Anal. Chem. 1994,66, 2497-2504
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Cyclic Ketone Mixture Analysis Using 2 1 Resonance-Enhanced Multiphoton Ionization Mass Spectrometry Dale R. Nesselrodt and Tomas Baer’ Department of Chemistty, The Universiw of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3290
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2 1 resonance-enhancedmultiphoton ionization (REMPI) has been used for qualitativeand quantitativeanalysis of cyclic ketone mixtures. A multidimensional analysis technique has been employed to eliminate the need to separate mixture components prior to analysisby time of flight mass spectrometry (TOF/MS). The separation step is unnecessary when the analyte is introducedinto the mass spectrometerin a molecular beam. The high degree of ro-vibrationalcooling of the analyte in the molecular beam reduces spectral congestion, which permits selective ionization of each mixture component. A new approach to this multidimensional technique permits the analysis of mixtures containing geometric and stereoisomers. Our method utilizes the electron signal as the means for detecting ions and employs a hard ionization scheme. In addition, accurate and precise quantitationwith 2 1 REMPI is demonstrated for nanomoles of analyte.
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Resonance-enhanced multiphoton ionization (REMPI), in which a real state of the molecule is excited at the one or multiphoton level, has been recognized as a powerful analytical tool for qualitative organic analysis. Many volatile low molecular weight substances have been studied via resonanceenhanced multiphoton i~nization.l-~Because REMPI has several distinct advantages over other more commonly used ionization methods, much effort has been directed toward introducing nonvolatile and biological analytes into the gas phase. Consequently, various large molecular weight compounds such as polynuclear aromatic hydrocarbons ( P A H s ) , ~ , ~ amino acids,8 peptide^,^ nucleosides,1° neurotransmitters,ll ~teroids,~~porphyrins,~~ andevencompoundsas largeasbovine Fax: 919-962-2388; e-mail address:
[email protected]. (1) Driscoll, J. W.; Baer, T.; Cornish, T. J. J . Mol. Struct. 1991, 249, 95-107.
(2) Cornish, T. J.; Baer, T. J . Phys. Chem. 1990, 94, 2852-2857. (3) Shang, Q.Y.; Moreno, P. 0.;Disselkamp, R.; Bernstein, E. R. J. Chem. Phys. 1993, 98, 3703-3712. (4) Zhu, L.; Johnson, P. J. Chem. Phys. 1993, 99, 2322-2331. (5) Li, S.;Bernstein, E. R.; Secor, H. V.; Seeman, J. I. Tetrahedron Lett. 1991, 32,3945-3948, (6) Lustig, D. A,; Lubman, D. M. In?. J. Mass Spectrom. Ion. Processes 1991, 107, 265-280. (7) Kovalenko, L. J.; Maechling, C. R.; Clemett, S.J.; Philippoz, J. M.; Zare, R. N.; Alexander, C. M. 0. Anal. Chem. 1992,64,682-690. (8) Karaiste, R. T. T.; Atkinson, I. M.; Shorter, J. A.; Knight, A. E. W.; Keene, F. R. Anal. Chem. 1993,65, 27762783. (9) Li, L.; Lubman, D. M. Appl. Spectrosc. 1988,42,411-417. (10) Li, L.; Lubman, D. M. In?. J. Mass Spectrom. Ion. Processes 1989,88, 197210. (11) Wang, A. P. L.; Li, L. Anal. Chem. 1992, 643769-775. (12) Grotemeyer, J.; Schlag. E. W. Eiomed. Enuiron. Mass Spectrom. 1988, 16, 143-149. (13) Zare, R. N.; Hahn, J. H.; Zenobi, R. Bull. Chem. Soc. Jpn. 1988,61,87-92.
