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Similarly, trans-cutaneous route is only effective for the potent non irritant drug and lipid soluble drug9 and poor patient acceptability of rectal, vaginal and ocular sites represents limitation for drug therapy. Therefore the need for development of nasal protein drug delivery system is necessary.
Mucosal surfaces such as nasal and buccal mucosa are potential sites for absorption of a variety of drugs and they provide certain advantages over other routes of drug delivery.
It avoids the first pass metabolism
Avoids the destruction of proteins in GIT
Existence of rich vasculature and large surface area of epithelium
Highly permeable structure in nasal membranes and better enzymatic flora are responsible for drug absorption.
Systemic drug delivery via the buccal mucosa, with drugs such as luteinizing hormone releasing hormone1,2 and Calcitonin3,4has been investigated. Encapsulation of model drug in to a mucoadhesive nanoparticles composed of Polyacrylic acid, Polyethylene glycol (PEG) and chitosan seems to be a promising strategy for mucosal drug delivery. It has received good attention in last few years as trans-mucosal penetration enhancer improving absorption of hydrophilic drugs. Inter polymer complex of PMAA-PEG-CHITOSAN nanoparticles posses' higher mucoadhesive property5, 6.
This complex has been developed as potential drug delivery system to determine the drug release from the nanoparticles. Metoprolol is used as model drug. It has great importance in the drug therapies of cardiovascular studies7.
Mucous membrane mainly concern to the absorption and secretion. Different body cavities are exposed to external environment and internal organ are lined by mucous membrane. Mucous membrane is found in different parts of the body. They are of different types according to their body parts comprise oral, GI, buccal, vaginal, oesophageal and bronchial mucosa
OVERVIEW OF THE ORAL MUCOSA
The oral mucosa is composed of stratified squamous epithelium, under this layer basement membrane and below this lamina propria. The innermost layer is the submucosa layer. Stratified squamous epithelia are similar to epithelium found in the body. Stratified squamous epithelium has mitotically active basal cell layers, which has number of differentiating intermediate layers to the super facial layers. These cells are shed from the surface of the epithelium10, 11. Buccal mucosa contain 40-50 cell layers thick epithelium where as sublingual epithelium contain somewhat less. Life span of epithelium cells has been estimated to 5-6 days12
Thickness of the oral mucosa vary depending upon the site, Buccal mucosa is about 500- 800µm, where as mucosal surface of tongue, hard and soft palate, and the gingivae measure about 100-200µm, epithelium composition also varies it may be keratinised or unkeratinised depending on the site in the oral cavity11. Keratinised layer of epithelium contains neutral lipids such as ceramides and acryl amides which are relate to the barrier function. Keratinised epithelium is relatively impermeable to the water. If epithelium is nonkeratinised such as buccal epithelia which do not contain acryl amides and small amount of ceramides but it contain neutral polar lipids such as cholesterol sulphate and glucosyl ceramides. Nonkeratinised epithelia are permeable to the water. Buccal mucosa has greater permeability about 4-4000 times than that of skin13.Depending upon the relative thickness and degree of keratinisation permeability of the mucosa decreases in order sublingual then Buccal and palatal. This is because sublingual mucosa is thin and nonkeratinised which is permeable to water but Buccal mucosa is thicker and nonkeratinised, where as palatal is keratinised which has intermediate thickness.
Mucus is the intracellular substance surrounded by oral epithelia. This epithelia consist of complexes of proteins and carbohydrate which plays a major role in cell-cell adhesion14.Mucus plays an important role in the bio adhesion of mucoadhesive drug delivery systems15 .due to its negative charge at physiological pH which is responsible in the mucoadhesion. At physiological ph mucus can form cohesive gel structure that binds to epithelial surfaces gelatinous layer10. The environment of the oral mucosa is watery so the choice of the hydrophilic polymers has importance in the oral mucosal drug delivery.
