(Hyphaene Thebaica) Leaves ﻣﯾﺛﯾ

Sudan University of Science and Technology College of Graduate Studies

Optimization of Reaction Conditions for Preparing Carboxymethyl Cellulose from Doum palm (Hyphaene thebaica) leaves أمثلة ظروف التفاعل لتحضير كربوكسي ميثيل السليلوز من أوراق الدوم

A Dissertation Submitted in Partial Fulfillment of the Requirements of the

Master Degree in Chemistry

Abdalwahab Abdalgadier Bsheer Ahmed

(BSc., Chemistry-Honours, SUST)

Supervisor: Dr. Essa Esmail Mohammad Ahmed

October 2020

Dedication

To who teach us the deeply meaning of life,

To who teach us how to be strong,

To my parent, teachers and friends

Acknowledgement First and foremost, I would like to thank Almighty Allah for completing this researh work. I would like to thank Dr.Essa Esmail Mohammed, my supervisor for support and suggestion.My thanks are extended to my colleagues and friends in the Department of Chemistry at Sudan University of science and technology. Finally, a lot of thanks go to Çankırı Karatekin University and Burdur Mehmet Akif Ersoy University in Turkey, especially Dr. Esra Demirdogen for their generous support and helping.

Abstract In the present study, the synthesis of carboxymethyl cellulose (CMC) from Doum palm (Hyphaene thebaica) leaves as a new source of cellulose was optimized and the produced CMC at optimum conditions was characterized by FTIR, 1H-NMR and TGA. Different solvent mixtures (Ethanol:isopropanol), varying temperatures and reaction time as well as different amounts of monochloro acetic acid (MCAA) were applied. The results showed that the highest degree of substitution (DS) was 1.16. This value was obtained under the following reaction conditions: solvent mixture of (1:1), reaction temperature of 65 ºC, reaction time of 3 hours and MCAA to cellulose ratio of 1:4.5 (moles). The produced CMC (DS1.16) has a creamy color and high water solubility (92%). The FTIR results confirmed the formation of CMC by the presence of carbonyl absorption peak at 1734 cm-1 of carboxymethyl substituent. The TGA results showed that CMC is less thermally stable compared to purified cellulose.

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المستخلص

فٍ هذِ انذراست حى ضبظ ظزوف ححضُز كزبىكسٍ يُثُم انسهُهىس يٍ أوراق َخُم انذوو كًصذر جذَذ نهسهُهىس و شخص كزبىكسٍ يُثُم انسهُهىس انًُخج فٍ انظزوف انًثهً بإسخخذاو طُف االشعت ححج انحًزاء وطُف انزٍَُ انًغُطُسً وانخحهُم انحزاري انىسًَ. حى اسخخذاو يخبنُظ يذَببث يخخهفت )اإلَثبَىل: األَشوبزوببَىل(، درجبث حزارة وأسيبٌ حفبعم يخفبوحت ببإلضبفت إنً كًُبث يخخهفت يٍ حًض انخهُك أحبدٌ انكهىرو. أظهزث انُخبئج أٌ أعهً درجت إسخبذال (DS) كبَج 7.... حى انحصىل عهً هذِ انقًُت فٍ ظم ظزوف انخفبعم انخبنُت: خهُظ انًذَب ).: .( ، درجت حزارة انخفبعم 76 درجت يئىَت ، سيٍ انخفبعم 3 سبعبث وَسبت MCAA إنً انسهُهىس .: 5.6 )يىل(. َخًُش كزبىكسٍ يُثُم انسهُهىس انًُخج (DS1.16) بهىٌ كزًٍَ وقببهُت عبنُت نهذوببٌ فٍ انًبء )29٪(. أكذث َخبئج طُف االشعت ححج انحًزاء حكىٌ CMC يٍ خالل وجىد قًت ايخصبص يجًىعت انكزبىَُم عُذ 835. سى -. نًسخبذل انكزبىكسٍ يُثُم. وأظهزث َخبئج انخحهُم انحزارٌ انىسٍَ أٌ كزبىكسٍ يُثُم انسهُهىس أقم اسخقزا ًرا حزارَبً يقبرَت ببنسهُهىس انًُقً.

Table of Contents Dedication ...... i Acknowledgement ...... ii Abstract ...... iii iv ...... انًسخخهص Table of Contents ...... v List of Figures ...... viii List of Tables ...... ix List of Abbreviations ...... x CHAPTER ONE ...... 1 Introduction and literature review ...... 1 1.1 Natural fibers ...... 1 1.2 Sources of natural fibers ...... 1 1.3 Chemical composition of natural fibers: Cellulose ...... 2 1.3.1 Source and structure ...... 2 1.3.2 Crystal structure and morphology of cellulose ...... 3 1.3.3 Isolation of cellulose ...... 4 1.4 Hemicelluloses ...... 5 1.5 Lignin ...... 5 1.6 Pectin ...... 5 1.7 Properties of natural plant fibers: physical properties ...... 5 1.8 Properties of natural plant fibers: Mechanical properties ...... 6 1.9 Industrial use of natural fibers ...... 7 1.10 Modification of cellulose ...... 7 1.10.1 Esterification of cellulose ...... 8 1.10.2 Alkylation (Methyl cellulose) ...... 9 1.10.3 Hydroxyalkylation ...... 9 1.10.4 Ionic functionalization ...... 10 1.10.5 Etherification of cellulose ...... 11 1.11 Carboxymethylation ...... 11

Figure 1.6: The reaction process for the synthesis CMC ...... 13 1.11.1 Molecular structure of CMC ...... 13 1.11.2 Degrees of substitution in CMC ...... 14 1.11.3 Applications of CMC ...... 14 1.2 Doum palm (Hyphaene thebaica) tree ...... 17 1.2.1 Description, distribution and uses...... 18 1.3 Previous studies on carboxymethylation of cellulose ...... 19 1.4 Objectives of the study ...... 21 CHAPTER TWO ...... 22 Materials and methods ...... 22 2.1 Sample collection and pretreatments ...... 22 2.2 Chemicals ...... 22 2.3 Chemical composition of Doum palm (Hyphaene thebaica) leaves ...... 22 2.3.1 Extractible contents ...... 22 2.3.2 Lignin content ...... 23 2.3.3 Holocelluloses content ...... 23 2.3.4 α-Cellulose content ...... 23 2.3.5 Hemicellulose content...... 24 2.4 Purification of cellulose from Doum palm leaves ...... 24 2.5 Optimization of carboxymethylation reaction of the purified cellulose ...... 24 2.5.1 Effect of solvent ratio on carboxymethylation reaction ...... 24 2.5.2 Effect of temperature on carboxymethylation reaction ...... 25 2.5.3 Effect of reaction time on carboxymethylation reaction ...... 26 2.5.4 Effect of concentration of MCAA on carboxymethylation reaction ...... 26 2.6 Determination of the degree of substitution (DS) ...... 27 2.7 Fourier transform infrared spectroscopy ...... 27 2.8 Nuclear magnetic resonance1H-NMR...... 27 2.9 Thermogravimetric analysis ...... 28 2.10 Viscosity measurements ...... 28

2.11 Solubility test ...... 28 CHAPTER THREE ...... 29 Results and discussion ...... 29 3.1 Chemical composition of Hyphaene thebaica (Doum palm) leaves ...... 29 3.2 Carboxymethylation of Cellulose ...... 29 3.2.1 Effect of various solvent mixtures on carboxymethylation reaction ...... 30 3.2.2 Effect of temperture on carboxymethylation reaction ...... 31 3.2.3 Effect of reaction period on carboxymethylation reaction ...... 32 3.2.4 Effect of amount of monochloroacetic acid (MCAA) ...... 33 3.3 Properties of CMC at DS 1.16 ...... 34 3.4 FTIR spectroscopic analysis ...... 34 3.5 Nucleur magantic reseonance ...... 36 3.6 Thermogravimetric analysis ...... 38 Conclusion and recommendations ...... 40