0003-2700/94/0366-2497$04.50/0 0 1994 Amerlcan Chemlcal Society
insulin14have been analyzed by REMPI mass spectrometry (REMPI/MS). Ionization achieved through REMPI schemes is attractive for several reasons. First, the ionization efficiencies of REMPI schemes can exceed that of normal 70 eV electron ionization by over 3 orders of magnitude.ls Furthermore, the narrow laser pulse provides an accurate and ideal start signal for measuring the ion time of flight (TOF) mass spectrum. Resolution as high as 20 000 has been achieved by TOF/MS.1”18 In addition, with someREMPI schemes, great control over the degreeof parent ion fragmentation is possible. For instance, laser power densities of IO6 W/cm2 can be used via a 1 + 1 REMPI scheme to softly ionized analytes, giving only a parent ion peak, which permits accurate molecular weight determinations.lO As the laser power density is increased, extensive fragmentation occurs, to the point where the bear C+ ion may appear as the base peak in the mass s p e ~ t r u m . ~The ~ . degree ~ ~ and ease with which the extent of fragmentation can be controlled is a unique characteristic of REMPI. Finally, because of the wavelength dependence of absorption, highly selective ionization of analytes is attainable. This is probably REMPI’s most important advantage over other ionization schemes. Thus, when laser power densities are kept relatively low in order to suppress nonresonant MPI, selective ionization of analytes is achieved without background interference. This latter feature is particularly useful for mixture analysis. Mixture analysis by most mass spectrometric techniques requires separating the individualcomponents prior to analysis. A separation step is necessary because a single mass spectrum containing peaks from every compound in the mixture would be uninterpretable. To accomplish separation, a variety of methods for sequentially introducing each analyte into the mass analyzer have been devised. The most popular techniques include gas chromatography mass spectrometry (GC/MS), high-pressure liquid chromatography mass spectrometry (HPLC/MS), and tandem mass spectrometry (MS/MS). (14) Grotemeyer, J.; Schlag, E. W. Org. Mass Specrrom. 1987, 22, 758-760. (15) Lubman, D. M.; Naaman, R.; Zare, R. N . J. Chem. Phys. 1980, 72,303& 3040. (16) Bocsl, U.;Neusser, H. J.; Weinkauf, R.; Schlag, E. W. J. Phys. Chem. 1982, 86, 48574863. (17) Opal, R. B.; Owens, K. G.; Reilly, J. P. Anal. Chem. 1985.57, 1884-1889. (18) Boesl, U.; Weinkauf, R.; Schlag, E. W. In?. 1.Mass Specrrom. Ion. hoc. 1992, 112, 121-166. (19) Zandee, L.; Bematein, R. B. J. Chem. Phys. 1979, 70, 2574-2575. (20) Bocsl, U.;Neusser, H. J.;Schlag, E. W . J .Chem. Phys. 1980,72,43274333.
AnalvticaIChemistry, Vol. 66, No. 15, August 1, 1994 2497
However, REMPI schemes which permit selective ionization can eliminate the separation step altogether as several researchers have demonstrated. For example, the REMPI absorption spectrum of [ 13C]benzene in a natural sample has been acquired without isotopic separation,21 and selective ionization of 12712in the presence of significantly greater quantities of 12711291 has been performed with REMPI.22 Beside selective ionization of isotopes, REMPI has been used to perform multidimensional analysis of aromatic mixtures. Lubman and co-workers have recorded the 1 + 1 REMPI optical spectra for each mixture component as a function of the parent ion’s mass.23,24The resulting threedimensional wavelength, mass, and intensity spectrum permitted accurate identification of each analyte without prior separation of the mixture into its constituent components. This same type of analysis can be performed on mixtures of methyl-substituted cyclic ketones and ethers, as will be demonstrated in this work. These compounds are particularly interesting because they are often found as structural units in large biological substances such as steroids and polyether antibiotics. Baer and Cornish have extensively studied the 3s n transition for five- and six-membered cyclic ketones and ethers in seeded supersonic molecular beams.2,25-27 This transition occurs near 50000 cm-I, which turns out to be more than two-thirds of the compounds’ ionization potentials, which are typically below 9.2 eV (74 200 cm-l). Hence, an efficient and convenient 2 1 REMPI scheme can be usefully employed. In this ionization scheme, two UV photons are simultaneously absorbed to promote a nonbonding electron on the oxygen atom to the 3s Rydberg state. Subsequent absorption of a third UV photon ionizes the molecule. Selective ionization is possible because the transition origins for the various methyl-substituted ketones and ethers are red and blue shifted by up to a few hundred wavenumbers from the unsubstituted compound’s transition origin.28 In addition, the absorption bands are sharp with fwhm’s of about 5 cm-l due to the extensive ro-vibrational cooling experienced during the supersonic expansion. Thus, selective ionization of a given ketone or ether in the presence of many others is possible when the compounds are cooled in a molecular beam. As noted above, some REMPI schemes such as 1 1 REMPI permit great control over the degree of fragmentation observed in the mass spectra. However, the lack of good tunable laser sources with high output powers below 200 nm has required that a 2 1 REMPI ionization scheme be used in the experiments described below. Although this scheme produces hard ionization even at low laser powers, giving much less control over the fragmentation pattern than 1 + 1 REMPI, molecular weight determinations from the parent ion mass peak are still possible. In principle, REMPI optical spectra can be obtained by monitoring either the total ionor the mass analyzed ion signal.
