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Multidimensional

phosphorimetry

Phosphorescence analysis of multicomponent samples can be enhanced by the rapid acquisition of multiparametric data. A new approach to phosphorimetry provides a sensitive and selective analytical technique through the acquisition of phosphorescence data in the form of an emission4xcitation matrix.

C.-N. Ho and I. M. Warner Colkge Station, Texas, U.S.A. Luminescence phenomena have been known and applied to chemical analysis for several decades. Lewis and Kashar had, as early as 1944, correctly attributed phosphorescence to an emission of visible light when a molecule relaxed from a triplet state to the ground singlet state. However, it was not until 1957 that its usefulness was demonstrated by Keirs and coworkersz. Later, in 1962, Parker and Hatchards performed an extensive and critical study on the merits and limitations of phosphorimetry as an analytical tool and compared it to fluorimetry. Since then, phosphorescence spectroscopy has gradually assumed a complementary role to the better known and popular, technique of fluorescence spectroscopy.

Fluorescence and phosphorescence

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Simplified energy level diagram for an organic molecule. Singlet (S) and triplet (T) electronic levels are shown as heavy horizontal lines; lighter lines are vibrational levels. Continuous vertical lines indicate transitions involving absorption or emission of radiation and dotted lines indicate non-radiative transitions. A = absorbance; F = fluorescence; P = phosphorescence; (R)ISC = (reverse) intersystem crossing; VR/IC = vibrational relaxation/internal conversion.

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Several reasons can be cited as to why phosphorimetry has not been as widely used and as popular as fluorimetry. First, direct excitation of a molecule from a ground singlet to populate an excited triplet state is ‘spin-forbidden’, and in practice usually occurs through an ‘intersystem crossing’ process from the lowest excited singlet. Therefore, phosphorescence lifetimes are considerably longer than those for fluorescence and are very susceptible to quenching processes under normal experimental conditions. Hence, for a long time; phosphorimetry was performed only under cryogenic conditions at liquid nitrogen temperature (77°K). Because of this ‘forbidden’ transition, fewer compounds exhibit ,phosphorescence than fluorescence. This apparent disadvantage, however, does confer a selectivity advantage on phosphorimetry. To obtain phosphorescence measurements, a phosphoroscope attachment is usually needed on a conventional fluorimeter in order to delay signal acquisition so that only the phosphorescence signal is recorded. These requirements, and the necessity to carry out experiments at low temperatures, create difficulties for many workers and prevent the routine application of phosphorimetry. However, phosphorescence has been observed at room temperature by appropriate manipulation of the environment and matrix of the phosphor of interest*. Thus, when compounds are constrained on adsorbent supports5, filter papersa, and in micellar solutions7, phosphorescence is observed at room temperature. A monograph on conventional phosphorimetry by McCarthy and Winefordners has been published. A description of the instrumentation, types of phosphoroscope, choice of solvents, optimization procedure for maximum sensitivity (based on signalto-noise ratio considerations), different modes of measurements (such as time-resolved) and several applications are discussed in detail. Until recently, phosphorimetric instrumentation had not changed much. In addition to the phosphorescence emission spectra, time-resolved phosphorescence has also been a popular mode of measurement. The mechanical phosphoroscopes have gradually been replaced by pulsed sources and latterly by pulsed lasers. Recently, Boutillier and Winefordners reported the use of pulsed laser excitation and exploitation of the heavy atom effect to improve detection limits in their analysis of drugs by time-resolved phosphorimetry. 0 1982ElrcvicrScienrilirPublishing Company

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Wilson and Miller’0 reported the technique of simultaneous time and component-resolved phosphorimetry, where a complete decay curve was acquired for each wavelength of emission. Thus, a complete emission spectrum was obtained at regular time intervals along the entire decay curve of the phosphor. This technique greatly enhanced the multicomponent capability of phosphorimetry because a solution with a mixture of components whose emission spectra overlap greatly can be resolved through differences in lifetimes. This work is convincing evidence of the inherent ability of an analytical technique to observe a number of parameters and can and should be fully exploited for the analysis of complex samples. Goeringer and Pardue’ * improved this technique and devised a rapid scanning spectrometer (using a silicon intensified target vidicon array detector). Since the imaging detector can acquire the entire emission spectrum very rapidly, the period needed to obtain a complete time and component-resolved spectrum is immensely reduced. In the studies of Wilson and Millerie, where a pulsed laser source and a photomultiplier tube detector were used, the emission monochromator had to be mechanically scanned. Thus, the

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data acquisition process for a phosphor with a 1ifetir;le of 30 ms took ‘almost an hour.’ With their rapid scanning spectrometer and regression methods of data analysis, Goeringer and Pardue successfully analyzed up to three-component mixtures of salts of organic acids which exhibited room temperature phosphorescence on filter paper. This approach, where a rapid scanning spectrometer is used in combination with powerful data reduction schemes, represents a major step towards achieving the rapid acquisition of a multiparametric phosphorescence database suitable for higher order data manipulation methodologies.

