1. INTRODUCTION

Bumetanide (BUM) is a loop diuretic of the sulfamyl category, (3-(butylamino)-4-phenoxy-5-sulfamoylbenzoic acid) most often used to treat heart failure. Most often used in the people whom high dose of furosemide or other diuretics are ineffective. The main differences between bumetanide and furosemide are the bioavailability and potency. Mostly 60 percent of furosemide is absorbed in the intestine, and these are substantial inter and intra-individual differences in the bioavailability (range 10-90%). About 80 percent of bumetanide is absorbed, and these absorption does not change when it is taken with food. It is said to be a more diuretics, meaning that the predictable absorption is reflected in a more predictable effect. Bumetanide drug are 40 times more potent than furosemide for patients with normal renal function. It is sometimes used for weight loss because, use as a diuretic, and removes water from body. The study of solid state chemistry of pharmaceutical solid focuses on all disciplines right from drug discovery to successful marketing. A clear understanding of the molecular structure can lead to a better design and control of the drug performance. Moreover, interest in the subject of pharmaceutical solids stems in the part from FDA's substance guideline that states appropriate analytical procedures to be used to detect polymorphic, cocrystals, hydrated or amorphous forms of the drug substance. Solid forms are usually more stable than liquid counterpart. The main factors which affect the oral route absorption are aqueous solubility, physical/chemical stability, and permeability. The fundamental parameters, i.e. aqueous solubility and gastrointestinal permeability which define oral drug absorption are used as the basis for the BCS classification scheme. These biopharmaceutical classes are defined as below figure.-01 Solubility and permeability parameters defined by FDA and according to it, 'A drug substance is considered highly soluble when the highest strength is soluble in 250 mL or less of aqueous media within the pH range of 1- 6.8 at 37 ?± 1?°C' and 'A drug substance is considered to be highly permeable when the systemic Bioavailability or the extent of absorption in the humans is determined to be 85 percent or more in to the administered dose based on a mass balance determination (along with evidence showing stability of the drug in the GI tract ) or in comparison to an intravenous reference dose'.

1 Bumetanide is the diuretics agent, belongs to BCS class II. A survey by Lin et al.3 revealed that almost 40% of marketed medicines and 90% of emerging new chemical entities suffer from poor solubility which allows them to be eliminated from the gastrointestinal tract before they get completely dissolved and absorbed into the blood circulation, thus results in low bioavailability, less potency, and higher dose strength. For these classes of drugs, higher dose strength would be required to ensure therapeutic concentration in blood but it may lead to adverse or toxic effects on the recipient's body. Thus, new solid forms of the API, which may overcome these problems without affecting the chemical nature of it, greatly reduces the risk, time and cost in development.

1.1. Types of solids forms: Longer range order shorter range order Crystalline Amorphous Liquid crystalline Single component multiple component Ionic non-ionic Polymorphs Salt Molecular adduct Solvates/Hydrates Cocrystal Solid is one of the four fundamental states of matter (the others are liquid, gas, and plasma). Solids molecules are closely packed. These are characterized by structural rigidity and resistance to changes in the shape or volume. Unlike a liquid, or a solid object does not flow to take on the shape of its container, nor does it expand to fill the entire volume available to it like a gas does. All atoms in a solid are tightly bound to each other, either in a regular geometric lattice (crystalline solids, which include metals and ordinary ice) or irregularly (an amorphous solid such as common window glass). Solids cannot be compressed within the little pressure whereas gases can be compressed within little pressure because in gases molecules are loosely packed. In materials science, polymorphism is the ability in a solid material to exist in more than one form or crystal structure. Polymorphism can potentially be found in the any crystalline material including polymers, minerals, and metals, and is compare to allotropy, which refers to chemical elements. Most pharmaceutical molecules are polymorphic. Polymorphism is an ability of a chemical compound to crystallize depending on the crystallization conditions in different crystal structures alias polymorphs. Molecules in the crystal structure of a Polymorphs are bonded by weak interactions (H-bridges, Van-der Waals forces, ??- ?? interactions). Amorphous materials have an internal structure made up of inter-connected structural blocks. This blocks can be similar to the basic structural units found in the corresponding crystalline phase of the same compound. Whether a material is liquid or solid depends upon the primarily connectivity between its elementary building blocks so that solids are characterized by a high degree of connectivity whereas structural blocks in fluids have lower connectivity. In pharma industry, the amorphous drugs are shown to have higher bioavailability than their crystalline counter parts due to the high solubility of amorphous phase. Moreover, certain compounds can undergoes precipitation in their amorphous form in vivo, and they can decrease each other's bioavailability if administered together.

1.2. Crystal Engineering: Crystal engineering developed over the past 65 years as a natural outcome of the interplay between crystallography and chemistry. Chemistry deals with the molecules while crystallography deals with regularly arranged molecules. The term 'Crystal engineering' was coined by pepinsky in the year 1955. According to pepinsky, crystallization of organic ions with a metal-containing complex ion of suitable sizes, charges and solubility's result in structures with cells and symmetries determined chiefly by packing of complex ions. These cells and symmetries are to be good extent controllable; hence crystals with advantageous properties can be engineered.'' Crystal engineering is an interdisciplinary area in chemistry, which bridges, chemistry and crystallography. In the present decade, research is mainly focused on controlling the directionality and strength of intermolecular interaction in the design of molecular crystals. A. I. Kitagorodskii gave the definition of the molecular crystal which state that, within a molecular crystal, it is possible to identify groups of atoms such that for every atom of a group, at least one inter atomic distance within this group is significantly shorter than the smallest inter atomic distance to an atom in another group.

