Anal. Chem. 1991, 63,744-746
744
interferences. Commercial software for data accumulation does not routinely do this. The GDMS technique has shown that it is capable of high-precision isotopic measurements of metallic samples, provided that molecular interferences are eliminated. Precision better than 0.07% RSD has been demonstrated with commercial data collection software that is not yet optimized for such work. With reasonable improvements, the attainment of precision better than 0.01% should be possible. Finally, the system of Pd metal containing isotopes of hydrogen has given insights into the gas-phase reactions in the negative glow region, which may be of interest in the study of catalysis and the chemistry of small metal atom clusters.
ACKNOWLEDGMENT We thank H. Harmon for preparing the Pd rod electrodes.
LITERATURE CITED
I
,
14) ‘
(5) (6) (7)
Harrison, W. W. J. Anal. At. Spectrom. 1980, 3 , 067-072. Sanderson, N. E.;Hall, E.;Clark, J.; Charalambous, P.; Hall, D. Mikrochim. Acta 1987, 1 , 275-290. Jakubowski, N.; Stuewer, D.; Vieth, W. Fresenius Z . Anal. Chem. 1888, 331. 145-149. Kina. F. L.: McCormack, A. L.; Harrison, W. W. J. Anal. At. Spectroih. 1988, 3 , 003-006. Loving, T. J.; Harrison, W. W. Anal. Chem. 1883, 55. 1526-1530. Scott, C. D.; Mrochek, J. E.; Scott, T. C.; Michaels, G. E.; Newman, E.; Petek, M. Fusion Techno/. 1990, 18, 103-114. De Bievre, P.; Barnes, I. L. Int. J. Mass Spectrom. Ion Process. 1985, 65, 21 1-230.
RECEIVED for review October 2, 1990. Accepted January 7 , 1991. Research was sponsored by the U S . Department of Energy, Office of Energy Research, under Contract DEACOS-MOR21400 with Martin Marietta Energy Systems, Inc.
CORRESPONDENCE Selective Detection in Pump-and-Probe Photolytic Fragmentation Fluorescence Spectrometry Based on Differences in Photofragment Electronic State Distributions Sir: In laser photolytic fragmentation fluorescence spectrometry (LP-FFS), nonfluorescent gaseous molecules are photolyzed with intense radiation from a pulsed laser, often a t 193 nm, to produce small (usually neutral) fluorescent fragments, such as CH, CN, C2, OH, or NH. Because virtually any analyte can be converted to fluorescent species in this manner, LP-FFS extends the analytical applicability of molecular fluorescence spectrometry to the many analytes that cannot be determined by conventional fluorescence measurements (1-4). Determinations via LP-FFS can be performed by using one or two lasers. In a “one-laser”experiment, a single pulsed laser is used to both produce and excite fragments. Only those fragments that are formed directly in emissive excited states in the fragmentation process or are excited by the tail of the photolysis laser pulse are detected. In many cases, the efficiency with which excited fragments are formed in a one-laser experiment is relatively small. This problem is remedied by performing a “two-laser” LP-FFS measurement, wherein photolytically generated fragments are promoted to emissive excited states by use of a “probe” beam from a pulsed dye laser (4,5). The principal advantages of 193-nm photolysis radiation in LP-FFS are the relatively high photon energy and (more important) the very high fluences obtainable from pulsed excimer lasers operating with ArF. The latter characteristic of excimer lasers is especially useful, because most UV photofragmentations proceed via absorption of several photons by the parent molecule and/or intermediate fragments (6, 7); use of high photolysis fluences enables partial or total saturation of these absorption transitions to be achieved, greatly diminishing the dependence of the fragment fluorescence signal on the photolysis laser fluence (8). When 193-nm photolysis radiation is used, “one-laser” LP-FFS spectra observed for structurally similar organic molecules usually are virtually indistinguishable (8). Thus,
193-nm LP-FFS does not appear inherently to be a highly selective technique. It has hitherto been tacitly assumed that selective determinations of individual analytes in multicomponent samples via LP-FFS could generally be achieved only by (a) chromatographic separation of sample constituents prior to fragmentation (9) or (b) expansion of samples into a supersonic molecular beam followed by selective photolysis of individual sample constituents using a tunable photolysis laser (analogous to the selective multiphoton ionization mass spectrometric experiments reported by Lubman and coworkers ( 1 0 , I I ) ) . In the latter case, it is normally necessary to use a dye laser as the photolysis source; the shortcoming of such a procedure is the much lower fluence available from a pulsed dye than an excimer laser. Herein we describe a third, previously unexplored, approach to selectivity in LP-FFS: the use of a probe laser to detect different state distributions of fragments formed by 193-nm photolysis. This approach is exemplified by a study of two isomers of molecular formula C3H,0: acrolein (H,C=CHCHO) and propargyl alcohol (HC=CCH,OH). A supersonic expansion is used in order to produce fragment fluorescence spectra having higher spectral resolution than is possible via effusive sample-introduction techniques.