GI TRACT MUCOSA:
Gastrointestinal tract (GIT) is made up of three different microscopic layers. Each layer plays an important role of peristalsis that is pinching motion of the intestine and functions of the digestion in gut. Innermost layer consist of the mucosa. Mucosa made up of the special cells known as epithelium cells which are arranged as single layers in the oesophagus and multiple layers in the stomach and intestine. It has function to reduce friction and form a protective barrier from the concentrated enzyme that is released in to intestine.
Mucosal layer made up of the thin layer of muscle tissue known as muscularis mucosa. It plays an important role in propelling nutrient in uniform direction from the lumen to the submucosa. Submucosa is encountered after mucosal layer which has main function to provide peristalsis to the gut. Third layer of the gut is serosa which is composed of connective tissue which gives strength to digestive tract16.
Fig.1.3- Transverse section of GI mucosa.
In the nasal cavity, nasal mucosa is lined to the surfaces of the middle and inferior conchae (turbinate bones) and their meatures. Nasal mucosa consists of three tissue layers. The luminal surface is made up of pseudo stratified columnar epithelium. In the columnar epithelium cells where many inter-mix flask-shaped goblet cells are present. All the epithelium cells are embedded in the basement membrane which attach to a thick layer of lamina propria. Connective tissues which are dispersed in the lamina propria have rich blood supply with many seromucosal glands.
Fig.1.1 - micrograph of respiratory Fig.1.2 - longitudinal section showing nasal mucosa mucosa
NASAL MUCOSAL DRUG DELIVERY SYSTEMS
Other bioadhesive nasal formulations
Nanoparticles are solid colloidal particles ranging from 200 nm. It also includes monolithic nanoparticles (nanospheres) in which drug is absorbed, dissolved, or dispersed throughout the matrix. Micro particles are colloidal particle in micrometer scale, generally in a size range from 0.2-100 µm. Size of nanoparticles (0.2-0.5 µm) are very small as compare to microspheres (30-200 µm)17. Nanoparticles consists of biocompatible and biodegradable materials such as polymers either synthetic or natural or solid lipids. They have less drug loading capacity as compared to microspheres18, 19. The drug loaded in the nanoparticles is usually released by different mechanisms such as diffusion, swelling, erosion, or degradation. Loading of drug in nanoparticles is on a surface or inside it. This depends on physicochemical properties of the drug. The release rate of a drug depends on the how the drug is to be administered19. EXPAND A BIT MORE, BE MORE CRITICAL IN YOUR DISCUSSION OF THESE FACTS
Advantages of nanoparticles:
High stability. Nanoparticles have the long shelf life.
High carrier capacity, combination of drug molecules can be incorporated in the polymeric matrix.
Usefulness of addition of both hydrophilic and lipophillic substances.
Utility of variable routes of administration, including oral administration and inhalation
Controlled drug release through the matrix can be designed by using different carriers20.
Nanoparticles has greater intracellular uptake compared to micro particle.
In terms of nature and charge properties nanoparticles influences the intestinal uptake by intestinal epithelia.
Nanoparticles prepared from the lipophillic polymer has seems to be higher uptake compared to nanoparticles prepared from hydrophilic polymer.
PREPARATION METHOD OF NANOPARTICLES:
There is no specific or universal method for the preparation of nanoparticles. The choice of specific preparation method and appropriate polymer rely on physiochemical properties of drug, its release characteristic and route of administration and which is determined by high drug loading capacity, encapsulation of drug and high product yield. Techniques employed for preparation of nanoparticles is classified into two groups. In first category nanoparticles are formed from the preformed polymers. This contains both water soluble polymer and water insoluble polymer of synthetic semi synthetic and natural origin. In second category nanoparticles are prepared from various polymerisation reactions of lipophillic or hydrophilic monomers.
Nanoparticles prepared from preformed polymers:
These nanoparticles are prepared by using preformed polymers having distinct advantages over nanoparticles prepared through the polymerisation of monomers. WHAT ARE THESE ADVANTAGES?? This contains use of polymers having well characterised physico-chemical properties WHAT ARE THESE PROPERTIES but there is lack of residual monomers or polymerisation agent such as initiator or catalyst and absence of reaction between drug and monomer.