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(21) Boesl, U.; Neusser, H. J.; Schlag, E. W. Laser Spectroscopy I V ; Walther, H.. Rothe, K. W., Eds.; Springer Verlag: Berlin, 1979; pp 164-174. (22) Lubman, D. M.; Zare, R. N. Anal. Chem. 1982, 54, 2117-2120. (23) Sin,C. H.;Tembreull, R.; Lubman, D. M.Anal. Chem. 1984,56,2776-2781. (24) Lubman, D. M.; Kronick, M. N. Anal. Chem. 1982, 54, 660-665. (25) Cornish, T. J.; Baer, T. Anal. Chem. 1990, 62, 1623-1627. (26) Cornish, T.J.; Baer, T. J . Am. Chem. Soc. 1988, 110, 3099-3106. (27) Cornish, T.J.; Baer, T.; Pedersen, L. G. J. Phys. Chem. 1989,93,6064-6069. ( 2 8 ) Cornish, T. J.; Baer, T. J . Am. Chem. SOC.1987, 109, 6915-6920.
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Analytical Chemistry, Vol. 66, No. 15, August 1, 1994
The advantage of the former is that the signal from all of the molecules in the mixture is observed. Such a total ion spectrum can be collected by monitoring either the electrons or all of the ions. The advantage of electron detection is that all electrons arrive within a few nanoseconds so that gating methods can be used to enhance the signal to noise ratio. Total ion monitoring of REMPI optical spectra only is feasible if the sample contains a limited number of components or impurities which absorb light in the region of interest. In this respect, a considerable advantage accrues in the use of UV rather than VUV photons. Fewer organic compounds absorb light near 400 nm as compared to 200 nm. With 200-nm light, the background electron signal overwhelms that from the analyte unless an ultraclean vacuum system is used. On the other hand, the electron or total ion signal is virtually free from noise due to pump oil and residual organics when the ionizing radiation wavelength is 400 nm. Consequently, a single REMPI optical spectrum displaying transitions for each mixture component, regardless of molecular weight, can be obtained from the wavelength-dependent electron signal. Once the transition wavelengths have been determined, REMPI mass spectra at each peak can be acquired under hard ionization conditions. In this way, the REMPI transition wavelengths and mass spectra are used to uniquely identify each component in a mixture. This approach contrasts with that used by Lubman and c o - ~ o r k e r s ,which ~ ~ , ~is~ based on determining parent ion masses through soft ionization and then obtaining photoion spectra for each analyte. Their technique is not applicable to mixtures comprised of equal mass isomers. As will be demonstrated below, our approach is applicable to mixtures of both geometric and stereoisomers and is less time-consuming than the alternate method. In addition, accurate and precise quantitation with 2 + 1 REMPI will be demonstrated. EXPERIMENTAL SECTION Instrumentation. A schematic diagram of the instrument used to collect REMPI optical and mass spectra is shown in Figure 1. The frequency doubled output of a Quanta-Ray Nd:YAG laser (DCR-2) pumps a tunable pulsed dye laser (PDL-2). DCM dissolved in methanol was used as the laser dye for all the experiments described below. The dye output and Nd:YAG fundamental were then frequency mixed using an angle-tuned KD*P crystal in a Quanta-Ray wavelength extender (WEX-1). The light generated from mixing occurs in the 392-407-nm range, which is just half the energy needed n transition near 50 000 cm-l. for the 3s A pellin-broca prism disperses the light from the WEX-1 and centers the UV light on a set of right angle turning prisms, which steer the laser light into the experimental chamber. A 30-cm focal length lens focuses the light into the ionization region of the spectrometer. All the steering and focusing optics are made from UV-grade synthetic fused silica. The time of flight mass spectrometer consists of two differentially pumped chambers. The lower chamber houses a Lasertechnics pulsed valve (Model LPV) with a 300-km nozzle, which is used to produce the seeded supersonic molecular beams. The valve is normally operated with an
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r
c
AG
(d
I
l
I
a Y
L
i
TP
.................................................. v ..
I
'FD
L d h y 72W
Dosc
392
393
I Gated
Integrator
1 ADC I 386-AT
Flgure 1. Schematlc diagram of the Instrumental apparatus: DCR, W Y A G pump laser; HQ, harmonic generatlon module; PHS, prism harmonic separator; POL, pulseddye laser: WEX, wavelengthextender: TP, rlght angle turning prism; L,30 cm f.1. lens; MS, llnear time of flight mass spectrometer; and PM, power meter.
