SUMMER TRAINING REPORT 2015

Undertaken At, Raj Pharmaceuticals Analytical and Research Laboratory

Under the guidance of, Dr. Rana Singh, Head of Analytical and Research Department

Benazir Shugufta SRM University

DECLARATION

This is to certify that the project entitled, Pharma Analytical Techniques, submitted by me in fulfilment of the requirements for the internship at the "Raj Pharmaceuticals" is a genuine work carried out by me under the supervision of my guide.

Date:

Mumbai

(Head, Analytical and Research Laboratory)

AKNOWLEDGEMENT

I take this opportunity to express my gratitude and special thanks to Dr. Rana Singh, the Head of Analytical and research laboratory in Raj Pharmaceuticals for guiding and allowing me to carry out my project at their esteemed work place and extending full support during the training.The internship opportunity I had here was a great chance for learning and professional development. I am also grateful for having a chance to meet so many wonderful people and professionals who led me though this internship period.I express my deepest thanks to [Rakesh Kumar], [Sr. Scientist], Ms. [Archana], [Asst. Researcher], Ms. [Shailja Bansal], [Jr. Scientist] and Mr [Rahul Singh], [Lab assistant] for their careful and precious guidance which were extremely valuable for my study both theoretically and practically. I perceive this opportunity as a big milestone in my career development. I will strive to use gained skills and knowledge in the best possible way.

Sincerely,Benazir Shugufta

INTRODUCTION

Pharmaceutical analysis is a branch of practical chemistry that involves a series of process for identification, determination, quantification and purification of a substance, separation of the components of a solution or mixture, or determination of structure of chemical compounds.The substance may be a single compound or a mixture of compounds and it may be in any of the dosage form. The substance used as pharmaceuticals are animals, plants, microorganisms, minerals and various synthetic products.The sample to be analysed is called as analyse and on the basis of size of sample, they can be classified as macro(0.1 g or more), semi micro (0.01 g to 0.1 g), micro(0.001 g to 0.01 g), sub micro (0.0001 g to 0.001 g), ultramicro (below 10-4 g), trace analysis(100 to 10000 ppm). Among all, the semi micro analysis is widely used.

Currently, the trend in the pharmaceutical industry and bioanalytical measurements is that the analytes of interest in very low concentrations must be measured from lower and lower sample volumes. This note presents various instrumental analytical techniques, sample preparation methods, in vitro and ex vivo systems which are frequently used in the drug R & D-, production- and quality control-processes and in pharmacokinetic, toxicity and drug metabolism studies.

TYPESThere are main two types of chemical analysis.1. Qualitative (identification) 2. Quantitative (estimation)

1. Qualitative analysisis performed to establish composition of natural/synthetic substances. These tests are performed to indicate whether the substance or compound is present in the sample or not. Various qualitative tests are detection of evolved gas, formation of precipitates, limit tests, colour change reactions, melting point and boiling point test etc.

2. Quantitative analyticaltechniques are mainly used to quantify any compound or substance in the sample. These techniques are based in :

(a) The quantitative performance of suitable chemical reaction and either measuring the amount of reagent added to complete the reaction or measuring the amount of reaction product obtained, (b) The characteristic movement of a substance through a defined medium under controlled conditions, (c) Electrical measurement, (d) Measurement of some spectroscopic properties of the compound.

Various types of Qualitative analysis:1.Chemical methodsa) volumetric or titrimetric methodsb) gravimetric methodsc) gasometrical analysis2.Electrical methods3.Instrumental methods4.Biological and microbiological1. Chemical methods

a) Titrimetric or volumetric method

It involves reaction of substance to be determined with an appropriate reagent as a standard solution, and volume of solution required to complete the reaction is determined. Volumetric methods require simple and less apparatus and they are susceptible of high accuracy.Various types of titrimetric methods are:i) Acid-base titrations (neutralization reactions)ii) Complex metric titrationsiii) Precipitation titrationsiv) Oxidation reduction titrationsv) Non aqueous titrations

b) Gravimetric methods

In gravimetric analysis, a substance to be determined is converted into an insoluble precipitate in the purest form, which is then collected and weighed. It is the time consuming process.In electro-gravimetry, electrolysis of the sample is carried out on the electrodes is weighed after drying.Thermo-gravimetry (TG) records the change in weight, differential thermal analysis (DTA) records the difference in temperature between test substance and an inert reference material, differential scanning calorimetry (DSC) records the energy needed to establish a zero temperature difference between a test substance and reference material.

c) Gasometrical analysis

Gasometry involves measurement of the volume of gas evolved or absorbed in a chemical reaction.Some of the gases which are analysed by Gasometry are CO2, N2O, cyclopropane, amyl nitrate, ethylene, N2, helium etc.

2. Electrical methodsElectrical methods of analysis involve the measurement of electric current, voltage or resistance in relation to the concentration of some species in the solution.Electrical methods of analysis include:(a) Potentiometry(b) Conductometry(c)Polarography(d) Voltametry(e) Amperometry

Potentiometry measures electrical potential of an electrode in equilibrium with an ion to be determined. Conductometry measures electrical conductivity of an electrode with a reference electrode while Polarography, Voltametry and Amperometry measures electrical current at a micro-electrode.

