304
Anal. Chem. 1986, 58,384-387
if some groups have much greater dispersion than others, then the use of normalized distances can help prevent three types of classification error that might otherwise be encountered: false failure to classify, incorrect classification, and falsely classifying a sample when the classification should have been ambiguous (even if the sample was correctly classified), and thus results in a more accurate analytical technique. LITERATURE CITED (1) Massie, D. R.; Norris, K. H. Trans. ASAE 1065, 8 , 598-600. (2) Ben-Gera, I.;Norris, K. H. J. Food Sci. 1988, 33,64-67. (3) Ben-Gera, I.; Norris, K. H. Isr. J. Agric. Res. 1088, 18, 117-124. (4) Wetzei, D. L. Anal. Chem. 1983, 55, 1165A-1176A. (5) Hrushka, W. R.; Norris, K. H. Appl. Spectrosc. 1082, 36,261-265. (6) Honigs, D. E.; Freelin, J. M.; HieftJe, G. M.; Hirschfeid, T. B. Appl. Spectrosc. 1083, 37,491-497. (7) Mark, H.; Tunneii, D. Anal. Chem. 1985, 57, 1449-1456. ( 8 ) Rose, J. R. Second Annual Symposium on Near-Infrared Reflectance Analysis, Technicon Instrument Corp., Tarrytown, NY 1982. (9) Shenk, J. S.; Landa, I.; Hoover, M. R.; Westerhaus, M. 0. Crop Sc;. 1981, 21, 355-358.
s. A. Ana/. Chem. 1877. 49. 297-302. wold, s.; Albano, C.; DunnlW. J.; Edlund, U.; Esbensen, K.; Geladl. P.; Heilberg, S.; Johansson, E.; Lindberg, W.; Sjostrom, M. I n "Chemometrlcs: Mathematics and Statistics in Chemistry" NATO AS1 series, Series C: Mathematical and Physical Sciences; Kowalskl, Bruce R., Ed.; D. Reidel Publishing Co.: Boston, MA, 1983; Vol. 138, Chapter 2, p 75. Gnanadesikan, R. "Methods for Statistical Data Analysis of Multivariate Observations"; Wligy: New York, 1977; Chapter 4. Wilkins, C. L.; Jurs, P. C. I n "Transform Techniques in Chemistry"; Griffiths, P.. Ed.: Plenum: New York. 1978 Anderson, T. W. "An Introduction to'Multiiariate Statistical Analysis"; Wiley: New York, 1958; p 105. Hald, A. "Statistical Theory wlth Engineering Applications"; Wiley: New Yojk, 1952; p 290. Mbndel, J. National Bureau of Standards, personal communication, Aug. 28, 1985. Ciurczak, E. 12th Annual Meeting of the Federation of Analytical Chemistry and Spectroscopy Societies; October 1985, paper No. 440.
(10) Mattson, J. S.; Mattson, C. S.; Spencer, M. J.; Starks, (11)
(12) (13) (14) (15) (16)
(17)
RECEIVED for review June 13, 1985. Accepted September 13, 1985.
Quantum Counting by Laser Dyes in a Broad Spectral Range. Including the Near-Infrared Region Eberhard Brecht* Central Institute of Genetics and Research in Cultivated Plants, Academy of Sciences of G.D.R., DDR 4325 Gatersleben, German Democratic Republic
Some laser dyes were examlned that could serve as quantum counters wlth an extended red response. The benropyryllum salts CZ 144 and CZ 682 dlssolved In dlchloromethane have hlgh molar absorptlvltles of 117 500 L mol-' cm-' at 667 nm (CZ 144) and 131800 L mol-' cm-' at 720 nm (CZ 682), relative quantum ylelds of fluorescence above 0.5, sufflclent stablllty, low changes In fluorescence lntenslty over the temperature range from 16 to 32 O C , as well as only small POlarlratlon effects. The laser dyes CZ 144 and, In a quallfled sense, CZ 682 are sultable quantum counters to correct excltatlon spectra of fluorescence up to 700 and 780 nm, respectively.
