Abstract
Lignite humic acid on reaction with formaldehyde and aqueous alkali led to a polybicyclo[3.3.1]nonane. This was characterized spectroscopically using mass spectrometry, FT-IR, powder X-ray diffraction, TEM, and NMR studies (1H-NMR, CP-MAS-SS-NMR) which though challenging were useful for confirming the structure of the BCN polymer. The product showed an envelope peak in its MALDI-MS spectrum, based on which , , and polydispersity index have been calculated. Based on the NMR spectrum of intermediate, the presence of the N-formyl group has been shown in it. The new polymer could be useful in agriculture in water-deficient areas. Most papers on humic acids include only broad and general information like elemental analysis (occasionally TEM, SEM, TGA, DSC, etc.). Very complicated structures have been proposed by scientists earlier. Only in very recent years, it has been recognized that these are relatively small molecules which masquerade as supramolecular structures. It is pointed out that most papers do not put down the structure of the compound and provide no concrete proof for proposing such structures. The novelty of our work is that we have characterized the precise molecular weights based on mass spectrometry and NMR spectroscopy along with a well-defined structure. This is not the case in most other publications.
1. Introduction
The use of humic acids from soil, brown coal, and city solid waste has been engaging our attention for some years now [1–5]. Humic acid isolated from lignite is used as a growth promotant in agriculture. One such example of a commercial product is Agritone 4.5 successfully used by farmers in India [6]. It is increasingly becoming clearer that humic acids are small molecules [7] which masquerade as large supramolecular structures [8, 9]. Though it has been stated that novel chemical compounds could be isolated from lignite, so far, no such compound has been isolated from it. In the meantime, we came across [10] a paper on cross-linking of humic acid using formaldehyde and alkali. This humic acid contained aromatic phenolic groups which could lead to cross-linking via methylene (-CH2-) bridge formation. Such cross-linked humic acids are capable of capturing both water and cations efficiently, a result which could be useful in water-deficient areas like deserts worldwide. The identification and analysis of envelope peaks in mass spectra have been used to characterize both polymers and peptides [11–15].
We carried out a similar formaldehyde-alkali reaction with lignite humic acid and isolated a new product which was lachrymatory in nature, and it is proposed that this is due to removal of the formyl group. Based on the prominent signal centered at 8.310 ppm in its 1H-NMR spectrum, it has been shown to be a N-formyl derivative. This compound shows a mass spectral envelope peak pointing towards its polymeric nature. Acid hydrolysis of this led to a product, which was no longer lachrymatory in nature. Based on X-ray diffraction studies, the “monomer” is proposed to be a substituted bicyclo[3.3.1]nonane containing an exposed exocyclic double bond. This new compound is not aromatic in nature but is a bridged alicyclic compound, not known in chemical literature before. Thus, cross-linking via formation of methylene (-CH2-) bridge observed in aromatic phenols cannot occur in our case. It is proposed that, instead, a base-catalyzed olefin polymerization happens in our case leading to the formation of the substituted polybicyclo[3.3.1]nonane. The envelope peak of the poly-(3-amino-1,5-dimethyl-7-methylenebicyclo[3.3.1]nonane-3-carboxylic acid) is centered at 445.1 a. m. u. and attributed to the “dimeric” repeat unit a. m. u. (% error: 0.04%). Further, , , and polydispersity index have also been calculated. Computationally, a supramolecular structure with a cavity has been generated [16, 17]. Based on the NMR evidence, it is proposed that this cavity traps formaldehyde –OH- [OHCH2O-] making it lachrymatory. On treatment with dilute HCl, the product was no longer lachrymatory. The latter could be important in agriculture for retention of cations and water, in water-deficient soils, especially in deserts [18–20].
2. Experimental
2.1. Materials and Methods
All the reagents and solvents were purchased from Sigma-Aldrich (greater than 99.9% purity). FT-IR studies were performed using Bruker Tensor 27 FT-IR with diamond ATR cell in methanol solution. Initial 1H-NMR studies were done in the International Centre for Genetic Engineering and Biotechnology (ICGEB, New Delhi) and the Translational Health Science and Technology Institute, Faridabad (THSTI, Faridabad). 1H-NMR, 2D-NMR, temperature-dependent NMR, and solid-state NMR spectra were recorded using Bruker 500 MHz NMR instrument in D2O solution in Tata Institute of Fundamental Research (TIFR), Hyderabad. Mass spectrum of this compound was recorded in THSTI, Faridabad, and MALDI-MS spectrum was recorded in the Regional Center for Biotechnology (RCB, Faridabad) in methanol solution. Transmission electron microscopy (TEM) was also recorded using the G2 30-U twin microscope in methanol solution and mica-coated ITO grid. Single-crystal X-ray diffraction was recorded using Bruker Smart Apex II Ultra with sample characterizing temperature range of 80 K–500 K for crystal structure determination and electron density mapping. The specifications are as follows: rotating anode; molybdenum source; collimator diameter, 0.5 mm; maximum operating power, 45 kV 80 mA; detector, CCD detector (Apex II) with a fixed distance of 58.2 mm; and 3-axis goniometer with fixed Kappa axis. Powder X-ray diffraction was recorded using Bruker D8 Advance with sealed tube anode, copper source, maximum operating power of 40 kV 40 mA, and detector (LYNXEYE (1D)). Both single-crystal X-ray diffraction and powder X-ray diffraction were recorded at X-ray facility in Indian Institute of Science (IISc), Bengaluru.
