l-methionine is involved in the methylation of CIa to Cz and then to GI. This is consistent with the work of Lee et al. (20) and Daniels et al. (21).
LITERATURE CITED (1) M. J. Weinstein, G. M. Luedemann, E. M. Oden, G. H. Wagman, J. P.
Rosselet, J. A. Marquez, C. T. Coniglio, W. Charney, H. L. Herzog, and J. Black, J. Med. Chem., 6, 463 (1983). (2) G. H. Wagman, J. A. Marquez, and M. J. Weinsteln, J. Chromatog., 34, 210 (1968). (3) D.J. Cooper, P. J. L. Daniels, M. D. Yudis, H. M. Marigliano, R. D. Guthrie, and S. T. K. Bukhari, J. Chem. Soc., 1971, 3126. (4) G. H. Wagman, E. M. Oden, and M. J. Welnsteln, Appl. Microblol., 16, 624 (1968). (5) W. L. Wllson, G. Richard, and D. W. Hughes, J. Pharm. Sci., 62, 282 i, i a m P. B. Ghosh and M. W. Whitehouse, Biochem. J., 108, 155 (1968). B. K. Lee, R. G. Condon, G. H. Wagman, K. Byrne, and C. Schaffner, J. Antlbiot., 27, 822 (1974). H. Bethke, W. Santi, and R. W. Frei, J. Chromtogr. Scl., 12, 392 (1974). H. Reimann, D. J. Cooper, A. K. Mallams, R. S.Jaret, A. Yehaskel, M. Kugelman, H. F. Vernay, and D. Schumacher, J. Org. Chem., 39, 1451 (1974). J. Sherma and J. C. Touchstone, Anal. Lett., 7 (4,279 (1974). J. R. Benson and P. E. Hare, Roc. Nat. Acad. Sci. USA, 72, 819 (1975). J. F. Lawrence and R. W. Frei, Anal.,Chem., 44, 2046 (1972). R. W. Frei and J. F. Lawrence, J. Assoc. Offic. Anal. Chem., 55, 1259 (1972). F. Van Hoof and A. Heyndrickx, Med. Fak. Landbouwet, 38 91 1 (1973). F. Van Hoof and A. Heyndrickx, Anal. Chem., 46, 286 (1974). D. M. Beniamin, J. J. McCormack. and D. W. Gumu, Anal. Chem., 45, 1531 (1973). W. Wortmann and J. C. Touchstone, in “Quantitative Thin Layer Chromatography”, J. C. Touchstone, Ed., Wiley-Interscience, New York, 1973 F: Abe and K. Sameiima, Anal. Biochem., 67, 298 (1975). R. U. Lemieux and M. A. Barton, Can. J. Chem., 49, 767 (1971). B. K. Lee, R. T. Testa, G. H. Wagman, C. M. Liu, L. McDanlel, and C. Schaffner. J. Antibiot., 26, 728 (1973). P. J. L. Daniels, A. Yehaskel, and J. €3. Morton, 18th Meeting of ICAAC, Chicago, Ill., October 1978, Paper 45.
.-. .,.
0
24
48
72
96
FERMENTATION TIME
120
144
- HOURS
Figure 3. Change in gentamicin Cia, C2,and C, fraction during a fermentation with the addtion of Lmethionine at the start of fermentation
carbon dioxide on the results of quantitative TLC was carried out. No effect was noticed. The variation in the gentamicin complex component area response fraction during a normal fermentation is shown in Figure 2. Gentamicin Cla, Cz, and C1 ratios remained relatively constant throughout the run. When 1-methionine was added at the start of the fermentation (Figure 3), gentamicin C2 and C1 ratios changed continuously. No detectable gentamicin Cla, the least methylated of the components, was present during most of the fermentation. Essentially, only two components were present, C2 and C1, the mono- and dimethylated derivatives of C1,. The gentamicin C1 fraction grew a t the expense of gentamicin Cz. It would appear that
RECEIVED for review January 21, 1977. Accepted March 15, 1977.