Some instrumental parameters that have wavelength-dependent characteristics such as the light output of the lamp, the spectral function of monochromators, and the response of the detector distort the real fluorescence spectrum of a measuring sample. All these instrumental artifacts should be eliminated to enable the comparison of the excitation spectra to the corresponding absorption spectra as well as to determine the quantum yield and energy transfer from the emission spectra. The methods to correct excitation spectra that appear to be widely accepted involve the use of thermal detectors (thermopiles) and quantum counters. But in most cases thermal detectors show too low sensitivity, especially in the ultraviolet spectral region. Therefore, the application of quantum counters for correction of excitation spectra is recommended ( I ) . The most commonly used quantum counter is rhodamine B, which is covering the spectral region up to 600 nm. But in photosynthesis research such a quantum counter is needed whose available spectral range extends up to 700 nm and more. Recent work (2) has shown that the quantum yield of the polymethine dye 1,1',3,3,3',3'-hexa-
methylindotricarbocyanine (HITC)absorbing above 730 nm is too low, and it may not be useful on many fluorescence spectrometers. Therefore, a series of new laser dyes (3)were examined to find appropriate quantum counters. EXPERIMENTAL SECTION Instrumentation. Experiments were conducted by using a microcomputer-controlled fluorescence spectrometer J Y 3 CS (Jobin Yvon, France) fitted with a continuous 150-W xenon lamp, monochromators equipped with holographically engraved gratings, and a R928 S photomultiplier. Absorption measurements were performed by use of a Carl Zeiss Jena Model Specord M40 spectrometer. Reagents and Chemicals. Chlorophyll a and chlorophyll b were extracted from Lycopersicon esculentum, separated by 2-fold paper chromatography ( 4 ) , and dissolved in ethanol for UV spectrometry. Synthesis of laser dyes was followed by a manifold recrystallization (3). Dichloromethane p.A. used as solvent was dried with calcium chloride and distilled. Quantum Yield of Fluorescence. The comparative method (5) was used in order to determine the quantum yield of the laser dyes, but there is a need for compounds to serve as quantum yield standards in the near-infrared region ( I ) . Therefore, chlorophyll b was used as standard with a quantum yield of 0.095 (6). This standard was checked by determination of the quantum yield of chlorophyll a in ethanol, which we have found to be 0.25 f 0.07. This value shows a satisfactory agreement with 0.23 obtained by Weber and Teale (6) under similar conditions. Generally, the fluorescence signal was detected at 90' to the excitation beam. The bandwidths used were 4 nm for the excitation as well as for the emission monochromator but only 1 nm on the spectrophotometer. The dye solutions did not show any self-absorption effects for an absorbance below 9.06 at 465 nm (chlorophyll b), 393.5 nm (CZ 144), and 388.5 nm (CZ 682). With an excitation at the same wavelengths the emission spectra were obtained between 600 and 870 nm using a RG 1cutoff filter (VEB Jenaer Glaswerk) and corrected for the wavelength-dependent sensitivity of the detector system. The calculation of the quantum yields was performed by using the computer program FLU02 made by
0003-2700/86/0358-0384$01.50/00 1986 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 58, NO. 2, FEBRUARY 1986
i : 2 0 ~ 1 0 - ~ ~ ~ ~ ~ ~ ~ , ~ = 0 . ~ 5 m m 2:2.4 10'~MCZ682,d:O050mm
10
Flgure 1. Geometrical arrangement for the determination of correction curves of excitation (1) and emission spectra (2) by means of a quantum counter according to Jobin Yvon (JY 3 CS Manual, part II), modified. Abbreviations used are the following: A, attenuator; CF, cutoff filter; CM, toric mirror; L, collecting lens; M, diffusion mirror (MgO); S EM, emission entrance slit; S EX, excitation exit slit; TC, triangular cuvette.
