Increasing Developments In Films Biology Essay

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Recently there have been increasing developments in films with antimicrobial properties in order to improve food safety and shelf life. Active material such as chitosan and its derivatives play important role in preventing the outbreak of infection and many more health application. For example in food industry, chitosan have been use as food packaging due to its quality preservation of a variety of food and it can be formed into fibers, films, gels, sponges, beads or even nanoparticles (Rinoudo 2006).

Surface bio-contamination is a problem that contributes to outbreaks of community-acquired and nosocomial infection through episodic fomite transmission of disease and through persistent fomitic reservoirs. The extent to which fomitic reservoirs contribute to the overall extent of nosocomial infection is unknown, but fomites are known to play some role in the transmission of many infections and diseases. Faster surface die-off of pathogens on a surface can significantly reduce the amount of time that a fomitic reservoir is capable of transmitting dangerous microbacteria, fungus and etc, and can also reduce the average surface population of pathogens available for transmission to a susceptible host. Effective routine chemical disinfection is difficult because strong chemical solutions must be applied correctly and left in contact with surfaces for prolonged periods of time. Many materials are not applicable to such treatments, and many clinical environments do not accommodate them easily. Exotic metal-containing antimicrobial surface materials provide broad-spectrum antimicrobial activity through the controlled release of metal ions (Feied 2004).

Chitin is a nitrogenous polysaccharide. It physical form is inelastic, hard and white in colour. Chitin is one of major causes of surface pollution that pollute coastal area. The main source of chitin mainly came from the canning industries, from crab and shrimp shells. From these shells, they will be chemically process to remove all kind of unwanted material such as protein, iodine that cause the allergy reaction for certain people. After being processed, flake-shape-like chitin was produce. Then it can be further processed to form powder (Rudall & Kenchington 1973).

Chitosan can be obtained by deacetylation process of chitin. It has higher solubility properties compare chitin due to its chemical structure. It can be used as chelating agent. It widely use in biomedical due to its biocapability and antimicrobial properties. Chitosan has exhibited high antimicrobial activity against a wide variety of pathogenic and spoilage microorganisms, including fungi, and Gram-positive and Gram-negative bacteria. Furthermore It can be use in cosmetic, water purifying, photography, ophthalmology, basic material in artificial skin and even food nutrition (Blackwell 1973).

Scope of study

In this study, the chitin-chitosan composites doped with silver salt is prepared by solvent evaporation technique and the characteristic of composite-film produced are investigated using XRD and FTIR.

Reason of using antimicrobial coating

Surface contamination not only presents a health risk for those within the facility, but also has the potential to render a facility unusable for some period of time. If an outbreak is believed to be related to facility contamination, the social and financial impact can persist long after decontamination is complete (Feied 2004).

When facility contamination is recognized, it is when a disease recognized as outbreaks of community-acquired or nosocomial infection, as when an outbreak of respiratory disease leads to recognition of legionella growing within cooling towers and other moist locations (Peters et Al. 1994; O'Mahon et al. 1989). Clusters of neurologic symptoms due to facility contamination with stachybotrys and other mycotoxin-producing molds have led to the abandonment of many buildings (Hughes et al. 1986), but most healthcare facility contamination are not even better. In a healthcare facility, contamination events are common, as endemic, epidemic, or emerging illness frequently enters a healthcare facility and goes unrecognized for many hours or days (shen et al. 2003).

Biocontamination may be spread widely by the time the problem is identified. The everyday presence of pathogens on common hospital surfaces is well documented, and reducing the environmental reservoir is recognized as a positive step as part of an overall strategy to reduce nosocomial infections (Wenzel and Edmund 2003; Dix Control Today 7:10; Yamaguchi et al. 1994; Wilcox and Jones 1995; Neely and Orloff 2001; bonilla 1995; boyce 1994). Common pathogens can survive on surfaces for an extended time and can be transferred anywhere (Noskin 1995). In the same study bed rails supported both species for 24 hours, telephones and fingers for 60 minutes, and stethoscope diaphragms for 30 minutes. Common coronaviruses, known to be transmissible by fomites, are able to survive on ordinary environmental surfaces for up to 3 hours (Ijaz et al. 1991; Sizun et al. 2000). Recent evidence suggests that hospital environments are particularly likely to serve as a reservoir for Methicillin-resistant staphylococcus aureus (MRSA) and Vancomycin-resistant enterococcus (VRE) as compared with gram-negative bacteria (Lemmen et al. 1987).

Contamination was found on many surfaces in patient rooms as well as in nearby nursing stations and other parts of the hospital. Contaminated surfaces including computer mouse at the nursing station and the handrail of the public elevator (Dowell et al. 2001). Hospital locations such as the emergency department or the intensive care unit may be overloaded with very sick patients, making regular surface disinfection impractical (Roberts 2000).

Where constant manual disinfection of surfaces and objects is not practical, the selection of surfaces and disinfecting add-on material that provide sustained intrinsic or extrinsic antimicrobial activity may help to reduce the fomitic transmission of disease. Cross contamination can occur in a very short period of time, thus unless surface killing is complete and instantaneous, it will not prevent all kind of cross-contamination. Reduced transmission of pathogen can also occur if there is a reduction in the size of the fomitic reservoir, because for many pathogens, the clinical infection is directly related to the number of organisms to which a patient is exposed. Reducing the size of the fomitic reservoir can reduce number of pathogens reaching a susceptible host, maybe below the threshold needed for transmission of infection (Schoenbaum et al. 1985).

