D. W. Johnson,1 J. B. Callis,2 and G. D. Christian1
Instrumentation
University of Washington, Seattle, Wash. 98195
Rapid Scanning Fluorescence Spectroscopy T h e inherent sensitivity and wide applicability of fluorescence spectros copy have made it a valuable approach for analysis in clinical, environmental, and forensic chemistry. T h e growing use of the technique is amply demon strated in the number of texts (1-3), reviews (4), and feature articles (5-7) devoted t o fluorescence analysis. T h e most widely cited advantage of the fluorescence technique is its in herent sensitivity, which is due to the fact t h a t the luminescence photons are detected directly by transducers capable of detecting a single photon with a reasonable probability. As a re sult, concentration sensitivities for macroscopic assays frequently reach into the parts per trillion level, while number sensitivities for microscopic assays have been reported for as few as 100 molecules and even lower. Often, however, the detection limits for a particular fluorescence assay are set by sources other than the photon statistical noise of the sought-for fluorophores; instead, the limitation arises from interference due either to blank fluorescence or to the presence of other fluorescent species in the sample itself. While it certainly is true t h a t both the wavelength of excitement and the wavelength of detection of the emission may be varied t o minimize interferences, in practice, the broad band nature of fluorescence often ren ders these degrees of freedom ineffec tual. This means t h a t analysis of a sin gle component will be subject to inter ferences from other fluorescent com ponents, and t h a t the simultaneous analysis of several components will be frustrated by spectral overlaps. Thus, fluorescence analyses are generally characterized by extensive sample preparation so t h a t only the lumines cent species of interest is present when the fluorescence is measured. If multiple components are t o be deter mined, the sample preparation be comes more complex and may include separate solvent extractions of each component or chromatographic sepa ration prior to measurement. Clearly, development of instrumen tation that allows the simultaneous
1 2
Department ot Chemistry. Department of Pathology.
determination of multiple fluorescent components is desirable. In this paper, we first present the rationale for an approach we have taken for multicomponent fluorescence analysis, based on the unique spectral information contained in the emission spectrum and the excitation spectrum of each
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luminescent species. A new rapid scanning instrument based on this ra tionale, the video fiuorometer (8, 9), which can simultaneously acquire both types of spectra in as little as 17 ms, is then described. Finally, we com pare our approach with previous schemes for multicomponent fluores-
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Figure 1, Illustration of relationship of EEM to a compound or mixture's excitation and emission spectra Component 1 is zinc octaethylporphin (ZnOEP); Component 2 is free base octaethylporphin (H2OEP)
ANALYTICAL CHEMISTRY, VOL. 4 9 , NO. 8, JULY
1977 · 747 A
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1C and D we give an alternative repre sentation of the fluorescence spectra, as the "emission-excitation matrix" (EEM). Each matrix element, Mjj, represents the fluorescence intensity as a function of Xem, indexed by i, and of Xex, indexed by j . Since a table of numbers is difficult to visualize, we use contour m a p representations of the EEM's, with the contours repre senting iso-intensity values. In Figure 1C and D, a horizontal slice (row of the matrix) represents a fluorescence spectrum, while a vertical slice (col umn of the matrix) represents an exci tation spectrum. Obviously, the EEM's for one-component systems are highly redundant; fortunately, this re dundancy may be expressed simply as follows:
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Figure 2. Optical diagram of video fluorometer A: Side view of excitation beam. B: Top view de tailing emission path to camera
