Article pubs.acs.org/JPCA
Structural Investigation of Titan Tholins by Solution-State 1H, and 15N NMR: One-Dimensional and Decoupling Experiments
13
C,
Chao He,† Guangxin Lin,†,⊥ Kathleen T. Upton,† Hiroshi Imanaka,†,‡ and Mark A. Smith*,†,‡,§ †
Department of Chemistry and Biochemistry, University of Arizona, Tucson, Arizona 85721, United States Department of Planetary Science, Lunar and Planetary Laboratory, University of Arizona, Tucson, Arizona 85721, United States § Department of Chemistry, University of Houston, Houston, Texas 77204, United States ‡
ABSTRACT: Titan, the largest moon of Saturn, is enveloped in a reddish brown organic haze. Titan haze is presumed to be formed from methane and nitrogen (CH4 and N2) in Titan’s upper atmosphere through energetic photochemistry and particle bombardment. Though Titan haze has been directly investigated using methods including the Cassini mission, its formation mechanism and the contributing chemical structures and prebiotic potential are still not well developed. We report here the structural investigation of the 13C and 15N labeled, simulated Titan haze aerosol (tholin) by solution-state NMR. The onedimensional 1H, 13C, and 15N NMR spectra and decoupling experiments indicate that the tholin sample contains amine, nitrile, imine, and N-heteroaromatic compounds of tremendous import in understanding complex organic chemistry in anaerobic, extraterrestrial environments.
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INTRODUCTION Titan is the largest moon of the Saturnian system and also the only moon having a significant atmosphere in the solar system. The thick atmosphere of Titan is composed primarily of nitrogen (95−98%) and methane and contains trace amounts of many simple organic compounds including ethane, ethylene, acetylene, hydrogen cyanide, and even benzene.1,2 Titan has attracted the attention of many scientists because it is surrounded by reddish brown, presumably organic, haze. The haze is believed to be initiated by charged particle chemistry and ultraviolet radiation in the Titan ionosphere, finally leading to complex aerosol and condensate layers in the stratosphere. The haze is expected to be composed of very complex, mixed organic aerosols, which contain a significant percentage of nitrogen-rich organic molecules and hydrocarbons.2−5 The aerosols fall to surface of the Titan and may react with periodic liquid water, which could derive from frozen water6 melt pools due to meteoritic impacts7 and/or cryovolcanism8 to yield prebiotic species, such as amino acids, purines, and pyrimidines.9−11 The study of the organic chemistry that occurs on Titan would contribute greatly to our understanding of prebiotic chemistry and perhaps ultimately to an understanding of how life arose on Earth. © 2012 American Chemical Society
Tholins, the material produced in laboratory simulations of the processes believed to be active in the Titan haze production, have been prepared in the laboratory by electrical discharges or photochemical reactions in nitrogen−methane mixture.3−5,12−21 The tholins have been studied quite extensively by various methods including UV/vis spectroscopy,3 IR spectroscopy,3,4,14−16 UV/vis fluorescence,22 pyrolysischromatography,23 mass spectrometry,11,12,18,19,22−27 and nuclear magnetic resonance (NMR) spectroscopy.4,11,14−16,28 Much useful information of the tholin material has been obtained by these studies, but the complex mixtures still hold a lot of unanswered questions particularly as regards to definitive organic structural or functional group inventory. NMR spectroscopy is a high resolution technique that can be used to structurally analyze complex mixtures and large compounds with tremendous structural specificity.29 Compared to some other analytical techniques used to characterize tholins, NMR is a nondestructive method that can provide definitive structural data without resulting in the structural degradation of Received: February 17, 2012 Revised: April 10, 2012 Published: April 10, 2012 4760
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resolution (exact mass) mass spectrometry.11,19,24−27 In the present article, we report our continuing efforts to analyze the tholin samples initially using one-dimensional NMR techniques. Using 13C and 15N isotope labeled tholin, we have carried out the one-dimensional (1D) NMR experiments on the three nuclei (1H, 13C and 15N) and developed 1D decoupling NMR experimental methods to help define a functional group inventory of tholins. In a future publication, we will explore the expansion of this to multidimensional NMR methods for the more quantitative determination of refined structures.