4 He invoked a question, molecule to crystal, he stated that packing of molecular solids was largely governed by considerations of size and shape, the so-called principle of packing. Today, X-ray crystallography is a matured science and has a far-reaching impact on material characterization. The structural insights obtained from crystal structure analysis led to unprecedented developments in electronic devices, mineralogy, geosciences, material science, and pharmaceuticals. Detailed knowledge of accurate structural information of active pharmaceutical ingredients (APIs) is a prerequisite for rational drug design and synthesis of new chemical entities for the development of new medicines. As research progressed with time, the focus shifted to multicomponent molecular crystals (cocrystals). The knowledge obtained from the analysis of the crystal structures is used in the selection of conformer for cocrystal design. A successful cocrystal synthesis requires the understanding of supramolecular synthons that occur most frequently between the functional groups of cocrystals components. Hence, the design of cocrystals for a given molecule starts with analyzing the functional groups available on that molecule and finding complementary functional groups which would likely form predictable supramolecular synthons. Thus, the conformer selection in a cocrystal design strategy reinforces a more significant role of the knowledge of intermolecular interactions which is often drawn from X-ray crystal structure analysis. In a crystal, molecules are associated with a specific pattern of non-covalent interactions such as hydrogen bonds, halogen bonds,5 and ??-stacking. Over the past century, single-crystal X-ray diffraction has proven to be an important tool for unambiguous determination of crystal structure, and thus, assisted in ground-breaking analysis of material properties. Concerning cocrystals, structural characterization (a) establishes the reliability of cocrystal design strategy, (b) reveals hydrogen bind preferences of the functional groups, and (c) provides insights into structure-property correlation. Crystal engineering is a concept of great application and scope. Presently, it focuses on more practical applications such as pharmaceutical cocrystals and high energy materials, etc. Fig.1. Crystal engineering involves modification Of the crystal packing of a solid material by changing the intermolecular interactions.6 1.3. Non-covalent Interactions: Non-covalent interactions are ubiquitous in chemistry and are the primary source of stability for many molecular complexes in biological, pharmacological. Chemical, physical, and material sciences, etc. while traditional chemistry focuses on the covalent bond, crystal engineering and supramolecular chemistry exploit the non-covalent interactions which hold molecules together in a crystal lattice. These forces include hydrogen bonding, metal coordination, hydrophobic forces, Van-der Waals forces, ??-?? interactions and electrostatic effect.

1.4. Hydrogen Bond (strong and weak): Hydrogen bonds are electrostatic and play an essential role in stabilizing the molecular aggregates. Nernst first introduced the phenomenon of hydrogen bong formation in 1891.7 Bernel and huggins proposed the term Hydrogen bond in the year of 1935-36.8 In the year of 1939, Linus pauling defined the hydrogen bond as under certain conditions, an atom of hydrogen is attracted by rather strong forces to two atom instead of only one, so that it may be considered to acting as a bond between them. The more elaborative and expanded definitions were given by many scientists as the increased interest of research in this field, which include Pimentel and McClellan (1960),9 IUPAC stated that the hydrogen bond (designated as D-H---A, where acceptor A and donor D are electronegative atoms) is an attractive interaction between a hydrogen atom from a fragment or molecule D-H in which D is more electronegative is that H, and an atom or a group of atoms A, in the same or different molecule where there is evidence of bond formation. Depending on the nature of the donor and acceptor atoms which constitute the bond, their geometry, and environment, the energy of a hydrogen bond can vary between 1 and 40 kcal/mol. The hydrogen bond is not a simple interaction but a complex conglomerate of at least four component interaction types: electrostatics (acid/base), polarization (hard/soft), van der Waals (dispersion/repulsion) and covalency (charge-transfer). It is neither a strong van der Waals interaction nor a weak covalent bond. It is not even a strong directional dipole-dipole interaction. For geometrical parameters of hydrogen bond see figure-2. This interaction characterized through X-ray diffraction, neutron diffraction, NMR, FT-IR and RAMAN spectroscopes. Three types of hydrogen bond exist very strong, strong and weak(table 1.1).

11 Because of this dual nature (very strong/weak), it attract many scientists all over the world. Hydrogen bonds are electrostatic interactions, but the proportions of electrostatic character can vary. A more expanded proposed definition of a hydrogen bond is as any interaction X-H---A with a shallower energy/distance dependence should be termed as a hydrogen bond. Table No.01: Classification of Jeffrey11 for strong, moderate, and weak hydrogen bonds (the numerical data are guiding values only) Strong Moderate Weak interaction type strongly covalent mostly electrostatic electrostatic/dispersive bond length [?…] H---A 1.2-1.5 1.5-2.2 >2.2 lengthening of X-H 0.08-0.25 0.02-0.08 130 >90 bond energy [kcal/mol] 15-40 4-15

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Introduction

Recently, much attention has turned to the structural and electronic properties of carbon-based materials. At present, especially, graphene is the hottest topics in condensed-matter physics and materials science. This is because graphene has not only unusual properties regarding extreme mechanical strength, thermal conductivity and 2-diemensional films, but also peculiar electronic characteristics such as Dirac-particles with a linear dispersion, transport energy gap and simply absorption coefficient of lights (Geim & Novoselov, 2007; Nair et al., 2008). These unique properties mean it could have a wide array of practical uses. In addition to monolayer graphene, few-layer graphene has been extensively studied. For example, bi-layer graphene creates a band gap when an external electric field is applied (Castro et al., 2007; Zhang et al., 2009). Graphene sheets have been produced mainly by exfoliating graphene flakes from bulk graphite and depositing them on the SiO2/Si substrate. However, the size and crystalline quality are not easily controlled. Some groups have grown epitaxially graphene sheets on SiC(0001) (Hibino et al., 2010), however the graphene layers have been widely distributed in thickness. ((N. G. Prikhod’ko et al. RUSSIAN JOURNAL OF PHYSICAL CHEMISTRY B Vol. 8 No. 1 2014)

