1007
Anal, Chem. 1085, 57, 1907-1910
2L
-
22
-
20
-
18
-
ion peak. For ion mobility spectrometers using @Niionization, this peak serves as a convenient measurement of resolution and the value obtained provides an estimate of the minimum separation performance that can be expected. In most cases, where product ions drift with longer times than reactant ions, resolution will be greater.
ACKNOWLEDGMENT The authors are indebted to W. Siems for his review of the paper and his help in the final stages of preparation.
e-
i
Registry No. Methyl propionate, 554-12-1;methyl butyrate, 623-42-7;methyl caproate, 106-70-7;methyl caprylate, 111-11-5; methyl caprate, 110-42-9; methyl laurate, 111-82-0; methyl myristate, 124-10-7; methyl stearate, 112-61-8.
W 16: 14
-
12
-
a
I
LITERATURE CITED * 10
-I
12
16 18 20 DRIFT TIME (ms) 14
22
24
Figure 7. IMS resolution measured as a function of drift time at an entrance gate width of 0.2 ms. The numbers at each point identify the ion used to make the measurement and correspond to those listed in Figure 6 and in Table 11.
entrance gate width was 0.2 ms. For an entrance gate width of 0.1 ms the plot was similar in shape but higher in resolution. Judging from both actual and theoretical curves, resolution of ions with drift timesgreaterthan about 15 ms vary in resolution less than 10%. Thus, under normal operating conditions, resolution for ions with long drift times will be at a maximum and can be considered to be independent of ion drift velocity. In this paper, however, a more conservative approach has been followed, calculating resolution from the primary reactant
f l ) Revercomb. H. E.: Msson. E. A. Anal. Chem. 1975, 47, 970. i2j Spangler, G. E.; Colllns, C. I . Anal. Chem. 1975. 47, 403. (3) Karasek, F. W.; Kim, S. H. “Study of Technology Relatlng to Plasma Chromatography Sensing Tubes”; Flnal Report, Contract #8SU7700227, Unlverslty of Waterloo Research Institute, Waterloo, Ontario, Canada, Dec 1980. (4) Casslco, J. P.; Sickenberger. D. W.; Spangler, G. E.; Vora, K. N. J. Fhys. E, Scl. Instrum. 1983, 16, 1058. (5) Spangler, G. E.; Cohen, M. J. I n “Plasma Chromatography”; Carr, T. W., Ed.; Plenum Press: New York, 1984; Chapter 1. (6) Tou, J. C.; Boggs, G. U. Anal. Chem. 1976, 48, 1351. (7) Carr, T. W. J. Chromatogr. Sci. 1977, 15, 85. (8) Hagen, D. F. Anal. Chem. 1979, 51, 872. (9) Kim, S. H.; Spangler, G. E. Anal. Chem. 1985, 57, 569. (10) Balm, M. A. Ph.D. Thesis, Washington State Unlverslty. 1983. (11) Carroll, D. I.; Dzldlc, I.; Stlllwell, R. N.; Hornlng, E. C. Anal. Chem. 1975, 47, 1956. (12) Karasek, F. W.; Kim, S. H.; Hill, H. H., Jr. Anal. Chem. 1976, 48, 1133.
RECENED for review June 18,1984. Resubmitted April 8,1985. Accepted April 8, 1985.
Simultaneous Evaluation of 7-Hydroxycoumarin Excited States by Synchronous Luminescence Spectrometry Claudio G. Colombano and Osvaldo E. Troccoli* Laboratorio de Andlisis de Trams-Facultad de Ciencias Exactas y Naturales, Buenos Aires, Argentina
The usefulness of synchronous luminescence spectrometry (SLS) for the analysis of complex luminescence mixtures has been recently demonstrated. It has been employed In our laboratory to study the fluorescence behavior of a natural coumarin: 7-hydroxycoumarin (7HC). The SLS spectra obtained at dlfferenl pH values have demonstrated the capablltty of asslgnlng the varlous transittons to the equlllbrium forms of the 7HC. A new band with maximum at 431 nm (exclting at 345 nm) can be assigned to the catlonlc form at H,, = -6. Comparlsons are made between the expected (theoretical) Ams and the experimental values, for two values of AX: 20 and 65 nm.