Biocontamination can be prevented if the number of pathogens can be suppressed below the threshold value without killing the whole pathogens colonies. Thus a surface with antimicrobial properties is a suitable idea to reduce the number of pathogens and prevent various kind of microbacteria infection. Chitin-chitosan is among the leading candidates for antimicrobial coating due its antimicrobial properties. As long we have a surface that literally kill the pathogen, it should be enough to prevent biocontamination (Radun et al. 2003).

Study organization

First chapter explains the important of developing antimicrobial application. This chapter also will briefly show you the procedure that will be used in this study.

The second chapter explains the properties of the main material i.e, chitin, chitosan and silver salt in detail.

Chapter 3 explains the experimental method used in preparing and characterizing the processed composites.

Chapter 4 contains result from XRD, FTIR and tensile test. Comparison of 5 samples have been made in order to identify differential between samples

Chapter 5 will be explaining the patterns of the results and try to point out the reasons.

Chapter 6 contains conclusion that sum up all the procedures, observations, results, and the pattern of the result in shorter ways. In this part suggestion were made so that in future this work may be helpful to community.

Literature Review

Chitin

Chitin structure

Chitin occurs naturally as partially deacetylated depending on the source (Qurashi et al. 1992). They consist of 2-acetamido-2-deoxy-b-D-glucose through a β linkage (Muzzarelli 1973; Zikakis 1984; Mass et al. 1998; Illum 1998). Chitin can be degraded by chitinase. Its immunogenicity is rather low, despite of the presence of nitrogen. Chitin is highly insoluble and has low chemical reactivity. Chitin behaves like cellulose if compare it chemical reactivity. Sometime chitin also known as celullose with acetamido group attached at C-2 position instead of hydroxyl in celullose. Depending on its source, chitin occurs as two allomorphs, α and β forms (Muzzarelli 1973; Zikakis 1984). They can be differentiated by infrared and solid-state NMR spectroscopy together with X-ray diffraction. Both forms are insoluble in all the solvents, despite natural variations in their crystallinity structure. The α-Chitin is by far the most abundant. It occurs in fungal and yeast cell walls, in krill, in lobster and crab tendons and shells, and in shrimp shells, as well as in insect cuticle. Its applications have been limited due to its poor solubility properties, which limiting chitin to be modified. Despite of this limitation, it still can be use as wound dressing component and biodegradeable suture. The advantage of chitin suture compare to other suture is, it could withstand with bile, urine and pancreatic juice where the other suture cannot resist it (Kumar 2000).

Figure 2.1 Chitin molecular structure adapted from Kumar (2000).

Properties of chitin

The insolubility is a major problem that been faced in the development of processing and usage of chitin. An important mechanism is that a solid-state transformation of β-chitin into α-chitin occurs by treatment with strong aqueous HCl (over 7M) and washing with water (Tseng et al. 1995). β-chitin is more reactive than α form, an important property in regard to enzymatic and chemical transformations of chitin (Kubota 1997). Because of the solubility problem, only limited information is available on the physical properties of chitin in solution. A few papers discuss preparation of alkali chitin by dissolution of chitin at low temperature in NaOH solution. To make it soluble, the deacetylation has to be around 50% and, probably, that the acetyl groups must be regularly dispersed along the chain to prevent packing of chains resulting from the disruption of the secondary structure in the strong alkaline medium (Huang et al. 1988, Bhaskara et al, 1998). In some study, they found that changing in temperature and pH altering the structure of chitin thus altering the natural behavior of chitin (Kurita et al. 1993; Huang et al. 1988).

Chitosan

Chitosan structures

Chitosan is the N-deacetylation derivative of chitin. When the degree of deacetylation of chitin reaches 50%, it becomes soluble in aqueous acidic media and is called chitosan but still depending on its source. The solubilization occurs by protonation of the amines functional group (-NH2) on the C-2 position of the D-glucosamine repeat unit, whereby the polysaccharide is converted to a polyelectrolyte in acidic media. Chitosan is the only pseudonatural cationic polymer (Kumar 2000).

Figure 2.2 Chitosan molecular structure adapted from Kumar (2000).

Properties of Chitosan

Chitosan, a linear β-1, 4-D-glucosamine, is a biocompatible, nontoxic compound obtained by deacetylation of chitin, a natural structural component present in crustaceans. Several study showed that the inherent biocide properties of this natural carbohydrate polymer against a wide range of microorganisms such as filamentous fungi, yeast and bacteria (Bordenave et al. 2007). Chitosan has already been approved as a food additive in some countries, like Japan and Korea (Brody 2001). This biopolymer also shows interesting properties such as excellent film-forming capacity and gas and aroma barrier properties at dry conditions. These unique properties make it suitable material for designing food coatings and packaging structures (Cha & Chinnan 2004). In addition, chitosan is also a suitable material for antimicrobial active packaging technologies in improving the quality and safety as well as elongate the shelf-life of perishable foods due to its unique properties (Hirano 1999).

