100 °C 1 (1.9) 0.4 (0.8) 0.4 (0.8)
150 °C 1 (1.9) 1 (1.9) 3.8 (7.2)
200 °C 1.8 (3.4) 3.2 (6.1) 3.4 (6.5)
Infrared Spectroscopy of the Cyanide Supernatant and Polymer
Figure 2.6 shows IR spectra representative of the cyanide polymer (black trace) and supernatant (orange trace) obtained from a six-month room temperature reaction produced during this study. Infrared spectra of the cyanide polymer gathered in this study correspond well to reported literature.43-46
Within these spectra are three distinct regions of IR absorbance bands, which are common to the liquid and solid. In the higher energy portion of the IR spectrum, there appear two strong and broad absorbance peaks attributed as primary (and possibly secondary) nitrogen-hydrogen bond stretching. These absorbance bands are associated with the amine functional group contained within the supernatant and polymer. The nitrile functional moiety (carbon-nitrogen triple bond absorbance) in the supernatant and polymer can be located in the middle of the two spectra at roughly 2150 cm-1. The last discernable
pair of peaks represented in both fractions is located near 1640 cm-1 and is attributed to carbon-nitrogen double bond absorbances associated with imine functional groups of the cyanide organic products contained in the supernatant and composing the backbone structure of the cyanide polymer. The remaining absorbances lie within the fingerprint regions of the spectra. For the polymer, there is a complex hodgepodge of absorbance bands likely associated with variations in substitution of carbon-nitrogen single and double bonds. This region has been reported in the literature as being attributed to polypeptides or polyamidines.10 Further analysis of this region may be possible with chemometrics software to assist in identifying additional structural elements of the cyanide polymer; however, the intention of IR analyses of these reactions is qualitative in nature and intended to reveal changes in the bulk properties of the cyanide products as the reactions are exposed to increasing temperatures. Therefore, more involved software analysis of the IR spectra was not considered necessary.
Figure 2.6. IR spectra of supernatant (orange) and polymer (black) obtained from a 6-month, room temperature NH4CN reaction. Both supernatant and polymer contain primary and secondary amines, nitriles, and imines. The blue boxed region contains a series of complex absorbances typically associated with C-C and C-N single and double bond stretching and bending vibrational modes.
Figure 2.7 is a series of overlaid supernatant spectra that have reacted for 72 hours. Each spectrum represents a separate reaction heated incrementally by 50 °C to 200 °C. Peak (A) at 1631 cm-1 is the imine peak and is the same in all the spectra regardless of temperature. As the temperature is increased, four additional peaks develop within the spectra. The second peak (B) at 1585 cm-1 is attributed a splitting in the imine peak (A) associated with a change in the structure of the supernatant. With an increasing prominence of a new structural motif in the supernatant, some of the imine functional groups are bound to this new structure and as a result their absorbance is shifted to slightly lower energy. This results in a splitting in the imine peak (A) to produce imine peak (B) at higher temperatures. The remaining peaks (C, D, and E) are attributed to these changes in the structure of the supernatant as variations in the substitution patterns as well as single and double bond character in the supernatant.
Figure 2.7. IR spectra of supernatant from NH4CN reaction heated at different temperatures for 72 hours. Peaks (A) 1631 cm-1, (B) 1585 cm-1, (C) 1454 cm-1, (D) 1382 cm-1 and (E) 1350 cm-1 are labeled.
Figure 2.8 represents overlaid polymer spectra with increasing ammonium cyanide reaction temperatures. As the temperature is increased, the amine absorbance bands show
no significant changes. The most significant differences in the spectra occur in the fingerprint region at less than 1640 cm-1. Here, the prominent peak in the room temperature spectrum at 1640 cm-1 corresponds to imine groups and remains present throughout the temperature series. At higher temperatures, the imine peak becomes a shoulder to absorbance bands at lower wavenumbers that develop with increasing reaction temperature.
Figure 2.8. IR spectra of polymer obtained from NH4CN reactions heated at different temperatures for 72 hours. The imine peak at 1631 cm-1 is present in all of the spectra. However, at increased reaction temperatures, there is a shift in the intensity of the absorbance bands between 1600-1000 cm-1 as the reaction temperature is increased.