References ...... 41

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List of Figures Figure 1.1: Molecular structure of cellulose………………………………....…2

Figure 1.2 Morphology structure of cellulose……….………………………….4

Figure 1.3: Main reaction of cellulose………………………………..…..…….8

Figure 1.4: Reaction of vinyl ester-based cellulose esterification ………….….9

Figure 1.5: Functionalization of cellulose monolith by CCC/urea deep eutectic solvent …………………………………………………………………....……11

Figure 1.6: The reaction process for the synthesis CMC …………….…….....13

Figure 3.1: Effect of solvent mixtures on carboxymethylation reaction……...31

Figure 3.2: Effect of temperature on carboxymethylation reaction………...…32

Figure 3.3: Effect of reaction time on carboxymethylation reaction ...... 33

Figure 3.4: Effect of amount of MCAA on carboxymethylation reaction...... 34

Figure 3.5: The FT-IR spectrum of cellulose purified from Hyphaene thebaica leaves...... 35

Figure 3.6: The FT-IR spectrum of synthesized CMC of highest DS ...... 36

Figure 3.7: 1H-NMR of cellulose. ………….…………………………….…...37

Figure 3.8: 1H-NMR of CMC ……………….…………………………….….37

Figure 3.9: TG/DTG and DTA graphs for cellulose ………………….…...….38

Figure 3.10: TG/DTG and DTA graphs for CMC ……………….…………39

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List of Tables Table 2.1: Reaction parameters at different solvent ratio...... 25 Table 2.2: Reaction parameters at different temperatures ...... 26 Table 2.3: Reaction parameters at different reaction times ...... 26 Table 2.3: Reaction parameters at different MCAA concentrations...... 27 Table 3.1: The chemical composition of Hyphaene thebaica………………...29 Table 3.2: The DS values of CMC products …………………………….....30 Table 3.3: Physical properties of prepared CMC…………………………...34

List of Abbreviations

CMC: Carboxymethyl cellulose DS: Degree of substitution AGU: Anhydroglucose units MCAA/MCA: Monochloroacetic acid NMR: Nuclear magnetic resonance FTIR: Fourier transform infrared TG: Thermal gravimetric DTG: Deferential thermal gravimetric

DTA: Differential Thermal Analysis

CHAPTER ONE Introduction and literature review 1.1 Natural fibers Natural fibers are a renewable resource and have several advantages associated with them which include beside others their biodegradability and ready availability from natural sources (Rohan, et al., 2018). In terms of utilization, there are two general classifications of plants producing natural fibers: primary and secondary. The primary plants are those grown for their fiber contents like cotton, while secondary plants are those where the fibers come as a by-product from some other preliminary utilization like pineapple (Saheb and Jog, 1999). Natural fibers derived from plants mainly consist of cellulose, hemicellulose, lignin, pectin, and other substances (Turbak, et al., 1983; Thakur, et al., 1997; Yang, 2007). 1.2 Sources of natural fibers Natural fibers can be classified according to their sources into plant fibers, animal fibers and mineral fibers. Cellulose is the main component of plant fibers, several of which serve in the manufacture of paper and cloth. Plant fibers can be further categorized into the following types: seed fibers, leaf fibers, bast fibers or skin fibers, fruit fibers and stalk fibers (Reddy and Yang, 2005). Animal fibers are generally made up of proteins. Examples include silk, wool, angora, mohair, and alpaca. Animal fibers can be classified to animal hair (wool or hairs), Silk fibers, avian fibers (Chandramohan and Marimuthu, 2011.) Fibers obtained from mineral sources may be used in their naturally occurring form or after slight modifications. They can be placed in the following categories: asbestos, ceramic fibers or glass fibers (glass wool and quartz) and metal fibers (Chandramohan and Marimuthu, 2011).

1.3 Chemical composition of natural fibers: Cellulose 1.3.1 Source and structure Cellulose was first isolated by the Anselme Payenis in 1834. It is a substance that makes up most of a plant's cell walls, since it is made by all plants. It is probably the most abundant organic compound on earth, and its biodegradable polymer (Becher, et al., 2004, Tang, et al., 1996). Cellulose can be extract from woods, microbes, animals, and annual plants. This involves fiber (cotton), grasses (bagasse, bamboo), wood, algae (Valonica ventricosa), bast fibers, and microorganism (Acetobacter xylinum) (Nevell, and Zeronian, 1985). Also tunicates (marine animals) deposit cellulose in their cell walls, the purpose why the bacteria produce cellulose is indefinite, but some studies suggested that it is needful for their survival against UV light (Coffey, et al., 1995; Moon, et al., 2011). Cellulose is a polydispersity linear homopolymer, consisting of regio- and enantio-selective β-1, 4- glycosidic linked D-glucopyranose units (so-called anhydroglucose units [AGU]) (Figure 1.1). It has been shown by 1HNMR spectroscopy that the β-D-glucopyranose adopts the 4C1 chain conformation, the lowest free energy conformation of the molecule. As a consequence, the hydroxyl groups are positioned in the ring plane (equatorial), while the hydrogen atoms are in the vertical position (axial). The polymer contains free hydroxyl groups at the C2, C3, and C6 atoms. Based on the OH groups and the oxygen atoms of both the pyranose ring and the glycosidic bond, ordered hydrogen bond systems form various types of supramolecular semi-crystalline structures (Klemm, et al., 2005).

Figure 1.1: Molecular structure of cellulose

Cellulose has repeated units of glucose and therefore has three groups of hydroxyl in each unit. Thus influencing its structure with hydrogen bonds and van der Waals forces. Within adjacent cellulose chains Hydrogen bonding may act to define the properties of the chain like straightness, thermal stability and mechanical properties to the cellulose fibers (O’sullivan, 1997). Thus to understand these polymorphs and stability of this fiber the hydrogen bonds must be well understood within the Iα and Iβ structures (Moon, et al., 2011). Cellulose has equatorial hydroxyl groups to ring plane, the internal hydrogen bonds in cellulose called the (plane). This name is due to the fact that the hydrogen bonding between the intra- and inter chains is more prevalent within the (110) level in the triclinic structure and within the (200) level in the monolithic structure (Moon, et al., 2011). 1.3.2 Crystal structure and morphology of cellulose The arrangement of the macromolecules is not uniform through the structure of cellulose fibers. There exist two regions low (amorphous regions) and high crystalline order. The source of the sample and pre-treatment controls the degree of crystallinity of cellulose that usually between 40% and 60%. The wide-angle X-ray scattering (WAXS) and cross polarization magic angle spinning (CP-MAS) 13C NMR are used to estimate the relative amount of polymer in highly ordered region (Klemm, et al., 2005). The fiber morphology of cellulose controls of biological activity and many application of cellulose. The morphological scale is explained by three

3 structural units, elementary fibrils (1.5 and 3.5 nm), micro fibrils (10 and 30 nm), and micro-fibrillary bands (100 nm) (Chen, et al., 2011). For many years the crystallites of cellulose and the degree of crystallinity have been subject of wide studies (Fink, et al., 1995; Ganster and Fink, 2009). The pore structure can be reflecting to cellulose morphology and it is highly significant for enzymatic degradation and chemical reactions accessibility (Westermarck, 2000). For wide range of cellulose products applications pore structure diversity must be controlled like nonwovens with excellent imbibition features (Liu, et al., 2011).

