We analyzed the lignin-like polymer we produced to confirm our experiment is successful. We started from analyzing the content and structure by UV-visible spectroscopy. Second, we used NMR spectroscopy and Mass Spectroscopy to analyze information about the molecular structure and molecular weight. In the last stage, Thermogravimetric analysis is used to analyze the melting or decomposition point of our product. After completed these analysis above, we have a deeper understanding of the lignin-like polymer we made. These data also help us improve, or even discuss the application of our products.

Reaction experiment

Ultraviolet–visible spectroscopy, UV-Vis

  Ultraviolet–visible spectroscopy (UV–Vis) refers to absorption spectroscopy or reflectance spectroscopy in the ultraviolet-visible spectral region.The absorption or reflectance in the visible range directly affects the perceived color of the chemicals involved.The wavelengths of absorption peaks can be correlated with the types of bonds in a given molecule and are valuable in determining the functional groups within a molecule.

   Data revealed difference between samples with only coniferyl alcohol and samples with Laccase and Peroxidase. The absorption peak shifted to the left, comparing with simulation from study (P. J. Salazar-Valencia et al. 2005), the wavelength of β-O-4 linkage absorption peak is shorter. This results are similar to the results of the study, we believe our product contain β-O-4 linkage.

Figure1: UV-Visible spectrum of products

Figure2: Simulation of the electronic or ultraviolet spectra for the 3 Coniferyl Alcohol units, with β-O-4 and β-5 linkages. (P. J. Salazar-Valencia et al. 2005)

1. This UV/Vis diagram shows the range from 230um to 300um of peaks. Peaks of Laccase only and peaks of peroxidase & laccase are very similar.
2. Compare with coniferyl alcohol (reactant) and laccase (product), we can obviously find out the the reactant peak at 270um has shift to 250um.

  This result greatly proves our enzymes have reaction with coniferyl alcohol. Combine our prediction and dimer structure, we determine that the peak at 250um is beta-O-4 bond.

Nuclear Magnetic Resonance Spectroscopy, NMR

  Nuclear Magnetic Resonance (NMR) is based on quantum magnetic properties at the atomic scale. The method of NMR observation of atoms is to place the sample under a large external magnetic field.Using such a process, molecular science research, such as molecular structure, dynamics, etc., can be performed.

  In the NMR experiment, we measured the commercial lignin, coniferyl alcohol, and the products we made.

  First, we first dissolved the coniferyl alcohol in ethanol and measured NMR to obtain the 1H-NMR chart. The chemical shift of each H is derived by the structure of coniferyl alcohol.Then we predict the form of the bonds between each monomer and monomer, and take the product to measure 1H-NMR. Because of some peak changes and the others no changes, we find the most suitable bond to form of the product.

  As a result, our product concentration is too low, peaks are not very obvious, and we can't confirm the correct and proper structural analysis, but there are still some small peaks similar to lignin, which proves that we successfully made similar bonds and indirectly explained our reaction. The successful reaction of the enzymes.

Figure3: Chemical shift of coniferyl alcohol

Figure4: NMR Spectrum of coniferyl alcohol

Figure5: NMR Spectrum of commercial dealkaline lignin

Figure6: NMR Spectrum of Laccase-involved reaction product

Figure7: NMR Spectrum of Laccase & Peroxidase-involved reaction product

Thermogravimetric analysis, TGA

  Thermogravimetric analysis (TGA) is a method of thermal analysis in which the mass of a sample is measured over time as the temperature changes. A TGA can be used for materials characterization through analysis of characteristic decomposition patterns. It is an especially useful technique for the study of polymeric materials.

Figure8: TGA chart for measuring the weight loss

  According to the chart, there is a tendency of pyrolysis at 150~160 °C; peroxidase+laccase has a pyrolysis reaction at 340~400 °C; laccase has a pyrolysis reaction at 420~480 °C.

1. The boiling point of coniferyl alcohol is 163~165°C. Compared with the TGA chart, there is a trend change in the range of 150~160°C. It is this temperature that causes the structure to pyrolysis and the slope is more inclined.


2. The product is similar to lignin and has a pyrolysis interval at 200-400 °C.


3. The product of laccase and the product of peroxidase+laccase, which have different phase transition temperatures, can prove that the products are not the same, which indirectly proves that we have a successful reaction, and peroxidase has a effect on the reactants.

Reference

P. J. Salazar-Valencia, S. T. P´erez-Merchancano, and L. E. Bol´ıvar-Marin´ez. (2005). Optical Properties in Biopolymers: Lignin Fragments. Brazilian Journal of Physics, vol. 36, no. 3B.