lsiah M. Warner Gabor Paionay Mark P. Thomas Deoartment of Chemistrv
Analytical measurement techniques that use more than one parameter for a single measurement can be termed multiparameter techniques. Thus, the measurement of luminescence is inherently a multiparameter technique because even the simplest such measurements involve the exploitation of more than one parameter. For example, the measurement of the luminescence (phosphorescence or fluorescence) from a sample involves the simultaneous use of an absorption wavelength and a luminescence wavelength since a molecule must absorb energy at wavelength A, and be promoted to an excited state before luminescing at wavelength &. The meacan, sured luminescence intensity, l ~ then be represented as a function of hex and hem IL = f ( L , L A (1) Selective measurement of individual luminophores in simple (1.2) or even complex mixtures ( 3 , 4 ) can be achieved by varying these two parameters; a number of examples were detailed as early as 1969 i n a review article by Sawicki (4). Another parameter often used in luminescence measurement is the lifetime of the luminophore. The luminescence decay after initial excitation will generally follow first-order kinetics. The time for the decay M reach l/e of its initial value is a characteristic parameter of the molecule. Thus, the use of this parameter in combination with Equation 1 provides increased selectivity. A number of representative examples in which the decay time of luminescing swcies is used to achieve additionalieiectivity can be cited (5-7). 0003-2700/85/0357-463A$01.50/0 @ 1985 American Chemical Society
In recent years, novel advances in instrumentation (8)and data reduction strategies (9)have encouraged the simultaneous exploitation of multiple parameters to achieve selective measurements. Consequently, the selectivity of luminescence measurements can be enhanced through both
instrumental and mathematical approaches. Figure 1is a diagrammatic representation of several luminescence parameters that can be used for selective luminescence measurements. Clearly, many parameters of luminescence can be simultaneously exploited to achieve
Figure 1. Examples of luminescence parameters that can be exploited to achieve selective measurement ANALYTICAL CHEMISTRY, VOL. 57, NO. 3. MARCH 1985
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Flwn 2. Contour plot of an ernission-excitatlon mblx of 8.3 X cene in cyclohexane Data w e acqulrsd in 0 . 6 ~ ~a4 1 v wn
a high degree of selective measurement and, ultimately, speeific measurement. One obvious approach to additional selectivity is to obtain 1, in terms of three parameters
LLTECH
PLIED SCIENCE
1, = f ( L . L m P ) (2) where P is an arbitrary parameter selected from among the many listed in Figure 1. Furthermore, more than three variables could be used in Equation 2 to increase the selectivity of the measurement. A number of other parameters could a h he included (IO)in Figure 1,particularly under the miscellaneous category. However, we have necessarily limited our diagram to include widely used parameters and those that are increasingly being used for selective measurement. It should also be noted that some of the parameters listed in the miscellaneous category are really not characteristic parameters of the molecule in the excited state. However, these characteristic properties of the molecule cnn he combined with excited-state information to increase selectivity. This article discusses multidimensional luminescence measurement (MLM) and ita applications in analytical chemistry. The MLM approach involves the simultaneoususe of two or more parameters of luminescence for selective measurement. We term this approach multidimensional rather than multiparametric since, in this article, we will primarily emphasize data
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acquisition, display, and processing in a multidimensional format. There are a number of advantagesfor reduction of data using a multidimensional format (9).An example of such an approach was presented in a recent Apage article by McCown and Bright (II ) in which phase-resolved fluorescence intensity is derived as a function of synchronously scanned wavelengths and of detector phase angle. Our discussion in thin article will be divided into the four major classifications of spectral measurements, lifetime measurements, polarization measurements, and miscellaneous approaches, as depicted in Figure 1.The utility of MLM will he demonstrated using one or two examples in each classification.
