Oxidative Properties of Lignin & Similar Compounds
You have reached the page dedicated to the research completed regarding the oxidation of lignin monomers during the summer of 2018 with the Doc Kieft Summer Research Program. This research is completed by Zelinda Taylor under the direction of Dr. Bradley Sturgeon.
Lignin Compounds
Lignin Monomers
Abstract
The synthesis of lignin via these three monomers is completed via oxidative coupling. Each monomer is composed of a phenol. P-coumaryl alcohol has no methoxy groups, coniferyl alcohol has one methoxy group, and sinapyl alcohol has two methoxy groups. These methoxy groups contribute to the overall reactivity of the compound.
Oxidation of Monomers
Beaker reactions with varying reaction conditions were completed to analyze the oxidation of each monomer. For each monomer, 100 mL of a 2 mM standard solution of the monomer was made using 50/50 dioxane/pH 5 buffer. Three reactions were then completed with varying concentrations of hydrogen peroxide in the presence of HRP. The first reaction was composed of 5 mL of the substrate, 10 µL of hydrogen peroxide (1 mM final concentration hydrogen peroxide) and 5 µL of HRP. The second reaction was composed of 5 mL of the substrate, 5 µL of hydrogen peroxide (0.5 mM final concentration hydrogen peroxide) and 5 µL of HRP. The third reaction was composed of 5 mL of the substrate, 5 µL of hydrogen peroxide (0.25 mM final concentration hydrogen peroxide) and 5 µL of HRP. These reactions were then analyzed on the HPLC for oxidation products of each monomer.
Analysis of Monomer Oxidation
The reactions and the standards were analyzed on the HPLC using an acetonitrile (ACN) and water gradient for 25 minutes. The first 10 minutes were ran at 100% water, 10-20 minutes were ran at an 80% ACN/20% water gradient, and the remaining 5 minutes at 100% water. This method is saved as "pCou_100417_SAZT."
| Ferulic Acid | Ferulic Acid | P-coumaric Acid | Synapic Acid |
Immobilization of Enzymes
An immobilization of peroxidase from horseradish (HRP) was completed using Affi-Gel 10. This procedure is centered around the isolectric point of the enzyme. The immobilization was completed using a MOPS buffer (pH 7.0) and about 2 mL of Affi-Gel 10 beads. The absorbance of the initial HRP in MOPS buffer was 6.28. The initial concentration of HRP was 616 µM. A successful immobilization of the enzyme results in a smaller absorbance. The smaller absorbance means there has been greater coupling between the enzyme and the beads. The absorbance of the immobilized HRP was 1.94. The final concentration of HRP was 190 µM.
Oxidation of Lignin Monomers Using Immobilized HRP
Beaker reactions were completed using the immobilized HRP and analyzed on the HPLC. The reactions had varying concentrations of hydrogen peroxide. A 2 mM standard was made for each monomer with a volume of 100 using 50/50 dioxane/pH 5 buffer. The first reaction was composed of 5 mL of substrate, 10 µL hydrogen peroxide (1 mM final concentration hydrogen peroxide), and 50 µL of immobilized HRP. The second reaction was composed of 5 mL of substrate, 5 µL of hydrogen peroxide (0.5 mM final concentration hydrogen peroxide), and 50 µL of immobilized HRP. The third reaction was composed of 5 mL of substrate, 2.5 µL of hydrogen peroxide (0.25 mM final concentration hydrogen peroxide), and 50 µL of immobilized HRP. These reactions were then analyzed on the HPLC for possible oxidation products using the method "pcou_100417_SAZT" for 25 minutes. The results were then compared to previously collected HPLC data using HRP, opposed to immobilized HRP.
| Ferulic Acid with HRPi | P-Coumaric Acid with HRPi | Synaptic Acid with HRPi |
The sinapic acid produced unpredicted results. The sinapic acid reactions using HRP in pH 5 buffer and dioxane produced no product peaks. The sinapic acid reactions using HRPi in pH 5 buffer produced product peaks. New reactions (using the same method as above) using sinapic acid and HRP or HRPi in pH 5 buffer were made. The results were then analyzed on the HPLC.
| Synapic Acid with HRP | Synaptic Acid w HRPi |
ESR Detection of Radicals
Similar Compounds
Oxidation of 4-hydroxy-3-methoxybenzyl
The same beaker reactions used for the lignin monomers were completed with the 4-hydroxy-3-methoxybenzyl. A 2 mM standard was created with a volume of 100 mL using 50/50 dioxane/pH 5 buffer.
Analysis of 4-hydroxy-3-methoxybenzyl Oxidation
Oxidation of Vanillin
The same beaker reactions used for the lignin monomers were completed were completed with vanillin. A 2 mM standard was created with a volume of 100 mL using 50/50 dioxane/pH 5 buffer.