Anal. Chem. 1987, 59, 1105-1112
impurity absorption, for example, may militate against employing this region of the excitation spectrum. Therefore, the IFS values given in Table IV, while not maximal, are useful indicators for evaluating the relative sensitivities of the RPHPLC fluorescence detection method of the CBI derivatives. The principal differences in CBI values clearly reflect the effect of variation in af,since Ax(mar)and WllZ remain essentially constant throughout the series. The IFS values confirm the unsuitability of CBI bis-derivatized Lys and CAD, as well as TBBI-t-Bu and DMA-CBI-t-Bu, for fluorescencebased detection. These results compare favorably with similar studies with other commonly employed amine and amino acid fluorescence derivatives, most notably the dansyl analogues which are 10-fold less efficient emitters (afvalues decrease by an order of magnitude) in mixed aqueous solvents. The emission maxima are also shifted from 500 nm (organic solvents) to 580 nm (H,O) and the band width is decreased in water (6,7). Even with these adverse properties, picomole and lower detection levels have been reported for dansyl derivatives (6). The fluorescence quantum yields for the OPA/2-ME derivatives (7)were found to range from 0.33 to 0.47. However, the instability of these derivatives required that the af determinations be performed on the labeled amino acids generated in situ in the presence of 0.01 M aqueous sodium borate (pH 9.0). Under these conditions OPA/2-ME derivatives of amides of amino acid and small peptides have reduced fluorescence quantum efficiencies (@f C 0.04). The CBI adducts of amines, amino acids, and small peptides exhibit excellent fluorescence emission characteristics. The high quantum efficiencies for fluorescence, the constancy of the afvalues with variation in solvent composition, the high efficiencies in aqueous solutions, the relatively narrow emission values of both the UV and visible bands, and the high Ax(max)
1105
absorption bands greatly favor these derivatives as exceptionally promising candidates as fluorogenic labels for very sensitive fluorescence-based methods of analysis.
ACKNOWLEDGMENT This research was generously supported by funds from the Kansas Commission on Advanced Technology and Oread Laboratories, Inc. LITERATURE CITED (1) Srinivasachar, K.; Carlson, R. G.; Glvens, R. S.; Matuszewski, B. K. J . Org. Chem. 1988, 5 1 , 3978. (2) Matuszewski, B. K.; Givens, R. S.; Srinivasachar, K.; Carlson, R.; Higuchi, T. United States Patent Application, submitted 1985. (3) de Montigny, P.; Stobaugh, J. F.; Givens, R. S.; Carlson, R. G.; Srinivasachar, K.; Sternson, L. A.; Higuchi, T. Anal. Chem., preceding paper in this issue. (4) deMontigny, P.; Sternson, L.; Repta, A.; Stobaugh, J.; Higuchi, T United States Patent Application, submitted 1985. (5) Roach, M. C.; Harmony, M. D. Anal. Chem. 1987, 5 9 , 411. (6) Chen, R. F.; Scott, C.; Trepman, E. Blochim. Blophys Acta 1979, 576, 440-445. (7) Chen, R. F. Arch. Biochem. Biophys. 1987, 120, 609. (8) Chen, R. F.; Smith, P. D.; Maly, M. Arch. Biochem. Biophys. 1978, 189, 241-250. (9) Roth, H. Anal. Chem. 1971, 4 3 , 880. (IO) Melhuish, W. H. J . Phys. Chem. 1981, 6 5 , 229. (11) Meech, S. R.; Phillips, D. J . Pbotochem. 1983, 23, 193. (12) Lloyd, J. B. F. J . Chromatogr. 1979, 178. 249. (13) Parker, C. A. Photoluminescence of Solutions; Elsevier: Amsterdam, 1968; pp 252-258, 262-268. (14) Berlman, 1. B. Handbook of Fluorescence Spectra of Aromatlc Molecules, 2nd ed.; Academic: New York, 1971; p 405. (15) Calvert, J. G.; Pitts, J. N., Jr. Photochemistry; Wiley: New York, 1966; p 800. (16) Stobaugh, J. F.; Kristjanson, F.; Wong, 0.;Hayakawa, N.. unpublished results. (17) Singh, H. N.; Hinze, W. L. Analyst (London) 1982, 107, 1073-1087. (18) Jones, B. N.; Paabo, S.; Stein, S J . Li9 Chromatogr. 1981, 4 , 565-586.
RECEIVED for review March 25, 1986. Resubmitted July 7 , 1986. Accepted December 18, 1986.
Existence of Microdroplets and Dispersed Atoms on the Graphite Surface in Electrothermal Atomizers John McNally and James A. Holcombe* Department of Chemistry, University of Texas at Austin, Austin, Texas 78712
Concentration studies, time and spatially resolved absorbance profiles, and aerosol deposltlon vs. manual pipettlng studies have been obtained for Cu and Au. The activation energy of release for Cu Is 30 f 2 kcaVmd (and also represents a AH) and for Au is 71 f 3 kcai/mol. A strong copper-graphite Interaction accounts for the characteristic broader half-widths and a spatlaliy nonuniform atom dlstributlon on the rislng portion of the peak. The relatively weak metal-graphlte Interactions observed for Au account for the narrower halfwldths and a relatively uniform atom distrlbution during atomization. Data suggest that Cu desorbs from the graphite surface as individual atoms, whlch results in an apparent first-order release. For Au the formation of mlcrodroplets or hemispherical droplets with desorption occurrlng from the droplet surface or at the meta-raphite Interface Is proposed. An apparent order of release