Recently, lots of attentions have been made on chitosan as a potential polysaccharide resource (Nishimura et al. 1991). In some researches, chemical modification could be use to create chitosan as functioning derivatives. (Zikakis 1984; Mass et al. 1998; Illum 1998), but very few of them able to archived solubility in general organic solvents (Nishimura et al. 1991; Toffey et al. 1996) and some binary solvent systems (Kim et al. 1994; Crini et al. 1997; Knaul et al. 1997). Chitin and chitosan that have undergoes chemical modification improved its solubility in common organic solvents. (Knaul et al. 1997; Sakamoto et al. 1994).

Chitin-chitosan processing

Crab or shrimp shell and fungal mycelia is the easiest way to obtain chitin. Chitin production has good relationship with food industries since it is considered as waste. Other resources such as fermentation processes could produce chitosan-glucan and production of citric acid from asperglillus niger, mucor rouxii, and streptomyces that produce chitosan-glucan as it by-product. In the same time this treatment also removing the proteins and deacetylates chitin. Depending on the alkali concentration, some soluble glycan are (Sakamoto et al. 1994). Calcium carbonate and protein which present in crustacean shells are removed by dissolution process. Chitin that was produced then undergoes deacetylation in 40% sodium hydroxide at temperature of 120oC for 1-3 hours. 70% deacetylated chitosan was produced from this process.(Crini et al. 1997).

Figure 2.3 Deacetylation process of chitin to chitosan reproduced from Kumar (2000).

Physical and chemical characterization of chitin and chitosan

Polycharides, e.g. cellulose, dextran, pectin, alginic, agar, agarose and carragenans, are neutral or acidic in nature. Chitin and chitosan are examples of high basic polysaccharides (Luyen et al. 1995). Chitin and chitosan are heteropolymers compared to cellulose, which is homopolymer. Chitin are highy hydrophobic and insoluble in organic acid such as hexafluoroacetone, hexafluoroisopropanol and chloroalcohols (Urbanczyk et al. 1994). Chitosan, the deacetylated product of chitin, is soluble in dilute acids such as acetic acid, formic acid, etc. Pure D-glucosamine could be produced by hydrolysis process of chitin under concentrated. Depend on the extent of deacetylation 5 to 8% of chitin may contain nitrogen, where the nitrogen are mostly form primary aliphatic amino groups in chitosan. Chitosan have good complexing ability, therefore it can undergoes many reactions typical involving of amines group, of which N-acetylation and Schiff reaction are the most important. Metals such as Copper and silver have specific interaction with the amines groups (-NH2) on the chitosan structure. Chelating process depends on the physical condition of chitosan itself whether in powder, gel, fiber and film form. Better chelation effect is obtained for greater degrees of deacetylation of chitin. Thus chelating process is related to the amines groups (-NH2) content as well as its distribution (VaËšrum et al. 2005).

Chitosan derivatives can be considered as substituted glucan and easily obtained under mild conditions and. N-actylation with acid anhydrides or acyl halides have replace chitosan nitrogen with amino group. (Knaul et al. 1997; Sakamoto et al. 1994).

At room temperature, chitosan form aldimines and ketamines with aldehydes and ketones, repectively. Glucans with proteic and nonproteic amino groups can be produced by reaction of ketoacids followed by addition of sodium borohydride. From glyoxylic acid N- Carboxymethyl chitosan can be obtained. The present of bulky subtituent weakening the hydrogen bonds of chitosan and N-alkyl chitosans will swell in water although the present of hydrophobicity of alkyl chains and make chitosan could hold film forming properties. O-hydroxyethylated chitin is partially part of N-Glycol chitin and it is the first derivative with practical use. Other derivatives and their proposed uses are shown in Table 2.1 (Kumar 2000).

Table 2.1 Chitin derivatives and their proposed.

derivative

examples

Potential uses

N-Acyl chitosans

Formyl, acetyl, propionyl, butyryl, hexanoyl, octanoyl, decanoyl, monocholoroacetyl, dichloroacetyl, trifluoroacetyl, succinyl, acetoxynebzoyl

Textile, membranes and medical aid

N-Carboxyalkyl (aryl) chitosans

N-Carboxybenzyl, glycine-glucan (N-carboxyl-methyl- chitosan), alanine glucan, tyrosine glucan, serine glucan, glutamic acid glucan, leucine glucan

Chormatographic media and metal ion collection

N-Carboxyacyl chitosans

From anhydrides such as maleic, itanoic, acetyl-thiosuccinic, glutaric, cyclohexane 1,2-dicarboxylic, phthalic, salicylic

unknown

o-Carboxyalkyl (chitosans

α-Carboxymethyl,

crosslinked α-carboxymethyl

Molecular sieves, viscosity builders and metal ion collection

Sugar derivatives

1-Deoxygalactic-1-yl-, 1-deoxyglucit-1-yl-, 1-deoxyelibiit-1-yl, cellobiit-1-yl-chitosan, products obtained from ascorbic acid.

