOZz+analyses. We ruled out significant static quenching by comparing T and intensity quenching measurements. The equations relating intensity, T, and quenching in an optically dilute sample are (10) To/T
=1
+ &v[Ql
(54
I o / I = 1 + (KSV+ Keq)[QI + KsvKeq[&l2 (5b) Ksv = k270 (5c) where Ksv is the Stern-Volmer quenching constant, Kq is the equilibrium constant for formation of a nonluminescent donor-quencher associational pair, I is the total emission intensity, [&] is the quencher concentration, and kz is the bimolecular quenching constant. The subscript 0 denotes the value in the absence of the quencher. Equation 5a is the lifetime form, and eq 5b is the intensity form of the SternVolmer equation. Our intensity measurements, s's, taken at 100 Hz, are indistinguishable from the corresponding total emission measurements that would be obtained at very low frequencies (eq 3a). Only if Keq 0-'.Because of the heavy C1- quenching (0-lm== 7.4), A exceeded the maximum W1 only above 8 kHz. Above 8 kHz the reduction of quenching effe& becomes quite dramatic due to the sum-of-squares form of eq 3a. For less heavily quenched samples, the necessary frequency is correspondingly reduced. This reduction in sensitivity to quenching is accompanied by a loss of signal amplitude. For example, at 30 kHz the signal is reduced to 9% of the low-frequency value. However, the relative standard deviation is improved by a factor of 100. Method 2 gives excellent recovery of the true So(-9.6 mV) as judged by the mean and standard deviations, except at 100 Hz. This poor recovery at 100 Hz is better than one might expect, given the very small 6's. Of course, one would always try to work a t a frequency where 21rfr0 > 1.
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 8, JULY 1986
The 6’s for method 2 largely reflect noise in the 6 and amplitude measurements since there are no inherent systematic errors. There are very large 0’s a t low f s , which rapidly drop to a long plateau at higher f s . At the highest f s , u’s may increase. This dependence of the relative deviations in So on f can be rationalized by a balancing of factors. For simplicity we consider only two noise sources, deviations in 6 and statistical photon noise. First, we consider the effect of deviations in the 6. The deviations in 6 are largely independent of angle. At low frequencies small deviations in the small measured 6’s produce large deviations in 7 and, hence, large deviations in the calculated S,. At intermediate and high frequencies this effect decreases. At intermediate f s the larger 6’s yield more accurate 7’s. At high f s , So (eq 3) becomes largely independent of deviations in 6. Photon noise will, however, increase the 6’s at high f s for the following reasons: Since the total luminescence signal (dc plus ac) is independent off, photon noise is independent of f. As f increases, however, S decreases (eq 3a). Thus, a t high enough f s photon noise will dominate and the signal-to-noise ratio will decrease. As shown in Table I, however, the precision and accuracy are rather stable over a wide range in 2irfT0 (2-20). Therefore, method 2 is remarkably forgiving as to choice of operating f. This analysis ignores nonstatistical noise (e.g., source fluctuations, convection, and photochemistry) that would tend to delay the onset of the plateau with increasing
f. Comparison of Methods. Both methods 1 and 2 are highly successful a t extracting quenching-corrected intensities from heavily quenched samples. Errors approaching an order of magnitude in estimated concentrations are reduced to a few percent by either method. Our results show that methods 1 and 2 are well-suited for use with long-lived species such as UOz2+ and room-temperature phosphorescences. With a commercial 50-MHz LI amplifier and a low-cost A 0 modulator, however, T ~ ’ as S short as 30 ns yield 2 r f ~= 9.4, which is adequate even for heavily quenched samples (see figures). Method 1 requires no computations beyond those performed in the LI amplifier. The vector output of the amplifier is the desired signal. Further, one does not need to know 70; the only requirement is that A > @-le Method 1does introduce small systematic errors since S depends somewhat on even a t high frequencies. These errors do, however, depend predictably on @ and f . Thus, method 1 is especially suited to real-time on-line systems such as uranium recovery process control, where rapid response is important but large deviations in the extent of quenching are unlikely. Method 1 gives no warning if the necessary condition for its application fails (i.e., A > @-I), but even this limitation can be circumvented. The instrumentation for methods 1 and 2 is virtually the same. Therefore, in an automated instrument, it would be simple to periodically switch from the rapid responding real-time method 1 mode to the slower method 2 mode to verify that method 1 is still reliable. We show elsewhere (15) that method 1 is suitable for examining UOZz+luminescence in the presence of several quenching metal ions. These ions represent those that might arise from contamination in process control (e.g., from stainless steel) and in mineralogical or environmental samples. Method 1 was especially suitable for systems extibiting oscillatory photochemical behavior, which played havoc with data reduction by method 2. Systematic errors in method 1 can be minimized by raising f, but there is a practical limit. The amplitude of the measured signal decreases with increasing f , but the photon noise remains fixed. Thus signal-to-noise ratio will suffer if f increases too far. A somewhat different problem arises from the