the tholin. However, as with many other analytical techniques, simple one-dimensional NMR experiments can only provide limited insight when applied to complex mixtures. Even with the most sensitive NMR instrumentation, the complex mixture would lead to NMR spectra with a great deal of heterogeneous and homogeneous peak overlaps. As a result, few studies have been reported on the characterization of nascent tholin samples by NMR. In 1993, Sagan et al. analyzed the Titan and Jupiter I tholins by solidstate 13C NMR.4 They found that both tholins exhibit resonances consistent with aromatic and/or alkene carbons. In 2000, Clark et al. used solution-state 1H NMR (CDCl3 as solvent) to analyze the gaseous photolysis products of cyanoacetylene plasma. 28 They identified acrylonitrile (CH2CHCN) and acetonitrile (CH3CN) by comparison of their 1H NMR spectra with authentic samples. In 2004, Ramirez et al. reported a solution-state 1H NMR spectrum of D2O−DMSO soluble fractions of tholins generated by laserinduced plasma irradiation.16 Characteristic peaks were assigned to a range of compounds containing saturated and unsaturated aliphatic hydrocarbons, as well as aromatic hydrocarbons and nitriles, consistent with the IR spectrum of the same sample. However, their assignment of a benzonitriletype hydrogen to the signal at 3.38 ppm is subject to debate as benzonitrile is observed in 1H NMR to have peaks in the range of 7.3−7.6 ppm.30 Ruiz-Bermejo et al. reported solid-state and solution-state (dissolved in D2O) 13C NMR hydrophilic tholins using a CH4/N2/H2 atmosphere and spark discharges either in aqueous aerosols or in liquid water.14,15 In solid-state 13C NMR spectra, broad resonances were observed at 10−60 ppm, near 70 ppm, 115 ppm, and 129 ppm. They assigned the signals at 10−60 ppm to saturated carbons (amine or −CH, −CH2−, and −CH3 groups), the resonances near 70 ppm to hydroxylic carbons (C−OH) or alkyne groups, the peaks around 115 and 129 ppm to nitriles and alkenes, respectively. In solution-state 13 C NMR spectra, the previous broad resonances were resolved and a great number of resonances were observed. All resonances are assigned to six main types of carbons (CO, CN, C−O, C−N, CH/CH2, and CH3). Pillig et al. described solution-state 1 H NMR of derivatized tholin in CDCl3 produced by soft X-rays.11 The 1H NMR spectrum of the tholin sample is consistent with that of standard adenine. Combining GC-MS and 1H NMR, they provided evidence for nucleobase synthesis in the Titan atmosphere, confirming that the organic chemistry on the Titan surface can be very complex and extremely rich in prebiotic compounds. Clark et al. only analyzed gaseous products,28 while Pillig et al. investigated the derivatized tholins.11 The NMR studies on tholins4,14−16 demonstrate that tholins are very complex mixtures containing both saturated and unsaturated compounds. However, all these studies only provided the 1H or 13C spectra of tholins, and one of them4 was acquired even 20 years ago with low frequency NMR. A thorough NMR study of tholin with advanced NMR spectrometers is highly necessary. Ruiz-Bermejo et al.’s study shows that solution-sate NMR has higher resolution than solidstate NMR in analyzing tholins. Thus, we perform the solutionstate NMR study of tholin and report the initial onedimensional NMR result here. Hertkorn et al. have succeeded in analyzing marine dissolved organic matter and unresolved organic fractions in Earth’s atmosphere by multidimensional NMR coupled with very high resolution mass spectrometry.31 We have already reported extensively on our studies of tholin analysis using very high
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EXPERIMENTAL METHODS
Tholin samples were prepared by exposing a mixture of 5% 13 CH4 (13C 99.9%, Cambridge Isotope Laboratories) and 95% 15 N2 (15N 98%+, Cambridge Isotope Laboratories) to an AC electrical discharge in a high vacuum stainless steel/glass reaction chamber at 195 K. The flow rate is set such that the pressure of the chamber is held at 850 Pa. This leads to a volume flow rate that produces a discharge exposure time of about 2 s in a discharge current of approximately 100 mA. After 72 h of discharge flow, a red/brown film is deposited on the wall of the reaction vessel. We recognize that tholins have been observed to change in structure between pressures of 2300 and 13 Pa.3 Imanaka et al.3 investigated the effect of pressure on Titan tholin and found that the tholin formed at low pressure has a higher degree of unsaturation and that nitrogen is incorporated into tholin more efficiently at lower pressures. The simulations at different pressures may reflect the Titan haze layers at different altitudes.3 Moreover, Titan’s atmosphere and surface are at cryogenic temperature and temperature affects physical and chemical properties as well as chemical reaction. Our experiment maintained at 195 K may