Speclral measurements Theory. The discussion provided here is necessarily brief; either of two useful monographs (12,13)can he consulted for detailed discussions on the general theory of luminescence. Luminescence measurements have had widespread applications in the analytical studies of large, unsaturated, highly conjugated, organic molecules such as polynuclear aromatic compounds (PNks). Organic molecules generally have an even number of electrons. Therefore. the absyption and fluorescence transitions occur between singlet states. Phosphorescence generally involves an intersystem crossing to a triplet
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length. A similar conclusion can be drawn about variations in the excitation spectra with emission wavelength. In fact, the number of changes noted in the emission or excitation profile is an indication of the minimum number of luminescing species (rank) contrihuting to the matrix. Instrumentation. The utility of obtaining luminescence spectra as a function of multiple excitation and multiple emission wavelengths encouraged some researchers to digitize and generate such information by manual manipulation of data from luminescence spectrometers (16).Such studies demonstrated the need for an automated luminescence spectrometer. An early automated luminescence spectrometer was developed and described by Wampler and DeSa in 1971 ( 1 7). The data were digitized and could be output in a suitable form for postprocessing. An automated iustmment was also described by O'Haver et al. in 1973 (18).However, this instrument acquired data in analog form for plotting on an x-y recorder. Consequently, the data were not in a suitable form for postprocessing. In the same year, Holland et al. (19)described a completely automated system capable of simultaneous ahsorption and fluorescence measurements. This system was interfaced to a dedicated digital computer for data processing. Perhaps the most completely automated luminescence spectrometer was described by Haugen et al. in 1975 (20).This spectrometer was interfaced to a general-purpose computer and operated in a time-share mode. Six modes of operation were possible for data acquisition, and the data could be stored on magnetic tape for further processing. Commercially available instrumentation capable of similar operations has been reported. Despite the high degree of automation in the instrumentation described in the preceding paragraph, the acqui-
Figure 3. The contour plot of an €EM of an approximalely eqJillLorescen1 six-component mixture of the PNAs benzo[k]fluoranlhrene. 2.3-benzanthracene. lluoranthrene. perylene, 2-ethylanthracene. and pyrene in cyclohexane Dam were acquied in 0.6 s an a video litommeler (299)m a 64 X 64 lama1
state with subsequent radiative decay to the ground singlet state. Since this transition is quantum mechanically forbidden (long-lived), and therefore more subject to nonradiative decay processes in fluid solutions, phosphorescence is usually observed under cryogenic conditions. Some recent studies have provided useful approaches to room-temperature phosphorescence measurements (14). Two useful properties of most luminescing molecules provide the selectivity of Equation 1descrihed earlier. The first property is that for most pure luminophores, the observed emission spectrum is independent of the excitation wavelength. The second property is that the excitation spectrum is independent of the monitored emission wavelength. Thus, variations in the emission profile with variations in the excitation wavelength suggest that more than one luminescing component is present in a sample. A similar conclusion could he drawn from variations in the excitation spectrum with monitored emission wavelengths. Many early analytical investigators of luminescence measurements varied the excitation and monitored luminescence wavelengths to achieve selectivity (2,4).However, for complex molecules with broad-band absorption and emission spectra, the selectivity of this approach decreases rapidly as the number of components increases. Optimum selectivity is likely to occur with narrow-band excitation and with 4 6 6 1 * ANALYTICAL CHEMISTRY,
an emission resolution better than the bandwidth of the monitored components. In addition, the number of excitation and/or emission wavelengths used should equal or exceed the numher of emitting components 115). Figure 2, which illustrates the preceding point, is a contour plot of a typical one-component fluorescenceexcitation matrix. Note that, within the defined contour levels, this pure compound adheres to the two properties of excitation and emission previously discussed. A more complex, sixcomponent mixture is shown in Figure 3. It is visually ohvious that this is a multicomponent system because the emission profile changes several times with variations in the excitation wave-
Table 1. Chronology of developments in luminescence spectrometers imrumi ( Y e w noted in IHW.Iun) (a)
Tuner fluorometer (1964) Mluofluorometer(1974)
Computerized fluorometer (1975)
Video fluorometer (1975) Commercial fluorometer (1979)
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(a) These are selected examples from Ihe literalure. Omer examples were published around the
same lime.
(b) Most of Mse times are estimates from Uw inlamalion presented in the referenqedarticles. In general. &e times do rot incluat digitizalion, slorage. and processing lime.
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sition of luminescence data as a function of multiple excitation and multiple emission wavelengths was a timeconsuming process. Table I provides a chronology of the improvements in the data acquisition time of luminescence data. Note that a significant decrease in data acquisition time was not realized until around 1974, and in 1975 when a video fluorometer was developed at the Univ. of Washington (23). The video fluorometer’s decrease in data acquisition time was due to a novel concept of polychromatic illumination coupled with the rapid twodimensional data acquisition capabilities of an intensified vidicon detector (24).As is often the case with such developments, some trade-offs had to be made. In this case, the video fluorometer is less sensitive than conventional luminescence instrumentation. However, the sensitivity of the instrumentation could be increased by using the integrating capabilities of the vidicon detector with a concomitant sacrifice in rapid data acquisition. A video fluorometer similar to the one developed at the University of Washington has also been described (25, 26). The incorporation of shutters and cryogenic equipment for low-temperature operation has also allowed this instrumentation to be used for phosphorescence studies (28,29).