Figure 1.2 Morphology structure of cellulose

1.3.3 Isolation of cellulose The isolation occurs in two steps. The ﬁrst step is the pretreatment of the original material by a puriﬁcation and the second step is a separation. The particular pretreatment is based on the cellulose source and to a less degree on the required morphology of the starting cellulose particle for the second stage treatments. In the lignocellulosic materials, cellulose is embedded in a gel matrix composed of hemicelluloses, lignin, and other carbohydrate polymers (Chen, et al., 2011). The four basic separation approaches are mechanical treatment, acid hydrolysis, alkali treatment and enzymatic hydrolysis. These approaches can be used separately, though in practice to obtain the desired particle morphology several

4 of these methods are used in sequence or in combination (Hubbe, et al., 2008; Habibi, et al., 2010; Siro and Plackett, 2010). 1.4 Hemicelluloses Hemicelluloses are a group of complex heteropolysaccharides made up of various sugars (D-xylose, D-glucose, D-mannose, D-galactose, and L- arabinose) and sugar acids (D-glucuronic and D-galacturonic acids), depending on the plant species (Yang, 2007). 1.5 Lignin Lignin is a complex biopolymer composed of different amounts of three monolignols, namely p-coumarylalcohol, coniferyl alcohol, and sinapyl alcohol (Yang, 2007). 1.6 Pectin Pectin is acidic structure of heteropolysaccharide, the main component is sugar acid derived from galactose also galacturonic acid. Pectin is a biocompatible and biodegradable natural polymer widely used in the food industry as emulsifier, stabilizer, gelling, and thickening agent, also targeted drug delivery, wound healing and tissue engineering applications (Thakur, et al., 1997). 1.7 Properties of natural plant fibers: physical properties There are many important physical factors of natural fibers that affect their uses. These include ultra-structure, density, micro-fibrillar angle, cellulose content and its crystallinity and water or moisture absorption. The hierarchical structure of natural plant fibers gives the fibrous material excellent performance properties, i.e., high strength to weight ratio (Acha, et al., 2005). The mechanical properties of plant fibers are much lower when compared to those of the most widely used competing reinforcing glass fibers (Acha, et al., 2005) however, because of their low density, the specific properties (property to density ratio), strength, and stiffness of plant fibers are comparable to the values of glass fibers (Wambua, et al., 2003). Different natural plant fibers have varying density values (Petroudy, 2017).

Micro fibrillar angle (MFA) is very important physical factor for natural fibers and especially for their applications in many fields. MFA is defined as the angle micro fibrils make with respect to the fiber axis (Petroudy, 2017). The cellulose content of natural fibers and its crystallinity index are critical microstructural parameters that affect the mechanical properties of natural plant fibers (Reddy and Yang, 2005). Water or moisture absorption and swelling thickness is one of the natural plant fiber obstacles for different applications. This is an intrinsic property of natural plant fibers due to free hydroxyl and other polar groups existing therein and leads to decrease of the mechanical properties and the dimensional stability, whereas it may play a positive role in the biodegradability of bio-composites (Mukhopadhyay and Fangueiro, 2009). 1.8 Properties of natural plant fibers: Mechanical properties Natural plant fibers possess high strength and stiffness. Cellulose fibrils in all natural plant fibers have typically a diameter of about 10-30 nm, are made up of 30-100 cellulose molecules in an extended chain conformation, and provide mechanical strength to the fibers (Mohanty, et al., 2005). Elongation at break (%) also known as fracture strain, is the ratio between changed length and initial length after breakage of the test specimen. It expresses the capability of natural plant fibers to resist changes of shape without crack formation (Guo et al., 2015). Natural plant fibers capability to withstand a suddenly applied load is defined as impact strength and is expressed in terms of energy. In other words, the impact strength is defined as the ability of a material to resist the fracture under stress applied at a high speed (Petroudy, 2017). Flexural strength is the ability of a material to withstand bending forces perpendicular to its longitudinal axis. The flexural strength and modulus have an increasing trend with increased fibers loading and fibers content (Shanks, 2015).

In general, the strength and stiffness of plant fibers depend on the cellulose content and spiral angle, which the bands of microfibrils in the inner secondary cell wall make with the fiber axis (Faruk and Saine, 2015). 1.9 Industrial use of natural fibers After World War II, there was an enormous rise in the production of synthetic fibers, and the use of natural fibers significantly decreased. Recently, with rising oil prices and environmental considerations, there has been a revival of the use of natural fibers in the textile, building, plastics, and automotive industries. This interest is reinforced by economic developmental perspectives on the agro- industrial market and local productions, with emphasis on economic development and independence versus the use of imported materials (Al-Oqla and Sapuan, 2014). 1.10 Modification of cellulose Cellulose is a starting material for many usages. Chemical modifications of cellulose are frequently employed to tailor its structure and properties for specific applications (Dou, et al., 2003). Common chemical modification methods of cellulose generally including esterification, etherification, oxidation, and free radical polymerization (Fox, et al., 2011; Hokkanen, et al., 2016). Many efforts have been devoted to preparing cellulose-based materials of special architectures such as aerogels hydrogels, film, fiber and micells (Azzam et al., 2015; Sirvi , et al., 2016; Qiao, et al., 2015). Another reaction has recently received extensive attention in cellulose modification ―Click‖ reaction (Yang and Cranston, 2014; Luo, et al., 2015; Zhu, et al., 2016), because of their remarkable advantages, such as mild reaction condition, high conversion and no toxic metal catalysts. Hu, et al., (2015) synthesized allyl celluloses (AC); the reaction started homogeneously and ended heterogeneously, therefor the ability of the click reaction of AC was evaluated.

Figure 1.3: Main reaction of cellulose (Klemm, et al., 1998). 1.10.1 Esterification of cellulose Cellulose esterification under homogeneous conditions has received increased attention during the last several decades aiming for better conversion and distribution. The generally applied solvents in this process are N,N- dimethylacetamide/lithium chloride (McCormick, et al., 1985), tetrabutylammonium fluoride trihydrate and a mixture of dimethylsulfoxide (DMSO) (Heinze, et al., 2000), various ionic liquids (ILs), like, 1-allyl-3- methylimidazolium chloride and 1-N-butyl-3-methylimidazolium chloride (Swatloski, et al., 2002). Parameters like degree of substitution (DS) and degree of polymerization (DP) are important to characterize the modified cellulose. The ionic liquids used to dissolve cellulose with a degree of polymerization (DP) in the range from 290 to 1200 to a very high concentration (Barthel and Heinze, 2006; Ramos, et al., 2011).

Figure 1.4: Reaction of vinyl ester-based cellulose esterification (Hinner, et al., 2016). 1.10.2 Alkylation (Methyl cellulose) Conventional preparation by Williamson reaction with gaseous or liquid chloroform (SN2 type nucleophilic substitution). Below is an example of methylation of alkali cellulose with excess of CH3Cl, 40% NaOH used in the industrial procedure (heterogeneous reaction), DS 1.5-2.0 are produced commercially. The DS dependence of solubility of methyl cellulose (and ethyl cellulose), at DS 1.7-2.3. The solubility of methyl cellulose is temperature sensitive at ca. 50-60°C. It forms gels above a critical temperature and the gelation is reversible along the hysteresis loop. Methyl cellulose has some applications of building industry, cosmetics etc (Heinze, et al., 2018).

1.10.3 Hydroxyalkylation Hydroxyalkyl cellulose derivatives soluble in water and produced in a wide range of viscosity grades, their solutions are pseudoplastic (Clasen and Kulicke, 2001). Hydroxyalkylcelluloses are manufactured by reacting alkali cellulose with alkylene oxides (ethylene oxide or propylene oxide) at elevated temperatures and pressures in a mixture of organic solvents and water. The

9 hydroxyalkyl side chain can ―grow‖ further from the first substitution (Majewicz and Ropp, 1981). 1.10.4 Ionic functionalization Polymeric ionic liquids (PILs) as novel polymers had drawn much attention recent years. PILs were known as very effective phase-transfer media that enabled transport of both the anionic dye and cationic dye individually and simultaneously, owing to the ion pairs’ strong electrostatic interaction with polar molecules (Schüler, et al., 2013; Liebert, et al., 2008). PILs contained ionic liquid (IL) moieties either in their backbone or side chains; as a result, they combined attractive IL properties. It is generally accepted that cellulose could be dissolved in imidazolium-based ILs, (Zavrel, et al., 2009). Dissanayake, et al., (2018) reported that according to the interactions between cellulose and ILs, the PILs which combined both advantages of polymers and ILs was presumed to have some non-polar modification on cellulose nanocrystals. Cationic surface functionalization of cellulose with ammonium or amino functional groups has proven to have immense value for a wide range of applications, which include special properties as biodegradability, low cost and low toxicity (Prado, et al., 2014; Muqeet, et al., 2017). The most common cationization methods involve the introduction of an ammonium group by reacting the hydroxyl group of polysaccharide with 2,3- epoxypropyltrimethylammonium chloride in an alkaline solution (Pei, et al., 2013; Sehaqui, et al., 2015). An alternative method involves the generation of the reagent in situ from (3-chloro-2-hydroxypropyl) trimethylammonium chloride and sequential periodate oxidation (Prado, et al., 2014; Liimatainen, et al., 2014).