EXPERIMENTAL SECTION Apparatus. The instrumentation used for LP-FFS measurements has been described in detail elsewhere ( 3 , 5 ) . Briefly, radiation from a pulsed excimer laser operating at 193 nm
(Questek 2210, operating with ArF, 10-Hzrepetition rate, IO-ns pulse duration, 40-120-mJ pulse energy) is focused by a 15 cm focal length plano-convex fused-silica lens into a rectangular vacuum chamber of ca. 450-mL volume. The pressure in the vacuum chamber (evacuated by a turbomolecular pump) is typically 2 x Torr in the absence of sample. Counterpropagating 7-ns probe laser pulses are generated either by a NdYAG-pumped dye laser (Quanta-Ray DCR-SA10 and PDL-2 with WEX) or an excimer-pumped dye laser (Questek 2210, operating with XeC1, and Lambda-Physik FL-2000).
0003-2700/91/0363-0744$02.50/0Q 1991 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 63, NO. 7, APRIL 1, 1991
-.-3 m
~\ co
-
i oi
CH
C
v1
.-v1
11
1
C
.-0
I
pure a c r o l e i n
A ) Acrolein
745
E) Propargyl Alcohol
E
Y
349.6
367.9
3ab.e
404.5
422.8
44i.i
459.4
477.7
Emission Wavelength ( n m ) 350
366.7
400.2
383.4
416.9
Figure 1. One-laser LP-FFS spectra of (A) acrolein and (B) propargyl
alcohol produced by photolysis at 193 nm. For both compounds, the pressure was ca. lo-‘ Torr and the laser pulse energy was 50 mJ.
The angle between the focused ArF laser beam and the unfocused dye laser beam is 1 8 0 O . The beams are carefully overlapped to produce maxima1 fluorescence signals; the fluorescent “spot” produced by the overlapped beams is a rectangle of approximately 0.1 X 2.5 cm dimensions. Both laser beams are propagated at right angles to the expansion and intersect the expansion at a distance of 2.5 cm from the nozzle. Fluorescence is collected at 90’ to the laser beam paths and 90° to the expansion, collimated by a 12.7 cm focal length plano-convex lens, and focused on the entrance slit of a 1-m monochromator (Jobin-Yvon HR-1000) by a 15.2 cm focal length cylindrical lens. The monochromator typically is operated at a band-pass of 0.1 nm. Signals from a photomultiplier tube (Burle 8850) are processed by a boxcar averager (Stanford Research Systems SR-250). Samples, diluted with argon in a reservoir (typical reservoir pressure = 780 Torr), are expanded through an 0.5-mm circular orifice into the vacuum chamber via a pulsed valve (R. M. Jordan C-211SS) and control unit that generates ca. 200-ps gas pulses at a 10-Hz repetition rate. The pulsed-valve control unit triggers both the pulsed valve and the firing of the photolysis and probe lasers; the delay between the two lasers pulses is controlled by a variable-delay circuit. Chemicals. Acrolein and propargyl alcohol (Aldrich)were used as received. Laser gases were obtained from Spectra Gases and MG Industries.
Emission Wavelength ( n m )
Figure 2. Two-laser LP-FFS spectra of (top) pure acrolein and (bottom) pure propargyl alcohol, both at 10’’ Torr. The spectra were produced by photolysis at 193 nm and probe-laser excitation at 382 nm. The center spectrum was obtained, under identical conditions, from a 1/99 (w/w) acrolein/propargyl alcohol mixture. The feature at 382.0 nm in all spectra is scattered probe laser radiation.
