CHAPTER III

METHODOLOGY

This chapter presents the methods to be used by the researchers in this experimental

study.

RESEARCH DESIGN

The research study used experimental research design for gathering and interpretation

of data. This study will utilize the experimental method of research for the kinetic modelling of

glucose decomposition to 5 - hydroxymethylfurfural reaction mechanism through cellulose acid

hydrolysis of Cladophora Rupestris. To characterize the hydrolysed sample, the researchers

measured the content using High-performance liquid chromatography (HPLC). High-

performance liquid chromatography (HPLC) is a form of liquid chromatography to separate

compounds that are dissolved in solution. All experiments were duplicated.

PREPARATION OF RAW MATERIALS

The sodium hydroxide (AR, Uni-Chem), hydrobromic acid (AR, Sigma-Aldrich), glycerol,

sodium hypochlorite (Reagent grade, Crimson Enterprises) and LDPE film (50 micron thick,

Grand Majestic Mfg.) will used as received from chuchu

COLLECTION OF GREEN ALGAE (CLADOPHORA RUPESTRIS)

The Green algae (Cladophora Rupestris) that will be utilized in the experiment will be

collected from Ilocos chuchu lugar. Roughly 2-3 kg of green growth (Cladophora Rupestris) will

be utilized in the trial. The gathered green growth will be oppressed for recognizable proof by

the National Institute of Molecular Biology and Biotechnology (BIOTECH) at the University of the

Philippines Los Banos as Cladophora rupestris.

PREPARATION OF GREEN ALGAE (CLADOPHORA RUPESTRIS)

Cellulose will separated from C. rupestris following the strategy depicted by Siddhanta et

al. (2009) with slight adjustment. The green algae will be washed and air-dried. Afterwards, it

will be defatted with repeated extraction by means of methanol for a time of eight days at room

temperature.

CELLULOSE EXTRACTION BY ALKALINE PRETREATMENT

The defatted samples will undergo drying, then will be cured with 0.5 M NaOH, filtered,

washed until reaching neutral pH and will be dried. Afterwards, the powdered algae will proceed

to bleaching by soaking in 1 L acetate buffer at 60 0C for 3 hours. The bleached sample will be

washed until neutrality, filtered and dried.

Dried sample will then be re-suspended in 5% HCl (200mL) and subjected to boiling.

The slurry will be cooled and stand overnight at room temperature. The extracted cellulose

fibers will be collected, washed until neutral, and freeze-dried (-40°C, 3300 x 10-3 Mbar).

(Sucaldito & Camacho, 2017)

DILUTED ACID HYDROLYSIS OF CELLULOSE EXTRACTED FROM GREEN ALGAE

(CLADOPHORA RUPESTRIS)

The extracted cellulose fibers will undergo acid hydrolysis with 1 g per set-up using

constant acid concentration of 2% H 2SO4 with 1:8 solid-liquid ratio at varying temperature:

(600C, 900C,1200C, 1500C, 1800C, 2100C, 2400C, 2700C) and reaction time (20 minutes, 40

minutes, 60 minutes, 120 minutes, 180 minutes) to further determine the decomposition

mechanism of cellulose. The resultant slurry will then be diluted with room temperature

deionized (DI) water. Afterwards, it will undergo 5 cycles of centrifugation rotating at 3000 rpm

for 10 minutes to eliminate excess acid and water-soluble remains. The dispersion pH will be

maintained at pH 4.0. (Dussan et al., 2014)

EVALUATION OF 5 - HYDROXYMETHYLFURFURAL CONCENTRATION USING HIGH-

PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)

High Performance Liquid Chromotagraphy (HPLC) is an analytical technique used for the

separation of compounds soluble in a particular solvent. HPLC can be used in both qualitative

and quantitative applications, that is for both compound identification and quantification.

For analysis of the 5 - hydroxymethylfurfural, profiling along with an appropriate range of

calibration standards will be analyzed by HPLC testing at Industrial Technology Development

Institute Standards and Testing Division at the Department of Science and Technology (DOST)

with a test fee of Php 1,900.00 and Php 600.00 cost per additional analyte.

DEVELOPMENT OF DEGRADATION KINETIC MODEL OF GLUCOSE TO 5 -

HYDROXYMETHYLFURFURAL

The simplified model of cellulose degradation was as follows:

Cellulose k1 Glucose k2 HMF

According to the literature (Bradbury et al. 1979; Girisuta et al. 2007; Zhuang et al. 2009;

Lee and Wu 2012; Zhang et al. 2012), kinetic model of cellulose hydrolysis k1 is the degradation

rate constant of cellulose and degradation rate constant of glucose that contain k2..

MATLAB fitting will be obtained by applying equations to the experimental data under

different reaction conditions for cellulose degradation. The cellulose degradation rate

constant, k1, the rate of glucose degradation to 5-HMF generated constant, k2.. The activation

energy of the degradation of cellulose, glucose, and 5-HMF were obtained according to

Arrhenius SA equations (Li et al. 2009).

 Collection and Preparation of Samples

Alkaline Pretreatment

Lignocellulosic Profiling

Acid Hydrolysis

Determination of 5- hydroxymethylfurfural Concentration

(HPLC)

Alkaline Pretreatment

Flowchart of Procedures