Figure 1.5: Functionalization of cellulose monolith by CCC/urea deep eutectic solvent (Yang, et al., 2019). 1.10.5 Etherification of cellulose Cellulose ethers are prepared underheterogeneous environments by a slurry process which involves the hydrolysis of the alkylation agent that leads to a substance loss and creation of by-products. In the process, cellulose is swollen in a mixture of aqueous NaOH in the range about 15–50% and an organic solvent. NaOH acts as activator by weakening the hydrogen bonds between cellulose sheets, as a result making the individual cellulose chains on hand for chemical modification. Moreover, the alkali raises the nucleophilicity of the OH groups of cellulose. The organic solvent acts as a diluent agent to disperse cellulose, help spreading of the alkylation reagent, transmission heat through the reaction and facilitate recovery of the reaction product (Heinze, et al., 2018). 1.11 Carboxymethylation Among derivatives of cellulose, carboxymethyl cellulose (CMC) represents the most important ionic cellulose ether, widely used in many areas (Klemm, et al., 1998). It is a nontoxic, biodegradable and biocompatible polysaccharide; it is water soluble cellulose ether, also known as cellulose gum. CMC was first prepared in 1918 and in the early 1920’s produced commercially, now slurry processes is use to produced CMC in large scale (Varshney, et al., 2006). The cellulose extracted from various biomass resources can be converted to CMC. Many research efforts have shown the feasibility of preparing CMC from different resources which include, beside others, banana stem (Adinugraha, et al., 2005), paper sludge (He, et al., 2009), cotton stalk (Zhang, et al., 2011),

11 sago waste (Pushpamalar, et al., 2006), sugarcane bagasse (Ge, et al., 2013), pod husk of Cacao (Kukrety, et al., 2018), palm kernel cake (Bono, et al., 2009), oil palm Fronds (Tasaso, 2015), bean hulls (Ibrahim, et al., 2011), durianun rind (Rachtanapun, et al., 2012), ewinia chrysanthemi (Boyer, et al., 1984), eucalyptus globulus (Dapía, et al., 2005), date palm rachis and posidonia oceanica (Khiari, et al., 2011). CMC is produced commercially by reacting cellulose with sodium monochloroacetate in an alcohol-water-NaOH system, (Heinze and Koschella, 2005) which has an excess of alcohol (Mann, et al., 1998). There exist several methods for preparing cellulose ethers, including homogeneous carboxymethylation (Heinze, et al., 1999) rotating drum technique, fluidized bed technique (Durso, 1981) sheet carboxymethylation (Collings, et al., 1942) solvent-less method using a double screw press and a paddle reactor. Several properties of CMC based on three major factors: cellulose molecular weight, DS and also on the distribution of carboxyl group substituents along the cellulose chains. DS varies by modify the reaction conditions like NaOH concentration, molar ratio of monochloroacetic acid, reaction time and temperature (Kukrety, et al., 2018).

Figure 1.6: The reaction process for the synthesis CMC 1.11.1 Molecular structure of CMC The structure of CMC has the β-(1-4)-D-glucopyranose linkage, when comparing length of CMC molecules with native cellulose on average somewhat is shorter in CMC, with different derivatization giving areas of high and low substitution (Kulicke, 1996). CMC molecules like rod at low concentrations, it most extended, but at higher concentrations the molecules entangle to become a thermo reversible gel due to overlap and coil up. Also by reducing pH and increasing ionic strength, the viscosity will decrease as they cause the polymer to become more coiled (Khraisheh, 2005).

1.11.2 Degrees of substitution in CMC Most CMC with degrees of substitution (DS) in the range from 0.5 to 1.5 has been the subject of several scientific publications (Klemm, et al., 1996, Kasuya, et al., 2000). By using different methods to improve the DS of CMC earlier efforts have been made by various researchers. These methods include dissolving of the cellulose in dimethylsulfoxide (DMSO)/tetrabutylammonium fluoride (TBAF) (Ramos, et al., 2005), also by dissolving cellulose in DMAc/LiCl or via hydrolytically unstable cellulose intermediates (Saake, et al., 2002). However, a maximum DS could be achieved by one-step reaction. DS values of more than 1.5 could not be obtained by the usual heterogeneous reaction (Fox, et al., 2011). Moreover, direct chemical modification of cellulose is often challenging because of its high stability and hydrophilicity (Koschella, et al., 2006). Several protecting group like triphenylmethyl, thexyldimethylsilyl, dichloroacetates and trifluroacetate, have been developed for synthesis of CMC and derivatives, with regard to regioselective substitution and high DS (Xu, et al., 2011, Heinze, et al., 2001). 1.11.3 Applications of CMC (i) Construction industry CMC is used in most of the compositions of cement and building materials because it acts as a stabilizer and hydrophilic agent. It improves the dispersion of sand in the cement, and intensifies its adhesive action. It is also used as glue in upholstery (Krenkel, et al., 2004). (ii) Detergents The detergent industry is the largest consumer of CMC. Technical grade CMC compositions are most often used for soaps and detergents. CMC acts as an inhibitor of the redeposition of grease in the fabric after it has been removed by the detergent (de Lima, et al., 2005).

(iii) Pulp industry CMC coating reduces the consumption of wax in waxed paper and paperboard, ensuring less penetration of the wax into paper. Similarly, the consumption of printing ink is reduced as a result of the surface shine it gives. In addition, because it smoothes the surface, CMC makes paper more resistant to grease and improves the union between fibers, thereby improving the color of the paper. It is also used as a dispersant aid in the extrusion of fibers from the pulp and to prevent their flocculation (Yufang, et al., 2001). (iv) Agriculture In pesticides and water-based sprays, CMC acts as a suspending agent. It also functions as glue to attach the insecticide to the leaves of plants after application. Sometimes, CMC is used as an aid in the deterioration of certain fertilizers that are highly polluting (Raafat, et al., 2012). (v) Adhesives CMC is added to various compositions of glues and adhesives that are used for almost any material. It is widely used in the leather industry. Adhesives that join wood to other wood have been effectively made by combining CMC with starch and phenol formaldehyde (Daws, 1999). (vi) Cosmetics CMC is used in dental impression materials, and in toothpastes and gels. This water soluble ether serves as a thickener, stabilizer, suspending agent and former of films in creams, lotions, or shampoos, and is widely used in hair care products (Vecino, et al., 2017). (vii) Paint CMC is used in oil paints and varnishes. It acts as a thickener and suspends the pigment in the fluid (Dawn, et al., 2011). (viii) Oil industry

Crude or purified CMC is used in drilling sludge as a colloid thickener and is applied when removing the drill from the hole to avoid sediments (Batelaan, et al.,1992). (ix) Ceramics The majority of water-soluble ethers are used to join pieces of porcelain. They have good baking properties and CMC solutions create very little ash (Krenkel and Renz, 2008). (x)Textiles Crude CMC is used as sizing agent for fabric. CMC is also used in combination with starch in laundry operations. To give a better finish to fabrics in the manufacturing process, the fabric is impregnated with CMC and is then treated with acid and heat. It is also a very effective agent in fabric printing and as a thickening agent in paints and textile varnishes (de Lima, et al., 2005). (xi) Food CMC is used in food as an auxiliary agent in the churning of ice cream, creams and dairy products, as an auxiliary to form gels in gelatins and puddings, and as a thickener in salad dressings and fillings. It is also used as suspending agent in fruit juices, as a protective colloid in emulsions and mayonnaise, as a protective agent to cover the surface of fruits and as a stabilizer in ready- to- bake products. Because CMC is not metabolized by the human body, it has been approved for use in foods that are low in calories (Wüstenberg, et al., 2014). (xii) Pharmaceuticals CMC is used to coat tablets with high degrees of purity and low viscosity. CMC is insoluble in the acidic environment of the stomach but soluble in the basic medium of the intestine. It is also used for form gels, to transport the drug, to disintegrate tablets and as a stabilizer for suspensions, emulsions, sprays and bio-adhesive tablets which attach internally to the mucus of a body part (Kadajji and Betageri, 2011).