simulate the processes taking place in Titan’s atmosphere better. The reaction vessel is allowed to warm to room temperature during a 24 h period under vacuum to remove the lower molecular weight fractions and volatile components. The polymeric solids were isolated in a dry, oxygen-free glovebox, and the samples were stored under N2 atmosphere. Approximately 20 mg of the solid sample was put into 1 mL of DMSO-d6 (D 99.9%, anhydrous, Sigma-Aldrich). After 10 min, the solid was dissolved completely. The operation was carried out in a glovebox under anaerobic and anhydrous conditions at room temperature. The samples were kept in sealed NMR tubes and wrapped in foil to avoid exposure to air and light, respectively. The solution-state NMR experiments were carried out on Bruker DRX 500 and Varian INOVA 600 spectrometers. The Bruker DRX 500 spectrometer is equipped with a Bruker 5 mm BBO S2 probe with z-gradient, and the Varian INOVA 600 spectrometer is equipped with an H, C, N triple-resonance cryogenic probe with z-gradient. The 1D 13C spectrum was acquired on the Bruker DRX 500 spectrometer due to its excellent 13C sensitivity (BBO 13C probe coil). Other experiments were achieved on the Varian INOVA 600 spectrometer. Since our tholin samples are 13C and 15N isotope labeled, all nuclei in the sample are spin 1/2 nuclei. The 13C and 15N isotope labeled samples not only make it easier to obtain the 1 H, 13C, and 15N spectra but also provide an opportunity to observe the 1H−13C, 1H−15N, 13C−15N, 13C−13C, and 13 C−15N couplings some of which would be very difficult to 4761
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chemical environment conclusions, while leaving more detailed polymer study and the presumed relationship between the small molecules and polymers to later studies. According to the chemical shifts of the protons,32 the whole range of the 1H spectrum can be divided into three regions: 0− 2 ppm, 2−5 ppm, and 5−9 ppm. The peaks below 2 ppm are from the protons attached to sp3 carbon and many bonds away from any unsaturated or electronegative atom; the peaks between 2−5 ppm are from the protons in the H−C−X system (X could be N, CN, CC, CC, or CN) or H−N (sp3); the peaks above 5 ppm are from the protons connecting to carbon or nitrogen (sp2). The sample is fully 13C and 15N isotope labeled and this spectrum is without 13C or 15N decoupling, so we can observe the 13C−1H and 15N−1H couplings in this spectrum. The peaks at 3.35/3.59, 4.42/4.67, 5.34/5.48, and 6.53/6.68 ppm are obvious doublets caused by 13 C−1H or 15N−1H couplings, the coupling constants of which are 144.29, 149.65, 88.50, and 90.51 Hz, respectively. The coupling constants33a,34 indicate that the doublet peaks at 3.35/ 3.59 and 4.42/4.67 ppm come from the one-bond 13C−1H couplings, and the doublet peaks at 5.34/5.48 and 6.53/6.68 ppm come from one-bond 15N−1H couplings. The broadening of the peaks at 6.53/6.68 ppm could result from the proton exchange in N−H. There are also a number of small doublet peaks caused by 13C−1H or 15N−1H couplings. In order to determine the couplings and the nuclear connectivities directly, we perform 1H NMR decoupling experiments. Three 1H spectra are obtained by using different decoupling methods, which are shown in Figure 2. They are 1H spectrum
observe in natural abundance samples. We developed and applied decoupling experimental methods for the 13C and 15N isotope labeled tholin samples. For the 1D 1H NMR experiments, we can obtain different 1H NMR spectra by using different pulse sequences. Comparing these spectra, we can observe the 1H−13C and 1H−15N couplings, which help us to distinguish which hydrogen is bonded to carbon and which one is bonded to nitrogen. The one-bond 1H−13C coupling constants33a also indicate whether the carbon is sp3, sp2, or sp type. We can also obtain the 13C NMR spectra with different decoupling methods. From the splitting patterns of the peaks, we can learn more detailed structure information. For instance, in the 13C NMR spectrum without 1H decoupling, singlet, doublet, triplet, and quartet peaks present quaternary carbon, methine (CH), methylene (CH2) and methyl groups (CH3), respectively. The rule is also applicable to the 15N−1H coupling and 13C−15N coupling systems and can be used to estimate how many hydrogens bond to nitrogen and how many nitrogens bond to carbon.
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RESULTS AND DISCUSSION In this article, we describe the results of 1D and decoupling NMR experiments and discuss the functional group inventory of the tholin samples (mixtures) based on the chemical shifts of the three nuclei (1H, 13C, and 15N) and the coupling information. Figure 1 shows the solution-state 1H NMR spectrum of Titan tholin without 13C and 15N decoupling. The peak at 2.5
Figure 1. Solution-state 1H NMR spectrum of Titan tholin in DMSOd6 without 13C and 15N decoupling.