Graphene, a single layer of carbon atoms arranged in a hexagonal lattice, is a 2D material with outstanding physical properties. The successful isolation of graphene has drawn great interest for experimental investigations and has opened the route for a wide range of potential applications. Mechanically exfoliated graphene from bulk graphite has enabled fundamental investigations on the physical properties of graphene; however, this technique is not suitable for the integration in practical device fabrication processes nor for the synthesis of large surface area devices. For several applications, if one excludes active semiconducting devices exploiting the quantum properties of single carbon layers, a material composed of a few layers graphene (FLG) is also extremely promising. The overall characteristics of graphene ?lms, both single and FLG, such as size, crystallinity, continuity, homogeneity and fabrication reproducibility are mandatory for successful practical application. (R. Giorgi et al).

Synthesis methods

Synthesis of Graphene Films in a Flame

The results of studying the synthesis of graphenes in a premixed propane–oxygen–argon flame at atmospheric conditions are reported. The temperature of 900–950°C and exposure time of 5 min are demonstrated to be suitable for the synthesis of graphene films on a nickel substrate, which is preferable to a copper substrate. It is demonstrated that the formation of graphene layers on the substrate occurs vertically along the flame height, with subsequent changeover to a soot structure. It is shown that the minimum number of graphene layers (two or three) is observed at angles of inclination of the substrate relative to the vertical axis of the flame within 0°–30°. (N. G. Prikhod’ko et al. RUSSIAN JOURNAL OF PHYSICAL CHEMISTRY B Vol. 8 No. 1 2014)

Photograph of a nickel substrate with carbon struc_

ture.

The Ni(111) surface is the excellent substrate for growth of single-layer-graphene sheet with macroscopic dimensions. Graphene sheets with a 1 x 1 atomic structure grew up epitaxially by CVD or surface segregation techniques. We in-situ observed the graphene growth of mono-, bi- and tri-layer step by step using carbon segregation phenomena on Ni(111) by LEEM. The summaries are as follows;

1. One can grow the uniform monolayer graphene on Ni(111) by adjusting the temperature. No domain boundaries and wrinkles were detected by LEEM.
2. The second- and the third-layer graphene grew at the interface under the first and the second layers. Bi-layer graphene domains grew at least 100?m scale. The third-layer started to grow before the completion of second-layer at 1050K in this experiment. More precise control of temperature seems to be required to complete the second-layer before starting the third-layer growth.
3. Shape of the islands differed depending on the thickness; the first- and third layer islands exhibit hexagonal edges, while the second-layer islands possess dendritic edges.
4. The different shapes of the first, second and third-layer islands presumably originate from the interfacial-bond strength depending on the graphene thickness.
5. The number of nucleation sites of graphene growth is extremely small on Ni(111) surface, which is an important factor for growth of large single-domain graphene crystals.
6. Chemical etching the Ni substrate made it possible to separate macroscopic selfstanding graphene sheets.

Synthesis of Graphene Films on Copper Substrates by CVD of Different Precursors:

Graphene ?lms of the order of 1cm2 were grown on copper foil substrates by CVD using hydrogen/methane or hydrogen/argon/ethanol mixturesasgasprecursors. The growth processes were performed near 1,000?C both at atmospheric and low pressures. A system for the fast cooling of the sample, based on the fast extraction from the hot zone of the furnace, was implemented allowing for rapid decrease of the temperature below 600?C in few seconds. Samples grown under different conditions were analyzed by SEM, Raman spectroscopy and XPS with the aim to assess their characteristics and to re?ne the growth process. (R. Giorgi et al).

Scheme of the CVD reactor

Graphene growth process

SEM image of Cu substrate after graphene growth

Films consisting of less than 5 graphene layers have been grown by CVD both from methane and ethanol as precursors. The use of copper substrates has allowed the growth of large area continuous ?lms of the order of 1cm2; a wet procedure was followed for the transfer of the grapheme ?lms on to SiO2/Si substrates more suitable for their characterization. Pressure and growth time have been found to be the main process parameters affecting the thickness and the quality of the graphene ?lms. The grown ?lms exhibited good crystallinity, but resulted composed of different overlapping regions with different number of layer. Factors in?uencing the ?lm homogeneity and uniformity have been identi?ed in the substrate features. Future work will be focused on the optimization of substrate treatments, with the aim to achieve more uniform large area graphene ?lms with controlled structure: number of layers and crystallinity. The matching of the large copper grain size (upto 1mm) with controlled growth of graphene (single and FLG) remains an interesting goal and a high challenge. (R. Giorgi et al).

Large-Area Synthesis of Graphene Films on Copper Foils:

Large-area graphene films of the order of centimeters on copper substrates was grown by chemical vapor deposition using methane. The films are predominantly single layer graphene with a small percentage (less than 5%) of the area having few layers, and are continuous across copper surface steps and grain boundaries. The low solubility of carbon in copper appears to help make this growth process self-limiting. Graphene film transfer processes were also developed to arbitrary substrates, and dual-gated field-effect transistors fabricated on Si/SiO2 substrates showed electron mobilities as high as 4050 cm2V-1s-1 at room temperature. (Xuesong Li.)

Time dependence of experimental parameters: temperature, pressure, and gas composition/flow rate.

SEM images of graphene on Cu with different growth times of (A) 1 min, (B)

2.5 min, (C) 10 min, and (D) 60 min, respectively.