(xiii)Medicine The most innovative applications of CMC are in the area of medicine. CMC solutions are used to form gels that are used in heart, thoracic and cornea surgery. In thorax operations, the lungs are stapled and then covered with a solution of CMC to prevent air leaks and fluid ingress. In the field of orthopaedics, CMC solutions are used in lubricating the joints of the bones, most often in the wrists, knees and hips. The fluid is injected into these joints to prevent erosion, swelling and possible destruction of the cartilage attached to bones (Jorfi and Foster, 2015). (xiv) Other applications CMC is also used in the manufacture of diapers and sanitary products of this type. Because it is hydrophilic, CMC helps gelatinize liquid and promotes retention (Qiu and Hu, 2013). CMC can be used to remove heavy metals from wastewater through complexation (Fu and Wang, 2011). It was shown by Barakat et al., (2010) that this material is effective for capturing Cr(III), Ni(II), and Cu(II) from synthetic wastewater solutions. Another method that was used to derive an effective adsorbate for heavy metals was devised by Garai, et al., (2014) by synthesizing graft copolymers from cellulose biopolymer. Using the derived adsorbent, Pb2+, Zn2+, Cu2+, Cd2+, were adsorbed from wastewater. 1.2 Doum palm (Hyphaene thebaica) tree Kingdom Plants Subkingdom Vascular plants Superdivision Seed plants Division Flowering plants Class Monocotyledons Family Palm family Genus hyphaene Species Hyphaene thebaica (L. )– (doum palm).

17 https://www.itis.gov/servlet/SingleRpt/SingleRpt?search_topic=TSN&search_v alue=506725#null 1.2.1 Description, distribution and uses Doum palm is type of a palm tree belongs to the family name Arecaceae and the scientific name is "Hyphaene thebaica", also gingerbread palm, zembaba, mkoma, arkobkobai and kambash is vernacular names like doum palm (Orwa, 2009; Auwal, et al., 2012). The Doum palm tree is grows up to 17 m high. The trunk, which can have a thickness of up to 90 cm, branches dichotomously and has tufts of large leaves at the ends of the branches. The bark is fairly smooth and dark grey. The leaf stalks are about a metre long, sheathing the branch at the base and armed with stout upward-curving claws. The leaves are fan shaped and measure about 120 by 180 cm. Male and female flowers are produced on separate trees. The inflorescences are similar in general appearance, up to about 1.2 m long, branching irregularly and with two or three spears arising from each branch (Sharma and Sarkar, 1966). The Doum palm grows in the northern half of Africa, Mauritania and Senegal, and east to Egypt, Kenya, Tanzania and Sudan in the areas which contain groundwater also Sinai, Yemen and Saudi Arabia. It grows in valleys and at oases, but it is considered as drought-resistant and sometimes grows on rocky hillsides (Vandenbeldt, et al., 1992; Fletcher, 1997). Doum palm tree is useful plants. People along the Nile used leaves to rotate the baskets. Their fruits also contain antioxidants. It is also used for fuel wood (Hsu, et al., 2006; Moussa, et al., 1998). Leaves are probably the most important part of the Doum palm, providing the raw material used in brooms, basketry, coarse textiles, making mats, string, thatching and ropes (Orwa, et al., 2009). Also may be used as fuel and for making fishing nets by flogging of the roots. It is difficult to cut them by axe due to the high amounts of fibers in wood, ―Wood produced from the male palm

18 is considered better than that of the female‖. It is often used as supports in construction, wood panels, rail conveyors, wheel fence areas waterways, and construction of mills. Dried bark is used to produce black dye to wear leather (Kamis, et al., 2003) as well as some uses in alternative medicine, for example the root used in the treatment of schistosomiasis, and fruit helps to reduce blood pressure control (Orwa, et al., 2009). The antioxidant activity of the aqueous ethanolic extract of Doum leaves and its potential superoxide anion radical scavenging activity was studied by Eldahshan, et al., (2008) using xanthine/hypoxanthine oxidase assay. Four major flavonoidal compounds were identified by LC/SEI as; quercetin glucoside, kaempferol rhamnoglucoside, dimethyoxyquercetin rhamnoglucoside. In the same study (Eldahshan, et al., 2008) fourteen compounds were isolated and identified. These are 8-C-β-D-glucopyranosyl-5, 7, 4`-trihydroxyflavone (vitexin), 6-C-β-D-glucopyranosyl5, 7, 4`-trihydroxyflavone (isovitexin), quercetin 3-O-β4C1-D-glucopyranoside, gallic acid, quercetin 7-O-β4C1-D- glucoside, luteolin 7-O-β4C1-D-glucoside, tricin 5 O-β4C1-D-glucoside, 7, 3` dimethoxy quercetin 3-O-[6''-O-α-L-rhamnopyranosyl]-β-D-glucopyranoside (Rhamnazin 3-Orutinoside), kaempferol-3-O-[6''-O-α-L-rhamnopyranosyl]-β- D-glucopyranoside (nicotiflorin), apigenin, luteolin, tricin, quercetin and kaempferol. 1.3 Previous studies on carboxymethylation of cellulose Carboxymethylation of cellulose from various biomass resources were extensively studied and reported in the literature (Salmi, et al., 1994; Mann, et al., 1998; Stigsson, et al., 2006; Pushpamalar, et al., 2006; Mansikkamäki, et al., 2007). In conclusion, these studies have shown a great variation on the properties of the produced CMC products based on the origin of the cellulose material, various treatments during cellulose purification and the various carboxymethylation reaction conditions applied.

Stigsson, et al., (2006) has purified cellulose by slurry process method. Carboxymethylation of cellulose was done under an excess of sodium hydroxide to cellulose ration of 2.3 mol (NaOH/AGU) in a mixture of solvent (EtOH/isopropanol) and monochloroacetic acid to cellulose ratio of 1.3 mol (MCA/AGU). The results have shown that the optimum solvent mixture (EtOH/isopropanol) is 1:1 which gave the highest DS of 1.02. Pushpamalar, et al., (2006) have isolated cellulose from sago waste, by ground methods and the carboxymethylation of the isolated cellulose was optimized against temperature, concentration and reaction time. The optimized product has a large DS of 0.821 at pure isopropyl alcohol, reaction period of 180 min, 6.0 g of sodium monochloroacetate, NaOH 25% and reaction temperature of 45 ºC. Mann, et al., (1998) were studied the ratio MCA/AGU in carboxymethylation with selective structure method using 13C cross-polarization/magic angle spinning NMR. Ground cotton linters as was used as a source of cellulose in alcohol-water medium at low concentrations of NaOH. The molar ratio of 1:4 (MCA/AGU) was observed for the highest DS of 2. Salmi, et al., (1994) were studied the effect of the reaction temperature in carboxymethylation by measuring the degree of substitution as a function of the reaction time in a batch reactor at temperatures range 30-80ºC by using industrial cellulose. The results have shown that the reaction rate increased with temperature, and its achieved DS between 1.5 and 2 within 2 h at the highest reaction temperatures (60-80) ºC Mansikkamäki, et al., (2007) were purified cellulose from water Hyacinth (Eichhornia crassipes) and studied the effect of isopropyl alcohol and 2-butanol mixed solvent on the degree of substitution of carboxymethyl cellulose. Highest DS value of 2 was obtained when the conectration of NaOH were 10% and the composition of the solvent mixture was 50:50.

