938
Anal. Chem. 1985,57,936-940
(17) Engstrom, R. C. Anal. Chem. 1984, 56, 890. (18) Sleszynski, N.: Osteryoung, J.; Carter, M. Anal. Chem. 1984, 56, 130.
RECEIVED for review October 29, 1984. Accepted December
26, 1984. Acknowledgment is made to the donors of the petroleum Research Fund, administered by the ~~~~i~~~ Chemical Society, and to the National Science Foundation, Grant NO. CHE-8411000, for partial support of this work.
Flow Injection and Liquid Chromatography Detector for Amino Acids Based on a Postcolumn Reaction with Luminol Allan MacDonald' and Timothy A. Nieman*
Department of Chemistry, University of Illinois, 1209 West California Street, Urbana, Illinois 61801
A detector for amlno aclds Is based on suppression of chemiluminescence (CL) In the Co( I I)-lumlnol-peroxlde system. The scheme Involves two successive postcolumn reactlons, complexation followed by CL. Followlng the column, Co(I1) Is added to the effluent. The stream then enters a mlcroporous membrane cell where an alkaline lumlnol/peroxlde solution ls added. The CL emlsslon ls proportional to the free Co( I I ) concentratlon. When an amlno acid elutes, It complexes Co( I I ) and reduces the free Co( I I ) concentratlon;the CL Intensity then drops and a peak results. Detection llmlts depend upon the magnltude of the complex formation constants, and range from 0.004 to 20 nmol In flow Injection mode. Preclslon Is 1-4%. Compared to the classlcal nlnhydrin reaction, thls method Is faster, can be run at room temperature, can determine secondary amlno aclds wlthout varylng the reactlon or Instrument, has simpler Instrumentatlon, and, in favorable cases, has lower detection Ilmlts.
As evidenced by recent reviews (1-7), there is much interest in developing chemiluminescent (CL) techniques for detection in order to take advantage of the sensitivity the method can provide. As a lack of selectivity has been the major limitation of CL based determination, it has naturally been suggested that CL be applied for detection of separated species. A few examples exist for the use of the luminol (3-aminophthalhydrazide) reaction as a detector for metals separated as their chloro complexes on a strong anion exchange column (8-11). One of these approaches measured Zn and Cd by suppression of the Co(I1)-enhanced luminol reaction (11). There are also a few examples of solution CL as an HPLC detector for organic species (12-21). Direct reaction with lucigenin has been used for detection of ascorbic acid and dehydroascorbic acid (12). Energy transfer from peroxyoxalate CL reactions to fluorescent analytes has been the basis for several other detection schemes (13-19). An early example of this method is the detection of dansylated amino acids using bis(2,4,6-trichlorophenyl)oxalate and HzOzas the fluorescent excitation reaction (13). Another detector (20) was a spray detector based on energy transfer from OZ('Ag) generated by reaction of hypochlorite with hydrogen peroxide at pH 10. In addition to liquid-phase detection, gas-phase chemiluminescent reactions have been employed for detection of HPLC or GC separated species (22-28). Our interest has been to develop a CL detector for amino acids in flowing streams such as in an HPLC detector. Metal ion chelators such as citrate and EDTA are known to cause Present address: Clorox Technical Center, P.O. Box 493, Pleasanton, CA 94566. 0003-2700/85/0357-0936$0 1.50/0
suppression of metal ion enhanced luminol CL (29). Also, amino acids have been detected in a static system by suppression of CuWenhanced luminol CL (30,31). Our system monitors the CL from a steady flow of Co(I1) reacting with luminol and hydrogen peroxide in a alkaline solution. If an amino acid is injected into the Co(I1) stream, some of the Co(I1) is complexed and decreased CL light intensity results. The injected slug of amino acid analyte then results in a negative peak, and the analyte concentration may be determined from the size of that peak.