cence analysis and discuss the specific limitations of each, as well as the gen eral limitations of simultaneous analy sis. Rationale A frequently cited advantage of flu orescence spectroscopy over other spectroscopic techniques is that the observed spectral intensity is a func tion of two wavelength variables, the excitation wavelength, \ex, and the emission wavelength, Xem· If Xex is held constant and \em is scanned, a fluores cence emission spectrum results; if Xem is held constant while λ οχ is scanned, a fluorescence excitation spectrum is obtained. For the large organic mole cules of interest here, the relative shape of the fluorescence emission spectrum is independent of the excita tion wavelength; conversely, the rela tive shape of the excitation spectrum is independent of the wavelength at which the emission is monitored. If the absorbance of the sample is low enough and the spectral variation of the excitation source is properly ac counted for, the excitation spectrum will closely resemble the absorption spectrum. Excitation-Emission Matrix. Fig ure 1A and Β illustrate the two types of fluorescence spectra for two related compounds, zinc octaethylporphin (ZnOEP) and free base octaethylpor phin (H9OEP), respectively. In Figure 748 A ·
M = axy which says t h a t the E E M (M) is the product of three factors, a wavelength independent factor α which contains all of the concentration dependent variables and the outer product of a vector χ which represents the relative fluorescence spectrum and a vector y which represents the excitation spec trum. When a mixture of two or more components is examined spectrally, the emission spectrum is no longer in dependent of the excitation wave length, and the excitation spectrum is no longer independent of the monitor ing wavelength. In this case, conven tional spectra such as those of Figure 1A and Β are not adequate to describe the fluorescence intensity behavior, and an E E M representation is neces sary. As an example, in Figure I F we illustrate the E E M for a mixture of H 2 O E P and ZnOEP. Figure I E shows an isometric three-dimensional projec tion of the E E M of Figure IF; here, the z-axis represents the emission in tensity. Careful examination of Figure I E and F shows how useful they are for the analysis of multicomponent fluorescence data. In particular, one immediately recognizes t h a t there are unique spectral regions where each component absorbs and emits inde pendently of the other. Other features of importance in fluorescence spec troscopy are also readily visualized. First, because of Stokes law, no emis sion is observed for Xom < Xcx; second, scattered exciting light, a major inter ference in turbid samples, is easily dis tinguished because it is constrained to lie on a straight line in the E E M where Xex = Xem; and third, the "mir ror-image" relationship between exci tation and emission spectra is also quite easy to see. Weber (10) was the first to point out how the E E M could be of great value in the complete characterization
ANALYTICAL CHEMISTRY, VOL. 49, NO. 8, JULY
1977
of a complex fluorescent system. T h e E E M has been used to separate the contributions from tryptophan and ty rosine to the fluorescence of total pro teins (77) and to elucidate the com plex nature of Chlorella fluorescence (72). More recently, the E E M has been found to provide useful finger prints of fuels and oils (13, 14). T h u s far, very little has been done to derive quantitative information from the E E M , although recent stud ies in our laboratory show this is quite feasible. If we suppose t h a t the sample contains r components, then it is easy to show t h a t the E E M has the fol lowing form:
M = Σ.
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i.e., the observed fluorescence is sim ply a linear sum of the individual com ponents' fluorescence, provided that the absorbances are low and t h a t no energy transfer takes place between components. In the case where each component is a known substance and one has a set of calibrated matrices for each, it is straightforward to use an optimization routine such as a leastsquares fitting to determine the amounts of the components. Warner (7.5) has determined three-component mixtures experimentally by this tech nique; computer simulations of polycyclic aromatic hydrocarbon (PAH) mixtures show t h a t determination of nine components is quite feasible (16). Even in the case where the compo nents are unknown, it is possible to make some progress. In 1961 Weber (10) showed that the rank of the ma trix M was a lower bound on the num ber of independently emitting compo nents in the solution. For the case of two unknowns, Warner et al. (17) have shown that spectral information can also be obtained. Briefly, the eigenvec tors of the real symmetric square ma trices, MM' and M'M are determined; these are then simultaneously trans formed to spectral vectors by taking linear combinations of the eigenvec tors so that all elements are positive. T h e spectra of severely overlapping combinations of substances can then be determined in this way. In most cases, at least one unambiguous exci tation or emission spectrum is ob tained for each component. Acquiring EEM. Clearly then, it would be desirable to have the capa bility of routinely obtaining an emis sion-excitation matrix for many types of fluorescence analyses, especially where background or impurity fluo rescence is likely, or where it is desired to determine multiple components si multaneously. Unfortunately, with currently available spectrofluorometric instrumentation, acquisition of an E E M can be time consuming and tedi-