Additionally, there is a noticeable increase in absorbance bands between 1500 – 1000 cm-1 attributed to substitution and single/double bond variations in the C-C and C-N network composing the polymer structure. This region of IR spectra is also associated with vibrational stretching and bending modes for several different functional groups. This includes amines, amides, alkenes, aromatics, and imines. Similar to the peak development observed in the supernatant, the development of a new structural motif in the polymer may explain these increases in absorbances in the fingerprint region of the polymer spectra.
The last significant variation of the cyanide polymer due to increasing temperature is identified in a close-up of the cyanide peak around 2200 cm-1. Shown in Figure 2.9 is an enlarged view of the cyanide polymer nitrile peak obtained from heated reactions for 72 hours at different temperatures. Peak shifting is observed within the nitrile peak, absorbing at 2180 cm-1 in the room temperature spectrum to a higher absorbance frequency of 2200 cm-1 in the 200 °C spectrum. With this shifting a peak develops at 2170 cm-1, which is assigned as an isonitrile peak based on the literature where ammonium cyanide polymer was produced under similar contditions. However, this study by Mutsukura and Akita. was not analyzing polymers produced at different temperatures.45 An alternative explanation of this peak splitting of the nitrile functional group could be changes in bonding characteristics of the cyanide polymer as the temperature of reaction increases (e.g., a shift from one type or mixture of structures dominated by polyamidines, polypeptides, or polynucleobases to another structure). Likewise, this structural change is thought to have an effect on the nitrile peak. As the reaction with cyanide continues, free cyanide reacts with different motifs in the polymer to produce a nitrile functional group and the associated absorbance peaks having similar but slightly different absorbance energies. As the new structure forms, cyanide may react with and bind to new motifs. This is thought to result in splitting of the nitrile peak.
All of the observations shown are representative of changes in the bulk properties of the cyanide products with increasing temperature (for the NH4CN reaction); however, there are questions that still remain. What is happening on a molecular level in both supernatant and polymer? How do these changes affect nitrogen heterocycle production?
Figure 2.9. Close-up of IR spectra of polymer obtained from NH4CN reactions heated at different temperatures for 72 hours. A shift in absorbance from 2180 to 2200 cm-1 reveals the development of an additional peak at 2170 cm-1at higher temperature.
Thermochemolysis of Cyanide Polymer Using TMAH GC-MS
Polymer samples (>5 mg each) of the 72-hour temperature series were shipped to NASA Johnson Space Center (Houston, Texas) for thermochemolysis gas chromatography mass spectrometric analysis (TMAH-GC-MS). TMAH cleaves labile functional groups (e.g., amidine, amide, and imine) and methylates the resulting products in situ.7 As a result, methylated compounds are likely to be the result of derivatization; however, cleavage of naturally methylated compounds from the polymer cannot be completely ruled out. Shown in Appendix A is a complete tabulation of all the compounds detected in the mass spectral data of the polymer samples with fits >700 along with methylated and non-methylated structures of these compounds.
The total ion chromatogram obtained from the room temperature polymer sample is shown in Figure 2.10 and was analyzed peak by peak using the NIST 2014 MS database. Additionally, masses corresponding to methyl-derivatized nitrogen heterocycles were
searched for by plotting extracted ion chromatograms, which can result in better examination of the data.
Figure 2.10. GC-MS total ion chromatogram with thermochemolysis products of room temperature cyanide polymer labeled: (A) trimethylamine, (B) formamide, (C) N,N’-dimethylformamide, (D) aminoacetonitrile, (E) N,N’-dimethylurea, (F) trimethylurea, (G) diamide, (H) N,N’dimethyloxamide, (I) formyltrimethylurea, (J) 1-methyl-dihydrouracil, (K) 1,3-dimethyl-2,4,5-trioxoimidazolidine, (L) tetramethylurea, and (M) 4-dimethylaminopyridin-2-amine.