In recent work, thin Ni films and a fast-cooling process have been used to suppress the amount of precipitated C. However, this process still yields films with a wide range of graphene layer thicknesses, from one to a few tens of layers and with defects associated with fast cooling (5-7). Our results suggest that the graphene growth process is not one of C precipitation but rather a CVD process. The precise mechanism will require additional experiments to understand in full, but very low C solubility in Cu (23-25), and poor C saturation as a result of graphene surface coverage may be playing a role in limiting or preventing the precipitation process altogether at high temperature, similar to the case of impeding of carburization of Ni (26). This provides a pathway for growing self-limited graphene films. To evaluate the electrical quality of the synthesized graphene, we fabricated dual-gated FET with Al2O3 as the gate dielectric and measured them at room temperature. Along with a device model that incorporates a finite density at the Dirac point, the dielectric, and the quantum capacitances (9), the data are shown in Fig. 3. The extracted carrier mobility for this device is ~4050 cm2V-1s-1, with the residual carrier concentration at the Dirac point of n0=3.2×1011 cm-2. These data suggest that the films are of reasonable quality, at least sufficient to continue improving the growth process to achieve a material quality equivalent to the exfoliated natural graphite. (Xuesong Li.)

Synthesis of multi-layer graphene ?lms on copper tape by atmospheric pressure chemical vapor deposition method:

Graphene ?lms were successfully synthesized by atmospheric pressure chemical vapor deposition (APCVD) method. Methane (CH4) gas and copper (Cu) tapes were used as a carbon source and a catalyst, respectively. The CVD temperature and time were in the range of 800–1000?C and 10s to 45min, respectively. The role of the CVD temperature and time on the growth of graphene ?lms was investigated in detail via scanning electron microscopy (SEM) and Raman spectroscopy techniques. The results of SEM images and Raman spectra show that the quality of the graphene ?lms was improved with increasing of CVD temperature due to the increase of catalytic activity.

Multilayer graphene ?lms were successfully synthesized on the Cu tapes by APCVD method. The quality of the graphene ?lms was improved with increasing CVD temperature. The growth time does not much affect the number of layers of graphene ?lms. CVD temperature of 1000?C and CVD time of 30min are the optimum temperature and time for growing high-quality graphene ?lms on the Cu tape, respectively. The graphene ?lms were successfully transferred from the Cu tape to other substrates by wet etching Cu with a solution of iron(III) nitrate. (Van Tu Nguyen1. Adv. Nat. Sci. Nanosci. Nanotechnol. 4 (2013) 035012 (5pp)

Self-Standing Graphene Sheets Prepared with Chemical Vapor Deposition and Chemical Etching:

The growth mechanism of graphene layers on Ni(111) surface was studied. The in-situ observation of the graphene growth of mono-, bi- and tri-layers using carbon segregation phenomena on Ni(111) by low energy electron microscopy (LEEM) were reported, which is a powerful technique to investigate thin films in mesoscopic scale. We also fabricated the self-standing graphene sheets by chemically etching the substrate (Odahara et al., 2009). The chemical process to remove the Ni substrate makes it possible to prepare a self-standing graphene sheets, which are characterized by scanning electron microscopy (SEM) or transmission electron microscopy (TEM).

. Typical snapshots of LEEM images obtained as the temperature was decreased from 1200 K to 1125 K (images (a) to (d)). The observed area was 6?m field-of-view. Letter in each image indicates the time-lapse order. Two graphene domains were united to form one graphene sheet. Image (e) is a typical LEEM image of 100?m field-of-view. The surface was entirely covered with monolayer graphene. LEEM images were obtained at the primary electron energy of 3.5 eV. Image (f) is a typical ?LEED pattern observed in the graphenecovered surface. The orientation of the graphene was slightly altered because the sheet is curved.

Typical LEEM images of the graphene growth at different stages: (a)-(b) the first layer growth observed at 1125K, (d)-(f) the second layer at 1050K and (g)-(h) the third layer at 1050K. Image (c) is a typical ?LEED pattern of a 1 x 1 atomic structure obtained from the single-layer graphene-covered surface. Image (i) is the electron reflectivity-energy curves obtained from each area.

(a) A TEM image of a carbon aggregate on the Au mesh with squares 10 ?m × 10 ?m in area, (b) A magnified TEM image of the thinnest area of the carbon aggregate, and (c) its electron diffraction pattern.

A TEM image of the other area in the carbon aggregate (a) and its diffraction pattern (b). One can see clearly doublets of diffraction spots in (b), and new cabon-nano-tube like structures in (a). The hole was covered with double-layer graphene.

Typical Raman spectrum of the monolayer self-standing graphene sheets. Small defect-origin D peak was detected at ?1350 cm-1.

15. The SEM image of single graphene sheet at 5 kV and its LETED pattern (upper right).

The LETED pattern of single-layer graphene at (a) 1 kV and (b) 500 V.

The SEM image of folding double graphene sheet (a) and its LETED pattern at (b) 4 kV and (c) 2kV.

The Ni(111) surface is the excellent substrate for growth of single-layer-graphene sheet with macroscopic dimensions. Graphene sheets with a 1 x 1 atomic structure grew up epitaxially by CVD or surface segregation techniques. We in-situ observed the graphene growth of mono-, bi- and tri-layer step by step using carbon segregation phenomena on Ni(111) by LEEM. The summaries are as follows;

1. One can grow the uniform monolayer graphene on Ni(111) by adjusting the temperature. No domain boundaries and wrinkles were detected by LEEM.
2. The second- and the third-layer graphene grew at the interface under the first and the second layers. Bi-layer graphene domains grew at least 100?m scale. The third-layer started to grow before the completion of second-layer at 1050K in this experiment. More precise control of temperature seems to be required to complete the second-layer before starting the third-layer growth.
3. Shape of the islands differed depending on the thickness; the first- and third- layer islands exhibit hexagonal edges, while the second-layer islands possess dendritic edges.
4. The different shapes of the first, second and third-layer islands presumably originate from the interfacial-bond strength depending on the graphene thickness.
5. The number of nucleation sites of graphene growth is extremely small on Ni(111) surface, which is an important factor for growth of large single-domain graphene crystals.
6. Chemical etching the Ni substrate made it possible to separate macroscopic self standing graphene sheets. (Jian Ru Gong. 2011)