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Lifetime measurements Theory. As mentioned earlier, the luminescence lifetime is another parameter that can be used for multiparametric evaluation of the luminescence information. This can be an especially important property of the luminescing molecules if there is a significant overlap in the excitation and emission bands. In such cases, the lifetime of the luminophore may be a distinguishing factor. The luminescence lifetime is the time required for the luminescence intensity to decay to l l e of its initial value ( E ) Hence, . the luminescence intensity can be obtained as a function of monitored wavelength of emission and the observation time after termination of the excitation pulse, ti,
IL = f ( A e m , t i )
(
A complete mathematical descriptio] of the luminescence intensity would require an equation in the form of Equation 2
IL
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Practical considerations determine the approach to acquiring data in the form of Equations 3 and 4. For instance, two parameters can be fixed and the third one varied (30).Obviously, the specific instrumentation will determine the necessary acquisiCIRCLE 188 ON READER SERVICE CARD
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Figure 4. Typical time-reaoived ETM of 2-naphlhoic acid and pamino-benzoicacid Repinled horn ReIerwm a1
tion time and data format. Since several methods of determining the luminescence lifetime can he used, it is useful at this point to discuas briefly the theory of the two commonly used techniques (22).These methods are based on the use of two types of instrumentation: time resolved and phase resolved. In the time-resolved method, the sample is excited with a short-duration pulse of light, and the time-dependent decay of the luminescence intensity can be used to calculate the lifetime. In the phase-resolved method,the sample is excited with sinusoidally modulated light. The phase shift and demodulation of the emission, relative to the incident light, are parameters that can he used for calculation of the lifetime. Both methods require some calculations to obtain the luminescence lifetime of the luminophore. This is especially true for the second method if the sample contains more than one luminophore. In general, the excitation of a luminophore with an infinitely short pulse of light (6 function) provides the best approach to explaining the luminescence decay. The luminescence intensity as a function of time after the excitation pulse is assumed to he an exponential decay curve since the luminescence decay process usually obeys first-rate kinetics
Equation 6 indicates that a timeluminescence decay curve can be obtained if an ideal delta pulse excitation is used. Unfortunately, any practical instrument is nonideal and can provide only an excitation pulse of finite duration. In addition, the associated electronics will have a response function of finitewidth. Consequently, the use of a nonideal lifetime instmment will produce data that are a convolution of several response characteristics. A mathematical process is therefore needed to deconvolute the time decay curve. With an appropriate modulation frequencyfor the excitation beam,the luminermnce will also have an assoeiated modulation depending on the time delay. Thii property is used in phase-resolved luminescence measurements. The phase-yolved method uses a continuous sinusoidally modulated excitation beam.If the modulation frequency V, and degree of modulation of the excitation source are known, a simple derivation (12) of the phase difference, 6, between the excitation source and the fluorescence gives the lifetime, r, an 1 .r=-(tand ZXf
..
(6)
Similarly, the observation of the demodulation (M) is also suitable for calculation of the lifetime since
ip MZ
r = 2?rf wherelL(t) andlL(0) aretheintensities of the luminescence at time t and time 0, respectively. The parameter k is the first-order rate constant for the decay process.
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(7)
Quations 6 and 7 are valid if the emission of the luminophore is spectrally homogeneous. If the system is more complex, the calculation of the lifetimes is more complicated. ANALYTICAL CHEMISTRY, VOL. 57, NO. 3, MARCH 1985
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It should be apparent that each lifetime method has certain advantages and disadvantages. The selection of the proper method w i l l depend on several variables including the nature of samples, deviation of decay from ideality, and lifetime range desired (12). For this discussion, namely, use in multidimensional luminescence measurements, the format of the data must first be examined. Several choices in multiparametric representation of the luminescence data are available depending on the complexity of the data set. If one desires a two-dimensional format, the data can be acquired using either the emission spectra or the excitation spectra as a function of luminescence decay time while fixing the other parameter. In both cases, a two-dimensional data matrix is formed (30,31). The intensity data can be represented as an (rn X n ) matrix where rn is the number of wavelengths observed in each excitation or emission spectrum and n is the number of time intervals at which spectra are obtained. The time difference between the two excitation or emission measurements greatly depends on the lifetime of the luminophore in the mixture. Typically, this value may range from -0.1 ns (fluorescence)to the order of ms or s (phosphorescence). Obviously, the multidimensional representation can also he extended according to Equation 4. Here, the intensity data are represented as an (m X n X 0 ) matrix where m is the number of excitation wavelengths, n is the number of emission wavelengths, and o is the number of time intervals at