EXPERIMENTAL SECTION Instrumental Methods. Figure 1 shows a schematic of the flow system used for HPLC detection. The flow system used for flow injection analysis was identical with the system in Figure 1 with the exception of the column being removed. The mobile phase was pumped by an Altex 110 pump, equipped with a pulse dampener (Anspec H1302), followed by a Rheodyne 7010 injector with a 20-pL sample loop. The mobile phase was then mixed in a tee-mixer with a Co(I1) solution which was delivered from a pressurized reservoir (ca. 5 psi). A short delay loop followed before the solution was mixed with the precombined luminol/HzOz/KOH solution in the flow cell. The luminol/H202/KOHreagent solution was also delivered from a pressurized reservoir (Ominfit glass reagent reservoir bottles). The light intensity produced was monitored by a Hamamatsu R372 photomultiplier tube. Flow of reagent solutions was controlled with Nupro metering valves and was monitored using flowmeters (Fisher and Porter or Manostat). The flow cells used in the course of these studies were microporous membrane flow cells developed previously in this group (32, 33).
Separations were performed on a Whatman Partisil 10 SCX column using a mobile phase of 0.01 M acetic acid/sodium acetate buffer at pH 5.5 with a flow rate of 1.0 mL/min. Reagents and Solutions. Luminol (Aldrich), amino acids (ICN pharmaceuticals, National Biochemical, Mallinckrodt, or Sigma), and all other reagents (Analytical Reagent Grade) were used without further purification. The luminol/HzOz/KOHreagent solution composition was 10 mM luminol,' 20 mM Hz02,35 mM KOH, and 1pM EDTA. This solution was stable for about 1 week. Solutions of Co(I1) were prepared by serial dilution from a stock solution of M CO(N03)z.6H20. Solutions of KOH were prepared from Acculute solutions (Anachemia). Water purified by a Continental Millipore Milli-Q system was used for all solutions.
RESULTS AND DISCUSSION Figure 2 illustrates the theory of the mechanism of the response of this detector. If only uncomplexed Co(I1) enhances the luminol reaction, then the emission intensity would vary as the concentration of uncomplexed Co(I1). In the presence of a chelating ligand the fraction of free Co(I1) is given by the ligand concentration and the complex formation constants, 0 1985 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 57, NO. 4, APRIL 1985
COLUMN
4 lOOnM
I
T
- - 0
20 pR
937
LUMINOL H202 KOH
Flgure 1. Block diagram of flow system. 0 1
I "
1
I
1
J
10-6
10.8
[Histidine] ( M )
Figure 3. Effect of stock Co(I1) concentration on histidine working
curve.
IOO-
0
P
I"
00-
!
1 0
8
a 73
log [LIGAND]
of ligand concentration. regardless of the initial concentration of Co(I1). In terms of light intensity, a negative peak from the background intensity is observed due to the complexation of some of the Co(I1) by the analyte. For the purpose of this discussion, the peak is discussed in positive terms in that measurements are described as peak height or peak area. The maximum ligand concentration which can be measured by peak height is that ligand concentration which causes the free Co(I1) concentration a t the center of the analyte band to drop below the detection limit for Co(I1) pnder these conditions. As the ligand concentration is lowered, the working curve should follow the curve defined by a plot of fraction of Co(I1) remaining vs. ligand concentration. This curve is nonlinear; it is sigmoidal if the ligand concentration axis is logarithmic. The ligand detection limit is determined by the smallest change in free Co(I1) concentration which is measurable. Reagent Optimization and Composition. Figure 3 shows the effect of tlie stock concentration on the working curve for histidine. As the stock concentration of Co(I1) is increased from 1 nM to 100 nM, the working range, measured by peak height, increases. The detection limit is about the same using 10 nM and 100 nM Co(I1). The data for histidine concentrations below 2 X lo4 M using 1 nM Co(I1) are not shown as they were indistinguishable from the noise in the background intensity. These data can be explained in terms of the theory described above. The maximum measurable concentration (by peak height) should decrease with decreases in stock Co(I1) concentration. As the stock Co(I1) concentration is decreased, the fraction of this concentration which represents the detection limit ofqCo(I1) increases; therefore, the ligand concentration a t which the maximum peak height is reached is lowered. If the noise in the background light intensity were proportional to this intensity and the Co(I1) working curve were linear, the minimum detectable change in Co(I1) concentmtion would be independent of the stock Co(I1) concentration. By use of 10 and I00 nM Co(I1) these conditions are apparently upheld because the observed detection limit for histidine was approximately the same. With 1nM Co(I1) the noise in the
B
60-
._N 0
Figure 2. Hypothetical plot of free Co(1I)concentration as a function
0
8,
i
0
cocn) 3
o
40-
B
A
IOOnM IOnM InM
0
0
z
[I
20-
0 0 €!A
E n
1
1
1
J