ous. Suppose, for example, one wanted to use a conventional instrument to obtain the E E M shown in Figure I E spanning 200 nm at a resolution of 4 nm; this would mean t h a t 2 500 data points would be required. If one al lowed a rate of 1 s/datum, it would take almost three-quarters of an hour to accumulate the necessary data, clearly unacceptable for routine analy sis. What then can be done to speed up the data acquisition rate? One obvious possibility is simply to scan faster, using high-speed stepping motors (78) or galvanometrically scanned mirrors (19). This scheme would be impracti cal in most cases because the signalto-noise ratio (S/N) in fluorescence measurements is usually determined by the photon statistical noise. T h u s , with a conventional fluorometer, rapid scanning could only be done at the ex pense of the S/N and would therefore be limited to the most brightly lumi nescent samples. Another possible approach would employ either H a d a m a r d transform spectrometers (20) or interferometers to obtain the fluorescence data in "multiplex" form. This approach to rapid scanning spectroscopy has been the subject of considerable interest in recent literature (21-23). Both inter ferometers and Hadamard spectrome ters offer the well-known Felgett's ad vantage, in t h a t all wavelengths are observed simultaneously. T h e Michelson interferometer also possesses the Jacquinot or throughput advantage, since the entrance aperture does not have to be restricted to achieve a spe cific resolution. Unfortunately, the Felgett advantage is only achieved in cases where the spectrometer is detec tor noise limited—certainly not the case with UV-VIS spectrometers. T h e application of these systems to molec ular fluorescence also suffers a multi plex disadvantage. This disadvantage arises from the fact t h a t since weakly emitting components are added to gether with strongly emitting compo nents, the weakly emitting ones may be buried in the noise of the strongly emitting ones. Other disadvantages of multiplex techniques are the delicate mechanical elements involved and the need to transform the data before it can be received by the spectroscopist in familiar and meaningful form. T h e final approach, and the one which we have adopted, is to use an imaging detector as a multichannel photometer to observe all wavelength regions of the fluorescence at once. Talmi has reviewed the operational characteristics of available televisiontype detectors (24) and their applica tions to spectroscopy (25). He has con cluded that these devices can perform the function of a large array of photo-
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Figure 3. Production of EEM for hypothetical compound A: Hypothetical compound's excitation and emission spectra. B: Fluorescent cuvette as illuminated by polychromatic beam. C: EEM as viewed by SIT camera. D: Isometric projection of EEM as may be ob tained on oscilloscope or graphics terminal
multiplier tubes with comparable sen sitivity and considerably less bulk and cost. Several studies show t h a t the sil icon intensified target (SIT) vidicon with a UV fiber-optic faceplate offers a reasonable price/performance trade off. In their studies of atomic fluores cence, Busch and Morrison (26) have reported t h a t in the visible spectral re gion, an S I T vidicon has comparable per channel sensitivity to that of highperformance photomultiplier tubes. Westphal (27) has reported a dry icecooled S I T vidicon detector t h a t has the capability of single photoelectron detection and can accumulate photons for up to 5 h. He has developed cali bration procedures and software for producing photometrically correct im ages that, to our knowledge, are as ac curate and precise as any system using photomultipliers. Video Fluorometer T h e above discussion has served as our rationale for the development of a new fluorescence instrument, the video fluorometer (8, 9). This com puter-controlled instrument is capable