which spectra are obtained. The advantages of this approach are clear since it is very unlikely that two components will have the same lifetime as well as the same excitation and emission spectra. Instrumentation. Because of the short lifetime of most fluorophores, phosphorescence lifetime techniques were developed much earlier than fluorescence lifetime techniques. A pulse source phosphorescence lifetime technique was described in 1974 (32).A complete two-dimensional time-resolved technique, according to Equation 3, was reported as early as 1975 (33).The method was fairly tedious and complicated due to mechanical control of the emission monochromator. However, this instrument was able to acquire data in an emission-time matrix (ETM) format. A more sophisticated time-resolved phosphorescence spectrometer was developed by Goeringer and Pardue in 1979 (31).A silicon intensified target (SIT) vidicon was used to collect the data in the ETM format (Figure 4). However, this instrumentation did not exploit the excitation information because the sample was irradiated with undispersed light. A complete timeresolved technique was reported in 1982 (29).Since this instrumentation incorporates a polychromator to disperse the excitation source, a complete phosphorescence-excitation-time matrix (PETM) is obtained. Although the representation of such data in a four-dimensional format is difficult because of the number of variables (Figure 5 ) , it should be clear that this format provides the greatest informa-
tion. The development of comparable fluorescence instrumentation would require much more sophisticated techniques because of the much shorter time intervals (nanoseconds). Although a great deal of work has been done in that field (34).the unavailability of complete multidimensional fast-scanning instrumentation similar to that reported by Ho and Warner (29)is understandable because of the lack of superfast (response time in the order of ns) SIT vidicons. However, there are several methods available that are able to obtain the data in a fluorescence-excitation-time matrix (FETM) (33,35).It is interesting to note that the ultrafast, picosecond spectroscopic techniques belong to the general field of multiparametric measurement. Although these methods have been used for studies of ultrafast physical and chemical processes, the use of streak cameras (36)should also be applicable to ultrafast multidimensional fluorescence measurements. The phase-resolved approach differs significantly in implementation. Because the data are not easily represented in an ETM or FETM format, a different approach to multidimensional luminescence measurement is necessary. A good example of this is the use of synchronously scanned wavelengths as a function of phase angle (11).
Polarization measurements Theory. When a luminophore is excited with linearly polarized light, those molecules with their absorption dipoles aligned with the electric vector
L .Mure5. Time-resolved PMof a binary mixture of phenanthrene and triphenylene. (a)t = 0 s;(b) t = 5.5 s;(c) t = 12.5 s;(d) t = 17.5 8 Reprinted tan R&~O 2s 472A
ANALYTICAL CHEMISTRY. VOL. 57, NO. 3, MARCH I985
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Figure 6. Block diagram of fluorescencedetected circular dichroism spectrometer
of the polarized light have the highest probability of excitation. The rules of photoselection dictate that the luminescence resulting from this absorption is also polarized. Because these molecules remain in the excited state for a finite time before they luminesce and because they are free to rotate during this time, some depolarization will occur. The amount of depolarization due to rotational diffusion is dependent on the size and shape of the molecule as well as solvent viscosity and is often used in biochemistry to investigate many important processes such as ligand binding, denaturation, and aggregation (12). In some cases energy transfer is a major contributor to depolarization, and luminescence polarization experiments can yield information on the excited states of the luminescing molecule (12). In luminescence polarization measurements, spectra are obtained by scanning wavelengths selected by an excitation monochromator and measuring the luminescence intensity parallel ( ) and perpendicular ( I) to the polarization of the exciting light. The degree of polarization is then given by
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I,,11,1111,,,11A 474A
I I I I I I I I I 1
where Ill and I L are, respectively, the intensities of the components of the emission parallel and perpendicular to the polarization plane of the excitation beam. In much of the luminescence literature, polarization data are reported in terms of anisotropy, which eases the theoretical derivation of model systems. The anisotropy is related to the polarization and is defined as
Circularly polarized light can also be used for molecular excitation. When light is passed through a doubly refracting crystal that has been oriented so that the light traverses the crys-
ANALYTICAL CHEMISTRY, VOL. 57. NO. 3, MARCH 1985