Nanoparticles prepared from the water insoluble polymers
Solvent evaporation method -
Water miscible organic solvent for the preparation of biodegradable and non-degradable nanoparticles, solvent evaporation technique is widely used. In this method polymer matrix and active pharmaceutical ingredient or drug are dispersed or dissolved. In a volatile, water miscible organic solvent such as methylene chloride, chloroform, ethyl acetate oil in water emulsion is prepared by adding the solution or dispersion into an external aqueous phase containing an emulsifying agent with the help of suitable emulsification equipment. To prepare nanoparticles, the o/w emulsion is homogenised under high shear with conventional homogenisation equipment (micro fluidiser, sonication). Particle size reduction in the internal organic phase into a colloidal size range is done before precipitation of polymer. This method is limited only for the water insoluble drug.
Nanoparticles prepared from hydrophilic polymer
These nanoparticles are prepared by two different methods, either by w/o emulsification method or by coacervation method (aqueous phase separation technique)
W/O emulsification method:
Nanoparticles of hydrophilic polymers such as chitosan, gelatine and carbohydrate can be prepared by w/o emulsification technique. High shear homogenisation equipment or ultrasonification can be employed for the formation of emulsion in the nanometer size. Aqueous solution containing polymer matrix is emulsified in to an external water immiscible phase, like an oil or organic solvent. The polymer droplets solidify after removal of water. For efficient drug loading in to hydrophilic polymeric nanoparticles, the drug must be insoluble in the external phase. This technique has limitation that it applies only for the water insoluble drug.
This method is used for the preparation of nanoparticles from monomers in presence of surfactant; monomer is emulsified in an immiscible external aqueous phase containing surfactant. Micelles formed due to the surfactant and are capable to solubilise the monomer molecule. Thus resulting in polymerisation within micelles or with more soluble monomers in continuous phase where the Emulsion is stabilised due to adsorption of surfactant on monomer ion droplets, giving birth to polymeric nanoparticles21.
Applications of nanoparticles:
Table1.2- The different applications of nanoparticles 22.23
Fluorescent biological labels
Drug and gene delivery
Biodetection of pathogen
Detection of proteins
Probing of DNA structure
Tumour destruction via heating(hyperthermia)
Separation and purification of biological molecules and cells
MRI contrast enhancement
anti reflection coating
Tailored refractive index
In a cancer diagnosis's a light based sensors
To increase the density of storage media
To increase heat transfer from solar collectors to storage tanks
To increase the efficacy of the coolant in the transformer
Mechanical-improved wear resistance
Improved wear resistance
A polymer is a composition of molecules characterised by the regular or irregular repetition of one or more different types of chemical units and polymerisation is a chemical reaction in which monomer units are linked together to form a Polymer24.Polymerisation is mainly classified in two types it may be condensation or addition process, condensation involves the reaction of monomers associate with loss of small molecules, such as water, whereas addition reaction involves the addition of monomers without loss of water. In 1950 P.J.Flory has developed clear classification which depends on the actual mechanism involved in the polymerisation process. There are two main mechanisms of polymerisation, Chain polymerisation and Step polymerisation. Chain polymerisation is made up of mainly three stages, initiation, propagation, and termination. There are different types of chain polymerisations mechanisms such as free radical, ionic, complex, and ring opening25.
Acrylic acid is known as propenoic acid and methacrylic acid is known as the 2-methyl propenoic acid. These are basic component for the huge number of the derivatives like acrylamide, methacrylamide, acrylic ester and methacrylic ester. Methacrylic acid is employed as co monomers to improve special polymer properties.
Properties of methacrylic acid
Methacrylic acid is colorless liquid .It has penetrating odours similar to the odour of acetic acid. Prizmatic crystals are found at low temperature due to freezing of methacrylic acid. It has tendancy of spontaneous polymerisation which may be explosive. It can be stored in stainless steel, glass or ceramics. Polymethacrylic acid is colourless solid with glass transition temperature 130oC. Above 200-250oC it loses water molecule and form an insoluble cross linked polymer anhydrides. Its decomposition starts at 350oC.Solublity of poly (methacrylic acid) decreases with temperature. Concentrated aqueous solution of poly (methacrylic acid) is rheopectic.