3. Instrumental methods of analysis

Instrumental method involves measurement of some physical properties of the compound or a substance. These methods are employed for determination of minor or trace concentration of element in the sample.Instrumental methods are preferred due to their selectivity, high speed, accuracy and simplicity of analysis. Any change in the properties of the system are detected by measurement of absorbance, specific rotation, refractive index, migration difference, charge to mass ratio etc.

Spectroscopic methods of analysis depend upon measurement of the amount of radiant energy of a particular wavelength emitted by the sample.Methods which include absorption of radiation are ultra violet, visible, infra-red, atomic absorption, nuclear magnetic resonance spectroscopy etc.

Emission methods involve heating or electrical treatment of the sample so that the atoms are raised to the excited state to emit the energy and the intensity of this energy is measured. Emission methods include emission spectroscopy, flame photometry, flourimetry etc.

Chromatographic techniques and electrophoretic methods are separation methods for the mixure of compounds, but also applied for identification of compounds of mixures. Various chromatographic techniques are GC, HPLC, TLC, HPTLC, PC etc.Mass spectrometry involves vaporization of material using a high vaccum and the vapour is bombarded by a high energy electron beam. Vapour molecules undergo fragmentation to produce ions of varying size. These ions are differentiated by accelerating them in electrical field and then deflecting them in a magnetic field. Each kind of ion gives a peak in the mass spectrum.

4. Biological and microbiological methods

Biological methods are used when potency of a drug or its derivative cannot be properly determined by any physical or chemical methods. They are called bio-assays.Microbiological methods are used to observe potency of antibiotic or anti- microbial agents. In antimicrobial assay, inhibition of growth of bacteria of the sample is compared with that of the standard antibiotic. These methods include cup plate method and turbidometric analysis.

APPLICATIONS

Manufacturing industries require both qualitative and quantitative analysis to ensure that their raw materials meet certain specifications, and to check the quality of final product.

Raw materials are to be checked to ensure that the essential components are present within the predetermined range of composition and there are not any unusual substances present which might upset the manufacturing process or it may appear as a harmful impurity in the final product.

In the development of new products which contains mixtures other than the pure material, it is necessary to ascertain composition of mixture which shows the optimum characteristics for which the material has been developed.

Geographical surveys require analysis to determine the composition of soil sample and numerous rock samples collected from the field.

Most of the industrial processes give rise to pollutants which may cause health related problems. So quantitative analysis of air, water and soil sample should be carried out to determine the level of pollution and to establish the safe limits for pollutants.

EXPERIMENT 1: ULTRA-VIOLET VISIBLE SPECTROSCOPY

AIM: To apply Beer- Lambert relationship to an aqueous solution containing an absorbing substance and thus determine its respective concentrations.

INTRODUCTION AND THEORY:UV/Vis Spectroscopy is routinely used in the analytical chemistry for the qualitative and quantitative determination of different analytes, such as transition metal ions, highly conjugated organic compounds, and biological macromolecules.UV/Vis refers to absorption spectroscopy or reflectance spectroscopy in the ultraviolet visible spectral region (200-400 nm). Absorption of the ultra-violet radiations results in the excitation of the electrons from the ground state to higher energy state. The energy of the ultra-violet radiation that are absorbed is equal to the energy difference between the ground state and higher energy states (delta E = hf).Generally, the most favored transition is from the highest occupied molecular orbital (HOMO) to lowest unoccupied molecular orbital (LUMO). For most of the molecules, the lowest energy occupied molecular orbitals are s orbital, which correspond to sigma bonds. The p orbitals are at somewhat higher energy levels, the orbitals (nonbonding orbitals) with unshared paired of electrons lie at higher energy occupied orbitals.The basic principle of quantitative absorption spectroscopy lies in comparing the absorption of a sample solution with that of a set of standards under radiation of a selected wavelength through the application of Beer-Lamberts law. Beer-Lambert law states that: when a beam of monochromatic light is passed through a solution of an absorbing substance, the rate of decrease of intensity of radiation with thickness of the absorbing solution is proportional to the incident radiation as well as the concentration of the solution.The expression of Beer-Lambert law is-A = log (I0/I) = EclWhere, A = absorbanceI0= intensity of light incident upon sample cellI = intensity of light leaving sample cellC = molar concentration of soluteL = length of sample cell (cm.)E = molar absorptivity

Instrumentation and working of UV spectroscopy

Instrumentation and working of the UV spectrometers can be studied simultaneously. Most of the modern UV spectrometers consist of the following parts-

Light Source- Tungsten filament lamps and Hydrogen-Deuterium lamps are most widely used and suitable light source as they cover the whole UV region. Tungsten filament lamps are rich in red radiations; more specifically they emit the radiations of 375 nm, while the intensity of Hydrogen-Deuterium lamps falls below 375 nm.

Monochromator- Monochromators generally composed of prisms and slits. The most of the spectrophotometers are double beam spectrophotometers. The radiation emitted from the primary source is dispersed with the help of rotating prisms. The various wavelengths of the light source which are separated by the prism are then selected by the slits such the rotation of the prism results in a series of continuously increasing wavelength to pass through the slits for recording purpose. The beam selected by the slit is monochromatic and further divided into two beams with the help of another prism.

Sample and reference cells- One of the two divided beams is passed through the sample solution and second beam is pass through the reference solution. Both sample and reference solution are contained in the cells. These cells are made of either silica or quartz. Glass can't be used for the cells as it also absorbs light in the UV region.