of automatically acquiring an E E M spanning up to 240 nm in emission and excitation wavelengths at a spa tial resolution of 1 nm per point in a time as short as 16.7 ms. This capabil ity was brought about by a novel scheme for polychromatic irradiation of the sample cuvette, and the use of an S I T vidicon detector to measure all regions of the E E M simultaneously. Polychromator. An optical di agram of the video fluorometer is given in Figure 2. T h e excitation source is the usual high-pressure xenon arc lamp. T h e excitation monochromator is mounted on its side so that the long axis of the entrance slit is horizontal. T h e exit slit is removed, and the emerging spectral continuum of exciting radiation is refocused onto the sample cuvette, as shown schema tically in Figure 3B. Figure 3B illus trates the appearance of the cuvette as if it contained a hypothetical com pound whose excitation spectrum is given by Figure 3A. Three bright bands of fluorescence appear along the cuvette where the three most strongly absorbed components of the excitation
ANALYTICAL CHEMISTRY, VOL. 49, NO. 8, JULY
1977 · 749 A
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continuum intersect the cuvette. Ob viously, scanning the length of the cu vette with a photodetector would pro duce an excitation spectrum of the sample. A lens at 90° to the excitation axis focuses an image of the fluorescent cu vette onto the analyzing monochromator, which is mounted in the usual up right position. T h e exit slit of the ana lyzing monochromator has also been removed. Present at the exit slit plane is an inverted image of t h a t part of the cuvette subtended by the entrance slit, but due to the presence of the grating, it is dispersed horizontally into its components of emission. T h u s , for the hypothetical component of Figure 3A, the exit plane image would be expected to resemble t h a t of Figure 3C. Each "excitation" band along the vertical direction is now dispersed into its three bands of emission along the horizontal direction. T h e effect of this novel scheme is to produce the E E M for the sample without the necessity of mechanical scanning. Video Detector. To quantitatively capture the fluorescent image it seemed natural to use a UV sensitive S I T vidicon interfaced to a minicom puter. Early in the design stages of the system, we decided t h a t there would be advantages to operating the camera at standard television rates: A flickerfree readout would maximize the ease with which the operator could set u p
the system, align the optics, and verify its operation; the system would be compatible with standard television devices such as conventional monitors, video tapes, and video disks. Thus, data could be recorded at remote in stallations and taken to the central system for processing. However, oper ation of the television camera at stan dard television rates makes extreme demands on the computer interface. A noninterlaced raster scan of 241 lines is completed in 16.7 ms (including re trace time). If one wished to measure 256 points along each line, then one picture element (pixel) intensity value would have to be digitized and stored in memory every 200 ns, a time far shorter than the read-modify-write cycle of most minicomputers. Interface. James Gladden of the University of Washington, Chemistry Dept. Electronics Shop has developed a unique video data acquisition sys tem (Figure 4) which overcomes the aforementioned obstacle a t a very rea sonable cost. In his scheme, the data from the video signal are stored in a 64K 8-bit byte buffer memory assem bled from eight standard 8K micro computer memory units t h a t use inex pensive IK static RAM chips. Control of the flow of data into and out of the buffer store is accomplished by means of the pixel processor which contains a high-speed arithmetic unit. T h e pixel processor can add succes
750 A · ANALYTICAL CHEMISTRY, VOL. 49, NO. 8, JULY 1977
sive frames to memory for improve ment of the S/N and subtract succes sive frames for the purpose of blank correction. Also, adjacent pixels can be added together for smoothing the data and increasing the dynamic range by trading off spectral resolution. For example, the operator can specify an image to be stored as a 64 X 64 matrix with pixel intensity resolution of 32 bits, or as a 256 X 256 matrix of 8-bit pixels. T h e processor does arithmetic operations on 8 bytes from memory and 8 bytes from the current video sig nal in parallel. Since the required eight bytes from memory are taken from the eight 8K RAM units in paral lel, the read-write-modify cycle time of the individual static RAM chips is not exceeded. After completion of the arithmetic operation, the modified pixels are redeposited in memory and simultaneously converted to an analog video signal, so that the operator can view the data acquisition process in real time. In addition to summation as a method of S/N improvement, the processor can allow the signal to accu mulate directly on the target of the vidicon by blanking the scanning beam for a preselected interval. Accu mulating signal directly on the target has advantages for very low light lev els because there appears to be a pro nounced threshold effect for detection of the video signal, below which the detection efficiency is very low.