the producer of the fluorescence spectrometer. RESULTS AND DISCUSSION A solution of the fluorescent dye contained in a cuvette with a suitable geometry (for example, the triangular cuvette shown in Figure 1, part 1) should be able to absorb all incident exciting light. If the emission spectrum and the quantum yield are independent of the excitation wavelength, the measured fluorescence signal is proportional to the irradiance of the lamp. The whole correction graph can be obtained by variation of the excitation wavelength. With such an adjusted excitation system the detection part of a fluorescence spectrometer can be calibrated too. In modern fluorescence spectrometers, both excitation and emission monochromators can be driven simultaneously, so the light can be directed to the detector system via a diffusion mirror (Figure 1, part 2). The division of a spectrum recorded under these conditions by the spectral irradiance of the lamp provides a correction curve for emission spectra. Characteristics Required of a Quantum Counter. It is evident that only a few dyes can be used as quantum counters. They have to satisfy the following conditions extensively (2): absorption over a broad spectral range, ability to absorb all incident light, high quantum yield, quantum yield independent of wavelength, extreme purity of the dye, high degree of solubility, photochemical stability, low temperature coefficient of fluorescence, small polarization errors, and minimal reabsorption errors. Selected from a series of new laser dyes, two benzopyrylium salts, CZ 144 (3)and CZ 682, were examined with respect to their ability to fulfill the said conditions using a commercial fluorescence spectrometer. Figure 2 shows the absorption spectra of CZ 144 and CZ 682 dissolved in dichloromethane. Both the dyes are able to absorb within the entire wavelength range of interest with a high molar absorptivity, t, of 117 500 L mol-' cm-' (CZ 144) and 131 800 L mol-' cm-l (CZ682) at their absorbance maxima of 667 nm and 720 nm, respectively. The fluorescence properties of CZ 144 and CZ 682 are shown in Figure 3. At high concentrations the emission maxima at the wavelengths 698 nm and 744 nm are red-shifted. Further spectroscopic characteristics of both dyes are described elsewhere (3, 7,8). T o ensure that all incident light is absorbed by the dyes, their concentrations were increased stepwise and the excitation spectra were measured. Above 6 g/L (CZ 144) and 8 g/L (CZ
I
.
200
385
.
,
xx)
4M
.
,
.
500 WAVELENGTH
.
Bm
.
,
(nm)
.
I
1
m,
8(13
Flgure 2. Logarithmic representation of the molar decadic absorptivity, t (L mol-' cm-I), of the benzopyryllum salts CZ 144 (mol wt 516.9) and
CZ 682 (mol wt 539.1) in dichloromethane measured on a Carl Zeiss
Jena, Model Specord
M40,
spectrometer.
1.0-
h E x 394 nm i C 2 144) 389 nm IC2 682 1
09-
SEX SEm
4nm 4 nm 295 k Filter RG 1 JY 3 CS T
08-
1
600
C Z 1 4 4 f 2 W6MVli
650
/
/
mc
CZ682 ( 3 10-6M 1
750
WAVELENGTH lnml
\
\
800
850
Flgure 3. Averaged and corrected emission spectra of the benzo-
pyrylium salts CZ 144 and CZ 682 in dichloromethane.
682) the spectra did not show any essential changes. Quantum counters should have a sufficient quantum yield to ensure their use on many instruments. On the other hand, substances with a high quantum yield may exhibit too small of a Stokes shift and substantial polarization effects. We determined the quantum yields of CZ 144 and CZ 682 in distilled dichloromethane by comparison with chlorophyll b in ethanol for UV spectrometry. Introducing the correction factor for differences in refractive index between fluorescence standard and sample (9),we determined the quantum yields of CZ 144 and CZ 682 in distilled dichloromethane to be 0.75 f 0.15 and 0.59 f 0.23, respectively, at 20 f 1 "C. The high standard deviations can be interpreted as the influence of the low signal-to-noise ratio above 800 nm. In spite of this inaccuracy it can be concluded that the estimated quantum yields are high enough to be useful on most commercial fluorescence spectrometers. The quantum yield of the laser dyes used as quantum counters has to be independent of the wavelength. This independence could be confirmed for rhodamine B and CZ 144 by comparison with the energy profile measured by a highly sensitive thermopile (corrected to quanta by multi-
ANALYTICAL CHEMISTRY, VOL. 58, NO. 2, FEBRUARY 1986
386 lo/
0 90 8-
0 7-
JY 3 cs Triangular cuvette Slits 4 x 20 nrn h Em 640 nm (Rho B ) 750 nm ( C Z 144) 825nrn(CZ682) Filter RG 1
5 0 4
02
WAVELENGTH ( n m )
01
200
300
400
533
600
703
Figure 5. Excitation spectra of chlorophyll a in acetone and CZ 682 in dichloromethane. Corrections are performed by using the quantum counter pair rhodamine B/CZ 682 (see Figure 4).