Detector- Generally two photocells serve the purpose of detector in UV spectroscopy. One of the photocell receives the beam from sample cell and second detector receives the beam from the reference. The intensity of the radiation from the reference cell is stronger than the beam of sample cell. This results in the generation of pulsating or alternating currents in the photocells.

Amplifier- The alternating current generated in the photocells is transferred to the amplifier. The amplifier is coupled to a small servo meter. Generally current generated in the photocells is of very low intensity, the main purpose of amplifier is to amplify the signals many times so we can get clear and recordable signals.

Recording devices- Most of the time amplifier is coupled to a pen recorder which is connected to the computer. Computer stores all the data generated and produces the spectrum of the desired compound.

Diagram :

APPARATUS AND CHEMICALS USED:Apparatus: Spectrophotometer, Cuvettes, Measuring flasks, pipette, Beaker etc.Chemicals: Salicylic acid(0.1%), Acetate buffer(0.05M), Distilled water

PROCEDURE:i. Turn on the spectrophotometer and allow it to warm up for at least 2 mins. Then we determine the absorption spectrum using the standard acetate buffer.ii. Select one of the cuvettes for the blank solution (acetate buffer) and insert it into cell holder with the index line facing us to avoid scratching.iii. Turn the wavelength control knob to 265nm with blank solution to calibrate the spectrophotometer.Preparation of the solution:i. Prepare a stock solution of 0.1% salicylic acid by measuring 100mg of salicylic acid and dissolving in 100mL acetate buffer.ii. From the above stock solution perform serial dilution containing 0.05%, 0.025%, 0.01%, 0.005%, 0.0025%, and 0.001%. iii. Label the test tubes from 1 to 6. Then add sufficient amount of acetate buffer to make the volume up to 10mL.iv. Then prepare standard blank of 20mL acetate buffer.

Results summarised in following table:

Determination of absorbancei. Set blank solution in the cuvette and calibrate at wavelength of 290nm.ii. Insert cuvette containing the sample iii. Read and record the absorbance for all the solution iv. Determine the absorbance of unknown sample.

CALCULATIONS:

We Plot the graph of absorbance vs. concentration in excel sheet:

From the Graph,We obtain the equation below:Y= 3.7129 X + 0.006Where,Y= Value from y-axis i.e. the absorbanceX= Value from x-axis i.e. the concentration in Mol/Litre.Y= 0.042 (absorbance of unknown sample)Hence we obtain X=

RESULT AND CONCLUSION:The concentration of the unknown sample was found to be Hence we can conclude that UV/Vis spectroscopy is the best method routinely used in analytical chemistry for the quantitative determination of different analytes.

DISCUSSIONS:Application of uv/vis spectroscopy:- Identification of an unknown compound- An unknown compound can be identified with the help of UV spectroscopy. The spectrum of unknown compound is compared with the spectrum of a reference compound and if both the spectrums coincide then it confirms the identification of the unknown substance.

Determination of configurations of geometrical isomers- It is observed that cis-alkenes absorb at different wavelength than the trans-alkenes. The two isomers can be distinguished with each other when one of the isomers has non-coplanar structure due to steric hindrances. The cis-isomer suffers distortion and absorbs at lower wavelength as compared to trans-isomer.

Determination of the purity of a substance- Purity of a substance can also be determined with the help of UV spectroscopy. The absorption of the sample solution is compared with the absorption of the reference solution. The intensity of the absorption can be used for the relative calculation of the purity of the sample substance.

EXPERIMENT 2: HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

AIM: Quantitative determination of Paracetamol from its formulated tablets

INTRODUCTION AND THEORY:HPLC: - High-performance liquid chromatography(HPLC; formerly referred to ashigh-pressure liquid chromatography), is a technique in analytical chemistryused to separate the components in a mixture, to identify each component, and to quantify each component. It relies on pumps to pass a pressurized liquidsolventcontaining the sample mixture through a column filled with a solidadsorbent material. Each component in the sample interacts slightly differently with the adsorbent material, causing different flow rates for the different components and leading to the separation of the components as they flow out the column. Chromatographycan be described as amass transferprocess involvingadsorption. HPLC relies on pumps to pass a pressurized liquid and a sample mixture through a column filled with a sorbent, leading to the separation of the sample components. The active component of the column, the sorbent, is typically a granular material made of solid particles (e.g.silica, polymers, etc.), 250 micrometres in size. The components of the sample mixture are separated from each other due to their different degrees of interaction with the sorbent particles. The pressurized liquid is typically a mixture of solvents (e.g. water, acetonitrile and/or methanol) and is referred to as a "mobile phase". Its composition andtemperatureplay a major role in the separation process by influencing the interactions taking place between sample components and sorbent. These interactions are physical in nature, such as hydrophobic (dispersive), dipoledipole and ionic, most often a combination thereof.

Paracetamol: Paracetamol, N-(4hydroxy phenyl) acetamide has analgesic and antipyretic. It is commonly used for the relief of headaches, relief of fever, and minor aches and pains as well as for the management of more severe pain, where it allow lower dosage of additional nonsteroidal anti-inflammatory drug to be used their by minimizing over all side effect [1-3]. The main mechanism of action of paracetamol is considered to be the inhibition of cyclooxygenase (COX) and recent finding suggest that it is highly selective for cox-z [4].