argon backing pressure of 450 Torr and a seed ratio of less than 1%. The beam is skimmed approximately 1.5 cm downstream from the nozzle with a 300-pm skimmer. With the valve operated at 10 Hz, the lower chamber pressure was normally 5 X l e 5 Torr, while that in the upper chamber was approximately 1 X 1O-a Torr. The skimmed molecular beam is intersected perpendicularly in the upper chamber by the focused laser beam. Along the third perpendicular axis lie the electron extraction optics and a time of flight mass spectrometer with Wiley-McLaren space focusing To collect REMPI spectra, a strong electric field (100 V/cm) accelerates the electrons down a 10-cm flight tube to a dual 25-mm microchannel plate detector. The detector output is amplified and sent to a gated integrator and an analog todigital converter (ADC). Thesignal is then processed digitally with a 386-AT microcomputer. All the 2 1 REMPI spectra 'were obtained by collecting the total electron signal as the dye laser wavelength was scanned. Each point in the spectrum represents the average signal of 10 laser shots. Similarly, TOF REMPI mass spectra were obtained with an extraction field of 150 V/cm and an acceleration field of 2200 V/cm. The ions then entered a 10-cm field free drift tube before striking the microchannel plate detector. The amplified signal from the microchannel plates was sent to a LeCroy 7200 digital oscilloscopewith a 7242 plug-in module (400 MHz, 1 GS/s), which was used to collect and signal average the TOF spectra for 500 laser shots. Parent ion flight times were typically 4-5 ps, while fragment ion flight times varied between 1.5 and 4 ps. The digital oscilloscope recorded 15-20 sample points per peak. The TOF spectra were then digitally converted into mass spectra with the 386-AT microcomputer. The mass accuracy of the molecular ion peaks was f 0 . 4 amu of the actual value.
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(29) Wiley, W. C.; McLaren, I. H. Rev. Sci. Insfrum. 1955, 26, 1150-1157.
Q
I\
Re-AmP
+
394
395
398
397
-
398
I
399
Wavelength (nm)
1 REMPI spectra of the 3s Figure 2. 2 cyclohexanone and its monomethyl Isomers.
n transltlons for
Quantitation. Quantitative measurements were made as follows: After evacuating the pulsed valve inlet, a 1-pL plug of analyte (nanomoles) diluted in methanol was injected into the valve inlet through a rubber septum. Upon sample and solvent evaporation, the inlet system was filled to a total pressure of 450 Torr with argon. Cyclohexanone was used as an internal standard to account for differences in mixing between the analyte and argon carrier gas as the inlet was pressurized. A total of 5 min was allowed for partial diffusional mixing of the analyte and carrier gas. The REMPI signal was measured with the laser tuned to the internal standard's transition origin and digitally averaged for 1000 laser shots. Then, the laser was tuned to the analyte's transition origin, and its REMPI signal was collected in a similar manner. Finally, the valve was evacuated for 5 min before this process was repeated for the next sample. The ratio of analyte signal to that of internal standard was used to make either calibration plots or standard addition curves for the quantitation of unknown samples. Quantitation worked very well when nanomole injections of analyte were made. Using the procedure outlined above, the actual amount of analyte consummed during analysis is approximately 5% of the injected volume. Thus, even though the absolute detection limits for the cyclic ketones are determined to be in the tens to hundreds of picomoles range for our instrument, the practical detection limits for gaseous analytes are determined by the size of the inlet system. For our nonoptimized system, the detection limits were less than 10 nmol for the cyclic ketones. Reagents. The samples of cyclohexanone (CHO), 2-methylcyclohexanone (2-MCHO), 3-methylcyclohexanone (3MCHO), and 4-methylcyclohexanone (CMCHO) were purchased from Aldrich. They were uJed without further purification. Both cis- and trans-3,5-dimethylcyclohexanone (3,5-DMCHO) were purchased as a mixture of isomers from Pfaltz & Bauer. Pure samples of these stereoisomers were obtained from the mixture by preparative gas chromatography using an SF-96 column.
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RESULTS AND DISCUSSION REMPI Spectra of Methylcyclohexanones. The 3s n 2 1 REMPI spectra for cyclohexanone and its methylsubstituted geometric isomers are shown in Figure 2. The
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Ana&ticalChemistty, Vol. 66, No. 15, August 1, 1994
2499
li
I1
1
A+
I
+
1.*
Lrl,
I
A
I
0: Ill
+
0’
C
6,
m/z 5 5
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Flgure 4. Main fragmentation mechanism for cyclohexanone.
m/z
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-
Flgure 3. 2 1 REMPI mass spectra of cyclohexanone and its monomethyi isomers obtained at their 3s n transition origins. The insets showing the parent ion peaks have been expanded by a factor of 25.