unknown

Metal ion chelates

Palladium, copper, silver

Catalyst, photography, health products, and insecticides

Semisynthetic resins of chitosan

Copolymer of chitosan with methyl methacrylate,

acrylamidemaleic anhydride

textiles

Natural polysaccharide complexes, miscelleous

Chitosan glucans from various organisms

Hydroxy butyl chitin, cyanoethyl chitosan

Hydroxy butyl chitin, cyanoethyl chitosan

Hydroxyl ethyl glycol chitosan

Linoelic acid-chitosan complex

Flocculation and metal ion collection

Desalting filtration, dialysis and insulating papers

Enzymology, dialysis and special papers

Enzyme immobilization

Food additive and anticholestrolemic

Degree of N-acetylation

This parameter is the most important if we want to examine closely the ratio of 2-acetoamido-2-deoxy-d-glucopyranose to 2-amino-2-deoxy-D-glucopyranose structural units or chitin. This ratio has play important role in chitin solubility and solution properties. Non-toxic N-deacetylated derivative of chitin is other name for chitosan that universally accepted as where it becomes soluble in dilute aqueous weak acid such acetic acid. In chitin, the acetylated units become dominant. Chitosan is the fully or partially N-deacetylated derivative of chitin with typical degree of acetylation of less than 0.35. To understand this ratio, many analytical tools have been used(Radun et al. 2003; Muzzarelli 1973; Zikakis 1984 Mass et al. 1998; Illum 1998; Nishimura et al. 1991; Toffey et la. 1996; Kim et al. 1994; Crini et al. 1997; Knaul et al. 1997; sakamoto et al. 1994), which include gas chromatography, IR spectroscopy UV spectrophotometry, and gel permeation chromatography, first derivative of UV spectrophotometry, 1H-NMR spectroscopy, 13C solid stat NMR, thermal analysis and HPLC, separation spectrometry methods and more recently, near-infrared spectroscopy and many more(Tseng et al. 1995).

Molecular weight

Chitosan molecular weight distribution can be obtained using HPLC (Tseng et al. 1995a). Chitin and chitosan molecular weights (Mw) can be determined by light scattering (Kurita et al. 1992). Viscometry is a simple and rapid method for determinination of molecular weight.

Solvent and solution properties

Chitin is highly crystalline, with intractable material and has only dissolved in certain solvent. Polysaccharides with extensive hydrogen bonding are degraded before melting and it has been shown by chitin and chitosan (Zikakis 1984 Mass et al. 1998). Appropriate polymer concentration, solvent system, counterion concentration, pH and temperature effects on the solution viscosity should been known. As a general rule, the maximum amount of polymer is dissolved in a given solvent towards a homogeneous solution. Subsequently, the polymer is regenerated in the required form. A coagulant is required for polymer regeneration or solidification. The solvent and solution properties as well as the polymer used play important role in determine nature of coagulant (Urbanczyk et al. 1994; Kurita et al. 1993).

Application of chitin-chitosan

Antitoxicity applications

Gliadins, derived from peptides from wheat are well known to be toxic for in vitro cultured small intestinal mucosa. This peptide can be separated by using methylpyrrolidinone chitosan coupled to sepharose 6-B matrix. The peptide concentration was reduced to one hundredth of feed after elution through the chitosan-sepharose column (Sakamoto et al. 1994). Carboxymethyl chitin has been used to reduce the toxicity of positively charged liposomes containing strearyl amine in blood. The stearyl amine liposomes toxicity ere inhibited at carboxymethyl chitin concentration as low as 10-3 or 10-2% (Tseng et al. 1995). Stearyl amine liposomes and erythrocytes ghosts interact by aggregation and mixing of lipids followed by mixing of the internal constituents. Carboxymethyl chitin has been used as an inhibitor of lipid mixing between stearyl amine liposomes and erythrocytes ghosts. Mixing were reduced about 20-40% by carboxymethyl chitin. The inhibition was due to the electrostatic interaction between carboxymethyl chitin and stearyl amine liposomes [Tseng et al. 1995a]. A clinical haemoperfusion system using neutral macro rectilinear styrene divinyl benzyene based resin coated with diacetyl chitin was developed for the treatment of intoxication due to hypnotics, sedatives and antidepressents (Kurita et al. 1992).

Antibacterial agents

The growth of Escherichia coli was inhibited in the presence of more than 0.025% chitosan. Chitosan also inhibited the growth of Fusarium, Alternaria and Helminthosporium. The cationic amino groups of chitosan probably bind to anionic groups of these microorganisms, resulting in growth inhibition (Suzuki et al. 2001).

Blood anti-coagulants ( heparinoids)

Chitin and chitosan sulphates have blood anticoagulant and lipoprotein lipase (LPL)-releasing activities. Chitin 3,6-sulphate showed about two-fold anticoagulant activity and 0.1- fold LPL-releasing activity over those of heparin; the sulphate derivatives might be usable as heparinoids for artificial blood dialysis (Sathirakul et al. 1996).

Anti-thrombogenic and haemostatic materials

Chitosan fibres were found to be thrombogenic and haemostatic in an in vitro test, and N-hexanoyl and N-octanoyl chitosan fibres were N-hexanoyl and N-octanoyl chitosan fibres were as haemostatic material; N-hexanoyl and N-octanoylchitosan fibres are used as anti-thrombogenic materials (Paillet et al. 2001).