2.2. Synthesis of Polybicyclo[3.3.1]nonane
500 mg of lignite humic acid and 50 mL of 1 M NaOH solution were poured into a 250 mL of round bottom flask. The mixture 70 mL of 37% formaldehyde was added. It was then refluxed with stirring at 60°C for 2.5 h. In the next step, solution was allowed to cool down at room temperature and then filtered.
The filtrate was acidified with hydrochloric acid (HCl) followed by ethyl acetate extraction. The ethyl acetate fraction was kept in the refrigerator with anhydrous sodium sulphate overnight. Following filtration, the filtrate was subjected to the Buchi evaporation to obtain the colorless, water-soluble polybicyclo[3.3.1]nonane (yield 280 mg).
3. Results and Discussion
The proposed structures of the “monomeric” and the “dimeric” repeat unit are shown in (Figure 1).
(a)
(b)
This is based on the most intense peak observed at 445.1 in the MALDI-MS spectrum.
Single-crystal X-ray diffraction analysis reveals that the molecular formula of the proposed monomer is C13H21NO2 and the systematic name is 3-amino-1,5-dimethyl-7-methylenebicyclo[3.3.1]nonane-3-carboxylic acid with the molecular weight of 223.1512 a. m. u. (Figure 2).
(a)
(b)
The molecule can exist in the double chair, chair-boat, and double boat conformations which could interconvert fairly rapidly due to low energy barriers between the different conformers.
3.1. Mass Spectral Studies on the BCN Polymer
The MALDI-MS spectrum of BCN polymer dissolved in methanolic solution shows the envelope peak centered at 445.1 a. m. u. (Figure 3). This peak represents the “dimer” of 3-amino-1,5-dimethyl-7-methylenebicyclo[3.3.1]nonane-3-carboxylic acid (error percentage being 0.04% ( a. m. u.)). [21].
The periodic pattern of loss of 30 a. m. u. (N-formylated intermediate) and 14 a. m. u. (N) has been observed which confirms its formation.
3.2. Analysis of the Envelope Peak and Determination of and
The envelope peak obtained from MALDI-MS spectrum and its analysis shows the polymeric nature of the material. Based on the above, the number average molecular weight () and weight average molecular weight () and degree of dispersity have been calculated (Table 1). The formulas used are given below.
The charge of the polymer is calculated to be 14.83. .
The above calculation shows its polymeric nature and confirms that the total number of monomeric units involved is 140.
3.3. FT-IR Spectral Studies
The FT-IR spectral studies show the peaks of native lignite humic acid at 1040, 1530, 1544, 1620, and 1700 (C=O stretching); 2850, 2920, and 3400 (O-H stretching); and 3690 cm-1(O-H stretching) absorption bands previously [22]. The FT-IR spectrum of BCN polymer is shown in (Figure 4).
The methanolic solution of BCN polymer provides the absorption band at 1088.11 (C-O stretching); 1237.93, 1382.80, and 1469.13 (C-H bending); and 2028.29, 2788.71, 2830.11, 2923.03, and 2983.85 cm-1 (C-H stretching). The peaks obtained at 1650.22 (N-H), 1712.74 (COOH), and 3304.83 cm-1 (O-H stretching) confirm the presence of –NH2, –COOH, and -OH functional groups in this polymer, respectively.
3.4. NMR Spectral Studies of the BCN Polymer
3.4.1. 1H-NMR Spectral Studies
1H-NMR spectral studies showed the formation of the N-formyl derivative. Peaks are centered at 8.310 (s, H) for N formyl group (NHCHO), 2.21-2.30 (m, 2 H) for CH2, 1.99-2.11 (m, 2 H) for CH2, 1.70-1.83 (m, 2 H) for CH2, 1.56-1.67 (m, 2 H) for CH2, 1.12-1.40 (m, 2 H) for CH2, and 0.77-0.86 (m, 3 H) for CH3 group (Figure 5). The detailed spectra are shown in SI-1 to SI-2.
3.5. CP-MAS Solid-State NMR Spectral Studies on the BCN Polymer
FT-IR spectrum and the alkali solubility of the compound indicated the presence of the carboxylic acid groups. However, 1H-NMR solution studies could not provide evidence for the presence of the carboxylic acid group [23]. Hence, we undertook 13C-CP-MAS-SS-NMR studies using Bruker 500 MHz instrument at 8 kHz for BCN polymer, shown in (Figure 6).