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[Histidine] (M1
Figure 4. Normalized histidine working curves for several stock Co(1I)
concentrations. background intensity was proportionately larger, causing a degraded detection limit. T o summarize, the ligand working curve should have a wider working range but similar detection limits with increasing Co(I1) concentration. This theory agrees with the data in Figure 3. Three other matters related to the stock Co(I1) concentration should be noted. First, the peaks are wider for lower stock Co(I1) concentrations at high ligand concentration. This behavior is predicted by our model in that, as mentioned above, the detection limit for Co(I1) is a larger fraction of the lower stock concentration and therefore a wider portion of the analyte peak will cause maximum suppression. Second, the absolute level of the residual light, or that light remaining after virtually all (99.9%) of the Co(I1) is complexed, is constant because it is due to a combination of stray light and the CL emission due to reagent impurities and the leaching of the flow system components by the reagents. As the stock Co(I1) concentration decreases, the absolute level of the signal decreases, and therefore the residual light level becomes larger relatiue to the signal. Third, if the free Co(I1) concentration controls the CL intensity and if the fraction of free Co(I1) is dependent on ligand concentration, then the relative peak height (relative to the maximum observed at high ligand concentration) should be independent of stock Co(I1) concentration. Figure 4 shows the data of Figure 3 replotted as relative peak height. It can be seen that this hypothesis is supported. Figure 5 shows a plot of the ratio of [H202] to [luminol] vs. the peak CL intensity observed using 1 nM Co(I1). This experiment was performed on a four-channel stopped flow instrument which automatically prepared dilutions of stock solutions (34). The results show that, although there is a slight dependence on the absolute concentrations of luminol and
938
ANALYTICAL CHEMISTRY, VOL. 57, NO. 4, APRIL 1985 I
""I I I 0
I
1 I 2
I
I 4
l
I 6
I
i
1
8
hydrogen peroxide, the ratio of the two concentrations is more important in obtaining maximum CL. Optimizing the background light intensity is important as the sensitivity of the method increases with background intensity if the Co(I1) flow is held constant. Therefore the H202:luminolratio in the stock solution was 20 mM:lO mM. The absolute concentrations chosen were higher than those shown in Figure 5 as the stock solution would be diluted by the other reagents and the mobile phase. The optimal KOH concentration or flow rate was found empirically. Delay Length. The effect of the length of the delay between the point where the eluent and the cobalt are mixed to the point where these are mixed with the CL reagent solution was observed. Two competing effects were anticipated. The first effect was that band broadening would increase with delay time, thus diluting the sample. The second effect was that mixing of the analyte and the cobalt would be improved by increasing the delay length and that these solutions would have a longer time to react with each other before reacting with the reagent solution. Working c w e s for five amino acids were prepared using delay times ranging from 1.4 to 6.5 s. The results for these experiments showed a slight decrease in analytical signal at the longest delay time. At the shorter delay times no discernible pattern was present. With delays below 1 s, the signal decreases. With no delay Gust the 0.05 s in the mixer and connector to the flow cell) the signal is only onethird of that obtained with a 1 s or longer delay. Relative Response of Amino Acids. As further assurance that a complexation mechanism was in effect, the relative responses of a series of amino acids were mehured. The amino acids chosen and the logarithm of the highest order formation constant with cobalt were as follows: P-alanine, 3.58; glutamic acid, 8.46; a-alanine, 8.48; glycine, 11.0; and histidine, 11.9 (35). Figure 6 shows the results. In general, detection of the amino acids follow their formation constants. The only exception was that detection of glutamic acid was horse than would be predicted from its formation constant, especially compared to a-alanine. However, glutamic acid is much more acidic than a-alanine. Samples of M a-alanine and M glutamic acid were adjusted to pH 7 and tested. The results were then the same for both amino acids. Detection of histidine is much more sensitive than the other amino acids, even considerably better than glycine which has a similar formation constant with Co(I1). However, histidine is a tridentate ligand which could give it a kinetic advantage over the other amino acids tested which were bidentate ligands. This result agrees nicely with results obtained by Haapakka (36)when he examined the effect of denticity of ligands on the inhibition of cobalt enhanced electrogenerated chemiluminescence of luminol. He found inhibition in the order EDTA > iminodiacetic acid > glycine.