Sub-Part-per-Trillion Detection of Polycyclic Aromatic Hydrocarbons by Laser Induced Molecular Fluorescence J. H. Richardson” and M. E. Ando‘ General Chemistry Division, Lawrence Livermore Laboratory, University of California, Livermore, California 94550
Laser-induced molecular fluorescence is shown to be a sensitive and selective means for determining representative polycyclic aromatic hydrocarbons (PAH) In aqueous solutions. The limits of detection of benzene, naphthalene, anthracene, fluoranthene, and pyrene were found to be 19 ppb, 1.3 ppt (parts-per-trillion), lo0 kW) by either angle (29) or temperature (30) tuning to below 220 nm. Laser-induced fluorescence is a very sensitive technique for PAH detection. The detection limits for representative PAH have been extended sufficiently to consider PAH detection in ground water (25). The greater sensitivity and selectivity demonstrated by laser-induced fluorescence should herald further progress in those areas of biochemistry, pollution monitoring, and chemical reaction mechanistic and kinetic studies that have been hindered by insufficiently sensitive analysis. Finally, recent and future advances in second harmonic generation and UV lasers will further widen the class of compounds which can be studied by laser-induced fluorescence. The sensitive detection of simple aromatics as well as PAH will greatly improve the applications of laserinduced fluorescence as an analytical detector (e.g., chromatographic applications).
ACKNOWLEDGMENT We thank D. C. Johnson, L. L. Steinmetz, and B. W. Wallin for technical assistance, R. G. Komoto for consultation in the distillation considerations, and J. A. Paisner for the use of the lithium formate monohydrate doubling crystal. LITERATURE CITED (1)
G.G. WlbaUn, "Practical Fluorescence: Theay,Methods and Techniques",
Marcel Dekker, New York, N.Y., 1973. (2) P. Vlgny and M. Duquesne, Phofocbem. Photobiol., 20, 15 (1974). (3) C. M. O'Donnell and T. N. Solie, Anal. Chern., 48, 175R (1976).
(4) (a) S.Cova, G. Prenna and G. Mazzini, Histocbem. J., 6, 279 (1974); (b) T. Hirschfeld, Appl. Opt., 15, 2965 (1976). (5) B. T.Hargrave and G. A. Phillips, Envlron. Pollut., 8, 193 (1975). (6) J. G. Eden, B. E. Cherrington, and T. T. Verdeyen, I€€€ J . Quantum Nectron. qe-12, 698 (1976). (7) R. A. Nathan, G. D. Mendenhall, J. A. Hassell, and J. D. Wallace, Ind. Res., 17 (13),62 (1975). (6) J. U. White, Anal. Cbem., 48, 2089 (1976). (9) N. J. Harrick and G. I. Loeg, Anal. Cbem., 45, 687 (1973). (IO) H. V. Malmstadt, M. L. Franklin, and G. Horlick, Anal. Cbem., 44, 63A (1972). (11) R. J. Kelly, W. B. Dandliker, and D. E. Williamson, Anal. Cbem., 48, 846 (1976). (12) T. F. Van Gee1 and J. D. Winefordner, Anal. Cbem., 48. 335 (1976). (13) R . J. Lukaslewicz and J. M. Fitzgerald, Anal. Chem., 45, 511 (1973). (14) A. B. Bradley and R. N. Zare, J. Am. Cbem. Soc., 98, 620 (1976). (15) J. H. Richardson, 8. W. Wallin, D. C.Johnson, and L. W. Hrubesh, Anal. Cbim. Acta, 86, 263 (1976). (16) (a) R. B. Green, J. C.Travis, and R. A. Keller, Anal. Cbem., 46, 1954 (1976); (b) M. B. Denton and H. V. Malmstadt, Appl. Pbys. Lett., 18, 465 (1971);(c) L. M. Fraser and J. D. Winefordner, Anal. Cbem., 43,
D. W. Jones and R. S. Matthews, Prog. Med. Cbem., 10, 159 (1974). A. P. Bentz, Anal. Cbem., 48, 454A (1976). W. Giger and M. Blumer, Anal. Cbem., 46, 1663 (1974). M. Novotny, M. L. Lee, and K. D. Bartle, J. Cbromatogr. Scl., 12, 606
(1974). (25) F. P. Schwarz and S. P. Wasik, Anal. Chem., 48, 524 (1976). (26) S.Singh, W. A. Bonner, J. R. Potopowicz, and L. G. Van Uitert, Appl. Pbys. Lett., 17, 292 (1970). (27) J. B. Birks, “Photophysics of Aromatic Molecules”, Wiley-Interscience. New York, N.Y., 1970,pp 70-75. (28) J. Novak, J. Zluticky, V. Kubelka, and J. Mostecky, J. Cbromatogr., 76, 45 (1973). (29) C.F. Dewey, Jr., W. R. Cook, Jr., R. T. Hodgson, and J. J. Wynne, Appl. Pbys. Lett., 26, 714 (1975). (30) R. K. Jain and T. K. Gustafson, IEEEJ. Quantum ,Electron., qe-12, 555 (1976).