~
349 5
363
376 4
389 9
403 4
416 9
Emission Wavelength ( n m )
RESULTS AND DISCUSSION
Figure 3. Dependence of the two-laser LP-FFS spectrum of acrolein on the probe-laser wavelength. The photolysis-laser wavelength was 193 nm in all cases.
One-laser LP-FFS spectra of acrolein and propargyl alcohol, obtained by using 193-nm photolysis radiation, are shown in Figure 1. The major emissive fragments observed in both spectra are C2 (Swan band system a t 1434 nm and Deslandres-d’Azambuja band system a t 1410 nm), CH (431.4 nm), and CO (368.5 and 389.3 nm). Acrolein produces a more intense LP-FFS spectrum than propargyl alcohol under the conditions used in this work. While there are some differences in the relative fragment fluorescence intensities in the two spectra (e.g., the CH/C2 Swan band intensity ratio is greater for propargyl alcohol than acrolein), the spectra are sufficiently similar that determination of one isomer in the presence of the other is, a t best, difficult. However, dramatic differences are observed in the LP-FFS spectra of the two compounds when a probe laser is used. As shown in Figure 2, when acrolein is photolyzed at 193 nm, followed by probing at 382.0 nm (delay between photolysis and probe pulses = 400 ns), a series of emission bands is observed, with the most intense features occurring in the 358-363-nm region. The most intense features in the two-laser LP-FFS spectrum of acrolein are anti-Stokes (energy of emitted radiation greater than that of the probe-laser photons), suggesting that a t least part of the two-laser spectrum must result from probe-laser excitation of “hot” fragments. In contrast, under identical photolysis and probe conditions, no detectable fluorescence is observed for propargyl alcohol.
The series of emission bands observed for acrolein in the two-laser experiment is absent in the one-laser LP-FFS spectrum (compare Figures 1 and 2). The two-laser LP-FFS spectrum of acrolein is quite sensitive to small changes in the probe laser wavelength (Figure 3). The emission bands excited by the probe laser are assignable to vibronic transitions band system of C2. As shown in Figure in the C Ing A ‘nu 4, an energy-level diagram for C2 (12), this system of fluorescence bands is excited by absorption of probe-laser excited state radiation by C2 fragments formed in the A ‘nu as a consequence of 193-nm photofragmentation. A ‘nufluorescence From the absence of similar C from Cz in the two-laser LP-FFS spectrum for propargyl alcohol, we infer that the number density of Cz fragments in the A lIIU excited state produced by 193-nm photolysis of propargyl alcohol is a t least a factor of lo4 smaller than that produced under the same conditions from acrolein. Thus, although it is not evident from the one-laser LP-FFS spectra of the two compounds, 193-nm photolysis of acrolein and propargyl alcohol generates substantially different electronic state distributions of the Cz fragment. As noted in Figure 2, the two-laser LP-FFS spectrum of a 1/100 acrolein/propargyl alcohol mixture is identical with that of pure acrolein. The “discrimination factor” (11)for detection of acrolein in the presence of propargyl alcohol is a t least 2
-
-
Anal. Chem. 1991, 63,746-750
746
Cz Swan bands d % g - - > a 3 n , De sl a n d r e s - d ' Az a m buj a bands
C
C 'TI, - - > A 'TI,
most molecules under intense UV laser illumination, it is currently not possible to predict with any degree of confidence the photolysis conditions (wavelength, pulse energy, pulse duration) required to maximize such differences in the "two-laser" LP-FFS spectra of a particular set of closely related analytes. Current evidence strongly suggests, however, that the behavior described here for acrolein and propargyl alcohol does not represent an isolated instance; thus, numerous instances in which the phenomenon can be exploited analytically are anticipated. Fragment state distributions, as inferred from such "photolyze-and-probe" experiments, also can be utilized to make inferences regarding molecular photofragmentation mechanisms; the use of two-laser LP-FFS for this purpose for acrolein and its derivatives will be reported elsewhere (13).
