Avance 3 hd nmr

Samples were analyzed at 500 MHz using a Bruker Avance III HD NMR spectrometer equipped with a triple resonance inversus (TXI) ¹H [¹⁵N, ¹³C] probe head and x, y, z gradient coils. For ¹H spectra, 128 transients were acquired in 32K data points, with a spectral width of 10,504 Hz and an acquisition time of 3.12 s. The receiver was set to 64. The sample temperature was maintained at 300 K, and the H₂O resonance was pre-saturated by single-frequency irradiation during a relaxation delay of 4 s, with a 90° excitation pulse of 8 μs. Automatic shimming of the sample was performed on the deuterium signal. Fourier transformation, phase correction, and baseline correction were carried out automatically using Bruker Topspin software (V3.5).

The size and polydispersity (PDI) of PNC were determined by dynamic light scattering (DLS) (Malvern Zetasizer Nano ZS). To ensure stability during storage in Milli-Q® (MQ) water at 4°C, multiple measurements were taken over several days starting at Day 0 immediately after synthesis. Measurement settings are listed in (Supplementary Table 2). At least 10 batches for each PNC type were synthesized and size and PDI measured to ensure reproducibility.
Yield of PNC synthesis batches were measured with quantitative 1D ¹H nuclear magnetic resonance (NMR) spectra of resuspended PNC. Particles from each synthesis batch were centrifuged as previously described, and the pellet was allowed to dry overnight in a fume-hood to ensure complete removal of HFIP and DEE. Dried PNC were resuspended in a 200 μl solution of deuterated DMSO (Cambridge Isotope Libraries, Inc.) and 10 mM maleic acid (Alfa Aesar). maleic acid served as an internal standard for ¹H NMR intensity. All ¹H spectra were collected on an 18.8 T Bruker Avance III HD NMR with a 3 mm HCN CryoProbe. The relaxation delay (d1) was set to 20 s to ensure complete spin relaxation, and the pulse width was programmed for 30° pulses (36 (link), 37 (link)). Peaks in ¹H NMR spectra were fit using custom code in Wolfram Mathematica and compared to standard solutions to determine the amount of peptide within PNC batches.

Samples were solved in CDCl₃ or methanol-d₄, transferred to 507-HP-8 NMR tubes (Norell Inc, Morganton, NC, USA) and analyzed with an AVANCE III 600 NMR equipped with a 5 mm TBI CryoProbe (¹H-NMR 600.25 MHz, ¹³C-NMR 150.95 MHz, 298 K) or an AVANCE III HD NMR (¹H-NMR 400.13 MHz, ¹³C-NMR 100.63 MHz, 299 K) (Bruker Corporation, Billerica, MA, USA). Subsequently structures were elucidated on basis of 1D-¹H, 1D-¹³C as well as 2D-¹H,¹³C HSQC, ¹H,¹³C HMBC, ¹H,¹H COSY and ¹H,¹H NOESY experiments with TopSpin 3.5.b.91 pl 7 (Bruker Corporation).

One-dimensional (1D) proton (¹H)-NMR spectra was acquired using different pulse sequences on a 500 MHz Bruker Avance III HD NMR spectrometer equipped with a triple-resonance inverse 1H probe head and x, y, z gradient coils. A standard nuclear overhauser effect spectroscopy (NOESY) pulse sequence presat (noesygppr1d.comp) was used on both serum and lipid extract samples. On serum samples, NOESY was used to detect both signals of small metabolites and high-molecular-weight macromolecules such as lipoproteins. Additionally, a standard diffusion-edited (DIFF) pulse sequence (ledbpgppr2s1d) was used on serum samples to detect only high-molecular-weight macromolecules, such as lipoproteins. Pooled samples were used as a quality control sample and were included in each batch for qualitative assessment of repeatability by overlaying the raw spectra.

The Aloe muth-muth gel and whole leaf plant materials were chemically characterized with quantitative proton nuclear magnetic resonance spectroscopy (¹H-NMR), and spectra were obtained with a Bruker Avance III HD NMR (Bruker Corporation, Billerica, MA, USA). The quantities of marker molecules (i.e., aloverose, glucose, malic acid, lactic acid, citric acid, and whole leaf marker) in the Aloe muth-muth gel and whole leaf materials were determined according to a previously published method [12 (link)].