Figure 2. Solution-state 1H NMR spectra of Titan tholin with different decoupling methods: (a) with only 15N decoupling, (b) with only 13C decoupling, and (c) without 13C or 15N decoupling.
ppm is from the proton impurity in DMSO. The distribution of sharp and featureless broad signals are indicative of the relative concentrations of small molecules (10%) and larger polymeric structures in these mixtures. From the study of the sharply resolved small molecule signals, we can determine specific functionality in the range of small molecules, while in the broad features, at present, we only determine rough distributions of the chemical environment of the nuclei within the polymers. The first observation regards the apparent small distribution of clearly resolved molecules in these gross mixtures. Electrospray or laser ionization mass spectrometric studies indicate the typical presence of thousands of distinct parent molecules in tholin mixtures.24,27 In this article, we report the obtainable information from simple spectral analysis and coupling studies providing the small molecule functional inventory and polymer
with only 15N decoupling, 1H spectrum with only 13C decoupling, and 1H spectrum without 13C or 15N decoupling, respectively. Comparing these 1H spectra, both 13C−1H and 15 N−1H couplings are observed among the various protons. In the 13C and 15N isotope labeled tholin sample, all the protons should have 13C−1H or 15N−1H couplings, but some are difficult to observe because of high spectral overlaps. The coupling information of the easily resolved peaks is listed in Table 1. There are one-bond 13C−1H and 15N−1H couplings and also two/three-bond long-range 13C−1H couplings. The peak at 1.39 ppm has a one-bond 13C−1H coupling (127.2 Hz) and a two/three-bond long-range 13C−1H coupling (4.1 Hz), so it could be from the proton in methyl (CH3−C) or 4762
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Table 1. 13C−1H and 15N−1H Coupling Constants (J)a 1
H (ppm) 1.39 2.06 2.27 2.30 2.70 2.71 3.47 3.58 3.71 4.00 4.10 4.33 4.44 4.55
1
J (Hz)
JCH JCH JCH JCH JCH JCH JCH JCH JCH JCH JCH JCH JCH JCH
= = = = = = = = = = = = = =
127.2 133.1 133.7 132.3 138.6 139.2 144.2 147.3 142.4 143.3 154.3 152.5 153.5 149.2
2
J/3J (Hz)
JCH JCH JCH JCH JCH JCH JCH
= = = = = = =
4.1 4.8 5.6 6.1 3.8 4.0 7.0
JCH = 3.4 JCH = 3.2
1
H (ppm) 5.16 5.41 5.53 5.75 5.96 6.04 6.28 6.59 7.16 7.44 7.65 7.97 8.24 8.30
1
ppm are typical one-bond coupling constants between sp2-type C and 1H,33a which confirms that the peaks in this region are from protons bonded to sp2 carbon or nitrogen. The structures of these protons bonded to sp2 carbon or nitrogen change with the increase of chemical shift from 5 to 9 ppm. For instance, the protons at 5−7 ppm may be in alkenes (HCRCR2), and the protons around 7−9 ppm could be in aromatic or Nheteroaromatic species.32 Figure 3 is the solution-state 13C NMR spectrum of Titan tholin with 1H decoupling. The peak at 39.5 ppm (arrow) is 13
J (Hz)
JNH = 81.5 JNH = 88.5 JNH = 83.7 JNH = 85.0 JNH = 89.3 JNH = 90.6 JNH = 87.2 JNH = 89.8 JNH = 85.4 JNH = 87.1 JCH = 185.2 JCH = 182.4 JCH = 181.3 JCH = 186.2
a1 J reflects a one-bond coupling constant, while 2J/3J implies a two/ three-bond coupling constant.