tal in a direction perpendicular to the optical axis, the ordinary and extraordinary rays are not separated but travel at different speeds. When the rays exit the crystal, they are out of phase, and the resulting beam of light is said to be elliptically polarized. The ellipticity is then given by the angle, 0, whose tangent is equal to the ratio of the minor axis to the major axis of the optical ellipse. If the crystal is of a thickness such that the rays .exit with a phase difference of r J 2 for a given frequency, the light is circularly polarized (37).In circular polarization, the electric vector does not oscillate in one plane but actually is constant in magnitude and describes a circular motion about the axis of propagation either clockwise (righthanded) or counterclockwise (lefthanded). In 1895, A. M. Cotton (38)discovered that chiral molecules differentially absorb left and right circularly polarized light. This discovery laid the foundation for the field of circular dichroism (CD). Experimentally, the CD spectrum of a sample is obtained by measuring the difference in the intensities of transmitted left and right circularly polarized light. Today, CD is an accepted technique for the study of chiral molecules and has been used most often in determining structural interactions between biomolecules (39). A new technique has been developed within the past 10 years that uses the Cottou effect to analyze chiral fluorescent molecules. Fluorescence detected circular dichroism (FDCD) is related to the classical CD method much as the fluorescence excitation spectrum is related to the absorption spectrum. Turner and Tinoco (40) first presented the concepts and preliminary experimental results of FDCD in 1974. This method is based on the assumption that the experimentally corrected excitation spectrum of a luminophore has the same profile as its absorption spectrum. This assumption is generally valid in
ce witn
1 I
G UR _ .and kinetics. Research grade performance, appl
ions
I-----
the absence of effects such as energy transfer. In FDCD, the differential absorption of left and right circularly polarized light is detected by the relative intensity differences of the fluorescence light. Thus, the FDCD spectrum is related to the CD spectrum since the intensity of fluorescence is proportional to the intensity of absorbed light a t low absorbance. The major advantages gained by FDCD are greater sensitivity and selectivity. Selectivity is gained because not every chromophore in a chiral mixture will fluoresce. This technique allows the potential of isolating a single chiral fluorophore in a mixture of chiral chromophores for determination of the structure at that particular location. A theoretical discussion of the FDCD technique has already been published (41). Early publications cited some problems in the theoretical and experimental techniques (42-44), but these were later resolved (45).The first application of FDCD demonstrated the validity of the technique and showed the method’s usefulness as a bioanalytical tool (46). Instrumentation. Conventional spectrometers can be used to measure luminescence polarization by adding linear polarizers in the excitation and emission beams of light. Several commercial instruments are available with polarization attachments. Polarization spectra must be corrected to account for a wavelength-dependent polarization response inherent in the detection system (monochromator, optics, detector). There are several methods to calculate the instrumental correction constant, the G factor. Techniques for determining the G factor and further details on the theory of luminescence polarization and depolarization are given elsewhere (12). Many early experiments in the field of FDCD were done on conventional CD instruments that were modified by placing a photomultiplier tube at a right angle to the excitation beam. Recently a new multidimensional instrument has been described that acquires ellipticity as a function of multiple excitation and emission wavelengths through the use of an intensified diode array combined with a polychromatic dispersion system (47).A block diagram of this instrument is given in Figure 6. The instrument acquires complete fluorescence spectra at an excitation wavelength by illuminating the sample with left circularly polarized light and then with right circularly polarized light. The FDCD in terms of ellipticity spectra is acquired as a difference in these two measurements. The data are temporarily stored in the memory of a microprocessor-based interface that also controls the excita476A
ANALYTICAL CHEMISTRY, VOL. 57, NO. 3, MARCH 1985
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&ipi$i e q m entrol (DBC) Quantitative Line Profiles Real-time, Pacicle Analysis and Chemical Characterization Stored Ima e Particle Analysis and Chemical daracterization Linewidth Measurement Image Stora e and Retrieval from SEh? from Microprobe from STEM from Video Camera Image Processing Image Mathematics Arithmetic Oper,ations Boolean Operations Logical Operations Two Dimensional Point Filters Smoothing Difference Transforms Log Gamma Histogram Equalized Pan and Zoom Image Display True Grey Scale Display Scanned Transmission Image Collection and Processing Perlormance. not promises . . . the main reason Tracor Northern is the world leader in x-ray microanalysis, with more advanced mstems installed than all our competitor's cbmbined
World Leader in X-ray Microanalysis Systems T W R YORnEIy 2551 VEST ELTUNE HlGHHlV MIDDLETON, WlSmNSlN 9562-2097 (Boll) W-8511
CIRCLE 203 ON READER SERVICE CARD
ANALYT!CAL CMMISmY, VOL. 57. NO. 3. MARCH 1985
477A
X-RAY MICROANALYSIS SYSTEM ~~
AN10030istheresultofacarefully planned development program from a company renowned for its innovative approach to systems engineering. Computer technology never beforeavailable in a standard system brings to the analyst a powerful yet
'Advanced technology computer hardware. 'Winchester disk as standard. ' High resolution colour display. *Comprehensive software with user program * Superior range o spectrometers. *Convenient packaging.
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THE X-RAY ANALYSER YOU'VE ALWAYS PROMISED YOURSELF mr,Lt LT ohi n t m t r
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