Vnmrs 600 spectrometer

In total, 40 mg of polysaccharide was dissolved in 1 mL of 99.9% deuterium oxide and the sample was placed in NMR tubes. The ¹H NMR and ¹³C NMR spectra were recorded on an NMR spectrometer (Varian VNMRS 600 spectrometer, Palo Alto, CA, USA) [38 (link)].

Absorption spectra: Varian Cary 100 Bio Spectrophotometer with baseline correction. Emission spectra: Varian Cary Eclipse at 20 °C: Cuvettes: Quartz cells (10 mm × 4 mm). NMR spectra: Varian VNMR-S 600 spectrometer (¹H: 600 MHz, ¹³C: 150 MHz), equipped with 3 mm and 5 mm triple resonance inverse probes. NMR spectra were processed with the software MestReNova (Santiago de Compostela, Spain; ¹H NMR, HSQC, HMBC) and Sparky (NOESY) [56 (link)]. Circular-dichroism (CD) spectra: Chirascan CD spectrometer, Applied Photophysics.

NMR data were recorded with a Varian VNMR-S600 spectrometer [600 MHz (¹H), 150 MHz (¹³C)] at 35 °C. NMR spectra were processed with the software ACD/NMR Processor Academic Version 12.01 and are referenced to the corresponding solvent [δ(DMSO-d₅) = 2.50 (¹H) and 39.5 (¹³C); δ(CHCl₃) = 7.26 (¹H) and 77.2 (¹³C)]. Elemental analyses data were determined with a HEKAtech EURO EA combustion analyzer by Mr. Rochus Breuer (Organic Chemistry I, University of Siegen). Mass spectra (ESI) were recorded on a Finnigan LCQ Deca (U = 6 kV; working gas: argon; auxiliary gas: nitrogen; temperature of the capillary: 200 °C). Absorption spectra were obtained with a Varian Cary 100 bio spectrometer in quartz cells (10 mm) with baseline correction. Emission spectra were recorded in quartz cells (10 mm) with a Cary Eclipse spectrometer at 20 °C. CD-spectroscopic measurements were performed with a Chirascan spectrometer (Applied Photophysics) in quartz cells (1.0 mm). Melting points were measured with a Büchi 545 (Büchi, Flawil, CH) and are uncorrected.

UV spectra were obtained by a Milton Roy Spectronic 3000 Array spectrophotometer (Rochester, NY, USA). Optical rotations were measured on a JASCO P-2000 polarimeter (Kyoto, Japan). CD spectra were recorded using a Jasco J-815 CD spectrophotometer (Kyoto, Japan). IR spectra were recorded on a Nicolet^TM iS50 FT-IR spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) and Perkin Elmer FT-IR 1760X spectrometer (Boston, MA, USA). HR-ESI spectra were measured on a Bruker APEX II mass spectrometer (Karlsruche, Germany) and Agilent 6540 UHD Accurate-Mass Q-TOF mass spectrometer (CA, USA). NMR was recorded on a Bruker Advance NEO 400 MHz NMR spectrometer (Karlsruche, Germany) and a Varian VNMRS-600 spectrometer (Lexington, MA, USA). Medium-performance liquid chromatography (MPLC) and flash column chromatography (Flash CC) were performed by using a PuriFlash^® XS 420 (Advion Inc., NY, USA), Sepacore^® purification system (Buchi AG, Flawill, Switzerland), or a ceramic pump (VSP-3050; EYELA, Kyoto, Japan). Silica gel 60 (70–230 or 230–400 mesh ASTM, Merck, Darmstadt, Germany), LiChroprep^® RP-18 (25–40 μm, Merck, Darmstadt, Germany), and Sephadex^TM LH-20 (GE Healthcare, Amersham, UK) were used as a stationary phase material for column chromatography (CC). Organic solvents (commercial grade) were redistilled prior to their use as a mobile phase composition.

A detailed characterization of the commercial oligochitosan (OC) was undertaken in order to determine the chemical composition of the repetitive unit necessary to fix the quantity of nitrogen per acrylic acid (N/AA ratio) used in the materials synthesis. The deacetylation degree (DD = 83 ± 5%) was determined by solid state ¹⁵N NMR using a Varian VNMRS 600 spectrometer operating at 5 kHz using cross-polarization magic-angle spinning conditions [30 (link)]. The DD was calculated from the integration of the amide (δ = 101 ppm) and amine (10 ppm) peaks using the following formula:
Note that a similar DD was obtained from liquid ¹H NMR data recorded on a Bruker 400 MHz spectrometer using the method reported by Trombotto et al. [30 (link)].
The quantity of lactate/lactic acid present in the sample was calculated from the liquid ¹H NMR spectrum recorded in D₂O. The amount of water was deduced from elemental analysis. The molar composition of the repetitive unit constituting the oligochitosan was then: H-(C₆H₁₁O₄N)_0.83(C₈H₁₃O₅N)_0.17-OH, 1.31(C₃H₆O₃), 0.09(H₂O), where C₆H₁₁O₄N represents the deacetylated unit, C₈H₁₃O₅N the acetylated unit and C₃H₆O₃ the lactate ion.

A D₂O solution of 800 mg/L HGA was prepared (D₂O solution 1). Ten microliters of 1 M NaOH solution was added to 0.8 mL of this solution (D₂O solution 2). Ten microliters of 1 M NaOH solution with NaOCl·5H₂O (2 wt%) was added to 0.8 mL of D₂O solution of 800 mg/L HGA (D₂O solution 3). Three types of D₂O solutions were prepared and subjected to NMR measurements. All NMR data were recorded on a Varian VNMRS 600 spectrometer, operating at 599.936 MHz for protons. NMR spectra were acquired at 293 K using 5-mm NMR tubes. 4, 4-Dimethyl-4-silapentane-1-sulfonic acid (DSS) was used as an internal standard for the chemical-shift calibration. ¹H NMR spectra were recorded using the standard pulse sequence and setting the repetition time and number of scans to 13.4 s and from 16 to 64 scans, respectively.

Nuclear magnetic resonance data were collected on a Varian VNMRS 600 spectrometer operating at 599.87 MHz for ¹H, 150.84 MHz for ¹³C, and 60.79 MHz for ¹⁵N. Chemical shifts (δ in ppm) are given from the internal solvent and the partially deuterated residual DMSO-d₆ 39.5 ppm and acetone-d₆ 29.8 ppm for ¹³C; DMSO-d₅ 2.5 ppm and acetone-d₅ 2.05 ppm for ¹H. External nitromethane (0.0 ppm) was used for ¹⁵N references. The ¹⁵N chemical shifts were obtained from two dimensional ¹H,¹⁵N-HMBC experiments with gradient coherence selection, which were performed using a standard pulse sequence from the Varian pulse library. CH₃NO₂ was used as an external reference for the ¹⁵N chemical shifts. The 2D experiments gCOSY, zTOCSY, NOESY, gHSQC and gHMBC were run using the standard Varian software. All data were analysed using MestReNova 14.2.1-27684 (5 May 2021, Santiago de Compostela, Spain) software.