Working:The schematic of an HPLC instrument typically includes a sampler, pumps, and a detector. The sampler brings the sample mixture into the mobile phase stream which carries it into the column. The pumps deliver the desired flow and composition of the mobile phase through the column. The detector generates a signal proportional to the amount of sample component emerging from the column, hence allowing forquantitativeanalysis of the sample components. A digitalmicroprocessorand user software control the HPLC instrument and provide data analysis. Some models of mechanical pumps in a HPLC instrument can mix multiple solvents together in ratios changing in time, generating a compositiongradientin the mobile phase. Various detectors are in common use, such asUV/Vis, photodiodearray (PDA) or based onmass spectrometry. Most HPLC instruments also have a column oven that allows for adjusting the temperature the separation is performed at.

Diagram :

MATERIALS REQUIRED:Paracetamol reference standard, tablets of paracetamol 500 mg, HPLC grade methanol and water and 0.45m nylon membrane filter.

PROCEDURE:A) Instrumentation-The method development was performed with a cyber lab reverse phase high performance liquid chromatography separating system. Separation was achieved using a mobile phase methanol and water in the ratio of (65:35) at flow rate 1.0 ml/min. The eluent was monitored with a UV-Detector at a wavelength 243 nm. The column was maintained at ambient temperature and injection volume of 20 l was used. The mobile phase was filtered through 0.45m nylon membrane filter. The absorbance have been taken with same solvents methanol and water (65:35) and same wave length 243 nm. The absorbance was measured with 3 mL capacity quartz cuvette.

B) Preparation of Stock solution-The stock solution of paracetamol 100 ppm was prepared in mobile phase methanol and water (65:35). The solutions were filtered through a 0.45 m nylon membrane. The mobile phase was degassed with sonication instrument. The mobile phase was sonicated to 5 min.

C) Preparation of sample solutionTo preparation of sample solution, twenty tablets were weighed accurately. These tablets were powdered and mixed well. Taken an equivalent quantity of the powder was transferred into a small conical flask and extracted with mobile phase the extract was filtered into a 100 ml volumetric flask. The volume was maintained with same solvent. D) LinearityAccurately pipette volumes of 0.25, 0.5, 0.75, 1.0, 1.25 and 2.50 ml of paracetamol stock solution was placed in 5 mL volumetric flasks and diluted to 5 mL with mobile phase. These different serial dilutions were filtered through a 0.45m nylon membrane and sonicate. The each solution of 20 l was injected into the column in thrice and 3 mL each solution was used to absorbance. The calibration curves were obtained by plotting peak area and absorbance versus concentration.

E) SpecificityTo determine the specificity was taken excipients of the tablets in equivalent to the sample weight. The solution was prepared similarly to the sample solution. The solution was analysed as per the proposed method F) AccuracyThe recovery was checked at the three theoretical concentrations level 25, 50 and 75 g. The percentage recovery was calculated using the following equation: % Recovery = [A] 100 / [B]Where [A] is the net peak area of the drug in sample, [B] is the peak area of the drug in standard mixture.

OBSERVATION:Table 1: It shows summary of validation system suitability parameters

RESULTS AND DISCUSSION:High performance liquid chromatography (RP-HPLC) was successfully applied in control laboratories for determination in single dosage form. The results of validation show high performance liquid chromatography (RP-HPLC) technique is simple, linear, precise, accurate and selective. Hence the above method can be recommended for simultaneous determination of paracetamol from formulated products.

APPLICATIONS:HPLC has contributed to analytical solutions in diverse fields such aspharmaceuticals, foods, life sciences, environment, forensics, etc.Common application areas in pharmaceutical analysis are: Assay Related SubstanceAnalytical Method Validation Stability Studies Compound Identification Working StandardsCommon applications in foods are: Fat soluble vitamins (A, D, E and K) Water soluble vitamins (B-complex vitamins such as B1, B2, B3, B6, Folic acid, Pantothenic acid, B12, Vitamin C) Residual pesticides such as 2, 4-D and Monochrotophos. Antioxidants such as TBHQ, BHA and BHT. Sugars: Glucose, Fructose, Maltose and other saccharides. Cholesterol and sterols Dyes and synthetic colours. Mycotoxins such as Aflatoxins B1, B2, G1, G2, M1, M2and ochratoxin.

EXPERIMENT 3: ATOMIC ABSORPTION SPECTROSCOPYAIM: Determination of Fusidic acid in bulk powder and in pharmaceutical dosage form.INTRODUCTION AND THEORY:AAS:- The technique makes use of absorption spectrometry to assess the concentration of an analyte in a sample. It requires standards with known analyte content to establish the relation between the measured absorbance and the analyte concentration and relies therefore on theBeer-Lambert Law. In short, the electrons of the atoms in the atomizer can be promoted to higher orbitals (excited state) for a short period of time (nanoseconds) by absorbing a defined quantity of energy (radiation of a givenwavelength). This amount of energy, i.e., wavelength, is specific to a particular electron transition in a particular element. In general, each wavelength corresponds to only one element, and the width of an absorption line is only of the order of a few picometers (pm), which gives the technique its elemental selectivity. Theradiation fluxwithout a sample and with a sample in the atomizer is measured using a detector, and the ratio between the two values (the absorbance) is converted to analyte concentration or mass using the Beer-Lambert Law.