Enzyme immobilizer

Enzyme can coexist in various oligomeric forms is of major importance for its catalytic expression. Enzyme immobilization is a technique of significant practical utility, especially to enhance the catalytic potential, resistance to pH and temperature, and continued reusability. It is known that chitosan is an excellent base material for immobilization of several carbohydrate degrading enzymes, as it exhibits increased thermostability compared to the free enzyme (Tseng et al. 1995a). Urease has been immobilized covalently onto glutaraldehyde crosslinked chitosan membrane, especially to provide resistance to the influence of inhibitors, such as boric acid, thioglycolic acid, sodium fluoride and acetohydroxamic acid (Zaborska, 1995). Similarly, resistance to mechanical stirring of D-amino acid oxidase (a flavoprotein using FAD as cofactor) has been provided by enzyme immobilization on crosslinked chitosan matrix (Castillo et al.).

Silver

Silver structure

Silver also known as Argentums (Ag), which has 47 atomic numbers. Silver positioning in Group 11 period 5, which also known as transition series element. Silver have ductile and malleable properties compare to other metals. And it classified as metallic element. It has highest electrical conductivity compare to the other metal. But the downside is it highly cost to exquisite and greatly tarnished compare to other conductive metals (Kelly 1978).

Properties of silver

Silver has the highest thermal conductivity and with the highest optical reflectivity (Oman 1992). Silver also well known as metal with lowest contact resistance compare to other metal. Silver halides are photosensitive and widely use to record latent image that can later be produced chemically. Silv tarnishes when exposes to air or water with ozone or hydrogen sulfide but generally stable in air and water. The most common oxidation number is 1+ that is in silver nitrate ((silver nitrate: AgNO3). Silver also have oxidation number of 2+ (silver(II) fluoride: AgF2) and 3+ (potassium tetrafluoroargentate: K[AgF4]). Meanwhile Silver with oxidation number 3+ known to have good complexing ability (Edward & Peterson). Silver standard atomic number is 107.8682. Silver have two naturally stable isotopes that is 107Ag and 109Ag. 107Ag is the most abundant (Kelly 1978).

Medical advantages

Hippocrates said that silver have several unique abilities. Healing and anti-disease properties is also its abilities (Microsoft 2006). Ancient civilizations used silver container to store water, wine and vinegar to prevent it from spoiled. In early 1900s people would put silver coin in milk bottles to help it increase milk freshness period (Strong 2007). The exact effects of its medical advantage are not well known yet but theories existed. One of them is oligodynamic effect, which effect on microorganisms but still do not explain about antiviral effects.

Before antibiotics were found, silver compound were used to prevent the infection during World War I. silver nitrate solution was a standard during that time and then been replaced by silver sulfadiazine cream, which later become the standard of care until late 1990s (Chang et al. 1975). Other options like silver-coating dressings are used to improve effectiveness of silver sulfadiazine cream (Atiyeh at al. 2007).

Silver usage became less due to emerging of modern antibiotics. Recent research on silver as a broad-spectrum antimicrobial agent has emerged. Paired with alginate a biopolymer derived from seaweed, are designed to prevent infections as part of wound management system (Hermans et al. 2006). In 2007, a company named AGC Flat Glass Europe developed first antimicrobial glass to fight hospital-cought infection or nosocomial infection. The trick is that glass is covered with thin layer of silver (Lo et al. 2008).

Aims and Objectives

Objective:

Our first objective is to synthesis silver doped chitin-chitosan chitosan composite. There a lot of methods that can be used. We have to choose only one that suitable for our study. To synthesis a chitin chitosan silver composite that will fullfill our requirement.

Second, To identify the characteristic of silver doped chitin-chitosan chitosan composite by using characterization methods. From characterization methods we can identified type of material that are form, bonding between particle and thermal properties.

To test this hypothesis the following key objectives were undertaken:

To design a procedure to produce silver doped chitin-chitosan composite

To identify the variables during production.

To identify optimum ratio of silver to chitin composite for strength and antimicrobial properties.

To identify the effect of silver toward chitin-chitosan.

Methodology

Chitin fibre preparation

Chitin nanofibres were prepared as per the reported procedure (Yoshikawa et al. 1995).

Figure 3.4 Chitin powder (sigma-aldrich).

Briefly, 1g of purified chitin powder was hydrolyzed by adding acidic solution and stirred at 105 °C. Hydrocloric acid (HCl) obtained from Sigma-Aldrich was added followed by stirring for 2h at 105 °C until colloidal solution was formed. HCl with 3 Normality (N) was used. 3 N of HCl was obtained by diluting 30 ml of HCl with 30 ml of distilled water (Glass et al. 1994).The ratio of HCI to chitin was maintained at 30ml/g i.e. 30 ml of HCl for each gram of chitin. After hydrolysis process, suspensions were diluted with distilled water followed by centrifugation.

Figure 3.5 Scanspeed centrifuge.

These procedures is mainly to separate the unwanted component. The centrifuge (Scanspeed 1236MG) works using the sedimentation principle. The mixture will be separated or dispersed according to the different molecular weight. The centrifugation was set at 9500 rpm for 10 min and this process was repeated thrice. The suspension was then transferred to a dialysis bag and dialyzed against distilled water overnight until they were neutral. The pH of the suspension was adjusted to 2.5 by adding HCl. This was subjected to ultrasonication (DAIHAN) for 20 min. Ultrasonication generates alternating low-pressure and high-pressure waves in colloidal solution leading to the formation and violent collapse of small vacuum bubbles. This suspension displayed a colloidal behavior and the stability was attributed to the presence of NH3+ at the surface of crystallites resulting from the protonation of amino group (Rinaudo 2006).