methylene (CH2−C). According to the chemical shifts,32 the peaks below 2 ppm, including the broad multiplet and the sharp peaks, should be characteristic of the proton in saturated hydrocarbon compounds, which is confirmed by the coupling result of the peak at 1.39 ppm. The peaks at 2.06, 2.27, 2.30, 2.70, 2.71, 3.47, 3.58, 3.71, 4.00, 4.10, 4.33, 4.44, and 4.55 ppm have one-bond 13C−1H couplings, and some of them have two/ three-bond long-range 13C−1H couplings. The chemical shifts of the protons and the coupling constants32,33a suggest these protons are bonded to sp3 carbon, which connects to unsaturated or electronegative atoms, such as sp2 carbon (CC, CN), sp carbon (CN, CC), and nitrogen. The protons that have the two/three-bond long-range 13C−1H couplings should be bonded to sp3 carbon connecting to sp2 or sp carbon, and the protons that do not have two/three-bond long-range 13C−1H coupling may be bonded to sp3 carbon connecting to nitrogen. As the chemical shift of the proton increases, the sp3 carbon that the proton is bonded to could connect to more than one of these unsaturated or electronegative atoms. For instance, the proton at 2.27 ppm could be next to one sp2 carbon (H−C−CC), and the proton at 4.55 ppm could be next to two nitrogens (N−CH−N). There is an intense broad peak from 2 to 5 ppm in the 1H spectrum besides the sharp peaks listed in Table 1. Similar to the sharp peaks, the broad peak is also contributed to by the protons bonded to sp3 carbon, which connects to unsaturated or electronegative atoms. The alkynes (H−CC−R) should be observed to have peaks in the region of 2−3 ppm in 1H spectra, and the peaks should have large one-bond couplings (more than 200 Hz).33a The coupling constants of the sharp peaks in the region 2−5 ppm imply that these peaks are not from the protons in alkynes. However, we cannot exclude the possible contribution of the protons in alkynes to the broad peak. The protons of amine (RNH2, R2NH) could also contribute to the broad peak in the region 2−5 ppm.32 The peaks above 5 ppm are from the protons bonded to sp2 carbon or nitrogen, including some sharp and broad peaks. As shown in Table 1, the peaks at 5.16, 5.41, 5.53, 5.75, 5.96, 6.04, 6.28, 6.59, 7.16, and 7.44 ppm are bonded to sp2 nitrogen, and the peaks at 7.65, 7.97, 8.24, and 8.30 ppm are bonded to sp2 carbon. The coupling constants are consistent with the onebond 15N−1H and 13C−1H coupling constants.33a,34 The coupling constants of the peaks at 7.65, 7.97, 8.24, and 8.30
Figure 3. Solution-state 13C NMR spectrum of Titan tholin in DMSOd6 with 1H decoupling.
from the carbon in DMSO. According to the 13C chemical shift,33b the peaks below 25 ppm should be from saturated carbons, which do not connect electronegative atom directly. The peaks between 25−85 ppm are from saturated carbons bonded to nitrogens (C−N). As the chemical shifts of the carbons increase from 25 to 85 ppm, the number of the nitrogens bonded to these carbons could increase from one (R3C−N) to three (R−CN3). The peaks between 100−140 ppm are from the carbons in nitriles (R−CN), alkenes, or aromatic compounds (CR2CR2). The peaks near 120 ppm are characteristic of nitriles, alkenes, or aromatic compounds. The peaks at 100−115 ppm and 125−140 ppm may come from heterosubstituted and multiple-substituted species. The peaks above 140 ppm are from the carbons in imines or Nheteroaromatic compounds (CN). Chemical shifts of the carbons in CN structures occur near 160 ppm, and the shift range may expand to 140−190 ppm because of the substituents as well as multiple substitutions.33b The sample is fully 13C and 15 N isotope labeled, so, in the 13C spectrum, we can observe the 1 H−13C and 15N−13C couplings. Like the 1H spectra, the 13C NMR decoupling experiments are carried out to resolve the coupling information. Using the different decoupling methods, we obtain three 13C spectra, which are a 13C spectrum with 1H and 15N decoupling, 13C spectrum with only 1H decoupling, and 13C spectrum without 1H or 15N decoupling (Figure 4). After carefully comparing these 13C spectra with different decoupling methods, we find the 1H−13C and 15N−13C couplings of some sharp peaks. These peaks have different splitting patterns from which we can obtain detailed structural information. For example, the peak at 27.59 ppm is a quartet in the 13C spectrum without 1H decoupling and becomes a singlet with 1H decoupling, which should be from a CH3 group. The peak at 27.59 ppm is a doublet in the 13C spectrum without 15N 4763
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Table 2. 1H−13C and 15N−13C Coupling Constants (J) of Some Sharp Peaks in 13C Spectra and the Possible Fragments to These Peaksa 13
J (Hz)
C (ppm)
13.46/13.82 (1JCC = 54.04 Hz) 27.59 29.48/29.83 (1JCC = 53.46) 38.64/39.01 (1JCC = 54.61) 50.11 50.82 54.43 55.26 65.25 73.78 116.45 116.87/117.22 (1JCC = 55.10) 118.39
Figure 4. Solution-state 13C NMR spectra of Titan tholin with different decoupling methods: (a) with 1H and 15N decoupling, (b) with only 1H decoupling, and (c) without 1H or 15N decoupling.
