Anal. Chern. 1982, 54, 1202-1204
1202
RotationaIIy CooIed Laser-I nduced FIuorescence/ Gas Chromat ography Sir: Recently, we reported (1-3) on the potential of supersonic jets ( 4 ) for analysis. I t was demonstrated that the simplification and narrowing of laser excited excitation and fluorescence spectra of molecular analytes, provided by their expansion in a monatomic beam, permits substitutional isomers of polycyclic aromatic hydrocarbons to be easily resolved in simple mixtures (3). The dramatic rotational and vibrational cooling accompanying expansion affords vibronic absorption line widths = 1 cm-' for even very large molecules (5). The selectivity of this method was noted (3) to be significantly higher (210 times) than fluorescence line narrowing spectrometry in low-temperature solids (6-8). Thus, rotationally cooled laser-induced fluorescence (RC-LIF) possesses a superior selectivity. However, our earlier work did not address the question of whether RC-LIF can be developed into a quantitative and practical technique. Nor have its detection limits and feasibility of application to the direct analysis of real samples been experimentally investigated. The difficulties associated with quantitation of condensed phase mixtures when a nozzle operating in the continuous mode is employed have been discussed (3). In our opinion, utilization of a pulsed nozzle is not, by itself, a practical solution to this quantitation problem. In this paper we describe a practical method by which RC-LIF is made quantitative and address other questions, vide supra, pertinent to the analytical utility of RC-LIF. The method is essentially the coupling of a simple and inexpensive gas chromatograph (GC) with and in close proximity to the supersonic nozzle. T h e primary role of the GC is not to improve selectivity but to ensure quantitative transfer through the nozzle of molecular analytes. The ultrahigh selectivity still derives from RC-LIF and so the combination is appropriately referred to as RC-LIF/GC. With it the fluorescence intensity as a function of time for any component of a mixture is proportional to the amount of that material introduced into the GC. The RC-LIF/GC apparatus employed in this work is a modification of the continuous nozzle RC-LIF system described previously ( 3 ) . T o demonstrate the exciting capabilities of RC-LIF/GC, we selected mixtures of naphthalene, a-methylnaphthalene, and @-methylnaphthalenefor study. The GC column employed permits physical separation of naphthalene from the methylnaphthalenes. Data for laboratory prepared mixtures are first used to establish that RCLIF/GC is quantitative, highly sensitive, and practical (e.g., acceptable sample turn around time). Following this, the successful application of the technique to the direct determination and quantitation of the above molecules in a crude oil sample is demonstrated.
EXPERIMENTAL SECTION The continuous nozzle RC-LIF apparatus has been previously described in detail (3). Briefly, the excitation source is the frequency doubled output of a Quanta-Ray PDL-1 dye laser pumped by a DCR-1 Nd:YAG laser. Its beam crosses perpendicular to the beam from a supersonic expansion through a 150-pm nozzle. Crossing occurs 5 mm from the nozzle. The resulting fluorescence is collected at right angles to both beams by an f/2.5 lens and then focused into a 0.3 m, f/4.2 monochromator. For this work the monochromator setting was 341.5 nm with a band-pass of 7.5 nm. This wavelength corresponds to a region in which each of the molecules investigated exhibits fairly broad emission bands. Signals from both the monochromator phototube and a reference tube (for normalization) were detected with pulse stretching preamplifiers and measured by a Molectron LP20 laser photometer.
Figure 1illustrates the simple GC used as the sample insertion front end of the supersonic nozzle. A stainless steel KF-10 tee fitting had a rubber septum attached to one arm. Helium carrier gas was connected to the leg of the tee and the other cross arm was connected to the GC column. This consisted of an 8 in. length of 8 mm 0.d. Pyrex tubing packed with 3% OV-101, on 80-100 mesh Chromosorb (Alltech) and a 2-in. length of 1 mm 0.d. capillary tubing which terminated in the 150-pm pinhole. The tee connector injection port was wrapped with heating tape and three additional heaters were wrapped around the glass column. The temperature distribution along the length of the column is shown at the bottom of the figure. These temperatures were measured in the unpacked assembly by moving a thermocouple along the length. The actual operating temperature could therefore have varied slightly from that shown but appeared to be quite constant after -24 h as indicated by the constancy of retention times over several days of operation. The retention times did increase if the voltage applied to the column heaters was decreased. Stock solutions of naphthalene (Aldrich),a-methylnaphthalene (Aldrich), and @-methylnaphthalene (Fluka) in cyclohexane (Fisher, spectroquality) were prepared by weighing of the naphthalenes into volumetric flasks and addition of the solvent. Less concentrated solutions of the three materials and mixtures were prepared by dilution of the stock solutions. The sample of Wilmington crude was obtained from the NBS/DOE Analytical Characterization Group. A 0.5-mL aliquot of the crude was diluted to 1.5 mL with cyclohexane. Chromatograms were obtained by exciting the core of the supersonic jet with a selected laser wavelength and injecting a known volume (up to 10 pL) of the solution onto the column while monitoring fluorescence. The laser wavelengths used were 315.4 nm (P-methylnaphthalene SI origin band), 310.6 nm ( a methylnaphthalene SI vibration), 308.0 nm (naphthalene SI, g1 vibration) and 266.0 nm (into the dense S2 vibrational structure of all three molecules).