Table1.3- Properties of methacrylic acid is.
Boiling point (oC) at 101 kPa
Vapour pressure (kPa)
Density (g/ml) at 25oC
Heat of polymerisation(kJ/mol)
Solubility in water
Chitosan is good cataionic polymer. It is polysaccharide achived by deacetylation of chitin. It is a major constituent of exoskeleton of crustaceous water animal. It can be found in the yeast, mushrooms and tough outer shells of insects and crustaceans. Chitosan is naturally abundant polysaccharide and found to be non-toxic and biodegradable polymer.
Chitosan has general properties like chelation; it can be selectively bind to the cholesterol, fats, proteins, metal ions and tumour cells. It has affinity towards the proteins like trypsin. Other properties like inhibition of tumour cell, antifungal effect, acceleration of wound healing. It can stimulate immune system26. Ability of film formation as well as gel and matrix formation makes it useful for the preparation of solid dosage forms such as granules and micro particles27.
Fig.1.5-structure of chitosan
Chitosan has molecular weight (50,000-2,000,000 daltons) Chitosan is weak base having pka in a range of 6.2-728. Chitosan is insoluble in the water, alkali and organic solvent, but mostly soluble in organic solvent when it has the pH above 626.
The solubility parameters depend on degree of acetylation and pH. Highly deacetylated Chitosan are soluble at pH 6 where as low degree of deacetylated Chitosan is soluble at pH 928. Viscosity of Chitosan depends on the ionic strength of compound.
Table 1.4- Following table summarizes the properties of Chitosan:
Melting point 60-66oC
Glass transition temperature 155oC
High density at pH<6.5
Biodegradable to body constituent
Adheres to negatively charged surfaces
Safe and non-toxic
Form gel with polyanions
Low to high molecular weight range
High to low viscosity
Chelate certain transition metal
Reactive amino/hydroxyl group
Polyethylene glycol (PEG), polyethylene oxide (PEO), polyoxiethylene (POE) and all polyoxirane is linear polymer obtained by the polymerisation of ethylene oxide. Polyethylene glycol signify the polymer molecular weight less than 20,000where as PEO signify higher molecular weight and remaining PEO and polyoxirane having wide range of molecular weight. PEG having molecular weight 2000-20000 are being used in the organic synthesis. Such restrictions have been made by the physical properties. Molecular weight in this range is available as crystalline solid where as higher molecular weight having lower loading capacity and PEG having low molecular weight is available in the form of crystalline solids. PEG is soluble in the water, DMF, Dichloromethane, toluene, acetonitrile and insoluble in hexane diethyl ether, ter-butyl methyl ether and isopropyl alcohol. Some of the physical properties of polyethylene glycol have been described in the table29.
Table1.5- shows physical properties of PEG30.
Melting point (o C)
Specific gravity 20/20 o C
Viscosity at 99o
Solubility in water (20oC/wt %)
PEG is mainly used for their solvency action, water solubility and blandness. It is used in the preparation of solid dispersions of the drugs. Water insoluble drug can be dissolved in PEG due to its solvency action. PEG's water solubility helps the drug to dissolve in the body.
Due to its water-solubility, inertness, and composed of molecules of different sizes which can pass through the biological membrane at different speed have been permitted to serve in the studies of intestinal permeability.
Coating, Paint and Ink
Pigment binding capacity wide range of water solubility, hygroscopicity, stability and spreading ability makes the PEG useful in the surface coating, paints and inks. It is used in jet printing inks.PEG is used in manufacture of water resistant, air drying paints.
Cosmetics, toiletries and household products
In cosmetic Peg increases the stability of the product and reduces the greasy feeling in other cosmetics. Because of the baldness, water solubility and solvency action it has been used in the toothpaste, hydrophilic denture adhesives, antiperspirant, perfumes and cosmetic regimens. Ester derivatives of peg are useful in the fabric conditioner, bleaching compound and detergent.
