Unity 400 mhz spectrometer

¹H NMR spectra were collected using a Varian Unity 400-MHz spectrometer outfitted with a 5-mm broadband probe. 50–75 mg of biocrude were dissolved in deuterated chloroform containing 0.03% tetramethyl silane (TMS) as an internal reference. Samples were then filtered (0.22-lm PTFE) to remove any suspended particulates before loading into 5 mm diameter NMR tubes. ¹H spectra were acquired with a 90° pulse angle, spinner frequency of 20 Hz, sweep width of 8000 Hz across 32 transients.

General Information. Commercial reagents were used as received without additional purification. Melting points were determined with Electrothermal IA9100 micro-melting point apparatus and uncorrected. NMR spectra were recorded with Varian Unity-400MHz spectrometer using DMSO-d₆ as solvent and tetramethylsilane as internal standard. Chemical shifts were expressed as δ (ppm). Splitting patterns have been described as follows: s = singlet; d = doublet; t = triplet; q = quartet; dd = double doublet; m = multiplet. Analytical TLC was performed on Art. 5554 Kieselgel 60 GF254 produced by E. Merck and the spots of compounds were detected with UV light indicator irradiated at 254 and 366 nm. Art. 7734 Kieselgel 60 GF254 (70–400 mesh) made by E. Merck was used for column chromatography. The purity of compounds was determined with HPLC and elemental analysis (EA). HPLC analysis was performed on HITACHI Chromaster 5110 HPLC, fitted with a UV detector and an auto sampler. The compounds were tested on a Mightysil RP-18 GP 250-4.6 (5 mm) with mobile phase 10 mM KH₂PO₄:acetonitrile (35:65) pH = 2.89. The flow rate was 1 mL/min and the sample injection was 100 µL (5.00 mg dissolved in 1 mL DMSO, then diluted with methanol to 5 mL). The wavelength was set at 295 nm. Elemental Analysis was recorded on Heraeus CHN-O Rapid apparatus and the results were within ±0.4% of the theoretical value.

Commercial reagents were used as received without additional purification. Melting points were determined with the Electrothermal IA9100 micro-melting point apparatus and were uncorrected. NMR spectra were recorded with a Varian Unity-400 MHz spectrometer using DMSO-d₆ and CDCl₃ as solvent and tetramethylsilane as the internal standard. Chemical shifts were expressed as δ (ppm). Splitting patterns had been described as follows: s = singlet; brs = broad singlet; d = doublet; t = triplet; dt = double triplet; m = multiplet. The raw data of NMR for all compounds are shown in Supplementary Materials. Analytical TLC was performed on Art. 5554 Kieselgel 60 GF254 produced by Merck, and the compound spots were detected with a UV light indicator irradiated at 254 and 366 nm. Art. 7734 Kieselgel 60 GF254 (70–400 mesh) made by Merck was used for column chromatography. The purity of the compounds was determined with elemental analysis (EA). EA was recorded on an Heraeus CHN-O Rapid apparatus and the results were within ±0.4% of the theoretical value.

Human samples were measured using a Bruker 600 MHz spectrometer (Bruker BioSpin) operating at 600.13 MHz proton frequency. The 1D ¹H-NMR spectrum of each sample was acquired with water peak suppression pulse sequence (NOESYGPPR1D; Bruker), using 64 free induction decays (FIDs), 64k data points, a spectral width of 20.0306 ppm, a relaxation delay of 4 s, and a mixing time of 100 ms. The FIDs were multiplied by an exponential weighting function corresponding to a line broadening of 1 Hz before Fourier transformation, phasing, and baseline correction.
Monkey NMR spectra were measured with a Varian Unity 400 MHz spectrometer. FIDs were recorded as 64k data points with a spectral width of 8.000 Hz. A single 45° pulse was used with an acquisition time of 4.10 s and a relaxation delay of 2 s. The spectra were acquired by accumulation of 128 FIDs. The signal of the residual water was removed by a pre-saturation technique in which the water peak is irradiated with a constant frequency during 2 s prior to the acquisition pulse. An exponential window function with a line broadening of 0.5 Hz and a manual baseline correction were applied to all spectra.