Chitin fibre preparation work flow

Chitin powder

Adding 3 N HCl with constant stirring

Centrifugation process at 9500 rpm for 3 times at 10 min

for 2 hours at 105

Dilute with distilled water

Dialysis process for overnight

Adjusted pH to 2.5 by adding HCl

Ultrasonification process for 20 min at 42 Watt

Chitin solution in a colloidal form

Preparation of chitin-chitosan film doped with silver sulphate

1 g Chitosan of high molecular weight (Sigma-Aldrich, Degree of Deacetylation of 85%) was dissolved in 1% (v/v) acetic acid under stirring. Briefly, 1 g of chitosan was stirred in 100 ml acetic acid solution (1 ml of acetic acid + 99 ml of distilled water) for 3 hours until fully dissolved. This was followed by the addition of the 1/5 of chitin from dialysis bag volume and further stirred for 2 hours.

Figure 3.6 Chitosan of high molecular weight.

The chitin to chitosan ratio used was 2:8. Various weight of silver sulphate salt (Sigma-Aldrich) (0.1 g, 0.3 g, 0.5 g, and 0.7 g) was added to the chitin chitosan mixtures under continuous stirring until silver salt was fully dissolved. The pH was maintained in range of 2.50 to 3.25. The solution was then spread on the glass plate/petri dish left to dry in the oven at 40 for 8 hours. The dried film was peeled of the plate. To neutralize the remaining residual acid, the film was immersed into sodium hydroxide solution (Sigma-Aldrich). The film then was washed with distilled water repeatedly and dried for the second round in oven. The dried membrane was then placed in the dessicator for further characterization process.

Flow charts used in the preparation of chitin-chitosan doped film with silver sulphate

1g of chitosan of high degree of deacetylation

Colloidal from chitin solution

chitosan solution stirred for 3 hoursAdding Acetic acid solution

Chitin-chitosan mix solution stirred for 2 hours

Silver sulphate of various amounts salts (0.1 g, 0.3 g, 0.5 g, and 0.7 g)

Solution was mixed until all silver dissolved

The solution spread on a glass plate/ petri dish for oven drying

Immersed in sodium hydroxide solution to neutralize residual acid

Film washed with distilled water repeatedly

Keep in desiccators for characterization

Characterization methods

X-ray diffraction technique

The film was analyzed x-ray diffraction method (XRD) by ex-situ D8 Advance X-Ray Diffractometer at Combicat, University of Malaya. The CuKα radiation was set at 40 kv and and 20 mA. The data was collected with 2θ ranged from 50 to 900 with step size of 0.020 and a step time of 1s. Identification of phases was made by comparing the diffraction patterns of film with Joint Committee for Powder Diffraction Studies (JCPDS) standard of silver sulphate (321023), chitin and chitosan (401518).

The crystallites size was analyzed using peak broadening of the XRD profile using EVA Software. The peak can be estimated with Scherrer equation:

Fourier transforms infrared spectroscopy (FTIR)

Fourier transform infrared (FTIR) spectroscopy is a measurement technique for collecting infrared spectra. Infrared (IR) light is guided through an interferometer. After passing through the sample, the measured signal is the interferogram. The analysis of transmitted light will shows how much energy absorbed at ÆŸ each wavelength. Mathematical analysis of Fourier transform on this signal results in a spectrum identical to from conventional (dispersive) infrared spectroscopy (Demirdöven et al. 2000). The transmittance or absorbance spectrum showing at which IR wavelengths the sample absorbs. The details about the molecular structure of the sample can be determined by analyzing these absorption characteristics. This technique works better on samples with covalent bonds. More complex molecular structures lead to more absorption bands and more complex spectra. This method is very useful in determining the characteristic of very complex mixtures (Lau 1999).

Figure 3.7 The basic concept of FTIR reproduced from Demirdöven et al. (2000).

In this research FTIR equipement brand Thermo Scientific model Nicolet is10 was been used to identify spectra. The infrared sprectra were registered to FTIR connected to computer with OMNIC software for data processing. The samples were analyzed in KBr smartscan ranging from 4000 to 650 cm-1.

Tensile test

Instron is one of a device that can measure sample mechanical and physical strength. In this study tensile test was conducted to see the effect of silver sulphate toward chitin chitosan. The samples parameters have been fixed so that the result is accurate. This test is just to see the effect of silver. In this test samples will be stretch by Instron in Microtensile mode. The samples will be stretch according to the load. More load required if samples not elongated. The samples with 1.4cm in width, 2.8cm in length and 0.015cm was prepared. All the measurements were done by using Vernier caliper.

Figure 3.8 Instron for tensile test.

Results and discussion

Figure 4.9 Dried silver sulphate doped chitin-chitosan films.

From the experiment 5 samples was produced. All samples show different colour intensities. Film with higher amount of silver salt tend to be darker meanwhile film with least silver salt more transparent but not transparent as film without silver salt.

X-ray diffractions

Figure 4.10 X-ray diffactorgram of (a) chitin chitosan, (b) 0.1 g silver sulphate doped chitin-chitosan, (c) 0.3 g silver sulphate doped chitin-chitosan, (d) 0.5 g silver sulphate doped chitin-chitosan, (e) 0.7 g silver sulphate doped chitin-chitosan, (1) silver suphate reference peaks and (2) chitin chitosan reference peaks.