RESULTS AND DISCUSSION Figure 2 shows representative chromatograms of a mixture of naphthalene, a-methylnaphthalene, and @-methylnaphthalene excited at four different wavelengths. In the top chromatogram excitation was with the frequency quadrupled Nd:YAG laser a t 266.0 nm. At this wavelength all three molecules absorb and subsequently fluoresce. Two peaks are seen in the chromatogram. By comparison with the chromatograms of single-component solutions, the peak on the left is assigned t o naphthalene while the second peak is due to both of the methylnaphthalenes. The intensity of the latter is equal to the sum of the intensities of the two species when each is injected separately. In the bottom three chromatograms of Figure 2 excitation wavelengths appropriate for each molecule (see Experimental Section) were used on three different injections of the mixture. When the wavelength selective for naphthalene is chosen (Figure 2) only a single peak with a retention time characteristic of naphthalene is observed. Similarly, selective excitation of @-methylnaphthalene eliminates the a-methylnaphthalenecontribution to its GC peak and vice versa (under these conditions the naphthalene peak is absent). Single peak chromatograms for naphthalene and @-methylnaphthalenehave been obtained for mixtures containing a thousand-fold excess of a-methylnaphthalene. Significant improvements beyond this must await improvements in sensitivity, vide infra, since column saturation in the present system is the limiting factor. In final reference to Figure 1 we note that no solvent peak appears in the chromatograms since even a t 266 nm cyclohexane is nonabsorbing. Table I lists minimum and maximum amounts of the three compounds which could be detected in a single injection. The
0003-2700/82/0354-1202$01.25/00 1982 American Chemical Society
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Figure 1. Diagram of the RCLIF/GC apparatus. The portion enclosed by the dotted line foirms a simple GC. Temperature variation along the column is shown below.
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Figure 3. Chromatograms of a Wilmington Crude Oil sample: (a-d) chromatograms of the crude oil excited at the wavelengths indicated; (e) a laboratoly prepared mixture of naphthalene, a-methylnaphthalene, and @-methylnaphthaleneat the concentrations determined for the crude oll sample.
which the calibration curve, peak height vs. weight, becomes sublinear. The origin of this nonlinearity is being investigated. Suffice it to say that linear calibration can be extended to higher amounts by exciting vibronic transitions weaker than the origin, by detuning, or by reducing laser power. The data in Table K show that with the present RC-LIF/GC system and experimental procedure, a detection limit of 10 ng is achievable for the naphthalenic systems studied. However, the oscillator strength, f , for their SI states is very low, lo4, so that detection limits for strongly absorbing systems would be significantly improved, fluorsecence quantum yields being equal. Of course, for the stronger absorbing species saturation and photoionization effects will onset at lower laser powers. Recall also that the detection monochromator wavelength was chosen for convenienceto be in a region where all three molecules studied emit. This is, however, a region of weak emission and we estimate that sensitivity for each molecule could be easily doubled by choosing an optimum wavelength setting for each molecule. B y fur the largest increase in sensitivity could be gained by increasing the excitation duty cycle. With the continuous flow nozzle and pulsed laser used in this work the excitation duty cycle is Use of a pulsed nozzle synchronized to the laser pulses would increase the duty cycle by IO3. An additional factor of ca. 10 in sensitivity can be gained by utilizing a planar supersonic jet (9) rather than the circular nozzle employed in this work. We conclude that detection limits of -1 pg for molecules possessing a moderate fluorescence quantum yield like naphthalene's are readily achievable. Figure 3 shows a series of chromatograms obtained when a sample of Wilrnington Crude Oil is injected into the column. The bottom three traces are obtained using excitation wavelengths appropriate for @-methylnaphthalene,a-methylnaphthalene, and naphthalene, respectively. In each of these injections only a single peak with the retention time characteristic of that species is observed. In Figure 3d the excitation wavelength was 266.0 nm. The naphthalene and methylnaphthalene peaks are clearly seen in addition to a number of other peaks with longer retention times. Figure 3e shows a laboratory prepared mixture of naphthalene and both methylnaphthalenes at the levels determined to be present in the Wilmington Crude. Table I1 summarizes the amounts of the three compounds found in the Wilmington Crude, as well as the amounts of the other two compounds detected in each of the "pure" materials. The amounts of the three compounds determined for the Wilmington Crude have been independently determined by GC/MS. The values
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Flgure 2. Chromatograms of an equimolar mlxture of naphthalene a-methylnaphthalene, and @-methylnaphthalene. Excitation wavelengths used for each injection are indicated.
Table I. Detection Limits and Correlation Coefficients for Naphthalene, p-Methylnaphthalene, and a-Methylnaphthalene lower limit, g naphthalene @-methylnaphthalene a-methylnaphthalene
6.0 X 1.4 X 4.0 x
upper limit, g 1.2 X
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0.986 0.996 0.972
data yield a linear relationship between peak height and injected amount. Also included in the table is the correlation coefficient, obtained in a least-squares fit of peak height vs. weight injected. For naphthalene and a-methylnaphthalene the maximum amounts detectable were determined by column characteristics. Injection of larger amounts led to broadening and asymmetry of the chromatogram peak. The lower maximum observed for @-methylnaphthalenemarks the point at
Anal. Chem. 1982, 5 4 , 1204-1206
1204
Table 11. Amounts (mg/g) of Naphthalene, 0-Methylnaphthalene, and a-Methylnaphthalene Determined in Wilmington Crude Oil and in Each Other Wilmington naphCrudea thalene naphthalene
0.19
@-MN a-MN b
b
Finally, it seems likely that RC-LIF/GC will ultimately be recognized as a member of a class of methodologies referred to as jet spectroscopy/gas chromatography. A myriad of other exciting members can be envisaged including ones which utilize phosphorescence and photoionization rather than fluorescence.
ACKNOWLEDGMENT
(0.16) @-methylnaphthalene a-methylnaphthalene
0.53
0.045
5.0
(0.56) 0.26
