2.3 Analytical methods for structural characterization
Attenuated total reflectance-fourier transform infrared (ATR-FT-IR). ATR-FT-IR method can be found in previously published paper (Ngo, Hoh, Foglia, 2012). The spectra were collected at 64 scans with wavelength range from 4000 cm-1 to 500 cm-1.
Nuclear magnetic resonance (NMR) . All samples were analyzed via solution state NMR spectroscopy recorded on a 14 Tesla Agilent Technologies (Santa Clara, CA) DD2 NMR spectrometer using a 5 mm One NMR probe. The starting material, L-arginine ethyl hydrochloride, was dissolved in D2O (99.9% D) with sodium trimethylsilyl propionate (TSP) added as chemical shift standard. All other samples were dissolved in deuterochloroform (99.9% D). Due to its reduced solubility in chloroform, ethyl n -stearoyl arginate hydrochloride was dissolved in a mixture of d4-methanol (99.8% D) and deuterochloroform. All solvents and standards were purchased from Cambridge Isotopes Laboratories, Andover, MA. All spectra were measured at 25 °C. The 1H spectra had spectral widths of 12 ppm and were acquired with a 45° pulse angle. The 13C spectra had spectral widths of 240 ppm and were acquired using a 45° pulse angle with a 1 s relaxation delay. The residual13C resonance of the solvent was used as the internal chemical shift reference for these 1D spectra, which were acquired, processed using SpinWorks4 (v4.2.10 Kirk Marat, University of Manitoba, Canada) or Open VNMRJ (website).
Liquid chromatography-mass spectrometry (LC/MS). All analyses were carried out using a Nano-Acquity instrument (Waters Co., Milford, MA) equipped with an Acquity UPLC BEH C18, 1.7µm, (1 x 50mm) column (Waters Co.). The temperature was set at 45 oC and the following gradient was used at 80 µL/min: initial time to 2 min water:acetronitrile, 50:50; ramped with a linear gradient to 10 min to water:acetronitrile, 5:95; and returning to the initial conditions at 15 min. with 10 min equilibration time between injections. The solvents contained 0.1% formic acid. The column effluent was connected to a quadrupole time of flight mass spectrometer (Q-ToF) Synapt G1 (Waters Co.) running in positive mode with an electrospray probe. Mass spectra (MS) were obtained using a capillary voltage set to 3 KV, temperature at 350 oC, and cone voltage at 40V. The protonated molecule, ([M+H]+), was obtained with a collision energy set at 6 eV for MS and ramped between 10 and 30 eV for fragmentation, (MS/MS), of selected ions.
Orbitrap Q-Exactive Plus high-resolution mass spectrometer (Thermo Scientific, Bremen Germany) operating in electrospray mode with a resolution of 70,000 (FWHM). The ion transfer capillary was set to 275o C. The [M+H]+ was analyzed by MS/MS using a normalized collision energy at 35eV and with a resolution of 70,000 (FWHM).