The elemental analysis shows the XRD pattern for silver sulphate doped chitin-chitosan composite in figure. It shows the weak diffraction peak intensity at 2θ = 9° and 2θ = 19° which indicate the present of α-chitin and at 2θ = 20° which indicate the present of chitosan. These weak diffraction peaks may be the indicator for amorphous morphology of the chitin chitosan composite (samanta et al. 2009). The peak intensity at 2θ = 38° indicates the present of silver element. But it is not a sharp peak but rather a weak diffraction peak which indicates the addition of silver elements positioning inside composite with amorphous morphology. In sample with 0.1g of silver, the weak diffraction peak at 2θ = 38° could not be spotted. This may due the low concentration or it present may be concealed by chitin chitosan composite (Jin et al. 2009). As the amount of silver is increased, the diffraction peak at 2θ = 38° start to gain it intensity. As mention earlier the addition of silver do not affecting chitin chitosan morphology but rather it intensity. The peaks intensities of 2θ = 9° and 2θ = 19° start to decreased as the amount of silver increased. This suggest the silver may It has been reported earlier crystalline region of chitosan stabilized the structure of the hydrogen bonds between water molecules and the amino groups of chitosan, as the water molecules are observed chitin (Nigam et al. 2009). Using x-ray study we could find out crystalline degree of the samples. Using the following expression the crystalline index (CI) was determined according to the proposed method for cellulose and applied to these polymers:

CI (%) = [ I110 - Iam / I110 ] X 100

Where I110 is the maximum intensity (arbitrary units) of the diffraction (110) at 2θ = 19° and Iam is the intensity of the amorphous diffraction in the same unit at 2θ = 12.6° (Gustavo et al. 2004).

Table 4.2 Crystalline indexes of samples.

samples

Crystalline index, CI (%)

0.0g silver sulphate doped chitin chitosan composite

47

0.1g silver sulphate doped chitin chitosan composite

50

0.3g silver sulphate doped chitin chitosan composite

53

0.5g silver sulphate doped chitin chitosan composite

57

0.7g silver sulphate doped chitin chitosan composite

44

FTIR spectroscopy

Figure 4.11 X-ray spectrography of (a) chitin chitosan, (b) 0.1 g silver sulphate doped chitin-chitosan, (c) 0.3 g silver sulphate doped chitin-chitosan, (d) 0.5 g silver sulphate doped chitin-chitosan, (e) 0.7 g silver sulphate doped chitin-chitosan.

Table 4.3 Samples and it characteristic bands.

Samples ID

Wavelength (cm-1)

Functioning group

0.0g Silver sulphate doped chitin-chitosan composite

889, 949, 987, 1057, 1125,1148, 1306, 1368, 1534, 1554, 1588, 1617, 1648, 2155, 3254, 2858, 3243, 3740

Amine I, Amine II, Amine III, ,amide, alkene, carboxylic acid, aromatic, alkyl halides, nitro compound and alkyne

0.1g Silver sulphate doped chitin-chitosan composite

889, 949, 987, 1057, 1125,1148, 1306, 1368, 1534, 1554, 1588, 1617, 1648, 2155, 3254, 2858, 3243, 3740

Amine I, Amine II, Amine III, ,amide, alkene, carboxylic acid, aromatic, alkyl halides, nitro compound and alkyne

0.3g Silver sulphate doped chitin-chitosan composite

889, 949, 987, 1057, 1125,1148, 1306, 1368, 1534, 1554, 1588, 1617, 1648, 2155, 3254, 2858, 3243, 3740

Amine I, Amine II, Amine III, ,amide, alkene, carboxylic acid, aromatic, alkyl halides, nitro compound and alkyne

0.5g Silver sulphate doped chitin-chitosan composite

889, 949, 987, 1057, 1125,1148, 1306, 1368, 1534, 1554, 1588, 1617, 1648, 2155, 3254, 2858, 3243, 3740

Amine I, Amine II, Amine III, ,amide, alkene, carboxylic acid, aromatic, alkyl halides, nitro compound and alkyne

0.7g Silver sulphate doped chitin-chitosan composite

889, 949, 987, 1057, 1125, 1148, 1306, 1368, 1534, 1554, 1588, 1617, 1648, 2155, 3254, 2858, 3243, 3740

Amine I, Amine II, Amine III, ,amide, alkene, carboxylic acid, aromatic, alkyl halides, nitro compound and alkyne