1.4 Objectives of the study The main objective to optimize the preparation conditions of carboxymethyl cellulose from Doum palm leaves. The specific objectives were to: (i) Determine the chemical composition of Doum palm leaves. (ii) Purify cellulose from Doum palm leaves using a chemical method. (iii) Characterize the purified cellulose using FTIR, 1HNMR and TGA. (iv) Investigate the influence of the mole ratio between MCA/AGU, solvent mixture (ETOH: Isopropanol), temperature and reaction time on the carboxymethylation reaction. (v) Characterize the produced CMC at optimum conditions using solubility, viscosity, FTIR, 1HNMR and TGA.

CHAPTER TWO Materials and methods 2.1 Sample collection and pretreatments 1 kilogram of dry raw Doum palm (Hyphaene thebaica) leaves was collected from Khartoum (Khartoum State, Sudan).The sample was authenticated by Medicinal and Aromatic Plants Research Institute, National Centre for Research (Khartoum, Sudan).The leaves were cut into 1-2 cm pieces, soaked in tap water for 24-36 hours and blended using a household blender. The resultant material was air-dried and kept for further treatments. 2.2 Chemicals Clorox (Aqueous solution of 5% Sodium hypochlorite) was purchased from local market, Khartoum. Sodium Hydroxide pellets (Minnumum assay: 97%) was obtained from EMPLURA, India. Isopropanol (Mininmum assay: 99.0%) and ethanol (Minimum assay: 99.9%) were both obtained from Iso Lab, Germany. Methanol (Minimum assay: 99.5%) and acetic acid (Minimum assay: 99.9) were both purchased from ALPHA CHEMIKA, India. Monochloroacetic acid (Minimum assay: 99.0%) was obtained from BDH, England.Potassium bromide (FT-IR grade) was obtained from CDH, India. 2.3 Chemical composition of Doum palm (Hyphaene thebaica) leaves 2.3.1 Extractible contents The air dried sample of 5 g was weighed in an extraction thimble and placed in Soxhlet extraction unit. A mixture of isopropanol and xylene was used as solvent and extraction process continued for a period of five hours. After extraction the sample was rinsed with ethanol and hot water and dried up to constant weight at the temperature of 60 °C. The extractibles were calculated as a percentage of the oven dried test sample and the method has been repeated for each sample.

2.3.2 Lignin content Two grams of extracted sample were placed in a flask and 15 ml of 72% sulphuric acid was added. The mixture was stirred frequently for two and half hours at 25°C and 200 ml of distilled water were added to the mixture. Then the mixture was boiled for next two hours and cooled. After 24 hours, the lignin was filtered and washed with hot water repeatedly until becoming acid free. The collected lignin was dried at 105°C and cooled down in desiccator and weighed. The drying and weighing were repeated until constant weight. 2.3.3 Holocelluloses content Three grams of air dried flax fibre were weighed and placed in an Erlenmeyer flask and then, 160 ml of distilled water, 0.5 ml of glacial acetic acid and 1.5 g of sodium chloride were added successively. The flask was placed in water bath and heated up to 75°C for an hour and then additional 0.5 ml of glacial acetic acid and 1.5 g of sodium chloride were added. The additions of acetic acid and sodium chloride were repeated two times hourly. The flask was placed in an ice bath and cooled down below 10°C. The holocellulose was filtered and washed with acetone, ethanol and water respectively and at the end; sample was dried in oven at 105°C before weighed. 2.3.4 α-Cellulose content Two grams of holocellulose were placed in a beaker and 10 ml of sodium hydroxide solution (17.5%) was added. The fibres were stirred up by glass rod so that they could be soaked with sodium hydroxide solution vigorously. Then sodium hydroxide solution was added to the mixture periodically (once every five minutes) for half an hour and the mixture temperature was kept at 20°C. About 33 ml of distilled water was added in the beaker and kept it for an hour. The holocellulose residue was filtered and transferred to the crucible and washed with 100 ml of sodium hydroxide (8.3%), 200 ml of distilled water, 15 ml of acetic acid (10%) and again water successively. The crucible with α- celluloses was dried and weighed.

2.3.5 Hemicellulose content The content of hemicelluloses of flax fibre was calculated from equation: Hemicelluloses = Holocellulose – α-celluloses 2.4 Purification of cellulose from Doum palm leaves Purification of cellulose was conducted based on the method described by Adinugraha, et al., (2005) with slight modifications. In a typical experiment, 350 g of the dried Doum palm leaves were soaked in a suitable quantity of distilled water in 3L beaker for about 30 minutes and decanted. The wet fibers were cooked in 4% NaOH solution at ratio of fibers to NaOH of 1:10 (w/v) for 4 h at 80 ºC. This step was repeated several times till the color of supernatant was changed to pale yellow. The obtained slurry was filtered then washed several times using distilled water and bleached with 1.7% (w/v)Clorox solution for 1 h at 60 ºC two times till the color converted to white. The bleached cellulose was washed again several times using distilled water until the odor of hypochlorite could no longer be detected (by using iodine test) then washed with ethanol and dried at 60 ºC in an oven. 2.5 Optimization of carboxymethylation reaction of the purified cellulose Carboxymethylation reaction was carried out at different reaction conditions which are described in details in Tables 2.1 to 2.4 below. 2.5.1 Effect of solvent ratio on carboxymethylation reaction 1.5 grams of the extracted cellulose were suspended in 60 ml mixture of ethanol and isopropanol in volume ratio of 1:1, and stirred well. 15 ml of an aqueous NaOH solution (40%) (w/v) were added drop wise over a period of 30 min. The mixture was stirred for 1 hour at room temperature. 3 g of monochloroacetic acid were dissolved in 15 ml of the above alcohols mixture (1:1) and were added to the alkalized cellulose. The content was left for an hour at room temperature, the temperature was raised to 55 ºC and the reaction was allowed to continue for additional 2 hours under reflux. Neutralization of NaOH was done using a glacial acetic acid solution and the product was filtered and

24 washed by absolute ethanol several times to remove undesirable by products then dried at 60ºC in an oven, same methods applied by Adinugraha, et al., (2005) with simple modification. For purification, the dried CMC was dispersed in 60 ml of 95% ethanol and stirred for 5 minutes then 10 ml of 2M nitric acid were added and the mixture was agitated for 2 min. The mixture was then heated to boiling for 5 min and agitated further for 15 min and left to settle. After the solution was settled, the supernatant liquid was filtered and discarded. The precipitate was washed with 80 ml of 95% ethanol and further washing process was carried out with hot 80% ethanol at 60ºC, until the acid and salts were removed. The precipitate was washed with methanol and transferred to a beaker and heated until the alcohol was removed. The beaker with the precipitate was dried in an oven at 105ºC for 3 h. Two further experiments were done by keeping all parameters constant (As shown in Table 2.1) and changing the solvent ratio. The DS was determined in each case and base on the results the optimum solvent ratio was used for the other experiments. Table 2.1: Reaction parameters at different solvent ratio No Solvent ratio NaOH Temp MCAA Time /h (Isopropanol:EtOH) 40% /ºC /g S1 1:1 15 55 3 2 S2 1:2 15 55 3 2 S3 2:1 15 55 3 2

2.5.2 Effect of temperature on carboxymethylation reaction The carboxymethylation reaction was done at different temperatures 55, 65 and 75 ºC. Based on the results of DS from section (2.4.1), the solvent mixture was kept at 1:1 ratio. The remaining parameters were kept constant as shown in

Table 2.2 and exactly typical experimental steps were followed as explained in (2.4.1). Table 2.2: Reaction parameters at different temperatures No Solvent ratio NaOH Temp / MCAA Time /h (Isopropanol:EtOH) 40% ºC /g S4 1:1 15 55 3 2 S5 1:1 15 65 3 2 S6 1:1 15 75 3 2