Signal accumulation may be carried on until a preselected number of frames is acquired or until the most intense pixel exceeds an operator se lected threshold. On command from the operator, the buffer memory is mapped onto the unibus and may then be treated as ordinary memory by the PDP11/04 CPU. T h e data can be stored on floppy disk as a named bina ry file or transmitted to the remote CDC 6400 campus computer for anal ysis by a package of Fortran coded programs called SIM-1 (spectral inter pretation model, version 1). SIM-1 performs various data reduction algo rithms such as eigen analysis and least-squares fitting as discussed above. Programs to allow simplified data reduction and local graphical dis play are currently under development. Performance of Video Fluorometer. Preliminary results obtained with the video fluorometer in its present as well as initial forms illus trate its potential as a useful tech nique and also some problems to be overcome. T h e first question to be an swered concerns the spectral resolu tion. Using a medium-pressure mercu ry arc source and a scattering plate in place of the sample cuvette, we pro duced a linear scattered light pattern in which the 404-407- and 576579-nm mercury lines were easily re solved, even when the monochromators were adjusted to place the lines at the extreme edges of the field of view of the television camera. More critical studies with hollow cathode lamps as spectral sources revealed t h a t the axial and tangential focus points of the monochromator are somewhat dif ferent; there is noticeable chromatic aberration due to the use of simple spherical lenses; and there is vignett ing in the monochromators due to the inadequate size of the turning mirrors, which results in the response of the system being greatest at the optical center. Solutions to all of these prob lems fortunately should be quite straightforward using the newly devel oped concave holographically ruled gratings (28). We next studied the sensitivity and linearity of the system. Figure 5 dem onstrates the video fluorometer's sig nal enhancement capabilities of direct target integration and summation in memory for a 1 0 - i i M sample of perylene. Linearity of response of the in strument spans four orders of magni tude from 10~ l u to 10~ 6 M perylene. T h e capabilities of the video fluo rometer for performing multicomponent analysis are illustrated in Figure 6, which shows the E E M of a mixture of the five aromatic hydrocarbons, pyrene, perylene, anthracene, fluoran thene, and chrysene. Quantitative analysis of this E E M by use of stan-
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Figure 5. Illustration of signal enhance ment by integration of signal in memory and on target of camera A: Single frame of 1.6 X 10~ 8 M perylene. B: In tegration in memory; top 8 bits of 24-bit pixel in tensity word generated by summing 4096 frames of 1.6 Χ 10~ β Μ perylene. C: Integration on cam era's target for 15 frames, then summed to mem ory and repeated until preset intensity value has been reached
dard EEM's for each component dem onstrated quantification within 10%, an entirely satisfactory performance. Comparison with Other Multicomponent Systems T h e preliminary results reported here show the video fluorometer to be a potentially useful system for multicomponent analysis. One way to ob tain a realistic assessment of its capa bilities is to make a comparison with
7 5 2 A · ANALYTICAL CHEMISTRY, VOL. 4 9 , NO. 8, JULY 1977
existing fluorescence systems for mul ticomponent analysis. Shpol'skii Effect. A very straight forward approach t h a t has been wide ly used for polycyclic aromatic hydro carbons (PAH) employs the fact t h a t at temperatures below 80 K, many PAH compounds exhibit very narrow band (quasi-line) emission spectra (Shpol'skii effect) (29) when dissolved in n-alkane solvents or codeposited on a cold window with an inert gas such as argon (matrix isolation) (30, 31). T h e spacings of the sharp vibronic lines of the emission spectra are well correlated with vibrational frequen cies of the ground state as measured by infrared and Raman spectroscopy. T h u s , the low-temperature fluores cence spectra provide a unique finger print for identification and quantifica tion. T h e most comprehensive studies using the Shpol'skii effect have been reported by Kirkbright and coworkers (32, 33). Using synthetic mixtures, qualitative identification of up to eight components was performed. Quantitative studies proved more dif ficult, and a combination of a stan dard addition