The phosphorescence emission-excitation matrix An improved fluorescence instrument, which minimizes the need for cumbersome, extensive sample clean-up and separation before analysis, has been described by workers at the University of Washington’*. An ingenious illumination method was employed to achieve simultaneous multiwavelength excitation of a fluorescent sample. This illumination scheme uses a vertically dispersed polychromatic beam to directly excite the sample in the cuvet. A fluorophor absorbs the beam and bands of fluorescence emanate from the cuvet. Thusj information is provided instantaneously on the absorption (excitation) properties of the fluorophor. Spatial information along the vertical axis (cuvet) is maintained while each of these ‘excitation’ bands is dispersed horizontally into its component emission. Thus, a two-dimensional image of fluorescence intensity, as a function of exciting and emitting wavelengths, is provided at the exit plane of the emission polychromator. Fig. la illustrates this for a hypothetical compound. The fluorescence emission-excitation matrix (FEEM) can be represented by a compact mathematical expression. For a single component solution, M;j = cUX;yi

(1)

where Mij is an element of the FEEM representing the fluorescence intensity excited at wavelength Li and emission monitored at 4. The xi is proportional to the quanta of photons absorbed at Ai,Ji is proportional to the quanta of photons emitted at Aj, and cr is a concentration dependent parameter. If the experimental conditions are such that synergistic effects are negligible, the total luminescence intensity for a mixture of ‘r’ components will be the linear sum of the contribution from the individual components present, i.e.

(2) Fig. 1. The formation of an emission-excitation matrix (EEM). The hypothetical compound has two absorption bands and two emission beaks as indicated by the conventional plots at the left and bottom of the EEM. The cuvet image, obtained by illuminating the sample with a polyGhromatic beam, retains the absorption proJire spatially as shown. The emission is subsequently dispersed into its component peaks by the analyzing polychromator.

where k denotes the kth component, xk and yk are column vectors composed of ordered sets ofxi’s andyj’s respectively. The symbol T denotes matrix transposition. Using these expressions, linear algebraic

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ELECTRONIC

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formalism can be applied and computer algorithms have been developed for both qualitative’3.‘4 and quantitative analysis15,16 of fluorescence data. The phosphorescence emission-excitation matrix (PEEM) can be depicted in the same format as the FEEM. At room temperature in the FEEM (Fig. la), only fluorescence is observed in fluid solution, due to the deactivation of the longer lived triplet state by quencher molecules such as oxygen. However, at low temperatures, such as liquid nitrogen temperature (77”K), these quenching interactions are minimized and phosphorescence is readily observed as illustrated in Fig. 1b. Note that the PEEM in this diagram has the same general form as the FEEM. However, more spectral information is provided for the emitting molecule. Thus, the PEEM information should provide greater specificity and selectivity. Moreover, for the time of decay for different phosphorescence, compounds can be easily exploited. Hence, it is now possible to combine the two-dimensional image of an EEM with time. With a suitable experimental arrangement, we can obtain this time-EEM (TEEM) completely and rapidly. This three-dimensional database presents a wealth of information which, with appropriate data reduction strategies can produce an interesting and useful new approach to phosphorescence analysis. This is more useful since the algorithms previously developed for the FEEM can be easily applied to the PEEM.