decoupling and becomes a singlet with 15N decoupling, so this carbon should also connect to a single nitrogen. The coupling information indicates the peaks at 27.59 ppm are from a CH3− N group. However, we also find that some couplings can be observed on all the three 1D 13C spectra and cannot be decoupled by 1H and 15N decoupling, which implies that they are not 1H−13C and 15N−13C couplings. Considering that the sample is 13C and 15N isotope labeled, we believe these couplings are 13C−13C couplings and will be verified in future 2D studies. The coupling constants33c suggest that they are 13 C−13C one-bond couplings. The coupling information, including the coupling constants and the splitting patterns, is listed in Table 2. After analyzing the coupling information of the peaks in the 13 C spectra, we obtained some structural information and can propose some fragments (Table 2). The peak at 13.46/13.83 ppm is a typical methyl carbon (−CH3). This peak has a 13 C−13C one-bond coupling, so it should come from the structure of CH3−C. We think that the carbon at 13.46/13.83 ppm and the proton at 1.39 ppm are bonded to each other. The peak at 27.59 ppm is a quartet by 1H coupling and a doublet by 15 N coupling, so it is from a methyl group connecting to nitrogen (CH3−N). The peaks at 29.48/29.83 and 38.64/39.01 ppm have the same coupling pattern and they are all doublets by 13C coupling, triplets by 1H coupling, and doublets by 15N coupling, so they are from the similar structure (C−CH2−N). The peaks at 50.11, 50.82, 54.43, and 55.26 ppm are all triplets by 1H coupling and triplets by 15N coupling, so they are all from methylene groups connecting to two nitrogens (N−CH2− N). The peaks at 65.25 and 73.78 ppm are also triplets by 1H coupling, so they are also from methylene groups. What other atoms do these carbons connect to? If they connect to carbon, the 13C−13C one-bond coupling should be big enough to be observed (the 13C−13C one-bond coupling constant is usually 30−180 Hz dependent upon hybridization of the carbon33c). We did not observe 13C−13C one-bond coupling, so they are not bonded to carbon. There is only one possibility: they are bonded to nitrogens, and the 15N−13C one-bond coupling constant is too small33d to be observed. In some case, the 15 N−13C one-bond coupling constant is less than 0.5 Hz,35 while the linewidths of the peaks at 65.25 and 73.78 ppm are 4.6 and 2.4 Hz. Combined with the 13C chemical shifts,33b we think they are bonded to two nitrogens with a very small
121.37/121.72 (1JCC = 53.51) 157.58 158.11 159.23 159.52 162.79 172.06
1
1 1
1
possible fragments CH3−C
JCH = 131.26 1
JCH = 138.62 (q), JNC = 10.56 (d) JCH = 143.82 (t), 1JNC = 3.88 (d)
CH3−N C−CH2−N
JCH = 145.83 (t), 1JNC = 3.76 (d)
1
JCH JCH 1 JCH 1 JCH 1 JCH 1 JCH 1 JNC 1 JNC 1
= = = = = = = =
154.28 (t), 1JNC = 11.16 (t) 154.21 (t), 1JNC = 10.75 (t) 153.07 (t), 1JNC = 3.05 (t) 152.98 (t), 1JNC = 2.81 (t) 145.44 (t) 149.58 (t) 11.16 (t) 15.75 (d), 2JHC = 7.87 (t)
1
N−CH2−N
N−C−N CH2−CN
JNC = 12.97 (d), 1JNC = 9.90 (d), 1 JNC = 3.07 (t) 1 JNC = 15.50 (d), 2JHC = 7.08 (t)
CN4
1
NCN2
JNC JNC 1 JNC 1 JNC 1 JNC 1 JCH 1
= = = = = =
21.20 (q) 20.07 (q) 19.21 (t) 20.18 (t) 20.19 (t), 1JNC = 9.43 (d) 185.43 (d)
CH2−CN
NCN NCN2 NCH−N
a
The d, t, and q in the bracket mean that the peak is doublet, triplet, and quartet, respectively. The carbons corresponding to the chemical shifts are in italic type in the fragments.