background. Background (e.g., Raman and fluorescence in this work) contribute directly to the output and usually do not decrease with f; this has the effect of accentuating background contributions with increasing f. Finally, if short-lived samples are being studied, it may be impossible to modulate at the required f s , although phase-shift measurements have been successfully made at 250 MHz (16). The problem of inadequate high-frequency range may be more illusory than real, however. In many homogeneous fluorescence measurements, the short-lived excited states are only quenched by high quencher concentrations, which are rarely encountered. Exceptions include complex biological matrices that organize components into close proximity and thus yield static quenching. Long-lived luminescences such as room-temperature phosphorescences (17) can require quenching corrections, but their long 7 ’ s generally permit the use of inexpensive modulators and lock-in amplifiers. Method 2 requires more elaborate computations than method 1 and requires measurement of the unquenched 7 in the analytical medium and the instrumental zero-lifetime phase angle, but these measurements would only have to be made occasionally. This problem is also common to one pulsed method (5). Method 2 avoids the systematic errors of method 1 for higher quenching and will generally be more accurate. Method 2 will also have a lower detection limit, since the signal is not attenuated by the high frequencies and background contributions are less severe. As we show, both methods work well if quenching is the dominant error source. Sample backgrounds generally set detection limits on all methods. We have shown elsewhere that in the absence of quenching, PRS can provide appreciably lower detection limits than CW methods in the presence of large backgrounds (12, 15). We conclude by comparing our methods with the other approaches for reduction of quenching errors in luminescence measurements. Both of our methods have specific advantages. Although instrumentally more complex, our methods have simpler sample handling requirements than either the standard addition or dilution methods and more readily lend themselves to on-line real-time analysis. Our methods are instrumentally simpler and cheaper than pulsed single photon counting 7 methods. They are also likely to be more maintenance free than Blumlein laser systems. Except for the weakest samples, they also exhibit faster response. The pulsed methods do have greater sensitivity than our methods. Further, they work much better with samples that exhibit both quenching and scattering fluorescence backgrounds. Our methods, the pulsed approach and sample dilution, do place greater demands on system stability than does standard addition. With standard addition, it may be necessary to try several additions, but one is, in effect, calibrating the instrument with every measurement. With the other techniques, however, accuracy depends on the stability of the previously determined calibration curve. However, with modern electronics and a source fluctuation-correcting ratiometric system, daily calibration should be adequate. Our methods and the pulsed methods fail with samples exhibiting static quenching, which, fortunately, is likely to be relatively uncommon. Dilution and standard addition methods adequately handle static quenching. In the case of very heavily quenched samples, some degree of dilution or pretreatment is necessary with all methods. Method 2 provides such knowledge earlier than any other approach. ACKNOWLEDGMENT We thank a number of people for the kind loan of equipment that made this project possible. We thank L. B.
Anal. Chem. 1988, 58,1721-1725
McGown for preprints and M. Trkula for helpful discussions and assistance. We thank N. S. Nogar and D. D. Jackson for their support. JND especially thanks R. A. Keller and other members of CHM-2 for their kind hospitality during his stay at Los Alamos National Laboratory. Registry No. U O;', 16637-16-4;C1-, 16887-00-6.
LITERATURE CITED (1) Parker, C. A. Pbotoluminescence of Solutions ; Elsevier: Amsterdam, 1968. (2) Willard, H. W.; Merritt, L. L., Jr.; Dean, J. A.; Settle, F. A., Jr. Instrumental Metbods of Analysis; Van Nostrand: New York, 1981. (3) Kaminski, R.; Purcell, F. J.; Russavage, E. Anal. Chem. 1981, 53, 1093. (4) Bushaw, B. A. Analytical Spectroscopy; Lyon, W. S., Ed.; Elsevier: Amsterdam, The Netherlands, 1984; pp 57-62. (5) HleftJe, G. M.; Haugen, G. R. Anal. Cblm. Acta 1981, 723,255. (8) Jameson, D. M.; Gratton, E.; Hall, R. D. Appl. Spectrosc. Rev. 1984, 20,55. (7) Lakowicz, J. R . Principles of Fluorescence Spectroscopy; Plenum: New York, 1983. (8) McGown, L. Anal. Cbim. Acta 1984, 757, 327. (9) McGown, L. B.; Bright, F. V. Anal. Cbem. 1984, 56, 1400A.