Fluorescencebehavior of coumarin derivatives, as a powerful analytical tool for their characterization and evaluation, is well documented (1-8). It has also been shown that fluorescence spectra and yields are highly dependent on the pH of the solutions (41,and the distribution of the different species in the ground state causing this pH dependence has recently been studied (5). 0003-2700/85/0357-1907$01.50/0
One of the difficulties found in these studies is the overlapping of the emission spectra of the different species. Several workers (5,9-12) have proposed deconvolution methods to obtain the individual spectral contributions to the main fluorescence band observed at 453 nm. As previously described, the synchronous luminescence spectrometry (SLS) (13)involves driving both, excitation and emission monochromators, simultaneously at either a fixed wavelength interval (14,15) or a fixed energy interval (16). Raman solvent bands and Rayleigh scattering are thus avoided or diminished. Furthermore as the compounds show different Stokes shifts,the spectra so obtained are “clearer”and simpler, allowing the study of overlapped bands. The bands appearing in SLS spectra are narrower than those of fluorescence spectra, and one of the major advantages of SLS over fluorescence is that all spectra are plotted in the same wavelength scale. The SLS has recently been used for the analysis of complex luminescence mixtures (17-21)and it has proven its capabilities as a simple way to acquire analytical information. In this work, SLS is used to study the luminescence behavior of the 7-hydroxycoumarin (7HC) as a function of pH, and it will be shown that it is possible to assign different bands to 0 1985 American Chemical Soclety
1908
ANALYTICAL CHEMISTRY, VOL. 57, NO. 9, AUGUST 1985 2
1
4
IC I (U.i
5
A(nm)
Excitation spectra of 7HC at different pH (H,): (1) H , = -6 ( A , = 431 nm); (2) from H, = -4 to pH 3.5 (ha,,,= 431 and 453 nm); (3) pH 7.7 (he,,,= 453 nm); (4) pH 11 (Aern = 453 nm). Figure 1.
Unml
Figure 2. Emission spectra of 7HC at different pH (H,): (1) H, = -6 (hex= 345 nm); (2) H , = -4 (bX = 325 nm); (3) pH 0 (hex= 325 nm); (4) pH between 2 and 11 (hex= 325 and 366 nm).
the species either in the ground state or in the excited state.
EXPERIMENTAL SECTION Apparatus. Absorption spectra were obtained with a Zeiss DMR 11 spectrophotometer using a 2.5 nm spectral band-pass. Fluorescence measurements were performed on a Perkin-Elmer LS5 spectrometer whose output is automatically corrected for instrumental response by means of a Rhodamine B quantum counter and fitted with a Hamamatsu R928 photomultiplier tube. All pH measurements between 1 and 11 were done with a Metrohm E 253B fitted with a EA 120 combined glass electrode. Reagents. 7-Hydroxycoumarinwas obtained from different natural sources and purified to spectroscopic grade in the Natural Products Laboratory and its degree of purity was controlled by HPLC, IH NMR, and I3C NMR. All coumarin solutions were prepared by proper dilution of a 2.0 X lo4 M methanolic solution with bidistilled water (under quartz)-final methanol content was ca. 1%-to 2.0 X lo4 M. All chemicals used for pH controls were analytical or spectroscopic grade.
RESULTS AND DISCUSSION The absorption and luminiscence spectra were performed between 200 and 500 nm at different pH (Ho) values. Several acid solutions were used to adjust the solutions acidity at the desired levels: (i) H,= -6 to pH 5.5, HC104solutions; (ii) pH 7.7, KH2P04/NaOHsolution; (iii) pH 11, NaOH solution. Ho values were obtained according to Paul and Long (22). From the excitation (absorption) and emission spectra (Figures 1 and 2) it can be shown that the neutral form (N) is excited a t 325 nm with an emission band at 400 nm, while the anionic form (A-) wavelengths are 366 and 453 nm, respectively. The emission band appearing at 478 nm (pH 0) is assigned to a tautomeric form (T) which seems to be stable only in the excited state (5). All wavelengths are in good agreement with the literature (3-5). The band appearing a t 431 nm a t Ho= -6 has not been previously described, and it is probably due to a cationic form (C+)of the 7HC, when it is excited as a cation at 345 nm. The 4-methyl-7-hydroxycoumarin shows a similar behavior (23).
3
a 550
Figure 3. Synchronous emission spectra of 7HC at different pH (H,). Ah = 20 nrn (1) -6; (2) -2; (3) 0; (4) 2; (5) 5.5; (6) 7.7. Scale: from 2 to 5 (X50); 1 (X10); 6 (X5).