Petroleum chemicals and ceramics
PEG is used as corrosion inhibitor in the oil wells to obtain products. Its water solubility and lubricant property is useful in the ceramic industry. PEG is used as vehicle for the ceramic pigment and coatings31.
Free radical chain polymerisation
In the free radical polymerisation, a small amount of initiator is required for the initiation step. Mostly used initiators are potassium per sulphate, azobisisobutyronitrile (AIBN), benzoyl peroxide. There are four main processes for effecting free radical polymerisation -Bulk, suspension, solution, emulsion32, 33.
Monomer units and small quantity of additives are charged to the reactor; inert diluents are not used. Very viscous mass formed due to high degree of monomer conversion and in a low heat transfer rates. The rate of production in the bulk polymerisation reactor is often limited by this low rate of heat removal; it is a difficult process when very reactive monomers such as methyl acrylate, acrylic acid are used. These are often complex and expensive unit with higher power requirement for mixing32, 33.
The polymer may be formed either soluble or insoluble in the solvent. Sometimes monomers are liquid under the reaction conditions and are completely dissolved in the solvent; in other cases gaseous monomers are dispersed as the bubbles in the solvent, mass transfer rates will be affected by the degree of agitation and reactor pressure. Catalyst may be employed in the solution polymerisation. It may be soluble or insoluble in the reaction mixture. The viscosity of the solution is much lower than bulk polymerisation and heat transfer is improved. Choice of the solvent is important parameter because it may affect the properties of polymer and reaction rate32, 33.
Monomer is dispersed in the form of fine droplet in water or a suitable solvent with help of vigorous agitation. It is also called as the bead polymerisation, because polymer is in the form of granular spheres. This method is widely used in the formation of PVC. Suspending agent is added with the aim of dispersion of monomer and to prevent the agglomeration of droplet. The type and amount of the stabiliser affect the size of monomer droplet and final size of the polymer particle. Suspension polymerisation has the good heat transfer rates32, 33.
This is similar to the suspension polymerisation except that the particle size is smaller and emulsifier and other additives are added to stabilise the system. Emulsion polymerisation is quite complex. Emulsifier affects the polymerisation and particle size of nanoparticles32, 33.
Types of polymer:
There is a growing demand for the design and development of biodegradable and biocompatible polymeric matrices for controlled/targeted release of bio actives for food/ nutraceutical applications34. There are different types of polymer used in the formulation some of them are entitled here.
Trans mucosal routes such as nasal, oral and pulmonary routes have great importance in the drug delivery systems. However peptide and protein drugs are degraded before they reach the blood stream, this drug cannot cross the mucosal barriers. These problems can be solved by using mucoadhesive polymer as a carrier. Mucoadhesive polymer broadly classified in to two parts such as hydrophilic polymer and hydro gels,
Hydrophilic polymer is the water soluble polymer that swells indefinitely in contact with water and finally undergoes complete dissolution. Examples of the hydrophilic polymer are methylcellulose, HPMC, sodium carboxy methyl cellulose, carbomers, chitosan and plant gums. Hydro gels are water swell able materials, usually crosslink polymer with limited swelling capacity polymers such as poly (acrylic acid co acrylamide) copolymers, carrageenan, sodium alginate, guar gum etc
Thermoplastic polymer is relatively new category of polymeric material which is the separate group of the rubber. It does not require vulcanization so that it has many advantages over other chemically cross linked elastomers35. It has good solvent resistance, elasticity, tear strength, flex fatigue and high degree of biocompatibility. These properties are associated with soft segment. They are used in the wide range of medical applications. These block polymer consist of the soft elastomeric material with low glass transition material and hard elastomeric material with high glass transition temperature36. Proportion of hard and soft segment determines the overall properties of thermoplastic polymer. The thermal stability of this polymer is generally low, especially above its softening temperature37, 38. These polymers include the non erodible neutral polystyrene and semi crystalline bio erodible polymers, which generate the carboxylic acid group as they degrade for example poly anhydrides and poly lactic acid. Ethylene-propylene-dine/propylene are most recently used thermoplastic blends in the market, poly (methlmethcrylate) one of the most important thermoplastic material because of its high transmissibility and high weather ability39.
