Inova 500 nmr spectrometer

¹H NMR analyses were performed to characterize GelMA/Bio-IL composite hydrogels using a Varian Inova-500 NMR spectrometer. ¹H-NMR spectra were obtained from choline bicarbonate, choline acrylate (Bio-IL), GelMA prepolymer, and GelMA/Bio-IL hydrogels. Methacrylated groups were identified due to the presence of peak values at δ = 5.3 and 5.7 ppm. The decreasing rate for the C=C double bond signals $\left(-\frac{\partial \left(C=C\right)}{\partial t}\right)$ in methacrylate group of GelMA was associated with the extent of crosslinking of composite hydrogel as well as conjugation of Bio-IL to GelMA. This area decrease was calculated using the following equation: $$\mathbf{D}\mathbf{e}\mathbf{c}\mathbf{a}\mathbf{y}\mathbf{o}\mathbf{f}\mathbf{m}\mathbf{e}\mathbf{t}\mathbf{h}\mathbf{a}\mathbf{c}\mathbf{r}\mathbf{y}\mathbf{l}\mathbf{a}\mathbf{t}\mathbf{e}\mathbf{g}\mathbf{r}\mathbf{o}\mathbf{u}\mathbf{p}(\%)=\left(\frac{\mathbf{P}{\mathbf{A}}_{\mathbf{b}}-\mathbf{P}{\mathbf{A}}_{\mathbf{a}}}{\mathbf{P}{\mathbf{A}}_{\mathbf{b}}}\right)\times \mathbf{100}$$ where PA_b and PA_a represent the peak areas of methacrylated groups before and after photocrosslinking, respectively. Accordingly, PA_b − PA_a corresponds to the concentration of methacrylated groups consumed in the photocrosslinking process. ACD/Spectrus NMR analysis software were used to integrate the area under the peaks and all the data was analyzed with respect to phenyl group peaks at δ = 6.5–7.5 ppm.

The hydroxyl group content of the lignin samples was measured using quantitative ³¹P NMR spectroscopy. The samples (30–40 mg) were dissolved in 500 μL of anhydrous pyridine and deuterated chloroform (1.6:1, v/v). This was followed by the addition of 50 μL of chromium(III) acetylacetonate (5.13 mg/mL in anhydrous pyridine and deuterated chloroform 1.6:1, v/v) used as the relaxation agent and 27.5 μL of cyclohexanol (21.07 mg/mL in anhydrous pyridine and deuterated chloroform 1.6:1, v/v) used as the internal standard. Finally, 100 μL of 2-chloro-4,4,5,5-tetramethyl-1,3,2-dioxaphospholane (TMDP) was added to the vial. The solution was thoroughly mixed and transferred to a sealed 5-mm NMR tube. All NMR experiments were carried out at 298 K on a Varian Inova 500 NMR Spectrometer operated at a frequency of 500.13 MHz and equipped with a 5 mm broadband inverse probe. ³¹P NMR spectra were recorded with 32,768 data points and a spectral width of 60,606.06 Hz, with a relaxation delay of 5 s and 512 scans. All chemical shifts are reported relative to the reference compounds of TDMP with cyclohexanol, which produces a sharp signal at 145.15 ppm referenced from the water signal (132.2 ppm) for the phosphitylating reagent [28 (link)].

The hydroxyl group content of the lignin and modified lignin samples was measured using quantitative ³¹P-NMR spectroscopy (Varian Inova, Palo Alto, CA, USA). The samples were derivatized with 100 μL of 2-chloro-4,4,5,5-tetramethyl-1,3,2-dioxaphospholane (TMDP). Derivatized samples (30–40 mg) were dissolved in 500 μL of anhydrous pyridine and deuterated chloroform (1.6:1, v/v) and mixed with 100 L of a solution of N-hydroxy-5-norbornene-2,3-dicarboxylic acid imide (10 mg mL⁻¹) and chromium (III) acetylacetonate (5 mg mL⁻¹) as internal standard and relaxation agent. The solution was thoroughly mixed and transferred to a sealed 5 mm NMR tube. All NMR experiments were carried out at 298 K on a Varian Inova 500 NMR Spectrometer [60 (link)]. ³¹P-NMR spectra were acquired using an inverse-gated decoupling pulse sequence with a 90-pulse angle, 25 s relaxation delay, and 256 scans.