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-absorption and 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
1722
ANALYTICAL CHEMISTRY, VOL. 58, NO. 8, JULY 1986
Another factor that can affect the errors in the frontrviewed configuration is the variation of the emission spectrum. The degree of self-absorption, and, thus, the emission spectrum seen by the detector, varies with excitation wavelength. The effect can be quite pronounced and is easily demonstrated visually. The front-viewed emission of RhB is orange on excitation a t the absorption maximum a t 550 nm but deep red on excitation a t the absorption valley a t 440 nm. Since the PMT signal is the integral of the product of the emission spectrum seen by the detector and the detector sensitivity curve, the QC’s spectral response curve can then depend on the specific P M T characteristics. In contrast, back viewing should have a response that is nearly independent of the excitation wavelength. The high absorbance of the solution ensures that excitation occurs near the cell’s front face in a path length that is much shorter than the QC cell thickness. This front surfacing of the emission ensures that, regardless of changes in the penetration depth, the emission escaping the rear face must pass through virtually the entire thickness of the QC cell. Therefore, the percentage change in path length to the detector is much smaller on variation of excitation wavelength with rear compared to front viewing. Unfortunately, the higher degree of self-absorption with rear viewing can yield a less sensitive device. In a typical QC system, a filter is used over the PMT to restrict the detector response to the QC emission and reduce sensitivity to stray and scattered light. Varying the filter characteristics modifies the amount and spectral distribution of the emission striking the detector. Since the front-viewed configuration samples an emission spectrum that depends on excitation wavelength, the response of a front-viewed QC system will depend on the spectral characteristics of both the filter and the detector. The goals of this study were to explore the relative merits of front- and back-viewed QC’s and to determine the effect of different filters and detectors on the characteristics of front-viewed QC’s. As we will show, a front-viewed QC can be successfully used, but severe errors can result if an improper viewing filter is used.
EXPERIMENTAL SECTION Materials. Rhodamine B was purified as described earlier (6). The QC solutions were made up in reagent grade methanol at a concentration of 5 g/L. Tris(1,lO-phenanthroline)iron(II) perchlorate was from G. Frederick Smith Chemical Co; a 0.0004 M aqueous solution was used as a nonluminescent blank, in order to permit elimination of any background effects due to offset voltages or scattered light. Emission and Absorption Spectra. Corrected emission spectra were measured on an SLM spectrofluorometer. In order to ascertain the emission spectra seen by the front-viewingPMT, we measured spectra for the QC solution in the QC cell with front viewing. Excitation was at 440 nm and 550 nm, which corresponds to the dye’s absorption minimum and maximum. Since we did not have a front surface attachment, we set the QC cell so that excitation and viewing were at approximately45’ to the cell face. The cell was tilted so that specular reflected exciting light just missed the emission monochromator’s entrance slits. This geometry gave the best approximation to that used in the frontviewed QC system. Absorption spectra were measured on a Cary 17 spectrophotometer. The 5 g/L QC solution was measured in a 0.1-mm cell. Even with this thin cell, the solution absorbed so strongly that only part of the spectrum fell on scale. To get the remainder of the spectrum, the QC solution was rerun at a 1:16 dilution. System Overview. We wished to design an instrument that would permit precise and accurate determination of the response of front-viewed QC’s under different conditions. Since we have earlier calibrated a rear-viewQC system (6),we chose this system as a reference. Figure 1 shows a schematic representation of our final design. The same QC cell is viewed simultaneously by front- and rear-
1
I
nl
1
T Pi+y COMPUTER
Figure 1. Schematic representation of QC comparator: S, xenon lamp; MONO, excitation monochromator; QC, quantum counter cell: PMTI, WT2, photomultipliers; DRIVE, stepping motor and controller for wavelength drive; IVC, current-to-voltage converter; MUX, reed relay analog multiplexer; GPIO, general purpose parallel I/O port; DMM, dgital multimeter; HPIB, IEEE-488 interface card: COMPUTER, HP-85 microcomputer;optical paths (- - -): mechanical paths (-). Filters are not shown. viewing PMT’s. In this way, the relative response of the frontviewing PMT can be directly compared and ratioed to the rear-viewed standard. Excitation is at normal incidence to the cell, and both PMT’s are positioned off-axis so that specular reflected or transmitted light does not strike them. In order to increase the accuracy and ease of use, the data acquisition and reduction system was computerized. The excitation