These polymers include polyvinyl alcohol, polyamides, polycarbonates, polyalkylene glycols, polyvinyl ethers, ester and halides, polymethacrylic acid34. Polylactic acid is used in the tissue engineering application because it is biodegradable and one of the synthetic polymers approved by food and drug administration. PEG is a variable synthetic polymer used for 3D encapsulation due to its bio inert nature.
Fig.1.4: structure of metoprolol
(+)-l-isopropylamine-3-[4-(2-methoxyethyl) phenoxy ] propan-2-ol
Molecular formula: C15H25NO3
Molecular weight: 267.4
Metoprolol is available in three forms as fumarate, succinate and tartrate.
Molecular formula: (C15H25NO3)2C4H4O4
Molecular weight: 650.8
pH of the 10% solution in water ranging from 5.5 and 6.5 are stored in the airtight container. Protect from light.
Molecular formula: (C15H25NO3)2C4H6O4
Molecular weight: 652.8
It is white crystalline powder freely soluble in water, soluble in methanol and slightly soluble in alcohol. pH of 2% solution in water ranging from 7.0 to 7.6 protect from light.
Molecular weight: (C15H25NO3)2C4H6O4
Molecular weight: 684.8
It is a white crystalline powder or colourless solid. It shows polymorphism. It is very soluble in water, freely soluble in alcohol, in chloroform, and in dichloromethane, slightly soluble in acetone, insoluble in ether.10% solution in water has pH 6.0 and 7.0 store in airtight container at temperature of 25oC40.
PHARMACOLOGY OF METOPROLOL:
Metoprolol has grater affinity for β1 than β2 receptors. Metoprolol is selective β1antagonist.
Table1.1- Pharmacological/pharmacokinetic properties of β receptor blocking metoprolol
Membrane stabilising activity
Intrinsic agonist activity
Extent of absorption (%)
Oral bioavailability (%)-
Plasma t1/2 (hours)
Protein binding (%)
*Detectable only at doses much greater than required for β blockade
PHARMACOKINETICS OF METOPROLOL:
It is completely absorbed after oral administration. Due to the first pass metabolism it has relatively low bioavailability. It is widely distributed in blood stream. It crosses BBB (Blood Brain, Barrier) and placenta; it is distributed in to breast milk. Metabolism of Metoprolol is takes place in the liver in presence of CYP2D6 enzyme. It undergoes oxidative deamination, O-dealkylation, followed by oxidation and aliphatic hydroxylation. Metoprolol has the half life 3-4 hours but can be increased up to 7-8 hours in case of poor CYP2D6 metabolizes41.
Metoprolol is Cardio selective β blocker. It is noted to absence of intrinsic sympathomimetic activity and to have lack of membrane stabilising activity, membrane stabilising activity detectable only at high doses than required for β blocked
It is used in the management of hypertension, angina pectoris, cardiac arrhythmias, myocardial infarction and heart failure. It is also used in the treatment of hyperthyroidism. It is used in prophylactic treatment of migraine40.
CHAPTER 2 EXPERIMENTAL TECHNIQUES,
MATERIALS AND METHODS
2.1 Experimental techniques
Different techniques were used for the preparation of the nanoparticles. Various equipment and analytical methods were used for the determination of the physical, chemical, and mechanical properties of the nanoparticles.
2.1.1 SCANNING ELECTRON MICROSCOPY:
Primary electrons are focused through a small diameter electron probe that scanned cross wide specimen, this is based on the fact that electrostatic or magnetic fields applied in the right angle to the beam can change the direction of travel of electron. Scanning can be done in two perpendicular direction which forms square or perpendicular area of specimen known as raster. Secondary electrons from each point on the specimen are collected and an image of this area can be formed42.