2.5.3 Effect of reaction time on carboxymethylation reaction The carboxymethylation reaction was conducted at different reaction times2, 3 and 4 hours. Based on the findings of the DS from sections 2.4.1 and 2.4.2,the solvent mixture was kept at 1:1 solvent ratio and the optimum temperature was set at 65ºC. The remaining parameters were kept constant as detailed in Table 2.3 and exactly typical experimental steps were followed as explained in (2.4.1). Table 2.3: Reaction parameters at different reaction times No Solvent ratio NaOH Temp MCAA Time /h (Isopropanol:EtOH) 40% C /g S7 1:1 15 65 3 2 S8 1:1 15 65 3 3 S9 1:1 15 65 3 4

2.5.4 Effect of concentration of MCAA on carboxymethylation reaction The carboxymethylation reaction was carried out at different concentrations of MCAA. Based on the results of the DS from sections 2.5.1, 2.5.2 and 2.5.3 the solvent mixture was kept at 1:1 solvent ratio, the optimum temperature was set at 65ºC and the reaction time was set at 3 hours. The remaining parameters were

26 kept constant as detailed in Table 2.4 and exactly typical experimental steps were followed as explained in (2.5.1). Table 2.3: Reaction parameters at different MCAA concentrations No Solvent ratio NaOH Temp MCAA Time (Isopropanol:EtOH) 40%/ml /°C /g /h S10 1:1 15 65 3 3 S11 1:1 15 65 4.5 3 S12 1:1 15 65 6 3

2.6 Determination of the degree of substitution (DS) 0.5 g sample of CMC of each of the above products were ashed at 700°C for 15 to 20 min. The ash was then dissolved in 42 ml boiling deionized water before being titrated with 0.1 N H2SO4 until the solution reached a pH 4.4 (with the aid of a pH-meter). Boiling of the solution was done three times between repeated titration to expel carbon dioxide. The DS value was calculated from the amount of titrated acid (b/ml) and the amount of CMC (G/g), using below equation (Hong, et al., 1978).

DS= ( ) ………………………………... (2.1)

2.7 Fourier transform infrared spectroscopy Fourier transform infrared spectra (FTIR) of cellulose and CMC were obtained using Thermo Scientific Nicolet 6700 FTIR spectrometer (p/n 912A0637) equipped with diamond Smart Orbit ATR sampling accessory (p/n 840- 145300). The samples were analyzed within the range between 4000 to 400 cm−1 with a resolution of 4 cm-1. 2.8 Nuclear magnetic resonance1H-NMR 1H-NMR experiments were performed at temperature range from -80ºC to +130 ºC using a Agilent Premium Compact NMR instrument (DD2 600 MHz), with 5 mm 1H-19F/15N-31P PFG One NMR Probe, 300 W Broadband Linear

Amplifiers. The spectra were analyzed with VNMRJ rev. 3.2A software. DMSO was used as a solvent for cellulose and CMC samples. 2.9 Thermogravimetric analysis Thermogravimetric analysis (TGA) were used in order to get acquainted with thermal degradation process detecting the mass loss as a function of raising temperature in the range from 25 to 1000°C. The sample weight was 3.146 mg and the heating rate was 10 °C/min. Mettler Toledo® TGA/DSC 1 STARe System equipped with Gas Controller GC10 were employed in this investigation. 2.10 Viscosity measurements An aqueous solution of 1% (wt/v) of CMC having DS values of 1.16 was prepared for viscosity measurements. The experiments were performed using Brookfield DV3T Rheocalc (Brookfield Engineering Laboratories, INC., USA). 0.5 ml of CMC (DS 1.16) solution was transferred into the sample cup and cone spindle cp52 was used. The temperature was set at 25ºC and the shear rate was varied from 10 to 200 S-1. 2.11 Solubility test 1 g of CMC having DS values of 1.16 was dissolved in 20 ml distilled water and stirred for 6 h , then transferred to centrifuge tube and running under 5000 rpm for 20 mint using Eppendorf centrifuge 5702, after that the solution is decanted and the residue is weighed after drying, the solubility was calculated by using bellow equation. Solubility%= (initial weight /final weight) ×100 ………………….…....(2.2)

CHAPTER THREE Results and discussion 3.1 Chemical composition of Hyphaene thebaica (Doum palm) leaves The chemical composition of Hyphaene thebaica was studied and the results are presented in Table 3.1 below: Table 3.1: The chemical composition of Hyphaene thebaica Component Hyphaene thebaica Extractive (organic solvents) 9.05±1.34 Liginin 27.7±3.57 Holocelluloses 75.45±0.64 α-cellulose 36.6±2.80 Hemicellulose 40.8±4.04

As it is obvious from the table, the hollocelluloses represent about 75% of the fiber. In addition, the fiber contains somewhat higher organic solvent soluble components; 9.05%. Comparing the chemical composition of Hyphaene thebaica leaves with other natural fibers reveals that it has similar chemical composition regarding cellulose and to some extent the hemicellulose contents with ficus leaf fibers‖ Liginin=23.4%, α-cellulose=38.1%, Hemicellulose=30.5%‖ (Reddy, et al., 2016), corn stalk ‖ Liginin=7.3%, Holocelluloses= 82.1%,α-cellulose=39%, Hemicellulose=42%‖, napier grass‖ Liginin=10.8%,Holocelluloses=82.4%,αcellulose=12.4%,Hemicellulose=68.2% ‖, (Daud, et al., 2014). On the other hand, the bast fibers such as sisal‖ Liginin=10-14%,α-cellulose=66-78%, Hemicellulose=10-14%‖(Reddy, et al., 2014), have higher celllulose content and lower hemicellulose as well as lignin contents compared to the present work. 3.2 Carboxymethylation of Cellulose The carboxymethylation of cellulose purified from Hyphaene thebaica leaves was optimized using different reactions conditions; namely solvent mixture,

29 reaction time, reaction temperature and mole ratio of cellulose/MCAA. The DS values are shown in Table 3.2. The variation of DS with various factors is discussed below. Table 3.2: The DS values of CMC products Sample No DS Sample NO DS S1 0.58 S7 0.52 S2 0.41 S8 0.68 S3 0.39 S9 0.57 S4 0.48 S10 0.78 S5 0.59 S11 1.16 S6 0.50 S12 0.67

3.2.1 Effect of various solvent mixtures on carboxymethylation reaction The effect of solvent mixtures on the degree of substitution was studied at reaction conditions as shown in Table 3.2 from S1 to S3 (i.e., at 55 C, 2 h reaction time, and cellulose: MCAA ratio 1:3). The highest value of DS (0.58) was obtained when the ratio between isopropanol and ethanol are 1:1 in sample 1. The role of the solvent in the carboxymethylation reaction is to provide miscibility and accessibility of the etherifying reagent (MCAA) to the reaction centers of cellulose chain rather than glycolate formation. It is therefore, considered promoter for CMC and inhibitor for glycolate formation (Toğrul and Arslan, 2003). The discrepancies in the extent of carboxymethylation using various alcohol mixtures (ethanol and isopropanol) can be illustrated by taking in consideration their polarities and stereochemistry. The reaction adequacy increases as the polarity of the solvent decreases (Barai, et al., 1997). Mixture of isopropyl alcohol with ethanol was the best choice as it is evident from the DS values. This is in agreement with previously reported research works (Han, and Bhattacharyya, 1995; Khalil, et al., 1990). From the above findings, the

30 solvent mixture of 1:1 has given the highest DS value and this was used for the next carboxymethylation experiments.

0.7 0.58 0.6

0.5 0.41 0.39

0.4 DS 0.3

0.2

0.1

0 1:1 1:2 2:1 Isoprpanol:ETOH

Figure 3.1: Effect of solvent mixtures on carboxymethylation reaction 3.2.2 Effect of temperture on carboxymethylation reaction The variation of DS with reaction temperature is displayed in Figure 3.2. As it obvious from the figure, increasing the temperature increases the DS from 0.48 to 0.59 at 55 and 65°C respectively, and then decreases to 0.50 at 75°C. The first increase in DS value can be explained by the enhancement of carboxymethylation reaction over glycolate formation. On the other hand, the decrease of DS with further increase of temperature is due to the preference of glycolate formation which was clearly observed during experimental work. Similar findings were also reported by Patel, et al., (2018). From the above findings, the temperature 65 °C has given the highest DS value and this was used for the next carboxymethylation experiments.