coupling constant (N−CH2−N). These sharp peaks from 27.59 to 73.78 ppm are consistent with saturated carbons connecting to nitrogens (one or two). The carbon peak at 116.45 ppm is a triplet by 15N coupling, indicating that it is a carbon bonded to two nitrogens (N−C− N). This carbon should not be bonded to either a proton or carbon because the 1H−13C and 13C−13C one-bond coupling constants are big enough to be observed. It could be bonded to two additional nitrogens with a very small coupling constant. The peaks at 116.87/117.22 and 121.37/121.72 ppm are doublets by 13C coupling and doublets by 15N coupling, so they are from carbons bound to one carbon and one nitrogen. The chemical shifts and the coupling constants33b,d of these carbons suggest that they are nitrile structures (C−CN). They also have two small 13C−1H two-bond long-range couplings, indicating that there are two protons attaching to the adjacent carbon (CH2−CN). The peak at 118.39 ppm is multiplet, all the splitting from 15N−13C couplings, and is assigned to a quaternary carbon bonded to four nitrogens (CN4). The 15 N−13C coupling constants are different, so the nature of the four nitrogens must also be different. Two small couplings are one-bond couplings between sp3 carbon and sp3 nitrogen, and two slightly larger couplings are one-bond couplings between sp3 carbon and sp2 nitrogen.33d The peaks at 157.58, 158.11, and 162.79 ppm have three one-bond couplings from the nitrogens, so they are from carbons bonded to three nitrogens. According to the chemical shift,33b we think these carbons connect to one nitrogen with a 4764
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N signal is still very weak because the sensitivity of the 15N is much lower than 1H nuclei (1:1000). Therefore, only a few peaks are observed after extensive signal averaging, and the 15N decoupling experiment becomes difficult. Although there are only a few peaks in the 15N NMR spectra, they still contain some important structural information. According to the chemical shift,36 the peaks in the 15N spectra can be assigned to general functionality. The peaks between 0 and 80 ppm in Figure 5b are from the nitrogens in amines, including primary, secondary, and tertiary amines (R−NH2, R2NH, and R3N). Though different R-groups can lead to different chemical shifts, the amine-type nitrogen will be observed to have peaks in this region.36 These peaks correspond to the peaks at 25−85 ppm in 13C spectra. The peaks between 160 and 260 ppm (Figure 5a) are from imines or N-heteroaromatic compounds (CN) and nitriles (R−CN). The result of 15N spectra is consistent with the 1H and 13C spectra. Sagan et al.3 reported that polycyclic aromatic hydrocarbons (PAHs) are present in Titan tholins with an upper limit of 6%. Their solid-state 13C NMR suggests the unsaturated carbon (aromatic and/or olefinic) accounts 25% of total carbon.3 In our study, the peak integration of 13C NMR spectrum indicates that the unsaturated carbon accounts 50% of total carbon, demonstrating our tholin is more unsaturated than theirs. However, the 13 C NMR spectra and the decoupling information of our tholin sample show there is no resonance corresponding to pure PAHs. The high resolution mass spectrometry of both AC discharge24 and EUV25 generated tholins also shows essentially no nitrogen-free species with a high unsaturated degree in molecule range from 200 to 300 Da, which rules out pure PAHs in favor of nitrogen-containing aromatic species. Both 1H and 13C NMR spectra of our tholin sample indicate the presence of nitrogen-containing aromatic species, consistent with the high resolution mass spectrometry results24,25 and the low pressure tholins prepared by Imananka et al.4 Their IR spectra4 show the characteristics of N−H, C− H, CN, CN, and NCN bonds, which are all identified in our tholin sample by NMR study. The high resolution mass spectrometry supports the presence of amino and nitrile functionalities in the AC discharge24 tholin prepared in the similar condition to our tholin sample. The NMR results indicate that amine and nitrile functional groups are present in our tholin sample. By comparing the IR spectra of tholin and poly-HCN, Coll et al.5,21 suggested the base of tholins could be poly-HCN. Quirico et al.13 also found several similarities between tholins and poly-HCN by IR and Raman spectra. However, the chemical composition of tholins is likely to be considerably more complex than poly-HCN due to the large amount of possible polymerization reactions.13 Vuitton et al. recently compared poly-HCN to three tholin samples.37 The very high resolution and exact mass measurements coupled to MS/ HRMS techniques revealed that poly-HCN is at best a minor component of tholins and the similar appearance of the IR spectrum of tholins and poly-HCN was only attributed to a similar distribution of functional groups rather than a detailed structural identity.37 The solution-state NMR study on polyHCN29 indicates that almost all the protons in poly-HCN are bonded to nitrogen and that the polymer structures are polyimine chain-like systems, while the present solution-state 1D NMR results display that amine, nitrile, imine, and Nheteroaromatic compounds are present in the tholin samples,
