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).