low background signal indicates that nonresonant multiphoton ionization of the argon carrier gas, analyte, and methanol solvent is negligible with laser powers in the 0.5-2 mJ/pulse range. With the exception of the 2-MCHO spectrum, the spectra are dominated by a single sharp peak corresponding to the 3s n transition origin. The transition origin corresponds to the electronic transition between the ground electronic and vibrational state to the 3s ground vibrational state. The series of four evenly spaced peaks between 396 and 399 nm for 2-MCHO is due to the excitation of low-lying vibrational states in the upper 3s Rydberg state. The interaction between the methyl and carbonyl groups causes a geometry changeupon excitation of 2-MCHO, which creates large Franck-Condon factors for the excitation of lowfrequency (ca. 90 cm-I) ring modes. The transition origins for the various methyl isomers are red or blue shifted relative to that of unsubstituted cyclohexanone. The transition origins for 2- and 4-MCHO are red shifted by 543 and 6 cm-l, respectively, while that for 3-MCHO is blue shifted by 147 cm-l. Since the shifts in transition energy are large compared to the peak fwhm’s which are typically 5 cm-l, selective ionization of individual components in mixtures of these ketones is readily possible. In other words, mixture analysis can be performed without separating the components when laser powers in the range cited above are used for ionization. REMPI Mass Spectra of Methylcyclohexanones. The 2 1 REMPI mass spectra displayed here (Figure 3) are characteristic of hard ionization and are nearly identical to conventional 70 eV electron impact mass spectra for these compounds. Although great control over the extent of fragmentation is possible with certain ionization schemes such as 1 + 1 REMPI, the fluence necessary to populate the 3s state through simultaneous two-photon absorption is so great that parent ions will absorb additional photons and undergo extensive fragmentation. Fragmentation of the parent ion even occurs at 300-400 pJ/pulse, which is the threshold laser power for observing 2 1 REMPI signal for these compounds. This is not the case for 1 + 1 ionization. Some experiments have been performed with both one- and two-color 1 + 1 REMPI using a hydrogen cell to raman shift the dye laser wavelength into the near-VUV range necessary to observe the
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Analytical Chemistry, Vol. 66, No. 15, August 1, 1994
3s n transition. However, with the VUV powers available (10-20 pJ/pulse) at the 8th anti-Stokes line, the cyclic ether tetrahydropyran (THF) was the only compound of those tried that produced a 1 + 1 REMPI signal. The 1 1 REMPI mass spectrum displayed two peaks, the parent ion (100%) and the formaldehyde loss peaks (10%). Even though the parent ion peak is less than 5% as intense as the base peak in the 2 1 REMPI mass spectra above, accurate molecular weight determinations are possible when the signal to noise ratio is sufficiently large. A signal to noise ratio greater than 10 is evident for the parent ion peak after expansion of the vertical scale by a factor of 25. The fragmentation mechanism for six-membered cyclic ketones is well k n ~ w n . ~For ~ . cyclohexanone, ~~ the main fragmentation pathway (Figure 4) occurs by cleavage of the bond between the carbonyl carbon and one of the CY carbons. This cleavage produces a primary radical, which undergoes a hydrogen shift to create a more stable conjugated secondary radical. Subsequent cleavage of the bond between the @ and y carbons produces the resonance stabilized m/z 55 ion. This ion appears as the base peak in the cyclohexanone mass spectrum. If the methyl group remains with this ion for the methylcyclohexanones, the base peak may shift by 14 units to m/z 69. This is in fact observed for the case of 3-MCHO. On the other hand, for 4-MCHO, the methyl groups leaves with the neutral, and the base peak occurs at m/z 5 5 . As a result, the mass spectra of CHO and 4-MCHO are nearly identical, except in their parent peak positions. Although 2-MCHO displays a prominent m/z 55 peak, the base peak occurs at m/z 39. Figure 5 shows the REMPI mass spectra for both cis- and trans-3,5-dimethylcyclohexanone.The two mass spectra are virtually identical as would be expected for stereoisomers. Like 3-MCHO, the base peak occurs at m/z 69. Unlike the monomethyl-substituted cyclohexanones, these compounds yielded parent ion peaks with a signal to noise to ratio of about 3 even though the same laser power was used to collect all the mass spectra.
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Three-Dimensional REMPI Optical/Mass Spectra of Mixtures. Figure 6 is a 2 + 1 REMPI spectrum of a 2: 1:1 mixture of 2-, 3-, and 4-methylcyclohexanone. As is expected, the spectrum shows the transition origins for both 3- and 4-MCHO at 392.96 and 394.16 nm, respectively. The vibrational progression characteristic of 2-MCHO between (30) Silverstein, R. M.; Bassler, G. C.; Morrill, T. C. Spectrometric Idenlificrrtion ofOrgonic Compounds, 5th ed.;John Wiley Br Sons,Inc.: New York, 1991; Chapter 2. (3 1) McLafferty, F. W. Interpretafiono/MrrssSpectra,3rd ed.;University Science Books: Mill Valley, CA, 1980; Chapter 8.
I
I
I I
1
h
: I
.CI
O
10
20
+
30
40
50
60
70
A BO
80 100 110 120 130 140
m/z
Figure 5. 2 1 REMPI mass spectra of cis- and tran43,5dimethylcyclohexanone. The insets showing the higher mass peaks have been expanded by a factor of 25.
Figuro 7. Three-dimensional REMPI optical/mass spectrum of a 2: 1:1 mlxture of 2-, 3-, and 4- methylcyclohexanone. The 2 1 REMPI spectrum Is displayed on the wavelength and relative intensity axes, while the 2 1 REMPI mass spectra at selectedtransttronwavelengths are shown on the m/z and relative intensity axes.