This study provided evidence for the presence of carboxylic acid at 165.795, CH2OH at 86.424, OCH2 signal at 54.801, and the methyl group peak at 18.292 ppm. Solid-state NMR studies thus confirmed the new compound being a carboxylic acid.
3.6. Powder X-Ray Diffraction Studies
Powder X-ray studies are generally used for characterizing crystallinity of materials and determining dimensions of unit cell. The spectrum has intense and sharp peaks. It contains a good proportion of highly disordered materials as shown in (Figure 7).
The peaks at 2 values shows the plane (220), (002), (110), (101), and (111) and points towards crystalline graphite-like structure. The neighboring planes (002), (110), and (220) indicate the random lattice structure of the crystalline material. The three peaks (110), (002), and (220) show the saturated structure of the material [24, 25].
3.7. Transmission Electron Microscopy Studies
Transmission electron microscopy (TEM) has been extensively used by researchers as characterization tool for better understanding morphology at atomic scale (Figure 8).
The 3D morphology of BCN polymer has been shown to be polycrystalline in nature [26]. The size of an aggregate is 200 nm at 0.2 μm scale.
Preliminary water retention studies of BCN polymer were carried out by the addition of 4-5 drops of water to 44 mg of BCN polymer (Figure 9). Observation reveals that the polymer swelled and the weight increased up to 90 mg.
(a)
(b)
Based on the above experiment, 104% increment of weight in BCN polymer on addition of water has been observed. After leaving it to dry for 30 minutes at room temperature, it weighed 66 mg indicating water retention up to 50% [27]. Further water retention studies using DOSY-NMR experiments are planned.
4. Conclusion
A new substituted methylenebicyclo[3.3.1]nonane (BCN) polymer has been prepared from lignite humic acid by base-catalyzed process. Spectroscopic studies like mass spectrometry, FT-IR, powder X-ray diffraction, and TEM and NMR studies (1H-NMR, CP-MAS-SS-NMR) though challenging were useful for confirming the structure of the BCN polymer. Most useful was the analysis of envelope peak observed in its mass spectrum at 445.1 which helped us to calculate its , , polydispersity index (PD), degree of dispersity, and charge on polymer. The new polymer could be used in water-deficient areas.
Data Availability
The data used to support the findings of this study have been deposited in the figshare repository (doi:10.6084/m9.figshare.22682530).
Conflicts of Interest
The authors declare that they have no conflicts of interest.
Acknowledgments
Authors thank Dr. Alok Adholeya, former Director, TERI Deakin Nano Biotechnology Research Center (TDNBC), TERI Gram, Gwal Pahari, Gurgaon Faridabad Expressway, Gurugram, Haryana 122002, for the facilities. Authors thank the Executive Director, Regional Centre for Biotechnology (RCB), Faridabad, Haryana, India, for the research facilities. We thank Mr. Nagavara Prasad G and Mr. Vijay Kumar Jha for mass spectral experiments and FT-IR studies. We thank Dr. Dinesh Mahajan (THSTI, Faridabad) for his unconditional support. Authors thank Dr. Vipin Agarwal (Associate Professor) and Dr. Krishnarao Doddapuneni (NFHF-NMR facility in TIFR, Hyderabad) for 1H-NMR and CP-MAS-SS-NMR studies. Authors thank Dr. Neel Sarovar Bhavesh (ICGEB, New Delhi) for 1H-NMR studies. Authors thank Mr. Manurbhav Arya, Department of Applied Physics, Amity University, Noida, Uttar Pradesh, for the help in compiling this manuscript. Authors thank Mr. Puran Chandra Prusty, TERI Deakin Nano Biotechnology Research Center (TDNBC), TERI Gram, Gwal Pahari, Gurgaon Faridabad Expressway, Gurugram, Haryana 122002, India, for the help with the experiment. 3Ex-Adjunct Prof. (Hony.), Deakin University, Australia; TERI Deakin Nano Biotechnology Research Centre, Gwal Pahari, TERI Gram, The Energy and Resources Institute, Gurgaon Faridabad Road, Gurgaon, Haryana 122002, India; and NSIC Technical Services Centre, NSIC, Room No. 19, Okhla, New Delhi, and BEMMS, R.S. No. 12/7, Villianur, Perungalur, Puducherry 605007 (current lab addresses).
Supplementary Materials
SI-1: 1H-NMR spectrum in the region 2.0-11.5 ppm, which provides concrete evidence for the aliphatic/alicyclic nature of the compound. Since this 1H-NMR spectrum was recorded in D2O, the protons of NH2 and COOH get exchanged by deuterium and hence not observed in this spectrum. SI-2: expanded 1H-NMR spectrum in the region 1.44–2.04 ppm where signals for different -CH2- and -CH3 groups are observed. Detailed 1H-NMR assignments are included in the text of the manuscript. Scheme 1: schematic for synthesis of BCN polymer. (Supplementary materials)