Fig.2.1- working principle of SEM
There are two types of the scanning electron microscope depending on electron energies
U = Acceleration voltage.
E= Electron energies
Electron energies in the range0.5-5keV is called low voltage scanning electron microscopy and electron energy in the range of 5-50keV are the conventional SEM instrument43.
Table2.1- Advantages and disadvantages of SEM
It has highest working distance
It gives only surface detection
It has enormous depth of focus
No colour recognition ( electron images)
Charging of specimens found (sputter images)
Chances of beam damage
Inability to differentiate between component in flat surface
Clear View of surface feature
Inability to distinguish between worn fibres
Best handling and viewing facilities
DIFFERENTIAL SCANNING CALORIMETRY
DSC is a thermo analytical technique and there is zero temperature difference between a standard and substance when they are heated or cooled at predetermined rate. Transition temperature is used to plot the DSC curve. Sample size of compound normally ranges from 1-10 mg. This apparatus generally operates at a temperature -1800-7000c. For the cooling of the substance, nitrogen is generally used. Sometimes electrical cooling or forced air cooling is applied. Basic principle of this technique is phase transition means minimum or maximum heat flow through sample DSC is applied only when limited amount of sample is available.
Fig 2.2: Different parts and working of DSC.
Types of DSC
Heat flux DSC
Power compensation DSC
Phase transition, melting, glass transition these are very important parameter for the DSC. The transition involved energy changes which can be detected by DSC. Generally the sample holder temperature is increased by the function of time.
Application of DSC
Correct determination of transition temperature.
Reaction kinetics can be determined by using DSC.
Purity of pharmaceuticals can be determined by using DSC.
Metal alloys can be evaluated by using DSC.
Characterisation of lubricant and greases44.
Freeze drying is a stabilising process where frozen formulation and quantity of solvent is reduced by sublimation (called as primary drying). With consecutive stage of desorption (called as secondary drying process) and last stage is the lyophilisation which involves three different, unique and interdependent stages and are critical to achieve final product.
In this step temperature of the compound is reduced to freezing point of water, due to this bulk quantity of water is converted in to the ice and remaining solution or dispersion is freezed contained ice crystals in random network to the viscous liquid called as freezing concentrate45. Freeze drying depend upon the freezing of the compound because freezing of the sample fix the sample structure, size and shape and its final characteristics46, 47. Temperature required for freezing is -500 C to -800 C. Compound which is to be used for freezing is placed in shell freezer. Liquid nitrogen, Dry ice, or are used for the freezing of compound. In freeze drying technique compound should be cooled by using the mechanism of Triple point. Triple point theory means temperature at which the solid and liquid phases of the compound can co-exist. In that solid material is directly converted into a vapour phase48. Larger crystals are freeze by using process known as annealing. Annealing means variation in temperature higher to lower. By using this step large compounds can be easily be freeze dried.
The effect of temperature and mass transfer resistance on the primary drying rate is mathematically expressed49, 50.
Dm/Dt=sublimation rate per vial
Po-equilibrium vapours pressure of Ice of frozen product.
Pc-pressure of drying chamber
Rp- resistance of dried product layer
In this process heat is supplied to the compound but more amount of heat causes the structural changes in compound. Pressure is lowered for sublimation. During the primary drying initial phase 95 % water should be removed after sublimating the compound. In primary drying vacuum is used to control pressure.
In the secondary drying dried and the stable product is formed at the encircling temperature by removal of the residual moisture i.e bound, unfrozen water. This step is performed by the application of the heat. This is because small amount of the residual water which is remained after the primary drying cause the damage of the product. Higher vaccum also helps to remove bound water at temperature zero. The physical and chemical processes occurring during the freeze drying and their effect on the final product has been discussed by franks 51 is summarised the above step as shown in the figure
Under cooling Seeding Nucleation
Ice crystal growth
Morphology, size distribution
Vitrification Eutectic phase separation
STORAGE Moisture /temperature control
The freeze-drying cycle and important physical parameters that control freezing and drying behaviour (adapted from Franks, 1990).