ACKNOWLEDGMENT We thank Priscilla J. Gannicott for assistance in performing the experimental work. LITERATURE CITED ,
I
:
-
,
,
,
______..__
_--- - - - -
0 20
10
re Figure 4. Energy-level diagram for Cp.The upward arrows indicate state, that can be absorption transitions, originating in the A induced by a probe laser: the downward arrows indicate specific vi.- A (Deslandres-d'Azambuja) bronic transitions in the C emlssion system of C2 observed by probe-laser illumination following 193-nm photolysis of acrolein. (Adapted from data compiled by Huber and Herzberg ( 12) )
'n,
'ne
'nu
X lo3 (Le., acrolein can be detected in a mixture that is 0.05% acrolein and 99.95% propargyl alcohol). The limit of detection (LOD) for acrolein under these conditions (defined as the minimum pressure of acrolein needed to generate a Cz fluorescence signal at 384.6 nm equal to twice the standard deviation of the "blank" observed for the empty chamber) is 7 x lo-* Torr, which, assuming a chamber volume of 450 mL, translates into a LOD for acrolein of 10 pg. In summary, while 193-nm photolysis of isomeric nonfluorescent compounds usually produces the same major fragment species for each isomer, the electronic state distributions of these fragments may be substantially different for different isomers. Although such differences may be difficult or impossible to infer from the one-laser LP-FFS spectra of the compounds in question, they may be detected relatively easily by use of a second laser to probe the fragment states formed by the first laser. In view of the lack of detailed understanding of the photofragmentation mechanisms for
Rodprs, M. 0.;Asai, K.: Davis, D. D. Appl. Opt. 1980, 19, 3597-3605. Halpern, J. 6.;Koker. E. 6.;Jackson, W. M. Anal. Chem. 1983, 55, 2000-2002. Wehry, E. L.; Hohmann, R.; Gates, J. K.; Guilbault, L. F.; Johnson, P. M.; Schendel, J. S.; Radspinner, D. A. Appl. Opt. 1987, 2 6 , 3559-3565. Rodgers, M. 0.:Davis, D. D. Environ. Scl. Technol. 1989, 2 3 , 1106-1112. Schendel, J.; Wehry, E. L. Anal. Chem. 1988, 60, 1759-1762. Gedanken, A.; Robin, M. B.; Kuebler, N. A. J . Wys. Chem. 1982, 86, 4096-4101. Colson. S. D. Nucl. Instrum. Methods 1987, 8 2 7 . 130-135. Jinkins, J. G.; Wehry, E. L. Appl. Spectrosc. 1989, 4 3 , 861-865. Guilbault, L. F.;Hohmann, R.; Wehry, E. L. J . Chromatcgr. 1989, 475, 237-245. Lubman, D. M. Anal. Chem. 1987, 59, 31A-40A. Sin, C . H.; Tembruell, R.; Lubman, D. M. Anal. Chem. 1984, 56, 2776-2761. Huber, K. P.; Herzberg, G. Constants of Diatomic Molecules; Molecular Spectra and Molecular Structure, Vol. 1V; Van Nostrand Reinhold: New York, 1976; pp 112-114. S. c. Lee, B. J. Stanton, and E. L. Wehry, unpublished results.
Sang C. Lee Bobby J. Stanton E. L. Wehry* Department of Chemistry University of Tennessee Knoxville, Tennessee 37996
RECE~VED for review November 5,1990. Accepted December 28, 1990. This work was supported by the National Science Foundation under Grant CHE-8822722. Acquisition of the dye laser was via funds provided by the Science Alliance, a State of Tennessee Center of Excellence at the University of Tennessee-Knoxville.
TECHNICAL NOTES On-Line Detection of DNA in Gel Electrophoresis by Ultraviolet Absorption Utilizing a Charge-Coupled Device Imaging System King C. Chan, Lance B. Koutny, and Edward S. Yeung* Ames Laboratory-USDOE and Department of Chemistry, Iowa State University, Ames, Iowa 50011 INTRODUCTION Gel electrophoresis is a powerful technique for the separation of high molecular weight biomolecules such as proteins
and nucleic acids (1). In particular, agarose gel electrophoresis is widely used in the separation of DNA fragments in restriction analysis (2-4). The method most commonly used
0003-2700/91/0363-0746$02.50/00 1991 American Chemical Society