Diagram:

FUSIDIC ACID:- Fusidic acid is antibiotic derived from Fusidium coccineum, exerts powerful antibacterial activity against a number of gram-positive organisms. Staphylococci, including the strains resistant to penicillin or other antibiotics, are particularly susceptible to fusidic acid. tomic absorption spectrometric method is based on precipitation of the ion associates formed from the reaction of fusidic acid with silver nitrate, copper acetate or ferric chloride standard solutions. The formation and solubility of the solid complexes at the optimum conditions of pH and ionic strength values have been studied. The method depends on direct determination of the ions in the precipitate or indirect determination of the ions in the filtrate by atomic absorption spectroscopy.MATERIALS REQUIRED:Double distilled water, Fusidic acid, 0.025 M silver nitrate (0.524% W/V solution), 0.01 M copper acetate (0.2% W/V solution) and 0.01 M ferric chloride (0.18% W/V solution), Fucicort cream labeled to contain 20 mg fusidic acid and 1 mg betamethasone per each game of cream.PROCEDURE:1. Standard preparations - Stock solutions containing 10 g ml-1 fusidic acid was prepared in distilled water. 2. Working standard solutions containing 20-100 ng ml-1, were prepared by suitable dilution of the stock solutions with distilled water.3. Atomic absorption spectrometric method utilizing silver (Procedure A) To aliquots of fusidic acid stock solution (equivalent to 20-100 ng ml-1), two ml of 0.025 M silver nitrate solution was added The precipitates were washed with redistilled deionized water until free of silver (I). Direct method -The precipitates obtained above were dissolved in a minimum amount of dilute ammonia solution and completed to 25 ml with redistilled deionized water. Two ml of the resulting solutions was diluted to 25 ml with redistilled deionized water. Indirect method - The filtrates and washings were collected in a 100 ml volumetric flask and completed to volume with redistilled deionized water. Ten ml of the resulting solution was diluted to 100 ml with redistilled deionized water.A blank (omitting addition of drug) was prepared and the absorbance was measured at the flaming conditions; wavelength 328.1 nm, lamp current 7 mA, slit width 3.8 A, air flow rate 10L/min and acetylene flow rate 2.6 L/min. Silver (I) concentrations were calculated from a calibration curve.4. Atomic absorption spectrometric method utilizing copper (Procedure B) To aliquots of fusidic acid stock solution (equivalent to 20-100 ng ml-1), two ml of copper acetate solution was added. Solutions were well shaken, filtered (whatman No.44), and the precipitates were washed with redistilled deionized water until free of copper (II). Direct method - Precipitates were dissolved in a minimum amount of dilute ammonia solution and completed to 100 ml with redistilled deionized water. Five ml of the resulting solution was transferred into a 50 ml volumetric flask and completed to volume with redistilled deionized water. Indirect method - The filtrates and washings were collected in a 100 ml volumetric flask and completed to volume with redistilled deionized water. Five ml of the resulting solution was diluted to 100 ml with redistilled deionized water.A blank (omitting addition of drug) was prepared and the absorbance was measured at the flaming conditions; wavelength 327.7 nm, lamp current 7 mA, slit width 3.8 A, air flow rate 10L/min and acetylene flow rate 2.3 L/min. Copper (II) concentrations were calculated from a calibration curve.5. Atomic absorption spectrometric method utilizing iron (Procedure C) To aliquots of fusidic acid stock solution (equivalent to 20-100 ng ml-1), two ml of ferric chloride solution was added, shaken well and filtered (whatman No.44). The precipitates were washed with redistilled deionized water until free of iron (III). Direct method - The precipitates were dissolved in a minimum amount of dilute ammonia solution and completed to 25 ml with redistilled deionized water. Two ml of the resulting solutions was diluted to 50 ml with redistilled deionized water. Indirect method - The filtrates and washings were collected in a100 ml volumetric flask and completed to volume with redistilled deionized water. Five ml of the resulting solution was diluted to 100 ml with redistilled deionized water.A blank (omitting addition of drug) was prepared and the absorbance was measured at the flaming conditions; wavelength 240.7 nm, lamp current 7 mA, slit width 3.8 A, air flow rate 10L/min and acetylene flow rate 2.5 L/min. Iron (III) concentrations were calculated from a calibration curve.6. For pharmaceutical preparations -Evacuate the contents of five tubes and extract the fusidic acid contents with ethanol and complete as above.

OBSERVATION:i. Slightly alkaline (pH 7.8-8.3) alcoholic solutions of fusidic acid gave white coagulated precipitates with silver nitrate (procedure A), green bluish precipitates with copper acetate (procedure B) and reddish brown precipitates with ferric chloride (procedure C). ii. These precipitates form the basis of the micro-quantitative determinations of sildenafil citrate. Ag (I), Cu (II) or Fe (III) contents can be determined either directly in the precipitate or indirectly in the filtrate by atomic absorption spectrometry. iii. Composition of the formed complex The molar ratios of the formed chelates were studied. The method revealed 1:1, 2:1 and 3:1 fusidic acid to silver (I), copper (II) and iron (III), respectively. iv. The stability constants of the formed chelates were calculated using the following equations: = A/Aex CX / (CM A/Aex CX) (CL nA/Aex CX)nWhere is the stability constant of the formed chelate, M indicates metal, L indicates ligand, n =X/(1-X) where X is the mole fraction of the ligand at the maximum of the continuous variation curve. A/Aex is the ratio of the observed absorbance to that indicated by the tangent for the same wavelength. CM and CL are the concentrations of the metal and the ligand, respectively, Cx = CL/n = CM [19].