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+
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TaMo 1. TwePhoton Abrorptlon WavMngthr, 90 n Transition Emrgkr, Pork fwh”0, and Rdatlvo Trandtbn Ofbln ShmV for 8.kOt.d Cydlc Kotonor compound CHO 2-MCHO
i!k
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398
= 399
Wavelength (nm)
Figwe 6. 2 1 REMPI spectrum of the 3s n transitions for a 2: 1:1 mixture of 2-, 3-, and 4methyicyciohexanone.
396 and 399 nm is also identifiable. It is relatively easy to determine what compounds are present from the REMPI spectrum alone knowing that the sample is a mixture and knowing that mixture contains nearly equal portions of each component. However, without prior knowledge of the sample composition, this determination is much more difficult but is possible using the procedure below. The ideal analysis scheme is to collect three-dimensional mass, wavelength, and intensity data in real time, storing the TOF mass spectra as the 2 1 REMPI optical signal is collected. Computer control would permit rapid scanning of the optical spectrum until a preset signal level had been reached. At this point, a TOF mass spectrum would be obtained with a digital oscilloscope while slowly scanning through the optical transition. When the optical signal then falls below the discrimination level, the TOF mass spectrum would be stored and rapid scanning of the optical spectrum would be resumed until the next peak is encountered. In this way a three-dimensional optical/mass spectrum could be obtained. Good mass spectra can be obtained with 500 laser shots. Since each of the spectroscopic peaks in Figures 2 and 6 consist of about 100 laser shots, the scan rate over the peaks would have to be reduced by a factor of 5 . This should not markedly increase the total scan time. The limited availability of the digital oscilloscope prevented us from developingthis three-dimensional approach. Instead, the optical spectra were obtained first to locate the major absorption peaks. The mass spectra were then collected at
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3-MCHO 4-MCHO cis-3,J-DMCHO trom-3,5-DMCHO
two-photon aborption wavelength (nm)
transition energ (cm-l!
394.10 398.36 397.70 397.05 396.40 392.96 394.16 392.28 396.14 395.59 394.94
50749 50206 50289 50372 50454 50896 50741 50984 50487 50558 50640
E; (cm-1)
transition origin shift (cm-1)
5
0
2 2 3
543
5 5 6 5 5 5 4
-147
a
-235 262
I, Transition origin shifts are calculated relative t o the transition origin for CHO.
the various absorption peaks. A three-dimensional REMPI optical/mass spectrum like that shown in Figure 7 is constructed from the wavelength, mass, and intensity data. They and z axes display the REMPI optical spectrum of the mixture, while REMPI mass spectra at particular transition wavelengths are shown on the x and z axes. We have experimentally verified that the REMPI mass spectra of these six-membered cyclic ketones are invariant over transition wavelengths a t least 10 nm from the transition origin. (This is not necessarily the case for all compound^.^*-^^ ) Thus, the strong wavelength dependence of the mass spectra indicates that the sample is a mixture of compounds. Comparison of these mass spectra to those of the pure compounds (Figure 3) in combination with the transition wavelengths (Table 1) permits unambiguous determination of the presence of 2-, 3-, and 4-MCHO. The three-dimensional REMPI optical/mass spectrum shown in Figure 8 is from a commercially available sample of cis- and truns-3,5-DMCHO. The sample was purchased as a mixture of the two isomers with a rated purity of 94%. (32) Kinsel, G.R.;Segar, K. R.; Johnston, M. V. Urg. Mam Specrrom. 1987,22, 621632. (33) Carney, T.E.;Baer, T. J. Chem. Phys. 1981, 75, 477-478. (34) Parker, D. H.UIrrasemitioe Laser Spectroscopy, Kliger, D. S., Ed.;Academic Press: New York, 1983; Chapter 4.
Ana(ytIcaIChemist~y, Vol. 88, No. 15, August I, 1994
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5 1
I
Quantitative Analysis. As demonstrated above, excellent qualitative mixture analysis is possible using a 2 + 1 ionization scheme. Ultimately, quantitative analysis by 2 + 1 REMPI is desired; however, the nonlinear nature of the two-photon absorption step does not make it immediately obvious that quantitative work is possible. Theoretically, assuming a steady-state the 2 1 REMPI ionization rate should scale linearly with concentration according to the following relationship:
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Figure 8. Threedlmenslonal REMPI optlcai/mass spectrum of a commercially available sample of cls- and frens-3,5dlmethylcycl~ hexanone. The spectrum shows that cyclohexanone and 3-methylcyclohexanone are present as impurities.