wavelength excitation. The emission polychromator, in its normal upright position, similarly has no exit slit and with the SIT-vidicon detector placed on its focal plane, two-dimensional imaging is achieved. The camera control and data acquisition is achieved by use of an Optical Multichannel Analyzer system (OMA-2, PAR, Princeton NJ). The OMA-2 has serial and parallel communication lines to the Hewlett-Packard 9845T computer for further data reduction and graphics. To acquire a PEEM, the sample is first dissolved in a suitable solvent mixture (e.g., EPA-ether, isopentane, alcohol in a 5 : 5 : 2 volume ratio). The quartz cell containing the sample is slowly introduced into the dewar filled with liquid nitrogen. It is then excited with a polychromatic beam for a period of time. At a given time, the electronic shutter is closed to shut off the excitation beam. After a predetermined delay a camera scans and records the PEEM in the memory of the OMA-2 for subsequent processing. We should indicate here that there are subtle differences in our acquisition of the PEEM as compared to the FEEM. For fluorescence measurement, the excitation source is continuous. Thus, the observed fluorescence signal reaches steady state conditions before the data are acquired. For our PEEM experiments, we desire to cut off the excitation source completely so that only a phosphorescence signal is observed. Thus, a constant emission signal is no longer provided within the time span required for scanning a complete PEEM. The resulting PEEM, acquired in the conventional scanning mode will actually be a convolution of the phosphorescence excitation and emission information with time decay of the phosphorescence 4694

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Apparatus and data acquisition The video fluorimeter, which has been described in detail elsewhere”, can be modified slightly to obtain the PEEM. A block diagram of the instrumentation is shown in Fig. 2. The most important part of this scheme is the polychromatic illumination method. This is achieved by placing the excitation polychromator on its side so that the beam is dispersed vertically towards the sample providing multi-

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intensity. For instance, the video fluorimeter in a routine experimental set up takes approximately half a second (0.5 s) to acquire an EEM of 2500 data points. In order that the intensity (r) of phosphorescence does not vary by more than lo%, the lifetime of the phosphor should be 5 s or more. This can easily be calculated assuming exponential behavior and using the following equation:

Z, = Z,, e- +

(3)

To overcome this convolution effect and to circumvent the need for extensive experimental modifications to achieve an increased rate of PEEM acquisition, we elected to integrate the phosphorescence signal on the camera target after an appropriate delay time. The spectral information is then scanned at normal acquisition speed. The length of integration and the period of delay before integration actually begins can be varied to allow time resolution. Time resolution of the PEEM of a multicomponent mixture is particularly useful for the deconvolution of the mixture into its constituents by an algorithm which has been described in detail’4. Briefly, the ratio deconvolution involves obtaining a series of PEEMs for a mixture of components in which the concentrations are altered in each PEEM. Thus, for a mixture matrix Mj, with standard single component PEEM, Ni, Mi = ~ i=

where of n-l series given

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(4)

oii is a relative concentration term. A sequence ratio matrices can now be obtained from the of mixture matrices. Thus, the ratio matrix, Ri, is by Ri = ~ ~ii Ni/~ Ni. i=

(5)

i=l

I

These ratio matrices, for a data set with good signal/noise, should provide plateaux of heights aij in the regions of non-spectral overlap. The number of plateaux correspond to the minimum number of phosphorescent components in the mixture. From these series of mixture matrices and the ratio matrices, we can obtain a linear system and derive a solution for the components’ PEEMs: M*=AN* where A is the n equation (6) is

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Fig. 4. Isometric pentacene from coronene is fairly is noisier due to

plots of the deconvoluted PEEM of (A) coronene and (B) the mixture shown in Fig. 3. The deconvoluted EEM of smooth due to the higher signal level while that of pentacene the lower signal/noise ratio.

sary for ratio deconvolution. In Fig. 3, we show two PEEMs of a mixture of coronene and pentacene after two different delay times. These data were obtained using the instrumentation which has been described above. The deconvolution of this mixture into its components using the ratio algorithm is displayed in Fig. 4.

Conclusions The sensitivity and selectivity of luminescence methods have been recognized for several decades. However, the multicomponent capabilities of these techniques have only recently been systematically exploited and used for the chemical analysis of complex samples. Multidimensional phosphorimetry is only one of many methods of simultaneous multiparametric luminescence measurement. Such studies will obviously have to couple novel instrumental methods with mathematical algorithms for data processing. The optimum use of the large data sets generated by multiparameter techniques require this integration of mathematics and instrumentation. The preliminary PEEM data that we present here demonstrates the validity of our approach to multicomponent phosphorescence analysis. Further experimentation is currently in progress to improve the general usefulness of this approach for multicomponent analysis.

n matrix of ratios. The solution to

Acknowledgements

N* =

This work was supported Ofice of Naval Research Energy.

A-1

M*.

(7)

The PEEM with a mixture of components of different lifetimes provides the characteristics neces-

in part by grants from the and the Department of

References 1 Lewis, G. N. and Kasha,

M.