procedure and the use of an internal standard were required to ensure sufficient accuracy and pre cision. In many cases, interferences were noted. For example, 3,4- and 1,2-benzo(a)pyrene, dibenz(a)anthracene and perylene all interfered with the determination of the isomers of dibenzopyrene. T h e problems with Shpol'skii techniques are: N o t all compounds give Shpol'skii spectra; the spectra are not reproducible, and thus quantitative analysis is difficult; because of the extremely opaque na ture of the matrices, scattered light is a difficult problem; and because of the continuum spectra underlying the sharp lines, it is impossible to selec tively excite only one component. T h e use of the video fluorometer to obtain the E E M ' s for compounds in Shpol 'skii matrices would obviously be of great benefit, since greater resolution would be obtained. Derivative Spectroscopy. O'Haver and his coworkers (34, 35) have been exploring techniques for multicompo nent fluorescence involving derivative spectroscopy. T h e y found t h a t deriva tive spectra may be produced elec tronically or by wavelength modula tion with synchronous detection (34). Although both methods gave equal signal-to-noise ratios, the former was easier to implement. As has been noted for absorption spectroscopy, the derivative spectrum was found to be useful as a qualitative technique be cause of its capability of enhancing minor spectral features. As a quantita tive technique, derivative spectrosco py also showed promise. Green and
O'Haver (34) demonstrated that de termination of pyrene in the presence of excess anthracene was greatly im proved by use of the second derivative spectrum. A related but more powerful tech nique, called selective modulation, has also been introduced by O'Haver and Parks (35). Here, the excitation mono chromator is wavelength modulated as the emission monochromator is scanned. The resulting ac photocurrent is detected with a lock-in amplifi er. The central excitation wavelength is chosen so that wavelength modula tion will cross a maximum or mini mum in the excitation spectrum of the first component, while the modulation crosses a sloping portion of the second component's excitation spectrum. In this case, the fluorescence intensity of the first component will be modulated at twice the fundamental frequency, while the emission of the second com ponent will be modulated exclusively at the fundamental frequency. Thus, scanning the emission monochromator with the detection system locked onto the proper frequency produces the emission spectrum of the desired com ponent exclusively. The use of the two wavelength parameters in a derivative scheme can be quite useful, since a fingerprint spectrum can be deter
mined. The importance of this point was illustrated by IR analysis of three components. The major difficulty with derivative and modulation techniques is that they must find spectral regions where the derivative forms of the spectra for each component satisfy specific crite ria with regard to the form of the spec tral overlap and S/N, and thus the ap plicability is rather limited. In con trast, our approach, which simply uses the linear additivity of the spectra of the individual components, is always valid and is limited by the more fun damental considerations of signal-tonoise ratio of the measurement. Also, our system has the potential to com pute derivative, difference, and modu lation spectra in both wavelength di mensions simultaneously. Synchronous Excitation Tech nique. Lloyd (36) has introduced a novel technique for qualitative and quantitative analysis of mixtures, called the synchronous excitation technique. The synchronous spectra are obtained by locking the excitation and emission monochromator drives together at a fixed wavelength inter val, usually on the order of 20 nm, and then scanning them over a fixed inter val. The resulting spectrum usually contains only a few peaks which then
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The SYSTEMS are available with various electronics packages, PMT's, monochromator, sample chamber, excitation source and power supplies. The modular construction lends itself to numerous options which can be added as needed. Computer software program for deconvolution is also available PRA offers complete service with these systems, installation, on-site testing and training in use of the instrument. PRA, with its expertise gained through five years of research in designing these systems, will be happy to consult with you as to your requirements. These SYSTEMS can be seen at the ACS Exhibit in Chicago from August 29-September 2 at Booth 107. For further information, please contact:
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754 A · ANALYTICAL CHEMISTRY, VOL. 49, NO. 8, JULY 1977
Perylene
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Figure 6. EEM of mixture of anthracene, perylene, pyrene, fluoranthene, and chrysene
form a unique fingerprint for each compound. This technique has been applied to the identification and quantification of crude oils (37) and their partially fractionated compo nents. The synchronous scanning protocol is easily visualized on the EEM; it rep resents the fluorescence intensity measured along a line defined by \em = λ,.χ + Λ, where Δ is the number of wavelength units difference in the set tings of the monochromators. Essen tially, the synchronous scan concept simultaneously takes advantage of variability of both the excitation and emission spectra of fluorescent species. However, although this tech nique works well in cases where welldefined vibronic bands exist, as in the example mixture of Figure 1, it is blind to other regions of the EEM where better separation might be ob tained. Time-Resolved Spectroscopy. A potentially very powerful technique in multicomponent luminescence uses the fact that the lifetime of an excited state can be a useful distinguishing feature. Most of the effort here has been in phosphorescence lifetimes be cause the lifetimes of triplet states (10 μβ to 10 s) are much longer than those of excited singlet (fluorescent) states (100 ps to 500 ns). Instrumentation for time-resolved phosphorescence has been described by Callis et al. (38), St. John and
Winef'ordner (39), and more recently by Wilson a n d Miller (40). Generally, a pulsed radiation source is used for excitation, and the time dependence of t h e intensity is measured. It is also possible to gate the detector so t h a t a spectrum in a narrow-time window is obtained. Or, a complete set of spectral intensities as a function of time of the excitation may be obtained and displayed in a three-dimensional format. An alternative approach uses a sinusoidal modulation of the radiation source and phase sensitive detection to selectively enhance various components (41). Time-resolved phosphorescence spectroscopy has been used for q u a n t i t a t i v e studies of illicit drugs (42) and pesticides (43). Very recently, nanosecond fluorescence spectroscopy has received serious considerations from analytical chemists. T h e time-correlated single photon technique is described by Cline Love and Shaver (7), although no analytical applications are described. T h e advantages of time-resolved spectroscopy are numerous: Scattered light can be eliminated by a p p r o p r i a t e gating of the detector; weakly emitting long-lived c o m p o n e n t s whose spectra are buried in the photon noise of
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short-lived components can be distinguished; and components that are very similar spectrally often display different lifetimes and can thus be distinguished. The major disadvantage of using phosphorescence for time-resolved studies is the requirement that the sample be cooled, although in some cases, this may be circumvented (43). Fluorescence decay times as measured by the time-to-amplitude conversion method are frequently time consuming and tedious to perform when the luminescence is weak and/or spectral scans are to be performed. Time-resolved phosphorescence can be performed with the video fluorometer, if a shuttered or pulsed radiation source is available. A succession of EEM's with a time resolution of 16.7 ms may be taken following pulsed excitation, and placed onto video tape. These may then be read back into the computer and analyzed. For shorter decay times, the image intensifier section may be gated on for periods as short as 50 ns. Such data would provide three dimensions of resolution simultaneously and might prove exceptionally powerful. Extension to fluorescence lifetimes must await development of a polychromatic
Packed with performance, Titralyzer II expedites and improves your analyses. It features: fully proportional endpoint; interchangeable "quick-fit" buret; expanded-scale electrometer for difficult or exotic applications; pH/mV printout for vital information before&-after titrations. Plus calibrated slope control; positive sample identification; automatic temperature compensation; and continuous LED display of rate of titrant delivered. Optional recorder or titration computer.