From the known transition wavelengths for the stereoisomers (Table 1) and from comparison of the mass spectra with those of the purified compounds (Figure 6), the 3-D REMPI optical/ mass spectrum shows that both cis- and trans-3,5-DMCHO are present in the sample. It also shows that two impurities are present. The transitions observed at 394.10 and 392.95 nm are assigned to CHO and 3-MCHO, respectively. At least two different approaches could have been followed for acquiring this mixture's 3-D REMPI optical/mass spectrum. First, the REMPI 3s optical spectrum of the mixture is obtained followed by collection of REMPI mass spectra at selected wavelengths in the REMPI spectrum. This approach takes advantage of the fact that electrons are a universal method of ion detection. The second method involves determining what parent ions are present in the mixture using a soft ionization scheme. REMPI spectra are then obtained by monitoring the wavelength-dependent parent ion signal. In both cases, REMPI optical spectra and REMPI mass spectra are used to determine the mixture composition. However, the former method appears to be a better approach. Had the m/z 126 parent ions been used to collect the REMPI optical spectrum instead of the electrons, the impurity peaks would not have been observed since they occur for lower mass ions. They are observed in the optical spectrum in Figure 8 because the collection of electrons, which have the same flight time regardless of the parent ion mass, is a universal detection method. Two additional wavelength scans using the m/z 98 and the m/z 112 parent ions would have been required to observe the transitions due to the impurities. Since each wavelength scan takes approximately 30 min to obtain, while each mass spectrum takes only 1 min of acquisition time, the latter method of analysis is more time consuming. Likewise, this alternate approach would fail in the analysis of the monomethyl isomer mixture since only one parent peak at m/z 112 would have been observed. Consequently, only a single REMPI spectrum comprised of transitions for all three isomers like that in Figure 6 would have been obtained. At this point, the second approach fails to identify the individual compounds, losing its utility. Therefore, the method used in this study is less time-consuming and applicable to mixtures of compounds with the same mass, in particular, geometric isomers and stereoisomers. 2502
Analytical Chemistry, Vol. 66, No. 75, August 7, 1994
where dNi/dt is the time dependent ion current, No is the analyte number density in the interaction volume, cqscn is the two-photon absorption cross section for the 3s n transition, I ( [ ) is the incident light intensity as a function of time, ~ f - 3 is the one-photon absorption cross section to the final ionic state, and y is the nonradiative transition rate from the 3s state. Note that depending on the extent of competition between the radiative and nonradiative transition rates from the 3s state, the ion current may display a quadratic or cubic light intensity dependence. As a result, if the temporal intensity profile of the laser beam fluctuates, the total number of molecules undergoing 2 + 1 REMPI will vary enormously. For instance, assuming the ionization rate displays a quadratic dependence, a narrow laser pulse with twice the fluence of a temporally broader one will ionize four times as many molecules as the latter even if the total number of photons in each pulse is the same. Consequently, power meter readings cannot be used to normalize the 2 + 1 REMPI signal to the light intensity since the meter responds too slowly to record the temporal intensity profile for each laser pulse. Therefore, this nonlinear dependence on light intensity makes quantitative work more difficult with 2 + 1 REMPI than for other REMPI schemes in which the ionization rate scales linearly with light intensity. As a result, the reproducibility of each laser pulse plays a key role in determining the quality of analytical results obtained with 2 + 1 REMPI. For instance, we have found that quantitative 2 + 1 REMPI experiments could not be performed with our Lumonics excimer-pumped dye laser system because of poor pulse reproducibility. On the other hand, a Nd:YAGpumped dye laser system did permit accurate and precise quantitation. Figure 9 shows the linearity of the 2 + 1 REMPI signal as a function of the amount of 3-MCHO injected into the sample inlet. The regression line slope of 0.99 f 0.03 shows excellent linearity between sample concentration and the 2 + 1 REMPI signal. Thus, the signal response is linear with sample concentration over at least 2 orders of magnitude when nanomoles of analyteare used. The error bars represent f l u in the shot to shot signal ratio for each of the plotted values. In contrast, the averaged signal ratios for repeat injections of the same sample produced a relative standard deviation of less than 2%. Two calibration curves for the determination of an "unknown" sample of 3-MCHO are displayed in Figure 10.
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(35) Parker, D. H.; Berg, J. 0.; El-Sayed, M. A. Adunnces in Laser Chemistry; Zewail, A. H., Ed.: Springer Verlag: Berlin, 1978; pp 32&335.
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Amount of 3-MCHO (nmol)
+
Flgure 0. Linearity of the 2 1 REMPI signal ratio of analyte to internalstandard as the of amount of 3-methylcyclohexanone is varied over 2 orders of magnitude. Error bars represent f l u in the shot to shot signal ratio.
