The best and most simplified method for determining lignin, cellulose and hemicellulose content in biomass is by using the UV-spectrophotometric analysis by dissolving the biomass in ionic liquids such as [Bmin][Cl] which is an imidazolium type ionic liquid.

Hope this helps answer your question.

Professor Yehia Khalil

Yale University

USA

I have applied the method 1  to determine holocellulose and then on the holocellulose I have applied the method 2 to obtain alpha-cellulose (see HANDBOOK OF WOOD CHEMISTRY AND WOOD COMPOSITES)

Method 1: holocellulose determination

The sample should be extractive and moisture free

Apparatus

Buchner funnel 250 ml Erlenmeyer flasks 25 ml Erlenmeyer flasks Water bath Filter paper Chemical fume hood

Reagents

Acetic acid, reagent grade Sodium chlorite, NaClO2, technical grade, 80%

Procedure

To 2.5 g of sample, add 80 ml of hot distilled water, 0.5 ml acetic acid, and 1 g of sodium chlorite in a 250-ml Erlenmeyer flask. An optional 25-ml Erlenmeyer flask is inverted in the neck of the reaction flask. The mixture is heated in a water bath at 70°C. After 60 minutes, 0.5 ml of acetic acid and 1 g of sodium chlorite are added. After each succeeding hour, fresh portions of 0.5 ml acetic acid and 1 g sodium chlorite are added with shaking. The delignification process degrades some of the polysaccharides and the application of excess chloriting should be avoided. Continued reaction will remove more lignin but hemicellulose will also be lost (Rowell 1980).

Addition of 0.5 ml acetic acid, and 1 g of sodium chlorite is repeated until the wood sample is completely separated from lignin. It usually takes 6 to 8 hours of chloriting and the sample can be left without further addition of acetic acid and sodium chlorite in the water bath for over night. At the end of 24 hours of reaction, cool the sample and filter the holocellulose on filter paper using a Buchner funnel until the yellow color (the color of holocellulose is white) and the odor of chlorine dioxide is removed. If the weight of the holocellulose is desired, filter the holocellulose on a tarred fritted dics glass thimble, wash with acetone, vacuum oven dry at 105°C for 24 hours, place in a desiccator for an hour and weigh. The holocellulose should not contain any lignin and the lignin content of holocellulose should be determined and subtracted from the weight of the prepared holocellulose.

Method 2: alpha-cellulose determination

Extractive-free, lignin-free holocellulose is treated with sodium hydroxide and then with acetic acid, with the residue defined as α-cellulose. The soluble fraction represents the hemicellulose content.

Apparatus

A thermostat or other constant-temperature device will be required that will maintain a temperature of 20 ± 0.1°C in a container large enough to hold a row of at least three 250-ml beakers kept in an upright position at all times.

Filtering crucibles of Alundum or fritted glass thimbles of medium porosity.

Reagents

Sodium hydroxide (NaOH) solution, 17.5% and 8.3% Acetic acid, 10% solution.

Procedure

Weigh out about 2 g of vacuum-oven dried holocellulose and place into a 250-ml glass beaker provided with a glass cover. Add 10 ml of 17.5% NaOH solution to the holocellulose in a 250-ml beaker, cover with a watch glass, and maintain at 20°C in a water bath. Manipulate the holocellulose lightly with a glass rod with a flat end so that all of the specimen becomes soaked with the NaOH solution. After the addition of the first portion of 17.5% NaOH solution to the specimen, at five minute intervals, add 5 ml more of the NaOH solution and thoroughly stir the mixture with the glass rod. Continue this procedure until the NaOH is consumed. Allow the mixture to stand at 20°C for 30 min. making the total time for NaOH treatment 45 min.

Add 33 ml of distilled water at 20°C to the mixture. Thoroughly mix the contents of the beaker and allow to stand at 20°C for 1 hour before filtering.

Filter the cellulose with the aid of suction into the tarred, alkali-resistant Alundum or fritted- glass crucible of medium porosity. Transfer all of the holocellulose residue to the crucible, and wash with 100 ml of 8.3% NaOH solution at 20°C. After the NaOH wash solution has passed through the residue in the crucible, continue the washing at 20°C with distilled water, making certain that all particles have been transferred from the 250-ml beaker to the crucible. Washing the sample in the crucible is facilitated by releasing the suction, filling the crucible to within 6 mm of the top with water, carefully breaking up the cellulose mat with a glass rod so as to separate any lumps present, and again applying suction. Repeat this step twice.

Pour 15 ml of 10% acetic acid at room temperature into the crucible, drawing the acid into the cellulose by suction but, while the cellulose is still covered with acid, release the suction. Subject the cellulose to the acid treatment for 3 min. from the time the suction is released; then apply suction to draw off the acetic acid. Without releasing the suction, fill the crucible almost to the top with distilled water at 20°C and allow to drain completely. Repeat the washing until the cellulose residue is free of acid as indicated by litmus paper. Give the cellulose a final washing by drawing, by suction, an additional 250 ml of distilled water through the cellulose in the crucible. Dry the crucible on the bottom and sides with a cloth and place it overnight in an oven to dry at 105°C. Cool the crucible and weighing bottle in a desiccator for 1 hr before weighing.

Calculation and Report

Calculate the percentage of α-cellulose on the basis of the oven-dried holocellulose sample, as follows:

α-cellulose, percent = (W2/W1) × 100 W2 = weight of the oven-dried α-cellulose residue

where W1 = weight of the original oven-dried holocellulose sample

TAPPI Method

You can use TAPPI test methods.

Thankyou all for your replies.All of your suggestions are highly informative.

Is there any calculation method for determination of biomass components ?

The most simplified method can be for determining lignin via Klason liginin by concentrated acids solubilization and via hydrolyse and solublized cellulose content in biomass is by using the UV-Anthone spectrophotometric analysis of dissolved cellulose. the solubilized lignin need to be also determined via UV-spectrophotometric analysis .For lignin ,I would suggest the AcBr method. See Fukushima and Hatfield (2001), Extraction and Isolation of Lignin for Utilization as a Standard to Determine Lignin Concentration Using the Acetyl Bromide Spectrophotometric Method, JULY 2001 VOLUME 49, NUMBER 7 ACS, Journal of agriculture and food chemistry . we used a UV spectrophotometric assay.

After estimating both lignin insoluble klason , soluble acteyl bromide uv , lignins and celulose via uv anthrone and total organics materials and ash , the inorganics , the diferences can be hemicelulose , pectine , extratives or proteins depending on the biomass compostions .

see more details in our published paper , if your in india you get my Phd thesis P.V.Pannir selvam. 1983 IITD, India ,biochemical eng reseach center , Pretreatment and enzymatic hydrolysis of agricultural residues or from acess to my old published paper listed below

Catalytic solvent delignification of agricultural residues: Organic catalysts

Article Catalytic Solvent Delignification of Agricultural Residues: ...

Chesson's method is very simple and accurate method for determination of lignin, celliulose and hemicellulose of biomass. No HPLC required! Just sulphuric acid is used. Much more simpler than NREL and TAPPI. I have used this method for rice straw and get accurate results.

Please read the paper in attachment for the method: