Eca 500 mhz spectrometer

Synechococcus cultures used for excreted metabolite assays were grown photoautotrophically in 50 mL batches with an inoculation density of OD_730 nm = 2.0. Cells were washed twice with A⁺(−N) media to remove media nitrogen before inoculation in either nitrogen-replete or nitrogen-deplete media for experimentation. Aliquots of cell suspension (1 mL) were removed from the culture at appropriate time points and centrifuged to pellet cell biomass. The resulting supernatant was filtered through a 0.45-μm silicon membrane and the filtrate used directly for high-performance liquid chromatography (HPLC) and nuclear magnetic resonance (NMR) spectroscopy. A Surveryor Plus (Thermo Scientific, Waltham MA) HPLC equipped with an Aminex fermentation monitoring column [(150 mm × 7.8 mm); Bio-Rad, Hercules, CA, USA] was used for HPLC analyses. 25 μL of filtrate was injected for isocratic elution using 8 mM H₂SO₄ as the mobile phase with a flow rate of 0.5 mL min⁻¹ and a column operating temperature of 45°C. A refractive index (RI) detector operating at 50°C was used for metabolite identification and quantification. Metabolite peaks were integrated and quantified using standard curves constructed from serial dilutions of authentic standards (Sigma-Aldrich, USA). Proton NMR spectra were generated with a JEOL ECA 500 MHz spectrometer; 128 scans were used with water-suppression.

Isolated compounds were dissolved in DMSO-d₆ for structure elucidation via ¹H-NMR analysis. NMR spectra (Figs. S5 and S6) were taken on a JEOL ECA-500 MHz spectrometer (JEOL, Tokyo, Japan) and processed using MestReNova 6.1.0 (Mestrelab). All chemical shifts (Table S4) are referenced to the shift of the solvent signal.

To confirm the chemical structure, the samples were measured using ATR-FTIR (Attenuated total reflection-Fourier transformed infrared spectroscopy, Bruker TEMSOR 37, Bruker AXS. INC., Karlsruhe, Germany) over the range 650 to 4000 cm⁻¹.
The X-ray photoelectron spectroscopy (XPS, VG Multilab 2000 Spectrometer, Thermo Scientific, Waltham, MA, USA) spectra of the irradiated MD was confirmed compared with pristine MD. The XPS was equipped with an Al Kα X-ray and then deconvoluted N1s spectra were obtained using XPS PEAK software (University of Hong Kong, China).
The nuclear magnetic resonance (NMR) spectra of radiated MD was observed by 500 MHz ¹H-NMR (ECA 500 MHz spectrometer, JEOL, Tokyo, Japan) with averaged over 32 scans. Then, the MD and radiated MD solution was mixed in 0.5 mL Deuterium oxide-d solution, and NMR spectra were obtained.