Two different techniques of finding the best Ah are available: (i) the classical “trial and error” and (ii) the Lloyd et al. (14)predicting scheme. Several attempts were made in order to find out the optimum AA in the terms of sensitivity, and the results were checked against Lloyd’s predictions. Wavelength intervals between 10 and 150 nm were used, some of which included the difference between the excitation and emission maxima. Figures 3 and 4 show the spectra obtained with Ah = 20 nm and Ah = 65 nm, and different pH (Ho). Synchronous
ANALYTICAL CHEMISTRY, VOL. 57, NO. 9, AUGUST 1985
I909
Table I. Experimental and Theoretical Synchronous Emission Wavelengths and the Postulated Species Involved in Each Transition AA, nm
exptl
nm theoretical
65
395 409 412 419 445
394 405 413 418 440
20
A*em
nm
X*em,
325 325 325 345 366
nm
ground state
excited state N C+
400 430 453 431 453
N N N
A-
C+
C+
A-
A-
N N’ A-
N AAAA-
369 465
369 460
325 445
400 453
479
479
459
479
N’ A-
According to the working pH.
hsem 1 nm
A!m(nml
Figure 4. Synchronous emission spectra of 7HC at different pH. Ah = 65 nm (1) 1; (2) 2; (3) 5.5; (4) 7.7; (5) 11. Scale: 1 (X10); 2 (X8); 3 (X7); 4 (X0.5); 5 (X0.3).
nm.
spectra at very high acidities are shown in Figure 5. A blue shift is thus observed with decreasing the hydrogen ion concentration. Three transitions can be “followed”simultaneously when operating at pH values between 1 and 11 (Figure 4). The band maximum appearing in 395 nm shows a decrease in intensity when pH is raised, whereas the band at 412 nm increased ita intensity with pH. A new band at 445 nm appeared at pH 5.5 whose intensity is also pH dependent. When a Ah = 20 nm is used, the synchronous spectra are more complex but still well resolved. A 398 nm band is observed a t Ho = -6 which is supposedly due to the same transition as before. There is a band at 421 nm when the pH is higher than 7 and between these two acidities a two-band system can be observed. The sharp band a t 369 nm has a maximum intensity at pH 0 and the bands with peaks at 465 and 479 nm show a similar pH dependence as before. Emission spectrum of 7HC in neutral absolute methanol shows a unique band at 396 nm when exciting at 325 nm which has been assigned to the neutral species (N) (4,5) since there is little possibility of finding ionic species in such medium. Similarly, an emission band a t 400 nm (when exciting at 325
nm) appears in aqueous solutions (Figure 2) at pH 0 but less intense. Supposedly this band belongs to the neutral species though it disappears when the pH is raised or lowered; the emission band a t 431 nm (exciting at 345 nm) when working at lower pH values is assigned the C+species, while the band appearing at 453 nm (exciting at 366 nm) in strongly basic medium is due to the A- species. Synchronous emission bands were assigned through a study of the synchronous fluorescence intensity (If“) vs. pH (Ho = -6, -4, -2; pH 0,1,2,3.5,5.5,7.7,and 11) and assuming that for Ho= -6 and pH 11 there only were one single species and the whole range of mixtures between those extreme pH (Ho) values. Moreover, theoretical Ls were calculated after Lloyd, using hemS= 2~*,mh*,,(h*e, + A*,, - Ah)-’ (1) where Ls is the synchronous emission wavelength maximum, A*,, is the emission wavelength maximum, Axe, is the excitation wavelength maximum, and AA is the wavelength difference between excitation and emission maximum, as a way to confirm the former assignments. The results presented in Table I are in very good agreement with the experimental
Flgure 5. Synchronous emission spectra at high acidity. (1) H o = -6; (2) Ho = -2; (3) pH 0.
A x = 65
1910
Anal. Chem. 1985, 57, 1910-1912
Registry NO.7-HC (N),93-35-6;7-HC (C+),58048-06-9;7-HC (A-), 32942-70-4.
LITERATURE CITED
'C
A-
Flgure 6.
values, showing that it is possible to postulate the following equilibria (Figure 6). Attempts to assign a band to the tautomeric form (T)were unsuccessful due probably to the obscuring of that band, which should appear at 421 nm (AA = 65 nm) or 397 nm (Ah = 20 nm) according to eq 1with A*, = 325 nm and A*, = 478 nm, produced by the other bands. On the other hand, when AA = 153 nm (A*,, - A*, for the tautomer), eq 1predicts a band a t 478 nm, but as the neutral-anionic transition appears a t 471 nm and its quantum efficiency is greater than that of the neutral-tautomeric form transition (5). Again, it was not possible to observe that band. The experimental data for Aems are in very good agreement with the theoretical values from eq 1, and this fact could mean that the contributions of the different species to the main bands are negligible.