Advantages of freeze drying
Freeze drying is used to reduce thermal inactivation of the product and immobilize solution component.
Freeze drying minimizes the salting out of the proteins, changes in the distribution of component within drying and dried product.
Shelf life of the drug can be increased by reducing the moisture content in the sample which is possible by the freeze drying.
Freeze dryer is used to improve the sample solubility, shrinkage, unacceptable appearance, or loss of activity.
Due to the high surface and highly porous product having the problem of packing.
It is very slow and time consuming process.
X- RAY DIFFRACTION METHOD
This method is depending upon the scattering of x-rays by crystals. Crystal structure of different compound can be determined by this method.
When beam of x-ray is passing through the substance, the electrons composing the atoms of the compound become as a small oscillators. These electrons are oscillating at equal frequency as that of incident radiation. Scattered rays are arranged in a regular manner in a crystal lattice. If these waves undergo interference then they are said to be diffracted which is useful for the determination of crystal structure of the compound.
Component of XRD
Solid state semi conductor detector
Applications of XRD
It is used to locate the trace elements in the body such as Iodine.
An x ray photo shows the location of the bones in the body and it is radially used to determine broken bones.
Used to define shapes of veins and capillaries .
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
HPLC is an analytical technique used for separation and determination of several organic, inorganic and biological products. In HPLC liquid mobile phase and finely divided solid stationary phase is used for the determination of sample.
The principle of HPLC depends on polarity of the component. Polar stationary phase material is packed in the chromatographic column. The mixture of sample is injected in to the column. Elution in the column starts with the less polar solvent and less polar compounds elute first in this solvent. Polar component are eluted after in the polar solvent, thus less polar are separated first than the polar component. Polar component has more affinity to the stationary phase than the less polar solvent
Fig2.3-different parts of the HPLC with their functions.
Instrumentation of HPLC
For the detection of the Drug loading in to the nanoparticles HPLC is used. For this purpose we used auto sampling instrument. Online hplc instrument consist of the solvent delivery system such as pump and gradient maker, injection system, column system, and detector(52)
Mobile phase is used for carrying eluent from stationary phase so that it can be separated properly. There are two types of elution
1) Isocratic elution: In which composition of mobile phase solvent is constant.
2) Gradient elution: In which composition of mobile phase solvent changes.
Pumps are used to produce constant and accurate flow without pulsation which helps to minimise detector noise at the range of flow rate of HPLC. Pumps are able to operate at a high pressure in the micro particulate column. It is adaptable to gradient eluation.(52)
a) Generate high pressure up to 6000 psi
b) Pulse free flow
c) Resistance to corrosion due to solvents
d) To obtain different flow rates from 1 to 10 ml/min.
Optimal separation of compound is depends upon media characteristics, column used and packing method of columns.(53)Columns are available from 10 to 30 cm in length with internal diameter of 2 to 5 mm. They are made up of stainless steel. The efficiency and selectivity of column relies on partition coefficient and is given as
Concentration of solute in stationary phase
Concentration of solute in mobile phase
Generally UV visible detector, flour meter, refractive index monitor or electrochemical detector is used for analysis by HPLC.
Applications of HPLC
In Qualitative and Quantitative analysis of biological compounds
Analytical method validation is important parameter for the biological sample assay which includes assay methods for quantitative analysis and other parameter like stability of analyte in the matrix,accuracy, precision, specificity and reproducibility.(54) Mc dowall has discussed the future role of automation in hplc assay of biological samples(55)
HPLC technique is broadly applicable to the analysis of surfactant. Analysis of pure surfactant and their solutions are easily done by using the HPLC(56).
In Separation of complex mixtures
It is useful for analysis of non volatile and thermally unstable samples.
HPLC is useful In clinical analysis of blood and biological fluids.
HPLC is used in pharmaceutical industry for isolation and identification of drug formulations.
It is used in analysis of polymorphs, isotopes and isomers.
Preliminary development of microparticles (put size range in nm) initially was undertaken by attempting various methods