RECEIVED for review January 21,1977. Accepted March 15, 1977. Work performed under the auspices of the U.S. Energy Research and Development Administration under contract No. W-7405-Eng-48. Reference to a company or product name does not imply approval or recommendation of the product by the University of California or the U.S. Energy Research & Development Administration to the exclusion of others that may be suitable.
1693 11971) ,.-.
.I.
(17) J,-C:Wright, Water& Sewage Works, 123, 46 (1976);presented at 3rd Annual FACSS Meetina. Philadelohia. Pa.. Nov. 1976. (18) G. F. Kirkbright and C.-GG. delima, Analyst, (London),99, 338 (1974). (19) T. Ya. Gaevaya and A. Ya. Khesina, J. Anal. Cbem. USSR, 29, 1913
,f I,q-7.A.,j.
(20) J. M. Harris, R. W. Chrisman, F. E. Lytle, and R. S.Tobias, Anal. Cbem., 48, 1937 (1976).
Micro Sampling and the Use of a Flow Cell for Coherent Ant i-Stokes Raman Spectrometry L. B. Rogers, J. D. Stuart,’ L. P. Goss, T. B. Malloy, Jr.,* and L. A. Carreira’ Department of Chemistry, University of Georgia, Athens, Georgia 30602
A micro optical cell has been developed to measure coherent antldtokes Raman spectra of organic molecules at low concentrations. The cell Involved the use of standard glass melting point capillaries whose small internal volumes allowed for the precise focusing of the two tunable dye lasers used In the excitation process. A detection limit of 1 ng/pL has been obtalned for the molecule trans-&carotene. No slgnal distortion was evident when solutions flowed through the capillary at 0.1 to 10 mL/min or 9 to 900 cm/s. As a result, its use as a cell in a speciflc detector for hlgh resolutlon, llquld chromatography appears to be feasible.
Coherent anti-Stokes Raman spectroscopy (CARS) is a nonlinear optical technique which has been applied to obtaining Raman spectra with very high efficiencies (1-17). One can obtain by the CARS technique photon efficiencies of up t o 1 in lo2, as compared to efficiencies of 1photon scattered in lo6 to 10’ incident photons generally detected by spontaneous Raman spectroscopy. Since the laser beams can be focused on small volumes, this technique should .be feasible for high resolution liquid chromatography for which the detector volume is typically 10 yL. The CARS process is illustrated schematically in Figure 1. CARS involves the use of two tunable dye lasers set at frePresent address, University of Connecticut, Storrs, Conn. 06268. ‘On leave from Department of Physics and Department of Chemistry, ~ i ~ ~State i University, ~ ~ i ~~ i~ ~i ~State, i ~~ ~i 39762.
quencies w1 and w2.When these two laser beams cross in the sample at the phase-matching angle 8, coherent anti-Stokes emission at w3 = 2wl - o2is generated through the third-order nonlinear polarization. This laser-like beam, w3, appears on the opposite side of the pump beam, wl,from the Stokes beam, w2,a t an angle 8’. The intensity of this signal is greatly enhanced when the frequency interval, w1 - w2 = A, is equal to a Raman-active molecular vibrational frequency. Raman spectra are normally obtained by fixing the frequency of w1 and varying the frequency of w 2 in order to change A. The CARS technique offers not only higher efficiencies but also essentially complete rejection of fluorescence since w 3 is both spatially and temporally removed from the fluorescence signal. That is, the CARS emission a t w3 occurs a t a higher frequency than either of the exciting frequencies. Hence, very efficient (up to 1 in 10’) discrimination of the natural fluorescence of the molecule is possible because molecular fluorescence involves the emission of energy a t lower frequencies than the excitation process. Also, the fluorescence occurs over 4n steradians whereas the CARS signal is emitted in a laser-like beam a t a particular angle 8’. Therefore, the fluorescence can be greatly reduced by observing the beam through a pinhole that is well away from the sample. In our particular setup, the beam passed through an iris 30 cm from the sample. This would correspond to an f value of approximately 100. The maior drawback to the CARS techniaue has been the intensity of the background emission resulting from the nonresonant, third-order susceptibility of the solvent.This i ~ usually ~ ~~ i.allows detection limits for solutes of only about 0.05 M. Recently, Chabay, Klauminzer, and Hudson (15) have ANALYTICAL CHEMISTRY, VOL. 49,
NO. 7,
JUNE 1977
959