The calculated stability constants for the formed chelates (Table 1) are ranging from 111.1482 x 10-7 to 179.2123 x 10-7 indicating good stability of the formed chelates.

(v) Quantification and validation of assay proceduresAnalysis of pharmaceutical formulations:The proposed atomic absorption spectrometric procedures were applied to the determination of fusidic acid in Fucicort Cream.

RESULT:Fusidic acid was successfully quantified in the pharmaceutical dosage forms.

DISCUSSIONS:Applications of Atomic Absorption Spectroscopy water analysis (e.g. Ca, Mg, Fe, Si, Al, Ba content) food analysis analysis of animal feedstuffs (e.g. Mn, Fe, Cu, Cr, Se,Zn) analysis of additives in lubricating oils and greases (Ba,Ca, Na, Li, Zn, Mg) analysis of soils clinical analysis (blood samples: whole blood, plasma, serum; Ca, Mg, Li, Na, K, Fe)

EXPERIMENT 4: THIN LAYER CHROMATOGRAPHY

AIM: Analysis of drugs in pain relievers by thin layer chromatography:1) Determine the Rf value2) To identify the components in the analgesic tablet by TLC comparison with standard compounds.

INTRODUCTION AND THEORY:Background: We sometimes refer to painpills in terms of their brand name, rather than their generic name, which is also different than their chemical one. We sometimes ask for Tylenol or Advil to mean pain drug, when in actuality, these drugs contain two different painkillers, acetaminophen and ibuprofen, respectively, and these work in different ways in the body. Some people refer to any pain killer as simply aspirin, while aspirin itself is a generic name for Bayer. This is most likely due to aspirin being the only pain drug available for many years.Thin layer chromatography (TLC) A method chemists use to separate one chemical from another using their differences in polarity. There are several components to TLC: Stationary phaseIn order to separate chemicals, we have to have a platform on which to separate them. In our case, the platform is a thin sheet of the polar chemical silica attached to a plastic support. The silica does not move in the process of chromatography, therefore, we call it stationary.

Mobile phase -A mobile phase is the solvent that carries the chemicals through the stationary phase. A small spot of chemicals wont move on silica alone, but if we add a solvent, it can draw the chemicals up through the paper using capillary action.

Point of originthe spot where you put your chemicals on the stationary phase.

Developing After putting chemicals on the point of origin, the chromatogram can be developed, that is, the mobile phase can be pulled through the stationary phase to separate the chemicals.

Retention factor (Rf)The distance our chemicals move during chromatography is typically less than the distance the mobile phase moves. We can measure the difference in distance by using the Rf value. This is the distance a chemical moves on our silica (stationary phase) during our separation divided by the distance the mobile phase moves. If I use water as a mobile phase, sugar as my solute, and silica as my stationary phase, my Rf value will always be the same in this system. Changing the mobile phase to alcohol will change the Rf for the sugar, because sugar has a different affinity for alcohol than it does water. The following formula is used to determine Rf:

TLC works off of affinities. If a chemical has a higher affinity for the silica plate and a low affinity for the solvent (which would happen if the compound is quite polar) it will stick to the plate (it wouldnt migrate far). If the chemical has a low affinity for the plate and a high affinity for the solvent (which would happen if it is quite nonpolar), it will fly up the plate with the solvent, because it hates being in contact with the polar silica, but is happy being in contact with the solvent.

Analgesica pain killing drug. This could be aspirin. This could be morphine. All pain killers are considered analgesics. Antipyretica drug that reduces a fever. Antiinflammatorya drug that reduces swelling. Tylenolgeneric name is acetaminophen, chemical name is N(4hydroxyphenyl) acetamide. Acetaminophen is both an analgesic and an antipyretic. Bayergeneric name is aspirin, chemical name is 2acetoxybenzoic acid. Aspirin acts as an analgesic, antipyretic, and an antiinflammatory, which makes it a very good overall pain reliever for many ailments. Aspirin also has the ability to prevent blood from clotting, which is why it is so widely used in people prone to heart disease. Advilgeneric name is ibuprofen, chemical name is 2(4isobutylphenyl)propanoic acid. Works mostly as an antiinflammatory drug, but acts as an analgesic when pain is associated with swelling. Caffeinenot sold as is, but a component of pills. Not an analgesic, but a stimulant and diuretic (makes you urinate). Can help a body absorb other drugs more quickly; it is commonly found with analgesics to provide faster pain relief.

MATERIALS REQUIRED:Beaker, silicaplates, aspirinsolution, watchglass, Acetaminophen solution, ibuprofen solution, caffeine solution, filter paper, Unknown drugs solution, ruler, capillaries.(*Solvent is 25:1:1 ethyl acetate: ethanol: acetic acid)

PROCEDURE:1. You will start this experiment by determining the chromatographic properties of the drugs in their pure form. Obtain a TLC plate and very gently draw a line about 1 cm from the bottom of the plate using a dull pencil on the side that has the silica. 2. On the line, draw 4 small lines and label the lines T (for acetaminophen, Tylenol), A (for aspirin), I (for ibuprofen), and C (for caffeine).3. Touch a clean capillary tube to the acetaminophen solution to draw up about 12 cm of solution. Being careful to not disturb the silica, touch the capillary to the silica on the T line. 4. Repeat the process using clean capillaries each time for the remaining 3 solutions.

5. Add the developing solvent to a depth of about 0.5 cm to TLC chamber. When all 4 spots on the TLC have dried, place the, spots down, in the beaker.6. Allow the solvent to reach within a half cm to the top of the plate and pull it out of the beaker. Immediately mark with a line the spot where the solvent stopped. This is called the solvent front and you will need it for measurement. Lay the TLC plate with silica side up until it dries.7. When the plate is dry, you can visualize the spots. The chemicals are present in very small quantities, and are not coloured, however, they absorb UV light. The silica contains an indicator dye that glows green under UV light.8. Where you see spots, lightly circle with a pencil. Set aside your plate for measurements at the end of the experiment.

CALCULATIONS:The unknowns are: Anacin, Excedrin, Motrin, Tylenol

The known compounds are Aspirin, Caffeine, Acetaminophen, and Ibuprofen.Measure the distance the spots moved in mm, measuring from the bottom line (where you initially spotted them) to the middle of where they ended up. Use this value to determine the Rf values for each.

The distance from starting point to the end point of the reading 1 is 5cm while for reading 2 is 5.1 cm.

The above calculations shows that the drug contains caffeine and aspirin.

RESULT :The drugs in pain relievers were analysed and the unknown drugs were successfully identified by TLC comparison with several known compounds. The compounds identified are Caffeine and Aspirin, hence the drug is Anacin.

EXPERIMENT 5: FTIR SPECTROSCOPY

AIM: Quantitative analysis of Ibuprofen in pharmaceutical formulations through FTIR Spectroscopy.

INTRODUCTION AND THEORY:The principle ofFTIRis based on the fact that bonds and groups of bonds vibrate at characteristic frequencies. A molecule that is exposed to infrared rays absorbs infrared energy at frequencies which are characteristic to that molecule. In a molecule, the differences of charges in the electric fields of its atoms produce the dipole moment of the molecule. Molecules with a dipole moment allow infrared photons to interact with the molecule causing excitation to higher vibrational states. Diatomic molecules do not have a dipole moment since the electric fields of their atoms are equal. DuringFTIRanalysis, a spot on the specimen is subjected to a modulatedIRbeam. The specimen's transmittance and reflectance of the infrared rays at different frequencies is translated into anIRabsorption plot consisting of reverse peaks. The resultingFTIRspectral pattern is then analysed and matched with known signatures of identified materials in theFTIRlibrary.Working :To understand the powerfulness and usefulness of FTIR spectrometer, it is essential tohave some background information of dispersive IR Spectrometer. The basic components of a dispersive IR spectrometer include a radiation source, monochromator, and detector. The common IR radiationsourcesare inert solids that are heated electrically to promote thermal emission of radiation in the infrared region of the electromagnetic spectrum. Themonochromatoris a device used to disperse or separate a broad spectrum of IR radiation into individual narrow IR frequencies.Generally, dispersive spectrometers have a double-beam design with two equivalent beams from the same source passing through the sample and reference chambers as independent beams. These reference and sample beams are alternately focused on the detector by making use of an optical chopper, such as, a sector mirror. One beam will proceed, traveling through the sample, while the other beam will pass through a reference species for analytical comparison of transmitted photon wavefront information.After the incident radiation travels through the sample species, the emitted wavefront of radiation is dispersed by a monochromator (gratings and slits) into its component frequencies. A combination of prisms or gratings with variable-slit mechanisms, mirrors, and filters comprise the dispersive system. Narrower slits gives better resolution by distinguishing more closely spaced frequencies of radiation and wider slits allow more light to reach the detector and provide better system sensitivity. The emitted wavefront beam (analog spectral output) hits the detector and generates an electrical signal as a response.Detectorsare devices that convert the analog spectral output into an electrical signal. These electrical signals are further processed by the computer using mathematical algorithm to arrive at the final spectrum. The detectors used in IR spectrometers can be classified as either photon/quantum detectors or thermal detectors.Diagram :

Ibuprofen:

PROCEDURE:IR analyses were performed in a Bruker FTIR IFS 68 equipment.Quantitative anaylsis of solutions of ibuprofen in chloroform were performed in a cell with CaF2 windows and variable optic pathway.Quantitative analysis of solid samples were performed through thin wafers with KBr.A) Liquid Samplei. Two salt plates were rinsed using chloroform and wiped dry.ii. A sample of liquid paraffin was taken and added to one of the salt plates.iii. The two plates held together by capillary action were then mounted in the beam path of the spectrophotometer.iv. The results were printed as a graph.

B) KBr pellets for solid sample:i. Approx. 1mg of ibuprofen sample was ground until they form fine powder.ii. KBr was added and mixture was made to the ratio 1:9. iii. To make KBr pellet the die pin from the storage container was removed. The collar was placed on lower anvil.iv. Powdered KBr was then placed in the collar unitl all the surface of the lower anvil was covered evenly.v. The die set is then carefully placed in hand press.vi. After aboubt 20 sec open the Kbr handpress and the pellet infrared spectrum was measured.