double bond and to the other two nitrogens with single bonds (guanadyl, NCN2). The peaks at 159.23 and 159.52 ppm are triplets by 15N couplings, so these carbons are bonded to two near equivalent nitrogens. From the chemical shift, they could connect to two nitrogens by double bonds (NCN). We only observe one 1H−13C one-bond coupling for the broad peak at 172.06 ppm, so this carbon is bonded to one proton and should not be bonded to carbon (the 13C−13C one-bond coupling would be large enough to be observed33c). The chemical shift implies this carbon should have a double bond to nitrogen. The remaining bond is connecting to another nitrogen (NCH−N). Because this peak is broad (the line width is 18.6 Hz), the 15N−13C one-bond couplings are too small to be observed.33d The five sharp peaks at 157.58, 158.11, 159.23, 159.52, 162.79, and 172.06 ppm are all from the carbons having double bonds with nitrogen, proving that the peaks in this region are from the carbons in imines or Nheteroaromatic compounds (CN). The results of 1D 13C NMR decoupling experiments show that there are few carbon−carbon bonds, but there are many carbon−nitrogen bonds forming a multitude of functional groups (single, double, and triple bonds). Since there is such a variety of carbon−nitrogen bonding, the nitrogen should have a broad range of magnetic environments. The 15 N 1D NMR experiments were carried out to demonstrate this. The chemical shift range of the 15N is very wide (from 0 to 900 ppm, referenced to liquid ammonia). There are only three elements (carbon, hydrogen, and nitrogen) in our sample, so the range of functionality is somewhat limited, and the chemical shift range of the 15N should be comparatively narrow (from 0 to 400 ppm). On the basis of the result of 13C decoupling experiments, we predicted the 15N chemical shift in the sample could be between 0 and 300 ppm. Even though the chemical shift range is reduced by more than half, the NMR spectrometer still does not have enough power to cover it in one experiment. For this reason, two 15N NMR experiments are carried out on the Varian INOVA 600 spectrometer using the cryogenic probe. Two 15N NMR spectra of Titan tholin with 1H decoupling are shown in Figure 5. Each spectrum covers a 200 ppm range. One is from 340 to 140 ppm (Figure 5a), and the other one is from 160 to −40 ppm (Figure 5b). Though our sample is 15N labeled, the
Figure 5. Solution-state 15N NMR spectra of Titan tholin in DMSO-d6 with 1H decoupling (chemical shift reference of 15N, δN (NH3) = 0 ppm): (a) 340 to 140 ppm and (b) 160 to −40 ppm. 4765
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Notes
suggesting that poly-HCN is not an effective analogue of Titan tholins. Ruiz-Bermejo et al.14,15 prepared two hydrophilic tholins using CH4/N2/H2 atmosphere and spark discharges either in aqueous aerosols or in liquid water and acquire their solid-state and solution-state 13C NMR spectra, the resonances on which correspond to CO, CN, C−O, C−N, and C−H functional groups.14,15 Our tholin exhibits resonances corresponding to CN, CN, C−N, and C−H functional groups. The CO and C−O but no CN functional groups exist in their hydrophilic tholins due to the presence of aqueous aerosols or liquid water when preparing tholins. Our tholin is prepared in an anaerobic and anhydrous condition; thus, it does not include oxygen-containing groups but has abundant nitrogen-containing compounds. A large number of nitrogen-incorporated and nitrogen-substituted compounds are detected in the tholin sample, of great significance in understanding complex organic chemistry in anaerobic, extraterrestrial environments. The present 1D NMR study gives new structural information on the tholins. The 1H, and 13C NMR spectra and decoupling experiments show that (1) the protons are bonded to both saturated and unsaturated carbons and nitrogens; (2) a very small amount of saturated hydrocarbon compounds are present in tholin samples; (3) the sp3, sp2, and sp carbons are all existing in the tholin samples, and the ratio of saturated and unsaturated carbon is 1:1; (4) there is an extensive range of carbon−nitrogen (single, double, and triple bonds); and (5) the carbon may connect to one, two, three, and even four nitrogens. The 15N spectra support this richness in functionality. The result of the 1D NMR spectra and the decoupling experiments indicate that amine, nitrile, imine, and Nheteroaromatic compounds are present in the tholin samples, and the signal integration of the 1H and 13C spectra shows that these nitrogenous organics are major components of the tholin samples.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
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REFERENCES
We gratefully acknowledge support of this work through NASA Exobiology Grant NNG05GO58G and the NASA Astrobiology Initiative, through JPL subcontract 1372177. We also recognize the assistance of Dr. Tamara Munsch in the very early portion of this study.
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CONCLUSIONS In this study, we prepared and analyzed 13C and 15N isotope labeled Titan tholins. Using 1D 1H, 13C, and 15N spectra and decoupling NMR experiments, we provide further insight into the chemical composition of tholins. The chemical shifts of the 1 H, 13C, and 15N all point to amine, nitrile, imine, and Nheteroaromatic functionality being present in the tholin samples. Some structural fragments, such as CH3−C, CH3− N, C−CH2−N, N−CH2−N, CN4, CH2−CN, NCN2, N CN, NCN2, and NCH−N, are identified using the decoupling experiments. A large number of nitrogen-incorporated and nitrogen-substituted compounds are detected in the tholin sample, contributing greatly to our understanding of organic chemistry that occurs on Titan as well as the anaerobic, extraterrestrial environments. A more systematic multidimensional NMR study is currently in progress in our laboratory and will provide more quantitative determination of refined structures in the future.
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AUTHOR INFORMATION
Corresponding Author
*Phone: 713/743-3755. Fax: 713/743-8630. E-mail:
[email protected]. Present Address ⊥
SABIC Innovative Plastics, 1 Lexan Lane, Mount Vernon, Indiana 47620, United States. 4766
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