Applications of large scale grapheme

Graphene for nanoelectronics

Graphene shows a glaring ambipolar electric field effect whereby charge carriers can be tuned continuously between electrons to holes. Single layer graphene atop a thermally grown SiO2 layer on a highly doped Si substrate may serve as a prototype of a field effect transistor. Under this arrangement, SiO2 play the role of an insulating layer, so a back-gate voltage can be applied to vary carrier concentration (figure 11b). Early graphene FET devices demonstrated by Novoselov exhibited dopant concentrations as high as 1013 cm–2and achieved a mobility that could exceed 10,000 cm2 /Vs (Novoselov, Geim et al. 2004). This translates into ballistic transport on submicron scales. The room-temperature mobilityis limited by impurities or corrugations of the graphene surface, which means that it can still be improved significantly up to the order of 105 cm2 /Vs (Bolotin, Sikes et al. 2008; Du,Skachko et al. 2008).

Electrons present in graphene act like mass-less comparable particles controlling a majority of its electronic properties. Among the most important results of such uncommon diffusion relation can be seen in the case of half-integer “Quantum Hall Effect” and the unavailability of localization, which can be very essential for graphene-based field effect transistors, (FET), (Geim and Novoselov 2007). Mechanical exfoliation of highly ordered pyrolitic graphite (HOPG) or high purity graphite flakes can lead to the generation of graphene crystals with very few flaws, which in turn show high movability of the charge carriers. Figure 12 shows scanning electron microscopy (SEM) and atomic force microscopy (AFM) of the grapheme based device reported in the literature as having the highest electron mobility to date (Bolotin, Sikes et al. 2008). The graphene film was obtained by mechanical exfoliation of graphite on Si/SiO2 substrate in which the oxide layer below the grapheme was etched in order to obtain a free-standing graphene flake connecting the metal electrodes.

Electrical measurements of resistivity vs. gate voltage show the intrinsic ambipolar characteristics of graphene. It was also established that the transfer characteristics of the device is greatly improved after undergoing a high-current annealing procedure to remove contaminants from the graphene surface. Mobility ? for this device reaches an exceptional value of 230,000 cm2/Vs measured at the highest carrier density n = 2x1011cm-2. Such high mobility would in principle favor high frequency performance. Furthermore, graphene devices pursuing high frequency have demonstrated encouraging characteristics, exhibiting a cutoff frequency fT of 26 GHz, which is the frequency at which the current gain becomes unity and signifies the highest frequency at which signals are propagated (Lin, Jenkins et al. 2008). Only recently, P. Avouris and collaborators reported the fabrication of graphene FETs on SiC substrates with cutoff frequency of 100 GHz for adevice of gate length of 240 nm and using a source-drain voltage of 2.5 V (Lin, Dimitrakopoulos et al.). This fT exceeds those previously reported for graphene FETs as well as those for Si metal-oxide semiconductor FETs for the same gate length (~40 GHz at240 nm) (Meric, Baklitskaya et al. 2008; Moon, Curtis et al. 2009).

CVD graphene for macroelectronics: Transparent conductive films

Another intrinsic property of graphene is its transparency. A single sheet of grapheme absorbs only 2.3 % of the incident light. Such combination of high conductivity and lowlight absorption makes this material an ideal candidate as a transparent conductive film. It isvery tempting to use the unique properties of graphene for technology applications even beyond graphene FET applications. Composite materials, photo-detectors, support for biological samples in TEM, mode-lockers for ultrafast lasers and many more areas wouldgain strongly from using graphene for non-FET purposes.

Graphene applications in photovoltaics

Photovoltaic cells: Graphene vs ITO

Solar energy harvesting using organic photovoltaic (OPV) cells has been proposed as a means to achieve low-cost energy due to their ease of manufacture, light weight and compatibility with flexible substrates. A critical aspect of this type of optoelectronic device is the transparent conductive electrode through which light couples into the device. Conventional OPVs typically use transparent indium tin oxide (ITO) or fluorine doped tin oxide (FTO) as such electrodes (Peumans, Yakimov et al. 2003). However, the scarcity of indium reserves, intensive processing requirements, and highly brittle nature of metal oxides impose serious limitations on the use of these materials for applications where cost, physical conformation, and mechanical flexibility are important.

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INTRODUCTION

Drug Delivery has become an interesting in the present day bringing together Material Science Engineering, Mechanical Engineering, Bio-medical Engineering and Pharm-ology together. This method helps to provide an effective way to use a compound to achieve a therapeutic effect in humans and animals. The technique involves modification of drug release profiles, absorption, distribution and elimination for the benefit of improving product efficacy, safety, as well as patient compliance and convenience.

