3 trimethylsilyl propionic 2 2 3 3 d4 acid sodium salt tsp

Deuterium oxide 99.9%, sodium dihydrogenphosphate hydrate (NaH₂PO₄·H₂O), disodiumhydrogen phosphate dihydrate (Na₂HPO₄·H₂O₂), and 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt (TSP) were purchased from Sigma-Aldrich (Milan, Italy). Deuterated EDTA d-16 98% was obtained from Product Cambridge Isotope Laboratories, Inc.
Phosphate buffer solution was prepared as follows: 49.7 mg of sodium dihydrogenphosphate hydrate (0.36 mmol), 202.9 mg of disodium hydrogen phosphate dihydrate (1.14 mmol), and 0.92 mg deuterated EDTA d-16 (0.003 mmol) were dissolved in 10 mL of water. The pH was checked at 7.1. The solution was distributed into 5 mL aliquots and then lyophilized. Each aliquot was dissolved in about 1 mL of D2O and lyophilized again. Finally, the buffer was dissolved in 5 mL of deuterium oxide with 0.002% TSP (12 mM).

Sodium alginates were purchased and used as received from two commercial manufactures: Sigma-Aldrich (St. Louis, MO, USA) and Panreac AppliChem (Darmstadt, Germany), hereafter referred to as SA1 and SA2 respectively. Zinc chloride (≥ 97.0%, bioreagent for molecular biology suitable for cell culture), calcium chloride (≥ 93.0%) and graphene oxide were acquired from Sigma-Aldrich and used without further purification. Guluronate and mannuronate carbohydrate standards were provided by CarboSynth (Compton, Berkshire, UK). Glucose, trifluoro acetic acid, triethylenetetraminehexaacetic acid (TTHA), deuterium oxide (D₂O, D-99.9%) and 3-(Trimethylsilyl)-propionic-2,2,3,3-d4 acid sodium salt (TSP) were supplied also from Sigma-Aldrich.

In order to study the complex formation and its stoichiometry, the Job method was performed. The experiments were carried out using a Shimadzu UV-2450 spectrophotometer. The Job method was performed by adding an aqueous host solution to an aqueous guest solution whose concentration varied from 0 to 1.0 mol fraction. The absorbance of the solutions was measured at a wavelength of λ_max = 286 nm for the NaSal-Na₄EtRA system and λ_max = 296 nm for the NaSal-β-CD.
An NMR titration was performed at 298.15 K using a Varian of 400 MHz spectrometer with a 5 mm probe. Residual solvent (HOD) at pre-saturation was used to perform the spectra; 24 k data points covering a spectral width of 8 kHz were obtained, the radiofrequency excitation pulse was 45° and the scan repetition time was 15 s to allow for complete relaxation. The resonance of the Si-(CH3)3 signal in TSP was used as internal reference, assigning it a shift value of 0 ppm. Samples were prepared in D₂O (Eurisotop, 99.9%) as solvent, and tracer amounts of 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt (TSP), from Sigma. The NMR titration was performed by adding a carrier aqueous solution (8mM) to sodium salicylate aqueous solution (2.9mM), during the entire titration process, no precipitate was observed [29 (link)]. The results were analyzed using the HypNMR software [13 (link)].