Amount of 3-MCHO (nmol) Flgure 10. Calibration curves for the quantitative analysis of 3-methylcyclohexanone showing the effect of laser power irreproducibiiity. The upper curve (solidcircles)was obtained with laser powers 0.2 mJ/pulse greater than that for the lower curve (open circles).
The curves are plots of nanomoles of 3-MCHO injected versus the signal ratio of 3-MCHO to C H O in the sample. C H O was used as an internal standard to account for differences in mixing with the argon carrier gas as the inlet system was pressurized. Signal intensity variations greater than 10%have been observed when no internal standard was added. Thus, the standard samples used to create the calibration plots consisted of dilute solutions of 3-MCHO and CHO in methanol; a fixed amount of CHO was added to each sample while the amount of 3-MCHO was varied. The procedure used to analyze them was fully described in the Experimental Section. The REMPI signal ratio of 3-MCHO to CHO was then plotted as a function of nanomoles of 3-MCHO injected. These two calibration curves were prepared on consecutive days. The 14% difference in the slopes is a result of the different laser powers of 1.3 and 1.1 mJ/pulse. Therefore, the accuracy of quantitative results based on calibration curves depends greatly on laser power reproducibility. To test the accuracy of the calibration curve method, an "unknown" sample of 3-MCHO was prepared. In addition, equations describing the two curves were obtain by linear
regression. Correlationcoefficients of 0.9998 and 0.9982were obtained for the least squares fits of these two curves. The actual amount of "unknown" injected into the inlet was 414 nmol, while the amounts calculated from the equations for the calibration curves were 410 and 390 nmol, respectively. Thus, a 1-6% error in the actual amount of 3-MCHO injected was obtained by this technique. Standard addition experiments in which the unknown amount of 3-MCHO was determined by adding incremental amounts of 3-MCHO to the unknown while holding the internal standard concentration constant produced similar results to the calibration curve method; determinations of amounts injected were accurate to within a few percent of the actual value. One advantage of the standard addition technique over the use of calibration curves is that the day to day reproducibility of laser power becomes less important for obtaining accurate quantitative results since a new curve is generated for each unknown sample. The nanomole detection limits reported here could be improved significantly through several modifications to the existing experimental system. As mentioned previously, our sample inlet system is not optimized for the introduction of small analyte volumes. Since approximately 5% of the sample was consumed during analysis, the inlet system volume could be reduced by at least an order of magnitude with no reduction in the signal to noise ratio. Furthermore, the fwhm of the gas pulse from our valve is approximately 180 ps, while the laser pulse duration is 5-6 ns. Consequently, only a small amount of each gas pulse is actually analyzed. Faster pulsed valves ( ~ 1 ps 5 fwhm) have been developed which can reduce the amount of wasted sample by a factor of Using a valve producing a shorter gas pulse will improve the detection limits by another order of magnitude since one-tenth of the gas volume is required to give the same signal to noise ratio obtained with our current valve. Thirdly, detection limits could be improved by ionizing the molecular beam closer to the pulsed value nozzle where the gas density is between 100 and 1000 times greater than in the ionization chamber. Ionization would occur prior to skimming the molecular beam. This improvement should give another 2-3 orders of magnitude increase in detection limits. The combined effects of these improvements will extend the detection limits for gaseous samples into the tens to hundreds of femtomole range, which is comparable to those obtained with conventional mass spectrometric techniques.
CONCLUSIONS 2 + 1 REMPI/MS is a sensitive technique for the qualitative and quantitative analysis of cyclic ketone mixtures. Unlike most commonly used mass spectrometric methods such as GC/MS, REMPI/MS eliminates the need for mixture separation prior to introduction into the mass analyzer because of selective ionization. Selective ionization of mixture components is possible because of the high degree of rovibrational cooling in the supersonic molecular beam and n transition because of the red and blue shifts in the 3s
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(36) Gentry, R.W.; Giese, C.F. Reu. Sci. Instrum. 1978, 49, 595-600.
Ana&ticalChemistry, Vol. 86, No. 15, August 1, 1994
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origins of methyl-substituted cyclic ketones. A new approach to three-dimensional REMPI/MS using the REMPI electrons for ion detection and using a hard ionization scheme permits the accurate identification of components in mixtures consisting of geometric isomers and stereoisomers. In addition, the accurate and precise quantitation of mixture components is possible with nanomole samples using a 2 + 1 REMPI ionization scheme. These results will be extended to the analysis of larger biological compounds, which contain cyclic ketones and ether structures such as steroids and polyether antibiotics.
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ACKNOWLEDGMENT We gratefully acknowledge the T. J. Meyer group for use Of their LeCroy 7200 digita1 oscilloscope* We appreciate financial Support Provided by the NSF under Grant CHE9003797*
Received for review January 28, 1994. Accepted May 4, 1994.'
.Abstract published in Advance ACS Absrracrs, June 15, 1994.