Gelatin is a naturally occurring macromolecular and bio degradable protein that is obtained from skins, tissues and bones of animals. It has high water solubility, non toxicity, high mechanical strength and elasticity in dry state making it a perfect material for drug delivery. There are two kinds of Gelatin. Type A, with isoionic value of 7 to 9, is inferred utilizing solely corrosive pretreatment. Type B, with isoionic value of 4 to 5, is the aftereffect of an antacid pretreatment.[2]

The usual sources of Gelatin are bovine and pig skins and demineralized bones and hooves. This is a cause for problem for people tend to not consume gelatin based medicines due to religious reasons, mad cow disease and social reasons. To solve this, another source of gelatin was experimented for, extraction of Type B gelatin from fresh water fish L. rohita. In this project we look at its extraction, manufacturing of gelatin and the characteristic properties of the type B gelatin obtained.[1]

PROCESSING

Since there are two sorts of Gelatin, there are two fundamental assembling procedures of Gelatin: Alkaline and corrosive process. The subsequent items can be altogether different as far as piece and physical properties.[4]

Acid handled collagen is absorbed weaken corrosive nature of the acid and after that extricated at about pH 4 for gelatin producing. Non-collagenous proteins and mucoproteins of the tissue are isoelectric at this pH and are consequently not so much solvent but rather more promptly coagulated under the extraction conditions. Contaminants which are expelled along these lines rely upon the quality and birthplace of the raw material and the reproducibility of the producers process.[4]

When managing the basic procedure, the pretreatment of the collagen requires a drawn out absorbing the soluble arrangements (by and large, immersed lime-water). A decent measure of the pollutions (proteins and mucosubstances) are dissolvable at the oppressed pH and are separated. Gelatins from soluble base process will in general be cleaner than corrosive created gelatin, yet this variety might be because of the assembling process.[4]

Moreover to the challenges referenced beforehand, the produce of gelatin is liable to more confusions. For instance the aggregate number of carboxyl gatherings accessible for ionization relies on the extraction technique. Distinctive gelatins can have diverse proportions of acidic and essential gathering in this manner distinctive isoelectric focuses. Charged gatherings impact the cooperations between nearby gelatin atoms, between every particle and the dissolvable and between various parts of a similar atom, as the protein chains are adaptable. The degree of these factors change with pH, and are likewise subject to the aggregate ionic sythesis of the framework in this way an itemized depiction of the dissolvable and additionally that of the gelatin is a necessity.[4]

CHARACTERIZATION

Sample Preparation

Fish scales of L.Rohita having weight of 100-200g were collected at Guntur , Andhra Pradesh , India. The scales were removed using hands, kept in air tight bags and on ice for preservation. Gelatin was extracted using the alkali method. Dried scales were stirred in a solution of 5% NaCl at 30 min room temperature. This step was repeated twice and then the scales were stirred with 4% NaOH to remove the non-collagenous proteins from the scales. To remove lipids from the scales, iso-butyl alcohol was used. This step was repeated three times in a digital linear shaker. The final step was dimineralisation which was done using 0.5N EDTA at a pH of 7.66 for four different time periods of 12h, 2h, 2h and 1h. The scales were then dried on plastic trays and grinded to obtain the gelatin in powder form.[1]

Gelatin Yield

[1]The gelatin yield is given by the following equation

Yield of Gelatin=(Weight of dried Gelatin)/(Dry weight of Fish Scale)?—100

Determination of Gelatin pH

To determine the pH of Gelatin, prepare a 1% gelatin solution in distilled water and cool to 25?°C in a water bath. The pH is then measured.

UV-vis Spectroscopy Analysis

Ultraviolet and Visible absorption spectroscopy is the measurement of the attenuation of a beam of light after it passes through a sample or after reflection from a sample surface. Absorption measurements can be at a single wavelength or over an extended spectral range. UV spectroscopy is used for:

• Detection of functional groups
• Detection of Impurities
• Qualitative analysis
• Quantitative analysis
• Single compound without chromophore
• Drugs with chromophoric reagent

Ultraviolet absorbption spectra arise from transition of electron within a molecule from a lower level to a higher level. A molecule absorbs ultraviolet radiation of frequency(V), the electron in that molecule undergoes transition from lower to higher energy level. The energy can be calculated by

E_1-E_0=hV

The UV-vis absorption spectrum was recorded using a UV vis double beam spectrophotometer in the range of 200-400nm.

Fourier transform infrared(FITR) spectroscopy analysis

FTIR is a technique based on vibrational spectroscopy. AN infrared spectrum is obtained using fourier transforms. Fourier Transfroms help to represent a range of Sinusodial waves as a single summition function. Presently FITR has replaced the dispersive method as it has a higher signal to noise ratio.[3]

The IR enters the interoferometer. It has a beam splitter and 2 mirrors. The beam splitter splits the radiation, One beam strikes a moving mirror and one beam strikes a fixed mirror. The two beams then combine and intract with the sample and then go to the detector. The difference in wave length is plotted and then used to determine a fourier transform.[3]

100 mg of KBr and 2mg of Gelatin was mixed and placed in FITR equipment. FITR was performed at room temperature and measurements were taken.

X-Ray diffraction

To determine the Crystal structure, X ray diffraction was used. The operating voltage was 45kV and the operating current was at 40mA. The radiation used was Cu K?±.[1]

Scanning Electron Microscopy

Scanning Electron Microscopy was used to determine the surface morphology of Gelatin. Gelatin was coated with gold in a vaccum sputter and a photograph was taken.[1]

RESULTS AND DISCUSSION

Yield of Gelatin

The gelatin extracted was yellow and the weight percentage was 24% which is higher than the yield of red tilapia(7.81%) and black tilapia(5.39%)(found in previous studies)[1]

Characterization results

The UV-vis spectroscopy showed absorption at 224nm, indicating a presence of strong peptide bonds. Absorption at 210-240nm shows the presence of peptide chromophore. Since the operational range was 200-400nm, the results can be considered valid. Refer figure 2.[1]

Figure2. UV-vis absorption spectrum of fish scale gelatin

FITR measurement made it possible to identify the amino groups present. Also it helped to identify the bonds responsible for the structural and functional stabilization. Amino acids are made of amide bonds which can be identified by the specific amide band that is absorbed at the FITR measurement. [1]

Peaks(cm-1) Possible functional bands

3433 Hydrogen bond

1630 Amide-I

1565 Amide-II

1240 Amide-III

1460 Symmetric Bending

1380 Asymmetric Bending

Table 1- FITR results of Gelatin

Figure 3. FTIR spectrum of gelatin

Porosity is determined by Scanning electron microscopy analysis. Porosity characterization is the determination of open pores which determine properties like permeability and the surface area of the porous structure.The microstructure obtained shows that the polymer has an array of hollow cells. Higher density shows greater mechanical strength and the higher porosity shows a better biological environment. The SEM micrograph shows a decent balance between the two.[1] Refer Figure 4.