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756 A · ANALYTICAL CHEMISTRY, VOL. 49, NO. 8, JULY 1977
laser and a nanosecond shutterable image converter, and both seem perfectly plausible. Ultimate Limitations in Multicomponent Fluorescence Analysis
The most fundamental limitations in simultaneous fluorescence detection arise from the photon statistical noise. If two spectra overlap, the weakly emitting component's intensity must be greater than the photon noise of the strongly emitting components at the wavelength where the weak one is to be detected. For techniques that separate the components and analyze them sequentially, the most fundamental limitation is simply the dynamic range of the detector; for photomultiplier tubes this can be six orders of magnitude. The dynamic range of the video fluorometer's memory is only four orders of magnitude. Another limitation for multicomponent analysis is that the total absorbances of solutions must be less than 0.01 in all spectral regions. As more and more components occur in the solution, the chance for spectral overlap increases; therefore, the maximum allowed concentrations of individual components must be lowered, iiventu-
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ally, t h e allowed c o n c e n t r a t i o n s m a y become so low t h a t t h e y can n o longer be d e t e c t e d . Although one could relax t h e concept of linear additivity, t h e analysis of t h e d a t a would become m u c h more difficult, a n d iterative m e t h o d s of solution would have to be employed a t t h e expense of consider able complexity in t h e algorithms. T h e final limitation we m u s t con sider is one in c o m m o n with o t h e r flu orescence analyses. T h e r e m u s t be nothing in t h e solution which can q u e n c h t h e luminescence. F o r t u n a t e ly, t h e r e q u i r e m e n t of low absorbances usually is a d e q u a t e t o e n s u r e t h a t nei t h e r resonance energy transfer nor sig nificant collisional q u e n c h i n g will occur. However, t h e presence of nonfluorescent s u b s t a n c e s in t h e solution, such as trace a m o u n t s of heavy m e t als, could cause interference. Conclusions P r e l i m i n a r y d a t a show t h e video fluorometer t o have considerable promise for improving t h e . m u l t i c o m p o n e n t capabilities of fluorescence as says. O n e of its major limitations re sults from t h e n o n o p t i m a l geometry of excitation; t h u s , it is not as sensitive as conventional filter fluorometers when c o m p a r e d on a single m e a s u r e m e n t basis. Since m a n y fluorescence assays are limited by fluorescence in terference a n d n o t by t h e signal-tonoise ratio, this m a y be a worthwhile trade-off. A n o t h e r major d i s a d v a n t a g e of t h e use of a m u l t i c h a n n e l d e t e c t o r is t h a t all of t h e resolution e l e m e n t s m u s t be calibrated individually. T h i s can be a formidable job if t h e fluores cence d a t a are to be absolute, since each resolution e l e m e n t has its own u n i q u e wavelength d e p e n d e n t re sponse curve. In m o s t cases of r o u t i n e analysis, absolute spectroscopy is hardly needed, a n d calibration curves can be easily c o n s t r u c t e d from known c o n c e n t r a t i o n s of t h e sought-for s u b stances, a t a few optimized wave lengths. J u s t because t h e system mindlessly p u t s o u t huge a m o u n t s of d a t a does n o t imply t h a t use m u s t be m a d e of all of t h e m . A few o p t i m a l m a t r i x locations m a y suffice for cer tain t y p e s of analyses. A final disad vantage of t h e i n s t r u m e n t is its r a t h e r high cost (currently a b o u t $40 000). However, t h e recent a d v a n c e s in mi c r o c o m p u t e r technology a n d in solid s t a t e imaging devices also give cause for o p t i m i s m in this regard.
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References (1 ) C. A. Parker, "Photoluminescence of Solutions with Applications to Photo chemistry and Analytical Chemistry", American Elsevier, New York, N.Y., 1968. (2) G. G. Guilbault, "Practical Fluores cence: Theory Methods and Tech niques", Marcel Dekker, New York, N.Y., 1973.
The authors: from left to right, David W. Johnson, PhD degree candidate; James B. Callis, research assistant professor of pathology; and Gary D. Chris tian, professor of chemistry ANALYTICAL CHEMISTRY, VOL. 49, NO. 8, JULY 1977 · 757 A