0.60

0.58

0.56

0.54 DS 0.52

0.50

0.48

55 60 65 70 75 Temperature/°C

Figure 3.2: Effect of temperature on carboxymethylation reaction 3.2.3 Effect of reaction period on carboxymethylation reaction The influence of time on the value of DS was considered in the carboxymethylation at optimum temperature and best solvent mixture. As illustrated in Figure 3.3, it is very clear that the value of DS increased with reaction time until three hours (180 min) then decreased observably. The noticeable increase in DS with reaction time could be due to the governance of carboxymethylation reaction over glycolate formation while the apparent decrease in DS with further increasing the reaction time could be ascribed to the glycolate formation more than carboxymethylation. This formation of gel may slow reaction due to a decrease in accessing and penetration of etherifying agent to the interior of the cellulose and also the difficulty of stirring with increasing the viscosity of the medium. This is also in agreement with the results reported by Aprilia, et al., (2017).

0.70

0.68

0.66

0.64

0.62

0.60 DS 0.58

0.56

0.54

0.52

0.50 2.0 2.5 3.0 3.5 4.0 Time/Hours

Figure 3.3: Effect of reaction time on carboxymethylation reaction 3.2.4 Effect of amount of monochloroacetic acid (MCAA) The amount of MC was varied to examine it is effect on the DS value. solvent mixture of 1:1 (ethanol: isopropanol), a reaction temperature of 65 C and reaction time of 3 hours were applied. Figure 3.4 shows the dependence of DS on amount of MCAA. As can be seen from the figure, generally the trend shows an increase first with increasing amount of MCAA which followed by a sharp decrease with further MCAA amounts. The first increase could be attributed to the availability of MCAA in the proximity of cellulose molecules that enhances the formation of carboxymethylated product. The sharp decrease in DS value could be explained by the formation of glycolate that results from more than adequate amount of MCAA in the vicinity of cellulose molecules, which is in agreement with other reports as well Khalil, et al., (1990).

1.2

1.1

1.0

0.9 DS

0.8

0.7

0.6 1:3 1:4:5 1:6 Amount of MCA/mole

Figure 3.4: Effect of amount of MCAA on carboxymethylation reaction 3.3 Properties of CMC at DS 1.16 The purified cellulose from Hyphaene thebaica leaves was converted to carboxymethylcellulose (CMC) by carboxymethylation reaction using monochloroacetic acid in alkaline medium. The conversion percentage, color and solubility in water of CMC with DS 1.16 are presented in Table 3.2. Table 3.3: Physical properties of prepared CMC Parameter Conversion Color Solubility Viscosity of 1% (%) in water aqueous solution (25ºC) (25ºC)at shear rate 20 S-1 CMC 66 Creamy 92% 16

3.4 FTIR spectroscopic analysis Figure (3.5) shows the FTIR spectrum of cellulose purified from Doum palm leaves. It is evident that the broad absorption band at 3430 cm-1, is due to the stretching frequency of the –OH group as well as intra-molecular and -1 intermolecular hydrogen bonds. The band at 2898 cm is due to and CH2 -1 stretching vibration. The bands around 1431-1323 cm are due to –CH2 and CH bending and 1635 for –OH bending vibration, the band at 1060 cm-1 and 1166 is due to stretching and non-symmetric bridge C-O-C respectively. Finally

34 the band at 602 is due to C-OH out-of-plane bending (Kondo, 1997; Yano, et al. 1976). .

Figure 3.5: The FT-IR spectrum of cellulose purified from Hyphaene thebaica leaves The FTIR spectrum of CMC on the other hand is displayed in Figure 3.6. As it is evident from the Figure, exactly tyypical absorption bands were noticed for CMC product and the only characteristic difference was the presence of a well defined peak at 1734 cm-1 which is due to carbonyl C=O stretching vibration of carboxymethyl substituent. Moreover, the area between 1200 and 1000 cm-1 becomes more intense and clearly separated which could also be ascribed to C- O-C ether streching vibration originated from the carboxymethyl substituent.

Figure 3.6: The FT-IR spectrum of synthesized CMC of highest DS 3.5 Nucleur magantic reseonance Highly resolved 1H-NMR spectra of cellulose and CMC were obtained and the results are displayed in Figures 3.7 and 3.8. According to Fig. 3.7, the protons of the glucose unit appear at 4.65, 3.74, 3.75, 3.76, 3.77 and 3.04 ppm which belong to H1, H2,3,5,4 and H6 respectively. In addition, the signals at 5.40, 4.49 and 4.29 ppm are ascribed to the H6, H2 and H3 protons of hydroxyl groups in anhydroglucose units (AGU) in cellulose. The strong peaks at 2.5 and at 3.1-3.4 are due to DMSO and H2O respectively. This variation in chemical shifts could be attributed to different electronic environments of the carbon atoms in AGU that arise from bonding to electron donating or withdrawing atoms (C or O atoms).

Figure 3.7: 1H-NMR of cellulose.

Figure 3.8: 1H-NMR of CMC Figure (3.8) displays the 1HNMR spectrum of CMC. As can be seen from the spectrum, the peaks of the solvent DMSO were observed at 2.4-2.6 ppm while the one for water was appeared at 3.355ppm. The peaks of AGU protons (3.4- 3.6 ppm) and the acetate group protons (near 4.6 ppm) were suppressed by the

37 strong absorption peaks of DMSO and water and appear as shoulder peaks and in some cases were completely disappeared. 3.6 Thermogravimetric analysis TG/DTG and DTA analyses were carried out to investigate the thermal stabilities of the purified cellulose and the produced CMC (DS 1.16). As can be observed from Figure 3.9, TG/DTG curves have shown two stages of mass loss. o The first one occurred at temperatures less than 100 C which could be attributed to moisture loss. The accompanying mass loss at this stage reaches 6%. o Alternatively, the onset of the second degradation stage starts at about 200 C o and reaches it maximum at 340 C while the offset was happened at around o 400 C. The mass loss at the maximum of the stage is about 52%. The second decomposition of the cellulose could be ascribed to glycosidic links degradation o as well as to pyrolysis of α-cellulose. Heating the sample beyond 400 C and up o to 1000 C has produced a mass loss of 91%. The degradation of cellulose in this temperature range is due to further α-cellulose decomposition and charring.

Figure 3.9: TG/DTG and DTA graph for cellulose

TGA/DTG and DTA was done to check the thermal stability of CMC. In Figure 3.10 there is no serious loss in the weight of the sample up to 200 οC. Above 200 οC, a noticeable degradation starts at around 220 οC and continues up to 330 οC with the maximum degradation temperature at 280 οC. The accompaying mass loss was about 32%. Above 400 οC the samples continues to degrade gradually until the temperature reaches about 650 οC after which the degradation was sharply increased and reaches it is maximum around 880 οC. At this stage the sample was completely decomposed and the remaining mass was almost zero (El-Sayed, et al., 2011; Nada and Hassan, 2000).

Figure 3.10: TG/DTG and DTA graph for CMC

Conclusion and recommendations

In this research, CMC with DS of 1.16 was prepared from cellulose purified from Doum palm (Hyphaene thebaica) leaves as a new source of cellulose. The optimum carboxymethylation conditions were found: solvent mixture ETOH/Isopropanol (1:1), reaction time (3 hours), reaction temperature (65ºC) concentration of NaOH (40%) and amount of MCA to Cellulose (1:4). The FTIR results confirmed the formation of CMC. Furthermore, the TGA results showed the thermal stability of purified cellulose and prepared CMC and found is less stable in CMC compare to native cellulose. For future study, more attention should be given to the solvent effect using other solvents such as cyclohexane and water might give better results.

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