( 1944) J. Am. Chem. Sot. 66, 2100

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2’ Keirs, R. J., Britt, R. D. Jr. and Wentworth, W. E. (1957) Anal. Chem. 29, 202 3 Parker, C. A. and Hatchard, C. G. (1962) Analyst 87, 664 4 Miller, J. N. (1981) TrendsAnal. Chem. 1, 31 5 Shulman, E. M. and Walling, C. (1972) Science 178, 53 6 Vo Dinh, T., Walden, G. L. and Winefordner, J. D. (1977) Anal. Chem., 49, 1126 7 Cline Love, L. J., Skrilec, M. and Habarta, J. B. (1980) Ana!. Chem., 52, 754 8 McCarthy, W. J. and Winefordner, J. D. (1967) in Fluorescence, Theory, Instrumentation and Practice, (Guilbault, G. G. (ed.)), Ch. 10, p. 371, Marcel Dekker, New York 9 Goutilier, G. D. and Winefordner, J. D. (1979) Anal. Chem. 51, 1384 10 Wilson, R. M. and Miller, T. L. (1975) Anal. Chem. 47, 256 11 Goeringer, D. E. and Pardue, H. L. (1979) Anal. Chem. 51, 1054 12 Johnson, D. W., Callis, J. B. and Christian, G. D. (1977) Anal. Chem. 49, 747A 13 Warner, I. M., Callis, J. B., Christian, G. D. and Davidson, E. R. (1977) Anal. Chem. 49, 564 14 Fogarty, M. P. and Warner, I. M. (1981) Anal. Chem. 53, 259

Signal

processing

15 Ho, C.-N., Christian, G. D. and Davidson, E. R. (1978) Anal. Chem. 50, 1108 16 Ho, C.-N., Christian, G. D. and Davidson, E. R. (1980) Anal. Chem. 52, 1071 17 Warner, I. M., Fogarty, M. P. and Shelly, D. C. (1979) Anal. Chim. Acta, 109, 361 Isiah M. Warner received his B.S. degree in Chemistry from Southern University in Baton Rouge, Louisiana in 1968. He then worked as a Research Chemist at Battelle Northwest, Richland, Washington from June, I!%8 to September, 1973. In October, 1973, he entered the graduate program at the University of Washington where he worked under the research guidance of Professor Gary Christian and received his Ph.D. in Analytical Chemistry in 1977. Isiah Warner has been an Assistant Professor of Chemistry at Texas A &? M University College Station, TX77843, U.S.A., since June, 1977. Chu-Ngi Ho received his B.S. degree from Denison University in Granville, Ohio in 1975. In August 1980, he obtained his Ph.D. in Analytical Chemistry under the supervision of Professor Gary Christian at the UniversiQ of Washington. He is presentb a Post-Doctoral Research Associate with Dr Isiah M. Warner at Texas A @ M University.

techniques instruments

in analytical

Signal processing is a unifying principle useful in understanding the operation of analytical instrumentation. Through it new modes of operation may be found for existing analytical methods and entirely new methods may be discovered. John B. Phillips Carbondale, IL, U.S.A. Signal processing, broadly speaking, is any operation which refines information contained in a signal. By this definition much ofwhat analytical chemists do is signal processing. An analytical determination begins with a sample and ends with some desired information about the sample. During the process the chemist manipulates the sample and signals derived from it to select the desired information and discriminate against all other information. A signal is any physical parameter whose value has meaning; that is, a signal carries information. The deflection of a meter on a spectrophotometer is a signal. So is the current which drives the meter, the light which generates the current in a photocell, and the concentration of substance in a cuvette which determines the light intensity. Every manipulation of a signal such as the selection of a particular wavelength of light by a monochromator is, in the broadest sense, signal processing. This approach to understanding analytical instru-

mentation was not much used in the past because the straightforward signal processing involved could generally be understood without a unifying concept. However, new instruments currently being developed and some which have already been introduced include a great deal more sophisticated signal processing. Much of this sophistication is due to the advances made in electronics and to the computers which are now being used in instrument automation, but in some cases advanced signal processing concepts are also beginning to affect the chemistry of the instrument. The distinction between the processing of electronic and computer signals and the chemical reaction signal is becoming more and more blurred. For example, most nuclear magnetic resonance spectrometers, especially for isotopes other than ‘H, now include a computer for signal averaging and Fourier transform calculations. Digital signal processing in the computer improves data quality. It also makes possible a variety of new chemistries such as cross polarization and spin echo techniques which produce additional kinds of information. Here the signal processing has changed the chemistry ofthe instrument in subtle but important