Figure 4. SEM micrograph of gelatin extracted from fish scale

The XRD diffractogram shows a sharp peak with low intensity at 2??=7?° and a broad peak at 2??=19?°. This is usually assigned to the triple helical crystalline structure of gelatin.[1]

Conclusion

The study shows that L Rohita is a good source of raw material for Gelatin production. It can be determined that gelatin has the necessary porosity for drug delivery and has a triple helical crystalline structure. The FITR shows it chemical bonds. Thus Fish based Gelatin is an effective source of gelatin for drug delivery.

References

Merina Paul Das, Suguna PR, Karpuram Prasad, Vijaylakshmi JV, Renuka M. Extraction and characterization of gelatin: a functional biopolymer. Int J Pharm Pharm Sci 2017;9(9):239-242.

Gavasane AJ, Pawar HA (2014) Synthetic Biodegradable Polymers Used in Controlled Drug Delivery System: An Overview. Clin Pharmacol Biopharm 3:121.doi:10.4172/2167-065X.1000121

Materials Characterization: Introduction to Microscopic and Spectroscopic Methods by Yang Leng, Second Edition (ISBN No. 978-3-527-33463-6) John Wiley & Sons (2013)

Felix, Pascal Georges, "Characterization and correlation analysis of pharmaceutical gelatin" (2003). Graduate Theses and Dissertations. https://scholarcommons.usf.edu/etd/1365

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ABSTRACT

Chitin is the second most abundant natural polysaccharide after cellulose and is present in the crustacean exoskeleton like crab, shrimp, insects and fungi. It is the main structural component of the exoskeletons of the animals like insects and crustaceans. Crab, shrimp, squilla and fish scale waste is ideal raw material for chitin production. The present work is aimed at extraction of chitin from crab shells. The methodology include acid hydrolysis, demineralization followed by deproteinization step. The chitin produced is analysed by FTIR based on the interpretation of the spectrogram of the two samples of chitin synthesized in the present work, it can be said that all functional groups expected are seen. The applications of the chitin are numerous but the study is focused on dye adsorption ability.

INTRODUCTION

The shell fish industry which is prominent in all costal countries generates about 60,000 to 80,000 tons of waste (Muzzarelli et al,1986). Even though the wastes are biodegradable, the dumping off large quantities makes degradation process slow resulting in accumulation of waste overtime which is a major environmental concern. A quick and effective solution to this is recycling of shell wastes and extraction of commercially viable substances like chitin from them. Chitin on its own has various applications. Chtin is a natural polysaccharide of major importance. This biopolymer is synthesized by enormous number of living organisms and it belongs to the most abundant natural polymers, after cellulose (Rinaudo et al,2006).

Expelling of dyestuff into water resource system causes major threat to the environment. Adsorption is the cost effective and potential method to remove the dyes from the effluents. Effluents from various industries contain harmful coloring agents, which have to be removed to maintain the quality of the environment. Paper, fabric, leather and dyestuff production are some of the industries that release harmful effluents (Lin S, Lin Cet al,1993).The aim of the present study was to investigate the chitin adsorption capability on major industrial dye, Methylene Blue.

MATERIALS AND METHOD

Sample preparation- Crabs were collected from Dapodi fish market, Pune. Crabs inedible parts including head, body shells and tails were removed from the whole body for extraction of chitin. The crab shell were washed and air dried and used for extraction.

Extraction of chitin-

Crab shell waste Crab shell powder Demineralization Deproteination Extracted Chitin

Figure 1.Flow chart of basic steps of Extraction of Chitin.

Process I-

10 grams of sun dried crab shell waste was demineralized by adding 1.5 N HCl at room temperature for 1 hour. Acid was discarded and the shells were washed with distilled water until the pH is neutral. The shells were then de-proteinized with 0.5% NaOH at 100°C for 30 minutes. Protein solution was removed and washed thoroughly with distilled water and the pH was checked. The de-proteinization process was again repeated, for that 3% NaOH was added to the sample at 100°C for 30 minutes. After draining the residual proteins along with the effluents, the sample once again washed and the pH was observed till it was approximately near to neutral. Hence the chitin slurry was obtained. The excess water was removed. The alkali was drained off and washed thoroughly with distilled water until the pH is less than 7.5 and then dried at ambient temperature (30 ?± 2?°C)

Process II-

10 grams of crab shell waste were refluxed in 100ml of sodium hypochlorite (NaCLO)solution at 100oc for 10 minutes. The NaCLO solution was decanted and the powder was washed with distilled water. The above step was repeated once more. The sample was again refluxed in 50ml of 1M HCL at 75oC for 15 minutes. The solution was decanted and washed with distilled till it becomes neutral. The sample was then refluxed in 50ml of 1M NaoH(sodium hydroxide)solution at 100oC for 2 minutes to remove any protein residues. The solution was decanted and remaining sample was washed with distilled water till it becomes neutral. They were filtered off and placed in an oven at 60oC for a week.