system is described elsewhere ( 6 ) . The frontviewing PMT was an RCA 931B or a Hammamatsu R666, while the back-viewing PMT was the RCA C7164R used earlier (6). The C7164R was used since it was calibrated earlier with RhB. The 931 and R666 were used since they are readily available and exhibit wide differencesin spectral sensitivity. The PMT’s currents were converted into voltages by FET current-to-voltage converters (1 M feedback resistors). The signals were digitized with a Keithley Model 177 digital multimeter (DMM) and read over an IEEE-488 bus with a Hewlett-Packard HP-85 computer. The HP-85 also controlled an analog multiplexer that switched the two PMT signals into the DMM (13). The wavelength scan (350-600 nm) was computer controlled by a stepping motor drive. Data were taken every 10 nm. The QC cell was a 10-mm-path-length by 50-mm cylindrical cell (6). Neutral density filters were used over the front PMT to attenuate more intense emissions. The filters were Corning long-wavelength-passglass filters. A 2-58 cutoff filter (>646 nm) was used over the reference back PMT. Software. To measure the relative response of the front-viewed PMT, two sets of measurements were required. First, the responses of the front and back PMT’s to the QC were measured at every excitation wavelength. The system background (Le., scatter, cell fluorescence,dark current, and offset voltages) was then measured with the absorbing, nonluminescent iron complex solution replacing the QC. After setting the slit widths, PMT voltages, and the starting wavelength for the excitation monochromator,each spectrum was collected automatically by the computer. For each wavelength the computer advanced the monochromator to the next measurement wavelength and then measured both PMT signals. Data were stored on tape. The corrected relative rebponse of the front-viewed PMT was calculated from R(A) = [zf(A) - if(A)l/[zb(A)
- ib(A)]
(1)
where R is the corrected ratio, I‘s are signals with the QC in place, i’s are the blank readings, and the subscripts f and b refer to the front- and back-viewed reading, respectively. For display and comparison,R(X)’swere normalized to an average value of unity. All R(A)’s were highly reproducible. Normalized replicate spectra agreed to better than 0.5% at all points, and more typically better than 0.25%. Our reported R(A)’s are relative.to a rear-viewed 5 g/L RhB QC, rather than to an absolute standard. Since our reference is flat to f4% ( 6 ) ,the R(X)’s are not greatly in error. Correction
ANALYTICAL CHEMISTRY, VOL. 58, NO. 8, JULY 1986
I
Rhodamine
B
in
1723
CH30H
u W
i
m
Lz 0
m
A
nm
Figure 2. Absorption spectrum of rhodamine B in methanol in a 0.01-cm cell: (A) 5 g/L, (B) sample A diluted 16 times.
1001
to absolute units could be done with the known correction factors (6).
To determine the relative sensitivities of the front-viewed QC arrangement for each color filter, we measured the front PMT signal with each filter using 550-nm excitation (wavelength of maximum dye absorption signal). Because of the large signal variations with filter, neutral density filters were used to reduce signals to within the range of the preamplifier. Data are corrected for the attentuation of the filters. To determine the effect of PMT spectral response on the front-viewed arrangement, we replaced the 931B with an R666 for one set of measurements. The 931B has an S-4 response that falls off rapidly in the region of the strongest dye emission. The gallium arsenide R666 has a nearly constant quantum efficiency over the dye's emission. A 3-73 filter gave the largest defects in QC response with the 931B (vide infra) and was used over the R666.
0 X
nm
RESULTS AND DISCUSSION
Figure 3. (A) Corrected front-viewed emisslon spectra of rhodamine B (5 g/L) in methanol excited at 550 nm and 440 nm. The excitation wavelength is above each curve. The small peak at 550 nm on the 550-nm excitation spectrum is scattered excitation. (B) Measured filter transmission characteristics. From left to right the filters have the fdlowhg Corning color specification numbers: 3-73, 3-68, 3-66, 2-62, 2-61, 2-59, 2-58, and 2-64.
The absorption spectrum of RhB is shown in Figure 2. The absorbanceat the 550-nm maximum is about 100 times greater than at the 440-nm minimum. For the QC to function properly, all the exciting light must be absorbed by the dye. Even well on the tail of the absorption band at 600 nm, the absorbance in the 1-cm QC cell is about 4. Thus, over the excitation wavelength range studied, virtually complete absorption of the excitation occurs in a thin layer of the QC solution. The front-viewed emission spectra using excitation a t the absorption minimum and maximum are shown in Figure 3. There is a very pronounced 20-nm emission blue shift on varying the excitation wavelength from the region of minimum to maximum absorbance. This contrasts sharply with the much smaller variation for rear viewing (5). The observed effect is that expected by self-absorption of the dye emission by its strongly overlapping absorption spectrum (Figure 2). A t 550 nm the emission only has to escape through a thin dye layer (
