Quantitative 31P NMR Analysis of Lignins and Tannins

Quantitative phosphorus NMR represents one of the most significant breakthroughs in lignin and tannin analytical chemistry during the last three decades. This technique offer fast, reliable, and quantitative information for the various hydroxy group in the sample. These NMR methods have tremendous value in understanding the structure of lignans and tannins.

It has also been applied to a variety of other systems that have reactive hydroxyl groups. Begin by pre-treating 100 milligrams of the lignin or tannin sample by drying overnight in a vacuum oven at 40 degrees Celsius. After drying, rapidly transfer the sample to an anhydrous calcium sulfate desiccator until it reaches room temperature.

To prepare the sample for NMR spectroscopy, accurately weigh about 30 milligrams of the sample in a two milliliter vial equipped with a stirring bar. Next, add 500 microliters of the freshly prepared solvent solution to the sample vial and seal the vial with a cap. Using a micropipette, add 100 microliters of the internal standard solution to the sample vial, then magnetically stir the resulting dispersion at 500 rotations per minute.

When the sample is completely dissolved, transfer 100 microliters of tetramethyl dioxyphospholane or TMDP to the sample solution while working under the hood and seal the sample solution before placing the sample for vigorous magnetic stirring. At this point, the indicated reaction occurs on the lignin and tannin samples. Using a Pasteur pipette, transfer the sample solution into an NMR tube for the analysis.

If a yellow precipitate is observed in the sample, repeat the procedure by ensuring all possible moisture contamination is avoided. Load the sample tube into the NMR instrument equipped with a broadband probe and set the experimental parameters. Using the resonance frequency of deuterated chloroform, set the frequency in the spectrometer.

Shim the sample and tune the spectrometer before starting the acquisition. Start processing raw data from P31 NMR spectroscopy by performing Fourier transformation. Adjust phase by manual phase correction by expanding the processing tab and selecting phase correction and manual correction.

Correct baseline manually by carefully setting zero points after clicking processing and selecting baseline and multi-point baseline correction. For signal calibration, set the signal for the phosphorylated water at the chemical shift value of 132.2 parts per million by opening the analysis tab, then select reference in the reference tab. For signal integration, open integral in the analysis menu.

To normalize integration, set the internal standard to 1.0 by clicking on the peak to select edit integral and enter value 1.00 in the normalized tab, then perform spectrum integration according to the chemical shifts reported in the manuscript. Use the equation to calculate the molar concentration of internal standard, or IS, solution and utilize the calculated value to estimate the equivalent amount of the specific signal per gram of the sample. The spectrum quantification of various hydroxy groups in a softwood craft lignin derived with TMDP was recorded using 300 megahertz and 700 megahertz NMR spectrometer.

In the NMR spectra, sharp and strong peaks were detected at 144 and 132 PPM due to the internal standard and the hydroxylation of TMD respectively. The different signals of the hydroxy groups were evident in all of the quantitative P31 NMR spectra of lignins. In a quantitative P31 NMR spectrum of a tannin sample derivatized using TMDP, a characteristic signal from the different aliphatic OH pyrogallol and catechol units were well visible.

The comparison between NMR spectrum of lignin before and post-oxidation was recorded using a 300 megahertz NMR spectrometer showing the reduction in the intensity of peaks of hydroxy groups. The crucial steps are those pertaining to the drying and the weighting of the sample and the NMR processing parameters to be used. And most importantly, the phasing of the obtained spectra.

When this method is coupled with two-dimensional NMR and gel permeation chromatography, a very detailed picture of natural polyphenols may emerge, offering unprecedented structural information and new insights.