c
We determined the degree of polarization p , of concentrated dye solutions between 200 and 700 nm (CZ 144) as well as between 300 and 780 nm (CZ 682) and found only relatively small deviations. CZ 144 in dichloromethane (6 g/L) shows polarization values p , of +0.196 on excitation at 300 nm, +0.251 at 515 nm, and 0.226 at 651 nm. In the case of CZ 682 in dichloromethane (8 g/L) we found +0.032 at 306 nm, +0.018 a t 418 nm, and -0.059 at 774 nm. We set the detection system to view the long-wavelength tail (750 nm for CZ 144,825 nm for CZ 682) of the quantum counter in order to minimize errors due to reabsorption appearing a t higher concentrations of fluorescent dyes. Applications. The laser dye CZ 144 could be found to satisfy the most important conditions for a quantum counter to a greater degree than CZ 682. On the other hand the latter enables to correction of excitation spectra up to 780 nm. But this laser dye is well-suited in the whole spectral range, including the ultraviolet in combination with rhodamine B as shown in Figure 4. There are two possibilities to check the quality of a correction procedure: the comparison of the corrected excitation spectrum with the corresponding absorption spectrum and the investigation as to whether the sharp peaks of the light source can be eliminated. After choosing the last way, we found no additional peaks (positions are indicated by arrows in Figure 5) in the corrected excitation spectra of chlorophyll a and CZ 682 using the quantum counter pair rhodamine B/CZ 682 as shown in Figure 5. The same results can be obtained by a correction with CZ 144 as quantum counter.
CONCLUSIONS It can be concluded that the correction of fluorescence spectra up to 700 nm can be performed with the use of the laser dye CZ 144. Furthermore, by use of an electronically processed fluorescence spectrometer, a calibration curve can be composed of a short-wavelength part from rhodamine B and a long-wavelength one from CZ 682. In this way it is possible to correct any excitation spectra between 200 and 780 nm with suitable quantum yield. But some open problems remain. Quantum counters with an emission maximum above 780 nm should be researched. In this way excitation spectra of photosynthetic bacteria could be corrected by quantum counting too. Finally, the usefullness of quantum counters would be increased if they were available as solid blocks. Therefore, techniques should be developed to diminish the serious anisotropy of dyes if they are inbedded in acrylic copolymers or other optically clear materials.
ACKNOWLEDGMENT I am greatly indebted to P. Czerney for providing the laser dyes used in this study and to R. Hultzsch for Figure 2. The
Anal. Chem. 1986, 58, 387-394
author appreciates the technical support of U. Herrmann and K. Wilke. Additional fluorescence measurements have been performed by E. Birckner and K.-D. Dorfmann. I am very grateful to 0. Aurich and A. Meister for critical reading of the manuscript. Registry No. CZ 144,81190-25-2;CZ 682,99309-28-1;CH2C12, 75-09-2. LITERATURE CITED (1) Miller, J. M. “Standards in FluorescenceSpectrometry”; Chapman and Hall: London, New York, 1981.
387
(2) Dicerare, J. Perkin-Elmer Paper 1980, 637, 1. (3) Hultzsch, R.; Czerney, P.; Hermann, U.; Hartmann, H.; Wilke, K., DD Wirtschaftspatent 208501. (4) Sapozhinikov, D. 1. “Pigment Plastid Zelenikh Rasteniy i Metodika ikh Issiedovanlya”; Nauka: Moskau, 1964. (5) Demas, J. N.; Crosby, G. A. J . Phys. Chem. 1971, 75, 991. (6) Weber, G.; Teale, F. W. J. Trans. Faraday SOC.1857, 53, 646. (7) Brecht, E.; Czerney, P.; Hultzsch, R., DD Wirtschaftspatent 219303. (8) Hultzsch, R., submitted for publication in Jena . (9) Melhuish, W. H. J. Opt. SOC.Am. lW1, 51. 278.
