The aim of our protocol is to fractionate the biomass and extract lignin in a single step. Using this method, lignin can be recovered by simple filtration after the treatment without adjustment of pH, but simply by adding distilled water. The focus of this study is to evaluate the effect of this combined treatment of feedstock fractionation, its influence on lignin purity and yield, and its effect on the molecular weights and chemical functional groups in the extracted lignin.
Deep eutectic solution microwave process is an ultra fast, efficient, and cost competitive technology for lignocellulosic biomass fractionation and lignin recovery with high purity. Prepare the deep eutectic solution in a 500 milliliter round bottom flask as described in the text manuscript. Then place five grams of feedstock, 50 milliliters of deep eutectic solution, and a stirring bar in a microwave in a closed polytetrafluoroethylene reactor.
Close the microwave container with an appropriate cap and attach a temperature cap. Place it on the edge of the turntable, ensuring that it is constantly agitated and run the microwave for one minute. Using suitable gloves, take the container out of the microwave and let the mixture cool.
Prepare a homogenous anti-solvent solution and add 50 milliliters of this solution to the treated feedstock. Centrifuge the mixture for five minutes at 3, 000 times G.Filter the supernatant using a glass filter crucible. Collect the remaining cellulose residue and wash it by adding 25 milliliters of anti-solvent solution and centrifuging.
Add the filtered lignin-rich fraction and the filtered washes to a 500 milliliter round bottom flask. Evaporate the ethanol using a rotary evaporator at 50 degrees Celsius and 110 millibar. Then add 150 milliliters of deionized water to the concentrated liquor.
Precipitate the lignin by centrifugation. Collect lignin as a pellet and wash it four times with 25 milliliters of distilled water. Then lyophilize the lignin or dry it in an oven at 40 degrees Celsius.
Place the filter crucible in a muffle furnace at 550 degrees Celsius for four hours. When the oven cools to 150 degrees Celsius, remove the crucible and place it in a desiccator to cool. Then weigh it.
Add approximately 30 milligrams of lignin into a borosilicate glass tube. Then add one milliliter of 72%sulfuric to the sample and place it in a 30 degrees Celsius bath for 60 minutes. Remove the sample and transfer it to a 100 milliliter glass bottle.
Then add 28 milliliters of distilled water to dilute the acid to a concentration of 4%Put the glass bottle in an autoclave at 121 degrees Celsius for 60 minutes. Then remove the bottle from the autoclave and allow it to cool. To analyze acid insoluble lignin, filter the hydrolysate using a crucible under vacuum and collect the remaining solids in the glass bottle with deionized water.
Dry the crucible containing the solids by placing it in an oven at 105 degrees Celsius for 16 hours. Then cool it in a desiccator and weigh the sample. Place the crucible in a muffle furnace at 550 degrees Celsius for four hours.
Weigh the sample after drying it in a desiccator. For analysis of acid soluble lignin, measure the absorbance of the hydrolysate filtrate with a spectrophotometer at 205 nanometers using quartz cuvettes. To analyze chemical function of the extracted lignin, process the background single channel without sample.
Then adjust the parameters and place one milligram of the sample on the crystal. Press sample single channel and process the obtained spectra. To determine the molecular weight of the extracted lignin, dissolve three milligrams of the lignin sample in three milliliters of DMF with 0.5%lithium chloride.
Put the dissolved lignin in a vial and install the column proceeded by a guard column. Inject the sample into a high-performance liquid chromatography ultraviolet system. After analyzing the data, calculate the molecular weight of the extracted lignin as described in the text manuscript.
Weigh a 50 milligram sample of lignin in a borosilicate glass tube and add three milliliters of one molar sulfuric acid. Then heat the mixture for three hours at 100 degrees Celsius and cool it. Add one milliliter of 15 molar ammonium hydroxide and check the pH to ensure that it is neutral or alkaline.
Add one milliliter of 2-deoxyglucose to each sample as internal standard. Then add 400 microliters of this solution into special tubes containing 400 microliters of mixed solution. Add two milliliters of sodium borohydride dimethyl sulfoxide solution.
Close the tubes and incubate for 90 minutes at 40 degrees Celsius in a water bath. Remove the tubes from the water bath and add 0.6 milliliters of glacial acetic acid, 0.4 milliliters of 1-methylimidazole and approximately four milliliters of acetic anhydride. After 15 minutes, add 10 milliliters of distilled water.Cool.
And add approximately three milliliters of dichloromethane. After two hours, collect approximately one milliliter of the lower organic phase and inject it into a gas chromatograph equipped with a flame ionization detector capillary column. The lignin yield obtained with deep eutectic solution one oxalic acid was lower than the yields obtained with deep eutectic solution two lactic acid and deep eutectic solution three urea.
Lignin purity exceeded 70%for the three pre-treatments of the biomasses, except for deep eutectic solution three urea pretreatment of alpha leaves, Aegagropila, and almond shells, which gave a lignin purity of 65%The highest lignin purity exceeded 90%It was obtained with the deep eutectic solution one treatment. Lignin purity and yield data were subjected to principal component analysis, which showed the deep eutectic solution one treatment to be positively correlated with lignin purity and confirmed it to be the purest lignin with the lowest yield. The sugar content in lignin extracted using deep eutectic solution three was the highest and followed by that obtained from solutions two and one.
Similarly, the nitrogen content of deep eutectic solution one lignin extract was lower than that obtained from solutions two and three. The type of sugars in the extracted lignin were also characterized, depicting D-xylose and D-glucose to be the most abundant monosaccharides. The chemical functional groups present in the extracted lignans were investigated by FTIR spectroscopy.
Infrared spectra of the different lignans between 3, 500 and 800 reciprocal centimeters are presented here. The spectra of lignin extracted with deep eutectic solutions one and two show the stretching vibrations of unconjugated and conjugated carbonyl groups, which were absent in the three commercial lignans:raw, soda processed, and alkali extracted lignans. The carboxylic acid signal, C double bond O, indicates possibility of the conjugation of some lignin functions with acids during its extraction and solubilization with the microwave-assisted deep eutectic solvent treatment.
These results demonstrate the possibility of extracting value-added lignin of high purity from Mediterranean biomasses, which is presently undervalued and can help determine the optimal deep eutectic solvent while ensuring the purity of lignin.