I
io-)
I 10-1
[AMINO ACID] (M)
[H202] /[LUMINOL]
Figure 5. Optimization of [H,0,] /[iuminoi] ratio: luminol concentration is (0)1, (0)2, (A) 3, (A)4, and (X) 5 mM.
I
I
IO+
Figure 6. Working curves for five amino acids: (0)histidine, ( 0 ) glycine, (X) a-alanine, (0)P-alanine, (m) glutamic acid.
100 L
c 0 '
50 0
d 10-6
IO+
10-4
IO-^
.
[HISTIDINE] ( M i
Figure 7. Comparison of peak height (0)and peak area ( 0 )working
curves.
Peak Height vs. Peak Area. The working curves shown above were nonlinear with a limited pseudolinear working range. Figure 7 compares the working curve for histidine obtained using peak area with the curve obtained using peak height. As we have previously mentioned, there is a fundamental limitation of the working curve due to the fact that there is a detection limit for Co(I1). The dramatic increase in linear working range using peak area is probably somewhat due to increased diffusional band broadening. However, even in the absence of band broadening, the portion of the peak width for which the free Co(I1) concentration is below the detection limit increases at high analyte concentrations, and results in a wider peak with a flat top. From these data it can be seen that the increase in dynamic range from about 1order of magnitude to over 3 orders of magnitude more than outweighs any difficulty in obtaining peak areas when quantitative results are required. Selection of Chromatographic Mobile Phase. As with all postcolumn reaction schemes, the selection of a mobile phase which is compatible with the detection reaction is a critical step in the development of the method. The constraints that the Co(I1)-luminol reaction system for detection of amino acids imposes upon the selection of the mobile phase are that a noncomplexing buffer of low buffer capacity should be used. A standard method for separation of amino acids is to elute them from a strong cation exchange column using citrate buffers, pH, and ionic strength appropriate for specific applications (37). Citrate buffers are incompatible with CL detection for two reasons. First, citrate complexes Co(I1) (log kf = 25.3) (35),thus competing with the amino acid complexation of Co(I1). Second, the buffer is acidic while the CL reaction of luminol is optimized at a pH closer to 11 or 12. Although a polymeric column is often used in ion exchange separation of amino acids (38, 39), a silica-based Partisil column was available in the lab so it was used in this case.
ANALYTICAL CHEMISTRY, VOL. 57, NO. 4, APRIL 1985 injection
(2)
Figure 8. Sample chromatogram: (1) glycine, 10 nmol, (2) hlstldine, 2 nmol, (3)arginine, 10 nmol; mobile phase, 0.01 M acetic acid/acetate buffer at pH 5.5, 1.0 mL/mln.