RECEIVED for review June 4, 1985. Accepted September 5, 1985.
Determination of the Fraction of Organic Carbon Observable in Coals and Coal Derivatives Measured by High-Resolution Solid-state Carbon- 13 Nuclear Magnetic Resonance Spectrometry Edward W. Hagaman,* R. Rife Chambers, Jr., and Madge C. Woody
Chemistry Division, Oak Ridge National Laboratory, P.O. Box X , Oak Ridge, Tennessee 37831
The aromatic carbon fractions of coals, fa, calculated by fitting integrated lntenslty data from cross poiarization/magic angle spinning lacnuclear magnetic resonance spectra of coals to an expression that describes the magnetization behavior at long cross polarization times, tcp,and f a determined from a single spectrum recorded with t , = 1 ms show good agreement. This indicates that In single spectrum analysis, a 1 ms cross polarization contact time is a judicious compromise value consistent with nearly complete cross polarization from ail carbons that contribute to the observeble magnetlzation and with minimum errors introduced by differentla1 T,,(H) for the aromatic and aliphatic resonance bands. Spin counting experiments performed using mixtures of coal and a diamagnetic standard establish that the observab/e carbon magnetization in several coals of widely varying rank is derived from 40 to 60% of the organlc carbon In these subexperiment it is stances. By use of a 1a~14C-double-iabeiing shown that in chemically modified coals, the “Introduced carbon” and coal carbon fractions are detected dlscriminately and to a degree that Is dependent on the type of modlficatlon the coal has undergone.
Solid-state 13C nuclear magnetic resonance spectrometry, experimentally performed using the techniques of cross polarization (l), magic angle spinning (2-5), and dipolar de; coupling (I, 6),Le., CP/MAS-I3C-NMR, has proven to be a powerful spectroscopic method for the study of solid fossil fuels. The technique has been widely applied in the measurement of the apparent aromatic carbon fraction of coals, f,‘ (7-111, and coal macerals (11-14) and has been used to describe chemically induced structural modification of coals (15-1 7). Though this experiment can yield carbon resonance area ratios in agreement with atomic ratios for proton-rich dia0003-2700/88/0358-0387$0 l.50/0
magnetic organic compounds (18), quantitative reliability of the experiment in applications to coal and other solid fossil fuels has remained uncertain. This circumstance arises primarily from two characteristics of these heterogeneous, amorphous materials: a typically low H/C ratio, and the presence of a free electron spin density (ca. 1016-1019spins/g) associated with the organic matrix of the coal (19). The low H/C ratio and unknown hydrogen distribution allow the possibility that some carbons may be isolated from hydrogens and incapable of cross polarization. The presence of unpaired electrons and attendant electron-nuclear dipolar interactions can render a fraction of the carbon resonance signal invisible, broedened, and/or paramagnetically shifted (20). The consequences of electron-nuclear interactions in coals with respect to CP/MAS-13C-NMR measurements are being examined by dynamic nuclear polarization experiments (21-23). Several attempts to ascertain the quantitative reliability of CP/MAS techniques in coal analyses have appeared (7, 24-29). A lack of consensus emerges in these studies in part because the criteria by which coal spectra could be judged directly, Le., atomic ratios representing carbon distribution among resolved resonances in the spectra, are not known. Peripheral experiments, e.g., comparison of Block decay and CP-NMR spectra of coals, test some facets of the NMR response but cannot done constitute a proof of the quantitative response of the sample. The purpose of this article is to report two complementary CP/MAS-13C-NMR experiments in which the integrated intensity of coal spectra are compared to a signal area from an appropriate reference material. In one case the reference area is elicited from methyl groups that have been chemically attached to the organic matrix of the coal. In the second case it arises from a diamagnetic organic compound blended with the coal. From these experiments the fraction of carbon in a coal sample that contributes to the observable magnetization in the CP/MAS-13C-NMR experiment is estimated. A preliminary report of part of this work was presented at the 1985 0 1986 American Chemical Society