Wolfbeis, 0. S.; Fiirlinger, E . ; Kroneis, H.; Marsoner, H. Fresenlus' 2 Anal. Chem. 1983, 314, 119-124. Dienes, A.; Shank. C. V.; Kohn, R. L. IEEE J . Quantum Electron. 1973, OE-9, 833. Sherman, W. R.; Robins, E. Anal. Chem. 1988, 4 0 , 803-805. Fink, D. W.; Koehler, W. R. Anal. Chem. 1970, 42, 990-993. Moriya, T. Bull. Chem. Soc. Jpn. 1983, 56, 6-14. Yakatan, G. J.; Juneau, R. J.; Schulman, S. G. Anal. Chem. 1972, 44, 1044- 1046. Nakashima, M.; Sousa, J. A.; Clapp, R. C. Nature (London).M y s . Sci. 1972, 235, 16-18. Beddard, G. S.; Carlin, M. S.; Davidson, R. S. J . Chem. Soc., ferkln Trans 1977, 2 , 262-267. Miller, T. C.; Faulkner, L. R. Anal. Chem. 1978, 48, 2083-2088. Rechsteiner, C. E.; Gold, H. S.; Buck, R. P. Anal. Chlm. Acta 1977, 95, 51-58. Gold, H. S.; Rechsteiner, C. E.; Buck, R. P. Anal. Chlm. Acta 1978, 103, 167-173. Gold, H. S.; Rasmussen, G. T.; Mercer-Smith, J. A.; Whitten, D. G.; Buck, R. P. Anal. Chim. Acta 1980, 722, 171-178. Lloyd, J. B. F. Nature (London),Phys. Scl. 1971, 231, 64-65. Lloyd, J. E. F.; Evett, I. W. Anal. Chem. 1977, 49, 1710-1715. Vo-Dinh, T. Anal. Chem. 1978, 50, 396-404. Inman, E. L., Jr.; Winefordner, J. D. Anal. Chem. 1982, 54. 2018-2022. Lloyd. J. B. F. Analyst (London) 1975, 100, 82-95. Andre, J. C.; Bouchy, M.; Nlclause, M. Anal. Chlm. Acta 1977, 92, 369-378. Blackledge, R. D. J . Forenslc Sol. 1980, D5, 583-588. Lloyd, J. E. F. Analyst (London) 1980, 105, 97-109. Vo-Dinh, T.; Gammage, R. E.; Martinez, P. R. Anal. Chem. 1981, 53, 253-258. Paul, M. A.; Long, F. A. Chem. Rev. 1957, 57, 1-45. Drexhage, K. H. "Topics in Applied Physics"; Voi. 1, Dye Laser: Schiifer, F. P., Ed.; Springer: Berlin, 1973: Chapter 4. I
ACKNOWLEDGMENT Acknowledgments are made to A. B. Pomilio for the coumarins and to A. L. Peuriot for his assistance in literature searching.
RECEIVED for review October 22, 1984. Accepted April 12, 1985. We are indebted to CONICET, SUBCyT, and UBA for financial support.
Experimental Comparison of Relative Responses for Alternating Current and Square Wave Polarography with Irreversible and Nearly Irreversible Systems Ari U. Ivaska* Department of Analytical Chemistry, Abo Akademi, 20500 Turku 50, Finland
Donald E. Smith' Department of Chemistry, Northwestern University, Euanston, Illinois 60201
The relative responses of ac and square wave polarography were studied in four lrrevetslble or nearly ilreverslblesystems. The in-phase component of the fundamental harmonic ac current was compared wlth the net current of the square wave technique. The two methods were found to give very slmilar responses in all lour systems when the same frequency and amplitude were used in the pertubation wave forms.
We have noted that direct comparisons of data obtained by ac and square wave polarographic or voltammetric methDeceased (January 1985). 0003-2700/85/0357-1910$01.50/0
ods, using comparable experimental conditions (same input wave form frequency and magnitude, as well as the same reactant and electrolyte concentration, etc.) are essentially nonexistent. We think that this situation deserves some form of empirical study to determine whether the two alternating potential techniques produce similar or divergent Faradaic results. It appeared to us that it would be most useful to compare data responses for irreversible or nearly irreversible systems. Such measurements are the most challenging as far as charging current compensation is concerned. Published theory (1-4),supporting experimental results (5-IO), and a review chapter (11) have given clear evidence that ac polarography is applicable and useful with irreversible systems. Publications considering irreversible systems for square wave polarography are infrequent (12),but this is of no consequence to the present investigation. 0 1985 American Chemical Society