Table I. Detection Limits for Amino Acids by Chemiluminescence amino acid
detection limit, nmol Acidic
hydroxyproline aspartic acid glutamic acid
1 20 1
Neutral glycine a-alanine
0.4 0.4 2 0.01 1 2
@-alanine
cystine valine phenylalanine Basic lysine histidine arginine
0.4
0.004 0.04
939
maintained across the membrane (33);there is a large pH change from the mobile phase to the reaction zone. Second, the reagent flow rate would be regulated by the membrane so that the flow rate would be more stable than that regulation which could be obtained using the needle valves alone. Third, the reagent flow rate could be made very small (0.1 mL/min) to minimize both sample dilution and reagent consumption. The microporous membrane cell used in the work described was about 0.12 X 1.48 X 1.59 cm and was measured to contain about 85 pL. On the basis of resolution considerations a smaller cell would be desirable. A new cell was constructed with dimensions about 0.12 X 0.12 X 1.59 cm and was measured to contain about 20 pL. To accommodate the smaller cell the pressure on the reagent reservoir was increased from 10 psi to about 18 psi. Even at this pressure, the flow of reagent through the microporous membrane was reduced. Use of the smaller volume cell results in lower CL emission intensities, but on a relative basis the peak heights are virtually unchanged. Lower intensities would be predicted due to the smaller volume of emitting solution viewed by the detector. Exact predictions of expected signal levels are complicated by different reagent flow rates and residence times between the two cells. The reduced cell volume had no effect on the working range. CONCLUSIONS Compared to ninhydrin detection, this chemiluminescence detection approach is faster and takes place at room temperature without thermostating. Since detection is based on complexation, secondary amino acids are detectable without modification of the detection reaction or instrumentation, as is necessary with both the ninhydrin and o-phthaltildehyde schemes (39). The instrumentation is simple as no light source or wavelength discrimination device is required. As the detection limit for amino acids by this method is fundamentally determined by the magnitude of the formation constant of the amino acid with Co(II), changing the metal ion used to produce CL could alter detection limits. Both Cu(I1) and Ni(I1) enhance luminol CL and have larger formation constants with amino acids (35);the detection limit for Co(I1) is lower than those for either Cu(I1) or Ni(II), however. One could also consider using this CL detection scheme with a metal ion like Fe(II1) which has higher formation constants with oxygen containing ligands than does Co(I1); one then should be able to detemine species like carboxylic acids, ketones, or phenols. Registry No. Co(NO&, 10141-05-6;hydroxyproline, 51-35-4; aspartic acid, 56-84-8; glutamic acid, 56-86-0; glycine, 56-40-6; a-alanine, 56-41-7; @-alanine,107-95-9;cystine, 56-89-3;valine, 72-18-4; phenylalanine, 63-91-2;lysine, 56-87-1;histidine, 71-00-1; arginine, 74-79-3; luminol, 521-31-3.
In order to use the Partisil 10 SCX column, it is necessary to use an acidic mobile phase as aqueous solutions of pH >7.5 will dissolve the silica. An acetate buffer is acceptable for this CL detection scheme because log Kffor a Co(I1)-acetate complex is only 1.9 (35). Figure 8 shows a test chromatogram of glycine, histidine, and arginine. The conditions used are described in the figure caption. Of the amino acids tested, only histidine and arginine were retained under these conditions. Table I gives detection limits for a number of amino acids by this detection method; all values were measured without the chromatographic column. As illustrated earlier in Figure 7, the working range is about 1order of magnitude if peak heights are used and over 3 orders of magnitude if peak areas are used. The precision is 'I2% a t large peak heights, 2l/,% a t midrange peak heights, and 4'/,% at low peak heights. Flow Cell Considerations. The choice of a flow cell for CL detectors involves a trade-off. In order to obtain a measurable amount of light, a large volume of reacting solution must be viewed by the photomultiplier tube. Consequently, the flow cell dead volume is relatively large. However, the larger the dead volume is, the greater is the dilution of sample due to band broadening. The microporous membrane cell had been developed in our group for chemiluminescence detection in flowing streams and applied to determination of metal ions (32),glucose (33,40), and cholesterol (41). The cell offers several advantages for this present work. First, a stable pH gradient could be
LITERATURE CITED Seitz, W. R. CRC Crit. Rev. Anal. Chem. 1981, 13, 1-58. Wehry, E. L. Anal. Chem. 1982, 5 4 , 13tR-150R. Monola, H. A.; Mark, H. B. Anal. Chem. 1982, 5 4 , 62R-83R. Wehry, E. L. Anal. Chem. 1984, 56, 156R-173R. Monola, H. A.; Mark, H. 8. Anal. Chem. 1984, 56, 96R-112R. Majors, R. E.; Barth, H. G.; Lochmuller, C. H. Anal. Chem. 1984, 56, 300R-349R. Miller, J. N. Analyst(London) 1984, 709, 191-198. Hartkopf, A. V.; Deiumyea, R. Anal. Len. 1974, 7 , 79-68. Neary, M. P.; Seitz, W. R.; Hercules, D. M. Anal. Lett. 1974, 7 , 563-590. Delumyea, R.; Hartkopf, A. V. Anal. Chem. 1976, 4 8 , 1402-1405. Burguera, J. L.; Burguera, M.; Townshend, A. Anal. Chim. Acta 1981. 727, 199-201. Veazey, R. L.; Nieman, T. A. J . Chromatogr. 1980, 200, 153-162. Kobayashi, S.;Imal, K. Anal. Chem. 1980, 52, 424-427. Melbin, G. J . Liq. Chromatogr. 1983, 6 , 1603-1616. Sigvardson, K. W.; Birks, J. W. Anal. Chem. 1983, 55, 432-435. Kobayashi, S.; Sekino, J.; Honda, K.; Imai, K. Anal. Biochem. 1981, 712, 99-104. Honda, K.; Sekino, J.; Imai, K. Anal. Chem. 1983, 55. 940-943. DeJong, G. J.; Lammers, N.; Spruit, F. J.; Brinkman, U. A,; Frei, R . W. Chromatographia 1984, 18, 129-133.
