Avance dpx 500 nmr spectrometer

Nuclear magnetic resonance (NMR) spectra (1D and 2D experiments) were obtained using an Avance DPX-500 NMR spectrometer (Bruker BioSpin Corporation, Billerica, MA, USA) at 35 °C. The concentration of the samples was 15 mg of polysaccharide/mL of D₂O.
GLC-MS of alditol acetate derivatives was performed using a Hewlett-Packard 6850 (Agilent Technologies, Santa Clara, CA, USA) chromatograph equipped with HP-5MS capillary column (30 m × 0.2 mm) with a temperature gradient of 150 → 230 °C at 3 °C min⁻¹.
Electrospray ionization mass spectra (ESIMS) were recorded with an Impact II Q-TOF mass spectrometer (Bruker BioSpin Corporation, Billerica, MA, USA). All spectra were acquired in the negative-ion mode, precalibrated with a standard “HP-mix” (Agilent Technologies, Santa Clara, CA, USA) for negative-ion mode at default instrument settings. The samples were diluted with ACN:H₂O (1:1) to approx. 0.01 mg/mL and introduced into the mass spectrometer at a flow rate of 5 µL/min using a syringe pump (KD Scientific, Holliston, MA, USA). The mass spectrometry detection was performed using a CaptiveSpray (Bruker Daltonics, Bremen, Germany) ionization source at a capillary voltage of 1.3 kV. Collision-induced dissociation (CID) product ion mass spectra were recorded in auto-MS/MS mode with a collision energy of 43 eV. The precursor ions were isolated with an isolation width of 1 mass unit.

Nuclear Magnetic Resonance spectra were recorded using an Avance DPX-700 NMR spectrometer (Bruker Biospin AG, Fällanden, Switzerland) and/or an Avance DPX-500 NMR spectrometer (Bruker, Hamburg, Germany). 1H, 13C, 1D TOCSY (total correlated spectroscopy) spectra and two-dimensional (2D) spectra (correlation spectroscopy COSY, rotating frame nuclear Overhauser effect spectroscopy ROESY, nuclear Overhauser effect spectroscopy NOESY, heteronuclear single-quantum correlation spectroscopy HSQC, heteronuclear multiple-bond correlation spectroscopy HMBC) were recorded for solutions of poly- and oligosaccharides in D₂O at 35–40 °C. The concentrations of the samples ranged between 3–10 mg·mL⁻¹. Unfortunately, the structure of the HMP fraction could not be determined by NMR spectroscopy, due to insufficient spectral resolution for detailed interpretation.

The content of carbohydrates was determined according to the method of Michel Dubois et al. [36 (link)]. The absorbance was measured at 490 nm using a Power Wave XS microplate reader (BioTek, Winooski, VT, USA). The glucose (1 mg/mL) was used as a reference standard.
The content of the sulfate group was determined by the turbidimetric method after hydrolysis of the sulfated laminaran AaLs with HCl (1N) [37 (link)]. The absorbance was measured at 360 nm using a Power Wave XS microplate reader (BioTek, Winooski, VT, USA). K₂SO₄ (1 mg/mL) was used as a reference standard.
The molecular weight of sulfated laminaran AaLs was determined by size-exclusion chromatography (SEC), using an Agilent 1100 Series HPLC instrument (“Agilent Technologies”, Waldbronn, Germany) equipped with a refractive index detector and series-connected SEC columns, Shodex OHpak SB-805 HQ and OHpak SB-803 HQ, (“Showa Denko”, Tokyo, Japan). Elution was performed with 0.15 M NaCl aqueous solution at 40°C, with a flow rate of 0.4 mL/min. The dextrans of 5, 10, 50, 80, 250, 410, and 670 kDa (“Sigma-Aldrich”, Saint Louis, MO, USA) were used as reference standards.
The ¹³C NMR spectra of native and sulfated laminarans were obtained on an Avance DPX-500 NMR spectrometer (“Bruker BioSpin Corporation”, Billerica, MA, USA) at 50 °C. The sample concentration was 15 mg of polysaccharide/mL of D₂O.