MATERIALS AND METHODS 3.

1 Materials

The study was performed with various materials including rice husks, chemical reagents i.e. hydrochloric acid, sulphuric acid, phosphoric acid, diethyl ether, metal catalysts (aluminium chloride, aluminium oxide, silicon (IV) oxide and zeolite) of analytical grade and a 99% furfural standard. Chemicals and metal catalysts were obtained commercially on the local market and used without purification. The standard (furfural-99%) was sourced from SigmaAldrich GmbH in Germany. 3.2 Sample collection and preparation

The rice husks were collected from a local rice mill in Namayumba town in Wakiso District, washed with distilled water to remove the dirt and then dried under the sun. The dried husks were ground using a mechanical grinder, packed and then stored in plastic bags for further use. 3.3 Determination of the furfural yields using various metal catalysts

3.3.1 Furfural production The method described by Mansilla et al., 1998 (one stage production process) to produce furfural was used but with slight modifications. Rice husks (10 grams) were weighed using an electrical digital balance and put into a 250 ml round bottomed flask, the metal catalyst (1g of either AlCl3, Al2O3, SiO2 or zeolite) was added and then to the mixture, dilute mineral acid (100ml of 15% of either hydrochloric acid, sulphuric acid or phosphoric acid) was added. The flask with the mixture was corked and placed on an oil bath where it was refluxed for 90 minutes. The heating was carried out at a temperature range of 423K- 443K under atmospheric pressure conditions. The hydroxylate (contents of the flask after heating) was

cooled using a cold water bath, filtered and stored in a brown reagent bottle for further analysis. 3.3.2 Furfural extraction from the hydroxylate Furfural was extracted from the hydroxylate using the solvent (liquid-liquid) extraction method. In this method the hydroxylate (50ml) was measured using a measuring cylinder and put into a 250ml conical flask. An equal volume (50ml) of diethyl ether was added to the flask, the mixture shaken and then left to stand for about 10minutes. An aqueous layer and non-aqueous (organic) layer were obtained.The mixture was put into a 250ml separating funnel where the aqueous layer (bottom layer) was separated from the organic layer (top layer). The organic layer (ether layer) was put into a clean 250ml round bottomed flask placed on a stand and simple distillation was carried out on the organic layer to drive off the ether using an electric heating mantle (0-350C). The liquid sample (furfural) that remained in the flask was removed using a micro syringe that determined its volume and then placed in a 10ml pre-weighed measuring cylinder where its mass was determined. This procedure was done for all the different acid-catalyst combinations. The volume(s) and mass(es) of the extracted furfural were recorded and used to determine the density of furfural. 3.3.3 Density determination of extracted furfural The density of the extracted furfural was determined using the obtained masses and corresponding volumes as described in section 3.3.2. The following mathematical relationship was used. where: is the density, m is the mass and v is the volume.

3.3.4 Furfural yield determination Furfural yield from the different acid-catalyst combinations was determined using the following formula.

Where:

is the furfural percentage yield,

is the mass of furfural obtained, and

is the

mass of the rice husks used. 3.4 Characterization of the furfural produced

The extracted furfural was characterized using IR and UV Spectrophotometric methods. It was then subjected to GC-mass spectrometry to confirm the spectroscopic analytical results comparing the bands obtained with those of pure furfural. This section is a short description of the analytical methods employed. 3.4.1 UV- Spectroscopic analysis of extracted furfural A SHIMADZU UV-1700 CE double beam scanning spectrophotometer was used to scan the furfural standard diluted with diethyl ether for the calibration curve determination and the extracted furfural. The equipment was switched on and left to activate its settings. Both the sample cell and the blank cell were removed, washed with ether and then filled with ether. The cells were then put back into the spectrophotometer and the baseline for the absorption measurement was set. The spectrophotometer was set to scan the diluted furfural standard and the extracted furfural over a wavelength range of 200 500nm.

For the calibration curve determination, the 99% furfural standard (1ml) was diluted to 1litre using diethyl ether. A known volume (0.25ml) of the diluted furfural standard was further

diluted with ether (4-8ml) to make five stock solutions that were scanned and the absorbance over the selected range (200500nm) was noted. The calibration curve was then made from the results of the concentration and corresponding absorbance obtained. The furfural extracted by the different acids combinations was also diluted with ether (5ml) and then the different extracts were scanned over the selected wavelength range (200500nm). 3.4.2 IR Spectroscopic analysis of extracted furfural The IR spectra of the standard furfural and the extracted furfural were determined using a FTIR SPECTROPHOTOMETER (model: IR PRESTIGE 21) SHIMADU

CORPORATION. Other used apparatus were mortar, pestle, spatula, die, sodium chloride plates and hydraulic press. The process is discussed in the following subsections. (a) Sample preparation The mortar and pestle were washed with distilled water and dried in an oven at 100oC. The mortar and pestle were then removed from the oven and left to cool. The IR spectrophotometer was switched on and left to activate its settings. Potassium bromide powder (2 spatulas) was put into the mortar, finely grinded and then placed into a 15mm die. The die was evacuated to remove any water vapour using a force of about 10 tonnes on a hydraulic press. The die was taken apart and the formed transparent disc of Potassium bromide was carefully placed in between two polished flat sodium chloride plates (cells). The cells were squeezed together and quickly mounted in the FTIR disc holder (sample compartment) of the spectrophotometer for analysis. Drops of furfural standard (0.10ml) and the furfural extracts of the different acids (0.10ml) were added to potassium bromide powder (2 spatulas). The mixtures of different

combinations were one at a time subjected to the same treatment as described in the procedure above. (b) Determination of the IR spectrum. The disc that contained only potassium bromide when placed in the spectrophotometer was scanned over the wave number range of 500-4000cm-1. During the scanning, the background for the absorption measurement was set. The discs containing furfural standard and extracted furfural were also one at a time scanned (10times) over the same range (500-4000cm-1) to generate their IR spectra. 3.4.3 GC-MS analysis The GC-MS analysis was performed using a gas chromatograph 6890N (Agilent Technologies) coupled with a mass spectrometer 5975inert XL MSD (Agilent Technologies) and a capillary column HP-5 MS (30 m0.25 mm0.25m). The mass spectrometer was operating in the EI mode (70eV; m/z 50-300; source temperature 230oC and quadruple temperature 150oC). The carrier gas was helium with a constant flow rate of 1 cm 3 min-1. The injector temperature was 280C with a split ratio of 10:1. The initial GC oven temperature set at 50C, held for 2 min and programmed to obtain a temperature of 270C at 5C min-1. The furfural standards (1ml) were diluted with ether (1litre) and then with the different extracts of furfural (0.50ml) were put in cuvettes. The cuvettes were placed in the GC-MS instrument for analysis. Furfural was identified based on retention time and mass spectra of the MS spectral library data of the standard compound. The method used was as described by Rui et al. (2011)