940
Anal. Chern. 1985, 57,940-945
(19) Weinberger, R.; Mannan, C. A.; Cerchio, M.; Grayeski, M. L. J. Chromatogr. 1984, 288, 445-450. (20) Shoemaker, B.; Birks, J. W. J. Chromatogr. 1980, 209, 251-263. (21) HIIi, E. A.; Nelson, J. K.; Birks, J. W. Anal. Chem. 1982, 54, 541-546. (22) Birks, J. W.; Kuge, M. C. Anal. Chem. 1980, 5 2 , 897-901. (23) Getty, R. H.; Birks, J. W. Anal. Lett. 1979, 12(A5), 469-476. (24) Nelson, J. K.; Getty, R. H.; Birks, J. W. Anal. Chem. 1983, 55, 1767-1770. (25) Bruening, W.; Concha, F. J. M. J. Chromatogr. 1977, 142, 191-201. (26) Massey, R. C.; Crews, C.; McWeeny, D. J.; Knowles, M. E. J . Chromatogr. 1982, 236, 527-529. (27) Nord, P. J. Dlss. Abstr. I n t . 8 1982, 43, 419. (28) Yu, W. C.; Goff, E. U. Anal. Chem. 1983, 55, 29-32. (29) Burdo, T. G.; Seitz, W. R. Anal. Chem. 1975, 47, 1639-1643. (30) Pantei, S.; Weisz, H. Anal. Chlm. Acta 1975, 74, 275-260. (31) Pabivanets, B. I.; Ryabov, A. K.; Shnaiderman, E. M. Zh. Anal. Khlm. 1975, 30, 2439-2443. (32) Nau, V. J.; Nieman, T. A. Anal. Chem. 1979, 51,424-428. (33) Pilosof, D.; Nieman, T. A. Anal. Chem. 1982, 54, 1698-1701.
(34) Stieg, S.; Nieman, T. A. Anal. Chem. 1980, 52, 796-800. (35) Inczedy, J. "Analytical Applications of Complex Equilibria"; Haisted Press: London, 1976; pp 317-368. (36) Haapakka, K. E. Anal. Chlm. Acta 1982, 139, 229-236. (37) Niederwieser, A. I n "Chromatography: A Lab Handbook of Chromatographic and Electrophoretic Methods", 3rd ed.; Heftmann, E., Ed.; Van Nostrand-Reinhoid, New York, 1975. (38) Lee, D. P. LC, Lip. Chromatogr. HPLC Mag. 1984, 2 , 828-832. (39) Dong, M. W.; DiCesare, J. L. LC, Ll9. Chromatogr. HPLC Mag. 1983, I , 222-228. (40) Malavolti, N. L.; Pilosof, D.;Nieman. T. A. Anal. Chem. 1984, 56. 2191-2195. (41) Maiavolti, N. L.; Piiosof, D.; Nieman, T. A. Anal. Chim. Acta, in press.
RECEIVED for review August 20, 1984. Accepted January 7, 1985. This research was supported in part by the National Science Foundation (CHE-81-08816).
Laser- Induced Fluorescence Spectrometry of Aromatic Hydrocarbon Derivatives in Vapor-Deposited Parent Molecule Matrices Charles F. Pace and Jon R. Maple* Department of Chemistry, University of New Mexico, Albuquerque, New Mexico 87131
The analytlcal utlllty of a low-temperature parent molecule matrix for the characterlzatlon of mlxtures of aromatlc hydrocarbon derlvatlves by fluorescence detectlon has been assessed, using a prototype system consisting of chloronaphthalenes In a naphthalene matrix. By vapor deposltlon of the samples and the naphthalene matrix, it has been demonstrated that good preclslon, low detectlon limits, and highresolutlon excltatlon and fluorescence spectra can be obtained. Chloronaphthalenes can be detected at low nanogram levels from a two-photon excltatlon spectrum, whlie the twophoton excitation bandwidths are about 1 cm-' (fwhm) and are virtually independent of the position of substitution when the parent molecule matrix is used. With UV (slngle photon) excltatlon subplcogram detectlon Ilmlts have been reported and each chloronaphthalene In a 10-component mlxture can be selectlvely excited wlthout fluorescence Interference from the other components of the mlxture. The 9 % precision of the quantitative measurements Is limited only by the stablllty of the laser excitation source.
There is a considerable interest in developing highly selective analytical methodologies which are capable of precisely characterizing the composition of environmentally hazardous materials which contain a complex variety of aromatic hydrocarbon derivatives (AHDs). The interest in individual AHDs is due in large part to the fact that the carcinogenic or mutagenic nature of an aromatic compound is strongly affected by the number, position, and identity of substituents on the parent molecule (1-3). Since there is an enormous number of possible derivatives for each aromatic hydrocarbon and since trace concentrations of AHDs are often highly toxic, carcinogenic, or mutagenic, a suitable analytical methodology must be highly selective and sensitive. Fluorescence methods which utilize the Shpol'skii effect are particularly attractive because of the high-resolution fluorescence spectra that result from preparing many aromatic
hydrocarbons in low-temperature Shpol'skii (Le., n-alkane) frozen solutions (4). The fluorescence peaks are sharp enough to distinguish individual AHDs, such as methyl derivatives of phenanthrene, chrysene, pyrene, benz[a]anthracene, benzo[a]pyrene, and benzo[h]quinoline (5-8). Moreover, the high selectivity of laser excited Shpol'skii spectrometry (LESS) minimizes the need for fractionation of complex samples and retains the high sensitivity associated with fluorescence detection (4,7-9). Nevertheless, there are many examples of situations in which the Shpol'skii effect does not occur and the excitation and emission bands of individual AHDs in complex samples cannot be resolved. This is often the case when a substituent on a parent compound is highly polar, as in the case of monohydroxy1 derivatives of naphthalene (IO) and pyrene (11)or carboxyl derivatives of benzo[a]pyrene (12), for example. Furthermore, there are no reports in the literature of observations of the Shpol'skii effect for many environmentally hazardous AHDs, including hydroxyl, chloro, and bromo derivatives of naphthalene, biphenyl, dibenzofuran, and dibenzo-p-dioxin. Thus, it is highly desirable to find low-temperature matrix materials in which these and many other AHDs exhibit high-resolution excitation and emission spectra so that the high selectivity and sensitivity advantages of LESS can be retained. We have recently advocated the usage of aromatic hydrocarbon crystals for the analysis of AHDs in situations where a suitable Shpol'skii matrix cannot be found (13,14). By the choice of an aromatic hydrocarbon matrix material with a molecular size matched to the AHDs of interest, the AHDs can be incorporated into well-defined sites or vacancies in the crystal, thereby minimizing inhomogeneous broadening. For example, methylnaphthalene (MN) derivatives prepared by normal freezing in durene crystals exhibit high-resolution excitation and emission lines at low temperatures (13, 14). Moreover, even though the MN 0-0 transition energies are closely spaced, each MN in a complex mixture can be identified from a two-photon excitation (TPE) spectrum ( 2 4 ) . Both the T P E and the selective excitation methods are highly sensitive and have resulted in subpicogram detection limits
0 1985 American Chemical Society 0003-2700/85/0357-0940$01.50/0