Characterization of Chitin

Solubility Test - Chitin dissolves completely in 1% Acetic Acid. For the estimation of chitin produced the sample was taken out of the storage and weighed . Then the sample was put inside a clean beaker and 10 to 20 ml of 1% acetic acid was added to it. The solution was kept in shaker for 30 to 40 minutes. Then the sample was taken out and weighed, carefully( Abhrajyoti Tarafdar et al,2013)

FT-IR Spectroscopy: The samples were analysed by FT-IR spectroscopy in Istrumentation Centre Solapur university and the graph depicts wave number versus percent transmission.

(Pandharipande S et al,2016)

Dye adsorption by Chitin:Stock solution of the dye was prepared by taking 10mg of methylene blue powder and adding it to 1000ml of distilled water( Paula Szymczyk et al,2015).The pH of the dye solutions was adjusted using 1 N NaOH or 1 HCl.About 1g of extracted Chitin(adsorbent) was added to 100mL of dye solutions(adsorbate). A control was also maintained without addition of chitin. At specific time intervals, aliquots of 2-3 ml suspension were filtered and used to evaluate the adsorption of dye.The absorbance spectrum of the supernatant was subsequently measured using UV-Vis spectrophotometer. Concentration of dye adsorption was calculated by the absorbance value at 668nm.

Percentage of dye adsorption was estimated by the following formula:

% adsorption = 100 — [(CO €’C)/ C0] Where: C0 is the initial concentration of dye solution and C is the concentration of dye solution after Adsorption( S. Dhananasekaran et al,2015).

RESULTS AND DISCUSSION

Extraction of chitin from crab requires harsh chemical treatments. The crab shells even though contains majority of chitin, also has proteins and minerals. Proteins are removed by deproteinization and carbon and other salts are removed by demineralization( Badawy et al,2011).

Process I “ Solubility test for sample 1

Initial weight of chitin was measured to be 0.40 gram. Final weight of chitin after reaction with 1% acetic acid was measured to be 0.22 grams and hence the total dissolved weight of chitin was calculated to be 0.18 grams.

Proecss II - Solubility test for sample 2

Initial weight of chitin produced was measured to be 0.40 grams .Final weight of shells after reaction with 1% acetic acid was measured to be 0.20 grams and hence the total dissolved weight of chitin was calculated to be 0.20 grams. Therefore, it was observed that chitin produced employing Process II was more readily soluble in 1% acetic acid solution then that produced through Process I.

FTIR Analysis- The interpretation of FTIR analysis of the samples is done for the possible presence of functional groups and the details are given in Table 1(Dhananasekaran S et al,2016)

Table 1

Sr.no Standard chitin wavelength in cm-1 Crab chitin wavelength in cm-1 Groups

Sample-1 Sample-2

1. 3300-3250 3373 3278 N-H

2. 2891 2952 2920,2826 C-H

3. 1680-1660 1653 1647 C=O

4. 1560-1530 1560 1568 Amide

5. 1072 1017 1024,1094 C-0-C

6. 952 952 901 Amide III

7. 750-650 667,625 685,617 N-H

The FT-IR spectra of chitin isolate from crab shell are given in Figure 2 and 3

Theoreitically,?±-chitin is characterized by three characteristic amide bands appearing at 1650,1620, and 1550 cm-1.In this study we observed FTIR bands at 1653 cm-1 and 1560 cm-1 for chitin sample 1 in figure 2.Here peak at 1653 cm-1 corresponds to symmetrical deformation to vibration of amide I band stretching C=O and 1560 cm-1 Coressponds to N-H deformation of amide II(Muhammed, R., et al,2010)

FIG 2: FT-IR spectra of chitin (Sample 1) FIG 3: FT-IR spectra of chitin(Sample2)

In sample 2,Figure 3 the peaks are observed at 1647 cm-1 and 1568 cm-1 which corresponds to symmetrical deformation to vibration of amide I band stretching C=O and N-H deformation of amide II respectively. From interpretation of FT IR it can be said that all functional groups which are during synthesis have been identified in the form of peaks that include amide,carbonyl and hydroxyl groups.This indicates the successive formation of chitin biopolymer(Muhammed, R., et al)

Dye adsorption by Extracted Chitin

Graph 1:Effect of pH on removal of dye Graph 2:Effect of contact time on removal of dye

Graph 1 shows the relationship between pH values and percentage removal of dye .The readings were taken having varying pH between 4-9 and between intervals of 30 minutes. A result shows that the effectiveness of dye adsorption onto chitin was decreasing along with the increasing pH value. Here, the effect of dye adsorption is found maximum at pH7 i.e the neutral.

The dye removal percentage with contact time between dye and extracted chitin is shown in Graph 2. The range of observed contact time was 30 -180 minutes with the increment of 30 minutes. It is observed that with increase in incubation time the effectiveness of dye adsorption by chitin increases. The % removal was found to be maximum at 180 minutes at pH 7 such as after 3 hours compared to initial readings. The % removal was found to be 39% after 180 minutes.

CONCLUSION

Chitin is one of the most abundant biopolymers in nature and is a major component in the supporting tissues of organisms such as crustaceans, fungi, and insects. It has wide application in various fields. This study shows the production of chitin from crab shell. The FTIR and chemical characterization studies confirm the production of chitin. In this study removal of dyes by adsorption using crab shell(chitin) was investigated. This study monitored the ability of chitin for removing dyes from aqueous solutions. Interaction between the chitin and dye were found to be strongly dependent on pH of the solution. The maximum percentage of dyes reduction was obtained at an optimum contact time 180 minutes and optimum pH of 7.Crab shell chitin has been found to be comparatively better adsorbent because it can remove almost 39 % of dyes within 3 hours. Finally, the result of adsorption study, it is concluded that chitin can be used as a coagulant of dyes because of its higher adsorptive capacity, cost effectiveness, environment friendly behavior and availability in nature.

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