International Journal of Scientific & Engineering Research, Volume 5, Issue 9, September-2014 134 ISSN 2229-5518 IJSER © 2014 http://www.ijser.org Comparative Analysis of Sugarcane Baggase Using Different Methods Protibha Nath Banerjee Department of chemistry, School of physical sciences, The University of Dodoma, Dodoma, Tanzania e-mail: pbanabo@yahoo.com, protibha.banerjee@udom.ac.in Abstract - To find reliable analytical methods to ensure an accurate analysis of structural compounds in plants is always challenging due to structural diversity of constituents in different lignocellulosics. The non-cellulosic carbohydrate in sugarcane baggase was analysed by acid methanolysis followed by gc analysis. Our results showed that 3 hours of acid methanolysis is more sufficient to obtain high yield of monomeric pentoses and uronic acid units. To determine total carbohydrate content the optimum condition for acid hydrolysis was found by the comparison of reaction kinetics using different acids (sulfuric, trifluoroacetic and ortho-phosphoric acid) at different concentrations and different reaction time followed by HPLC and GC analysis. The lignin content was determined by conventional klason method as well as by AcBr method. The effect of perchloric acid on lignin determination by AcBr method has also been evaluated and the optimum condition for sugarcane baggase has been found. Acetyl group content in non-cellulosic carbohydrate was analysed after mild alkaline hydrolysis followed by hplc. Keywords— Analytical methods, HPLC, GC, Methanolysis, Non-cellulosic carbohydrates, Sugarcane bagasse ——————————  —————————— 1 INTRODUCTION Traditional methods for the characterization of the carbohy- drate composition in biomass are usually based on two stage hydrolysis experiment followed by HPLC analysis. (e.g. TAP- PI & NREL methods). However, in case of sugarcane baggase containing mainly xylan, where xylose units are linked to 4-O- methyl glucuronic acid (4OMeGlcA), the analytical procedure sometimes underestimate the (pentoses and 4OMeGlcA) car- bohydrate composition due to acid resistance of 1 2 linkages between 4OMeGlcA and xylose units. Moreover, due to dras- tic condition employed during acid hydrolysis stage, the hy- drolyzed monomeric sugar units, especially pentoses and uronic acids, can also be easily degraded. The hydrolytic pro- cedure and the subsequent analysis have been pointed out as problematic by various groups. [1,2] Other commonly used method for the depolymerisation of exclusively amorphous hemicelluloses involves acid methanolysis [3,4,5] when applied directly on wood samples without pretreatment such as delig- nification. However, little work on depolymerisation by acid methanolysis followed by the analysis of liberated monosac- charides on sugarcane baggase has been done (Ericka et al). Lignin, an integral chemical component of cell wall of ligno- cellulosic biomasses is commonly associated with the reduced digestibility of fiber. Simple, but time-consuming Klason lig- nin method used 72% sulfuric acid are gravimetric and results are affected by solubilisation of acid soluble lignin and (or) by contamination with proteins bound to lignin [6] or other con- densation products like polyphenolics from the cell contents. Alternatively lignin can be determined spectrophotometrically using AcBr method which was developed by Johnson et al and modified by Iiyama et al 7 where perchloric acid was used to accelerate the dissolution of the samples. Hence the objectives of this research was to evaluate • the optimum condition for the analytical hydroly- sis of sugarcane bagasse using different acids (sul- furic, trifluoroacetic acid, and ortho-phosphoric ac- ids) of different concentration and different reac- tion time, • the optimum condition for acid methanolysis of hemicelluloses and pectin in sugarcane bagasse, • the effect of the various concentration of perchloric acid and the time of digestion on lignin determina- tion in sugarcane baggase by AcBr method, meth- od for accurate acetyl group determination.. 2 EXPERIMENTAL 2.1 Biomass sample Sugarcane baggase from the experimental sugar factory of National Sugar Institute, Kanpur (India) was washed with water, air dried and then dried in oven at 65°C for 24 hours. The oven dried bagasse was grounded in a Wiley mill to parti- cles passing a 20-mesh screen. The sample was then extracted with ethanol/toluene in accordance with Tappi Method T204 om-88 (Tappi, 1988a, b). 2.2 Acid methanolysis followed by GC analysis The dried extracted-free sugarcane baggase (10 mg) was trans- ferred to a pear shaped flask and dried in a vaccum oven at 40°C for 1 hour. Two milliliters of 2M HCl in anhydrous methanol was added to each flask (Sundberg etal, 1996) and the samples were then kept at 105°C for 1, 2, 3 and 5 hours, respectively. Four different calibration solutions containing equal amount of the sugar monomers and uronic acids (except 4-O-MeGlcA) were also subjected to acid methanolysis under similar condition. The vessels (2, 3, 5 hrs) were shaken every hour to ensure uniform methanolysis. All samples were cooled to room temperature and neutralized by addition of 200 µl of pyridine. 4 ml of 0.1 mg/ml of sorbitol solution in methanol was added as internal standard to all the samples, the content was mixed and 1 ml of clear solution was then transferred to another flask. The methanol was then evapo- IJSER