Within this chromatogram, several compounds were identified with good matches to NIST library spectra including some previously reported in HCN polymers by Matthews and Minard such as aminoacetonitrile, cyanamide (as the dimethyl derivative), formamide (as the methyl and dimethyl derivatives), urea (as the dimethyl, trimethyl, and tetramethyl derivatives), adenine (as the methyl, dimethyl, and trimethyl derivatives), xanthine (as the trimethyl derivative, caffeine) and cyanuric acid (as the trimethyl derivative).7 Additional compounds were also identified in this study including (1) oxamide (as the dimethyl derivative), (2) dihydrouracil (as the methyl derivative) and hypoxanthine (as the dimethyl
derivative), which are in the class of pyrimidines and purines, (3) 2,4-diaminopyridine (as the dimethyl derivative), which is a 6-membered nitrogen heterocycle, and (4) 2,4,5- trioxoimidazolidine (as the dimethyl derivative) and 2,4-imidazolidinedione (as the trimethyl derivative), which are both 5-membered nitrogen heterocycles. The complex suite of TMAH thermochemolysis-derived compounds reinforces the concept that the cyanide polymer is complex and heterogeneous in nature. Table 2.3 lists the compounds identified in the room temperature polymer sample.
Shown in Figure 2.11 are overlaid total ion chromatograms of the four cyanide polymer samples reacted at increasing temperatures. In this figure, one can easily visualize the changes in compound production with increases in the reaction temperature. After establishing the identity of these compounds, thermochemolysis-derived compounds of the polymer can be tracked with respect to reaction temperature.
Figure 2.11. Total ion chromatograms of polymer samples synthesized at different reaction temperatures for 72 h that have been measured by thermochemolysis GC- MS. Aromatic and heterocyclic compounds are tracked throughout the entire temperature series: (A) benzene, (B) anthracene/phenanthrene, (C) m-di-t-butyl- benzene, (D) trimethyl-cyanuric acid, (E) 1,3-dimethyl-5,6-diamino-uracil, (F) 1,7- dimethyl-hypoxanthine, (G) N6,N6ʹ,N9-trimethyl-adenine, and (H) 1,3,7-trimethyl- xanthine.
31
Table 2.3. Compounds identified in room temperature polymer samples as determined by TMAH-GC-MS and comparison to NIST/EPA/NIH Mass Spectral Library.
32
Table 2.3 Continued. Compounds identified in room temperature polymer samples as determined by TMAH-GC-MS and comparison to NIST/EPA/NIH Mass Spectral Library.
Figures 2.12-2.15 provide an analysis of extracted ion chromatograms; there appears to be a general decrease in abundances (peak areas) of nitrogen heterocycles as the temperature of the NH4CN reaction increased. Figure 2.12 shows the reduction in purine nitrogen heterocycles that corresponds to increasing reaction temperatures. This trend extended to many other non-N-heterocyclic compounds as well (Figure 2.13). Displaying the reverse trend observed in Figure 2.14, there is an increase in abundance of aromatic products such as benzene and anthracene/phenanthrene as the temperature of the NH4CN reaction increased. Benzene has the most pronounced increase in abundance at 150 °C which then decreases at 200 °C. This production is still higher than that produced in the room temperature reactions. However, the sharp increase followed by a pronounced decrease in benzene production cannot be ignored. Reasons for this potential discrepancy could result from variation in sample size (1 – 3 mg) and/or to benzene product fragments from other compounds at the associated retention time (EI source may fragment compounds like m-di-t-butyl-benzene).
Figure 2.12. Relative peak area versus temperature plots of thermochemolysis products of cyanide polymer produced from heated ammonium cyanide reactions. Peak area was determined from the GC-MS extracted ion chromatogram at the appropriate m/z. Purine heterocycles decrease in abundance as reaction temperature is increased.
Figure 2.13. Relative peak area versus temperature plots of thermochemolysis products of cyanide polymer produced from heated ammonium cyanide reactions. Peak area was determined from the GC-MS extracted ion chromatogram at the appropriate m/z. Non-heterocyclic products decrease in abundance with increased reaction temperature.
Figure 2.14. Relative peak area versus temperature plots of thermochemolysis products of cyanide polymer produced from heated ammonium cyanide reactions. Peak area was determined from the GC-MS extracted ion chromatogram at the appropriate m/z. Aromatic compounds increase in abundance as the reaction temperature is increased.
To highlight these trends, a comparison of adenine (nucleobase) and benzene (aromatic) is shown in Figure 2.15. The extracted ion chromatograms of adenine are shown on the left and are all scaled to the room temperature extracted ion chromatogram. The extracted ion chromatograms of benzene are shown on the right and are all scaled to the
150 °C extracted ion chromatogram. It is possible that higher reaction temperatures (for NH4CN) have produced char, highly aromatic coal-like structure, from the cyanide polymer because multiple aromatic species were observed to increase with increasing reaction temperature.
The products that can be released from cyanide polymer are thought to play an important role in prebiotic chemistry on early Earth. One important observation from this study is that many of these products (e.g., Nitrogen heterocycles) are not “better preserved” in cyanide polymer form compared to the supernatant form (the latter will be discussed in the next section) and products that can be released from cyanide polymer will strongly depend on initial reaction conditions.
Figure 2.15. Comparison of extracted ion chromatograms of adenine (nucleobase) and benzene (aromatic) production in thermochemolysis products of NH4CN polymers. Relative peak area for adenine decreases while the relative peak area for benzene increases, both with respect to increasing NH4CN reaction temperature that produced the cyanide polymer.
Qualitative Identification of Nitrogen heterocycles Using HPLC
In this study, HPLC with UV detection was used for qualitative identification of nitrogen heterocycles. Initially, 15 nitrogen heterocycles were chosen for analysis as they are either known to be produced in room temperature cyanide reactions or are found in formic acid extracts of meteorites. Many nitrogen heterocycles, including nucleobases, have a significant UV absorbance at 260 nm or nearby. The exception to this was s-triazine whose lambda max is near 200 nm and has virtually no detectable absorbance at the monitoring wavelength of 260 nm. Therefore, triazines were not searched for in the supernatant samples due to current instrumental limitations. Triazines have been previously shown to be produced in cyanide reactions and have been identified in meteorite extracts.47
As most of the products of cyanide chemistry are produced in relatively low abundance, have similar chemical properties, and similar chromatographic retention times, optimization of the separation method is paramount. Without adequate peak separation, co- injection and comparison to standards will only reveal so much. After working on optimizing the chromatographic method, adequate separation of a mixture of 13 nitrogen heterocycles was achieved. This separation has two exceptions; co-elution of nitrogen heterocycles occurs in two separate peaks (uracil, 2,6-diaminopurine, and 6,8- diaminopurine around 28 min and adenine and xanthine around 32 min) in the standard nitrogen heterocycle chromatogram. Figure 2.16 below represents the chromatograms of four standard nitrogen heterocycle solutions. The retention times of these standards are used for compound identification, and comparison of these chromatograms identified which compounds co-eluted.
Figure 2.16. HPLC-UV chromatograms of four different mixtures of nitrogen heterocycle reference standards in 0.1 M NH4OH. Peaks are labeled as follows: cytosine (C), 5-hydroxyuracil (5h), orotic acid (O), 5-aminoorotic acid (5ao), uracil (U), 2,6-diaminopurine (2,6-DAP), 6,8-diaminopurine (6,8-DAP), guanine (G), hypoxanthine (H), adenine (A), xanthine (X), purine (P), and thymine (T).
The retention time, including standard deviation, for reference nitrogen heterocycles are shown in Table 2.4. Some nitrogen heterocycles are weakly absorbing at 260 nm such as 5-hydroxyuracil and 5-aminoorotic acid, which increases their limit of detection. The peak for orotic acid at 22.65 ± 0.02 min is sharp but elutes at an aberrant location in the blank chromatogram of 0.1 M ammonium hydroxide. In this region, a small peak elutes adjacent to the peak associated with the orotic acid standard. This is not considered problematic as the peak of orotic acid is strong, sharp, and easily distinguished from the aberrant background peak.
Table 2.4. HPLC-UV retention times of nitrogen heterocycles. Abbreviations for compounds are shown in parentheses. Standard deviation (SD) has been shown in minutes (min.) and seconds (sec.).
Retention Times of Nitrogen heterocycles
Nitrogen heterocycle Average (min.) SD (min.) SD (sec.)