1721
(IO) Demas, J. N. Excited-State Lifetime Measurements; Academic Press: New York, 1983. (11) Teale, F. W. J. Time-Resolved Fluorescence Specfroscopy in Biochemistry and Biology; Cundall, R. B., Dale, R. E., Eds.; Plenum: New York, 1983; p 59. (12) Demas, J. N.; Keller, R. Anal. Cbem. 1984, 57, 538. (13) Moriyasu, M.; Yokoyama, Y.; Ikeda, S. J. Inorg. Nucl. Cbem. 1977, 3 9 , 2205. (14) Yokoyama, Y.; Masataka, M.; Ikeda, S. J. Inorg. Nucl. Cbem. 1976, 36,1329. (15) Demas, J. N.; Jones, W. M.,in preparation. (16) Haar, H. P.; Hauser, M. Rev. Sci. Instrum. 1978, 49, 632. (17) Cline Love, L. J.; Skriiec, M.; Habarta, J. G. Anal. Chem. 1980, 52, 754.
RECEIVED for review June 14,1985. Resubmitted January 27, 1986. Accepted January 29,1986. JND thanks the University of Virginia for the award of a Sesquicentennial Fellowship, which helped make his sabbatical at Los Alamos possible. We gratefully acknowledge support by the Department of Energy (Office of Safeguards and Security) and the National Science Foundation (NSF 82-06279).
Luminescence Quantum Counters. Comparison of Front and Rear Viewing Configurations Gregory S. Ostrom and J. N. Demas*
Chemistry Department, University of Virginia, Charlottesville, Virginia 22901 €3. A. DeGraff*
Chemistry Department, James Madison University, Harrisonburg, Virginia 22807
A critical comparison of front- and rear-viewed iumiriescence quantum counter (QC) arrangements is presented. A precise and accurate automated instrument Is shown for intercomparing the two configurations under differing conditions of biocklng filters or with different detectors. We show that in front vlewlng, an Improper choice of biocklng fllter can yield a very nonuniform spectral response (factor of 3) even for a good QC material such as rhodamine 6. The errors originate from variable emission self-absorption with varying excitation wavelength. A rear-viewed configuration Is almost free of thIs error source, because the QC dye itself acts as a good filter. Methods of avoiding errors in different QC arrangements are discussed.
Determination of light intensities is a pervasive measurement in many physical and biological studies. A common device for measuring relative intensities is the luminescence quantum counter (QC) (1-12). A QC detection system consists of an essentially totally absorbing dye or luminescent screen viewed by an optical detector, usually a photomultiplier tube (RMT). If the dye absorbs all the incident light, the luminescence spectrum and quantum yield are wavelength independent, and the viewing geometry remains fixed, then the PMT's current is proportional to the number of incident photons on the dye and is independent of the excitation wavelength. Thus, over the wavelength range where these conditions are satisfied, the response of the QC system will be the same per incident photon and independent of the excitation wavelength. This property led to the adoption of
the term QC by Bowen (3). The laser dye rhodamine B (RhB), first introduced by Melhuish (4),is one of the most commonly used QC materials. His careful calibration coupled with the dye's relatively flat spectral response over the 250-600-nm range have contributed to its broad popularity. Recently, dyes with deeper red and near-IR responses have been proposed (10, 11).
A number of possible QC configurations with different cell designs or detector viewing geometries have been proposed and used (12). However, in many cases justification for the arrangement has been limited, nonexistent, or wrong. The two common geometries employ front or rear viewing. Each configuraton has potential advantages and disadvantages. Front viewing yields the greatest sensitivity. Typical QC dyes exhibit severe overlap of their emission and absorption spectra. As opposed to a rear-viewed configuration, the emission in front viewing has to pass through a shorter dye path length, and emission self-absorption is minimized. Unfortunately, variable penetration of the excitation into the solution at different excitation wavelengths yields differing degrees of self-absorptionand can produce insidious variations in response for front viewing, even for a perfect QC dye. For example, excitation at an absorption maximum minimizes excitation penetration into the dye solution; this reduces the total path length that the emitted photons have to travel to the PMT and maximizes the signal. However, at an absorption minimum, the excitation penetrates more deeply, the emission must exit to the detector through a thicker dye layer, and self-absorption reduces the signal relative to an equivalent excitation at the absorption maximum. This problem is most severe for dyes exhibiting strong self-absorption.
0003-2700/86/035S-1721!$01.50/00 1986 American Chemical Society