In FTIR spectrum we could see the sample have exhibits transmission valley at 1148, 1057, 987 and 889 cm-1 present at both chitin and chitosan, which explain the present of saccharide moiety. 3243cm-1 shows valley that corresponded to the vibration of N-H and O-H bond. Meanwhile, peak at 1588cm-1 is due to the -NH2 group which is present in both chitin and chitosan but mainly in chitosan. In addition the characteristic peak at 1588cm-1 start to decrease it transmittance percentage as amount of silver sulphate been added. This indicates the reduction of amino group on chitosan. Amide I was detected when the spectroscopy shows characteristic valley in the region of 1617 and 1648cm-1. This shows the present of chitin inside the samples. The stretching of C-N vibration of superimposed C=O group may attributed to the formation band at 1648cm-1 (Jin et al. 2009). This band only clears in sample with 0.1g, 0.5g and 0.7g of silver sulphate. The amino group inside the chain (C=O∙∙∙H-N) are bonded with half of the carbonyl groups through hydrogen bonds that is responsible for the vibration mode at 1648cm-1. Same bond plus another with the group -CH2OH was been produced at the side chain. The amide I band transmittance percentage at 1648cm-1 was reduced as the amount of silver increased. This could be explained by the removing of H2+ ion with Ag1+ ion. Due to these inter-chain bonds, the α-chitin structure exhibit high chemical stability. The characteristic of α-chitin from shrimp is shown with valley at 3243cm-1 and band at 3243cm-1 are shown by the valley in the region of OH and NH (3600-3000cm-1) (Gustavo et al. 2004). Characteristic peak of chitosan should be located at 3429cm-1 for hydroxyl group and 1592cm-1 for the amino group. Other major absorption band between 1220 and 1200cm-1 represented the free primary amino group (-NH2) at the C2 position of chitosan. The peak at 1384cm-1 represented the -C-O stretching of a primary alcohol group (-CH2-OH). The bending vibration in range of 1650 to 1000cm-1 start to intensify with the increasing amount of silver indicates the possible interaction between silver elements with amino groups of chitosan. The amide I (1648cm-1) due to stretching of C=O, II (3243cm-1) and III (1314cm-1) bonds start to decrease it transmittance with the additional of silver. Amide I, II, III and V are mostly present in chitin (Nigam et al. 2009). At 987cm-1 which represent the characteristic bands corresponded to chitin could be found. This band is due to vibration of CH3 which present in functioning group inside chitin. As the amount of silver increased the bond transmittance percentage start to drop. This may due to the substitution of silver elements (Kumar et al. 2009). When silver was added, the transmittance peaks of amide were not shifted to 3275cm-1 and 1589cm-1 as reported by Zhang Xu and his colleagues. In their research, they mentioned if that bands shift happen chitosan will be functioning as controller of nucleation as well as stabilizer for preparation of silver nanoparticles with present of reducing agents. 1534 and 1554cm-1 may show characteristic of nitro compound. 2155cm-1 represent alkyne but 2354cm-1 may be distinguished as alkyne due to its weak distortion of spectrometry. Unexpected wave band occurred at 1306 and 1368cm-1 that may represent alkyl halides functioning group.all the transmittance percentage of characteristic band decrease as the amount of silver been added. This may due to its unique complexing ability.

Tensile test

Figure 4.12 tensile test result for 5 samples

Table 4.4 Tensile test results.

samples

Load ,F (N)

Cross sectional area, Ao (cm2)

Initial length, Lo (cm)

Change in length , ΔL (cm)

Modulus young, E (N/cm2)

0.0g Silver sulphate doped chitin-chitosan composite

20

0.21

1.4

2.5

53.33

0.1g Silver sulphate doped chitin-chitosan composite

33

0.21

1.4

1.8

122.22

0.3g Silver sulphate doped chitin-chitosan composite

22

0.21

1.4

1.5

171.11

0.5g Silver sulphate doped chitin-chitosan composite

8

0.21

1.4

1.3

41.03

0.7g Silver sulphate doped chitin-chitosan composite

5

0.21

1.4

1.5

22.22

Figure 4.13 plotted graph of silver salts in 5 sample against calculated Modulus Young

Modulus Young can be used to measure stiffness of isotropic elastic material. It is the ratio of unaxial stress over unaxial strain in range of stress. By using equation below, Modulus Young can be determined.

As the amount of silver sulphate increased to 0.1 g the amount of load that can be handled also increase compared to sample with 0.0 g silver sulphate. In the other hand it could not handle amount of stress compare to the sample with 0.0g silver sulphate. Sample with 0.0g silver sulphate elongated almost 3mm compared to sample with 0.1g silver sulphate that only elongated less than 1mm. As the silver contain increased the load that could be handled by sample start to drop drastically. Sample with 0.3g silver sulphate only managed to hold 2/3 of load that could be handled by sample with 0.1g silver sulphate. Sample with 0.5g and 0.7g silver sulphate only could withstand load less than 10N. As the amount of silver increased the sample became more fragile and easily to rupture. This may caused by the excessive addition of silver. From XRD diffactorgram, the samples with higher concentration of silver sulphate start to lose it characteristic peak of chitin-chitosan but not the peak for silver.

Conclusion and Suggestions

By using several analytical techniques it is possible to analyze the structures and chemical composition of the samples. From these techniques the reactions undergo could be predicted. It is crucial to know the reactions involve if further study are to be done. By FTIR it is possible to identify different absorption bands from α-chitin and chitosan.

The chitin-chitosan morphology change as silver is present and it been prove by the XRD diffactorgram. The amount of silver shows significance role in changing the composite morphology. As the silver become excessive, the crystalline indexes start to decrease. It shows that silver when excessive can react with other functioning group and altered chitin-chitosan composite properties as the tensile test result have shown.

To see silver sulphate doped chitin-chitosan true potential further test should be done. As the crucial ratio of silver sulphate to chitin-chitosan that is 0.1g silver sulphate to 1g of chitosan has be known we could send it out for further testing for it antimicrobial properties and extended physical testing.

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