Cytosine (C) 19.75 0.01 0.89 5-hydroxyuracil (5hU) 21.15 0.01 0.35 Orotic acid (O) 22.65 0.02 0.97 5-aminoorotic acid (5aO) 25.14 0.01 0.64 6,8-diaminopurine (U) 28.12 0.03 2.08 Uracil (U) 28.18 0.04 2.53 2,6-diaminopurine (U) 28.57 0.01 0.73 Guanine (G) 29.66 0.04 2.28 Hypoxanthine (H) 30.87 0.04 2.30 Adenine (A) 32.13 0.04 2.48 Xanthine (A) 32.28 0.02 1.11 Purine (P) 37.11 0.08 4.80 Thymine (T) 38.54 0.04 2.38
In the following Figures 2.17 – 2.22, the nitrogen heterocycles that have been tracked through the different temperature series and time series reactions have been assigned specific labels. These labels are listed in Table 2.4 and are as follows: cytosine (C), 5-hydroxyuracil (5hU), orotic acid (O), 5-aminoorotic acid (5aO), guanine (G), hypoxanthine (H), purine (P), and thymine (T). There are two peaks associated with co- eluting nitrogen heterocycles that need special acknowledgement, the peak associated with uracil, 2,6-diaminopurine, and 6,8-diaminopurine have all been assigned as (U) and the peak associated with adenine and xanthine is assigned as (A).
Nucleobases in the Time and Temperature Reaction Matrix
All five of the canonical nucleobases (cytosine, uracil, thymine, guanine, and adenine) commonly found in Earth’s biosphere were determined in the supernatant using HPLC with UV detection at 260 nm. Cytosine was not detected above the instrument’s
limit of detection (10 picomoles on column) throughout the entire time and temperature reactions. Analysis for uracil was more complicated due to peak co-elution with 2,6- and 6,8-diaminopurine. However, there was a peak that corresponds to the retention time of these compounds in multiple HPLC-UV chromatograms, most noticeable at the 200 ˚C reaction temperature. Thus, at least one of these compounds among uracil, 2,6- and 6,8- diaminopurine was likely produced in the 200 ˚C NH4CN reactions. Thymine is difficult to discern. In multiple UV chromatograms, there is a peak near the thymine standard peak, which may be thymine. Interestingly, there is a very large peak near the retention time of thymine in the supernatant of the reaction conducted at 50 ˚C for 3 hours but this compound is likely not thymine because its lambda max is 295 nm (while thymine’s lambda max is ~260 nm).
Best matches for guanine were found in the 50 and 200 ˚C NH4CN reaction (both 3-hour reaction times). Also, there are peaks located within the standard deviation (0.04 min) of the retention time of guanine (29.66 min) in other reactions. Adenine and xanthine co-elute at almost the same retention time. There is a very closely associated peak to these standards in almost all supernatant samples. Furthermore, a distinct peak for the entire temperature series at 24 hours and 72 hours is easily observable. Adenine is typically the nucleobase synthesized in the highest abundance in cyanide reactions, so the presence of adenine here is not completely surprising. It’s worth mentioning that both adenine and xanthine were identified as thermochemolysis-derived compounds of the cyanide polymer by GC-MS and comparison with the NIST mass spectral library. Therefore, we conclude that both adenine and xanthine are likely to be present in many of the supernatant samples.
Figure 2.17. UV chromatograms of the supernatant isolated from NH4CN reactions heated at different temperatures for 3 hours. 20 °C is a 6-month room temperature NH4CN reaction. Compounds are cytosine (C), 5-hydroxyuracil (5h), orotic acid (O), 5-aminoorotic acid (5ao), uracil (U) co-elutes with 2,6 and 6,8-diaminopurine (2,6- DAP and 6,8-DAP), guanine (G), hypoxanthine (H), adenine (A) co-elutes with xanthine (X), purine (P), and thymine (T). Dashed lines are retention times of associated N-heterocycle standard peak.
Figure 2.18. UV chromatograms of the supernatant isolated from NH4CN reactions heated at different temperatures for 24 hours. 20 °C is a 6-month room temperature NH4CN reaction. Compounds are cytosine (C), 5-hydroxyuracil (5h), orotic acid (O), 5-aminoorotic acid (5ao), uracil (U) co-elutes with 2,6 and 6,8-diaminopurine (2,6- DAP and 6,8-DAP), guanine (G), hypoxanthine (H), adenine (A) co-elutes with xanthine (X), purine (P), and thymine (T). Dashed lines are the retention times of associated N-heterocycle standard peak.
Figure 2.19. UV chromatograms of the supernatant isolated from NH4CN reactions heated at different temperatures for 72 hours. 20 °C is a 6-month room temperature NH4CN reaction. Compounds are cytosine (C), 5-hydroxyuracil (5h), orotic acid (O),