OBSERVATION:

RESULT:The quantification of Ibuprofen through infrared spectroscopy was accomplished with the requirements of specificity, precision and accuracy in order to be used as a method for the quality control of pharmaceuticals.

EPERIMENT 6: POLYACRYLAMIDE GEL ELECTROPHORESIS

AIM: To analyse purified fluorescent protein by 12% PAGE.

INTRODUCTION AND THEORY:Gel electrophoresisis a method for separation and analysis of macromolecules DNA, RNA and proteins) and their fragments, based on their size and charge.The separation occurs on the basis of charge, electrophoretic mobility and molecular size.ELECTRICAL CHARGE: Many molecules like amino acids, RNA and DNA have naturally occurring positive and negative charges.Molecules with a negative charge (anions) will be attracted to the positively charged node (anode).Molecules with a positive charge (cations) will be attracted to the negatively charged node (cathode).The microscopic particles attach to one another forming tunnels that act as a sieve to separate the molecules. Small molecules can move faster than large molecules.ELECTROPHORETIC MOBILITY: Electrophoretic mobility is defined as the rate of migration (cm/sec) per unit field strength (Volts/cm) =Q/6rWhere- Electrophoretic mobilityQ-Net charge on the ionr- Ionic radius of the solute - Viscosity of the mediumPOLY ACRYLAMIDE GEL ELECTROPHORESISPolyacrylamide gels are synthetic gels and are tougher than agar and agarose gels. Polyacrylamide gels are optically clear (including UV transparency) and electrically neutral due to absence of any charged groups, in contrast to the presence of COO- and SO3- groups in agar and agarose gels. Moreover, Polyacrylamide gels can be prepared with wide range of pore size by the relative proportions of Acrylamide to bisacrylamide. Therefore, Polyacrylamide gels are widely used to resolve mixture of peptides, proteins and small molecular weight of nucleic acids.Polyacrylamide gels are chemically cross-linked gels formed by the polymerization of acrylamide with a cross-linking agent, usually N,N-methylenebisacrylamide. The reaction is a free radical polymerization, usually carried out with ammonium persulfate as the initiator and N,N,N,N-tetramethylethylendiamine (TEMED) as the catalyst. Although the gels are generally more difficult to prepare and handle, involving a longer time for preparation than agarose gels, they have major advantages over agarose gels. They have a greater resolving power, can accommodate larger quantities of DNA without significant loss in resolution and the DNA recovered from polyacrylamide gels is extremely pure (Guilliatt, 2002). Moreover, the pore size of the polyacrylamide gels can be altered in an easy and controllable fashion by changing the concentrations of the two monomers. Polyacrylamide gel electrophoresis is two types viz., Denaturing and non -denaturing (native) PAGE. In denaturing PAGE the protein are treated with the anionic detergent, SDS and -mercaptoethanol or DTT. The SDS molecules bind to protein by strong hydrophobic interactions and it has been estimated that 1.4g SDS binds per gram of proteins. Since each SDS carries a negative charge, a protein molecule with a molecular weight of 50,000(i.e) aprox.450 amino acids acquires 225 net negative charges.In order to break intra and inter disulphide bonds the protein are treated with -mercaptoethanol.Volume of Reagents Used to Cast Polyacrylamide GelsGel % 30% Acrylamide Water5x TBE APS TEMED

8 % 3.2 ml 6.4 ml2.4 ml200 l10 l

10% 4.0 ml 5.6 ml2.4 ml200 l10 l

12% 4.8 ml 4.8 ml2.4 ml200 l10 l

MATERIALS REQUIRED:1. Mini gel Apparatus (Vertical)2. D.C power supply with Cord3. Test tubes4. Eppendorfs5. Micropipettes and tips6. Gel storage bagReagents required:1. Acrylamide stock (30%)2. Separating Gel Buffer (1.5M Tris, pH 8.8)3. Ammonium per sulfate (10% APS)4. N, N, N, N- tetra methyl ethylene diamine (TEMED)5. Electrophoresis bufferPreparation of reagents:1. Acrylamide stock (30%) Acrylamide29.2gm Bisacrylamide0.8gm Distilled water100ml Dissolve the salts in 100 ml water2. Separating Gel Buffer (1.5M Tris, pH 8.8)Weigh 18.17gm of Tris in 90 ml of distilled water adjust the pH was adjusted to 8.8 using 1N hydrochloric acid and final volume make upto 100 ml3. Ammonium per sulfate (10% APS) Weigh 1gm of APS and dissolve in10ml of distilled water.4. 10X TBE Stock Solution (Tank Buffer)1X = 89 mM Tris base, 89 mM Boric acid, 2 mM EDTA) 108.0 g Tris base 55.0 g Boric acid 7.44 g Na2EDTA 2H20 Adjust volume to 1 liter with distilled water. Filter through a 0.45 m filter PH adjustment is not necessary.PROCEDURE:1. Clean the glass plates by soaking in a detergent and wash thoroughly with water and keep in hot air oven.2. Assemble the plates with spacers using petroleum ether (Vaseline) and clamp it with the help of clips.3. Prepare the gel mixture as shown in the table and immediately pour into the assembled plate.4. Insert the comb gently in between the glass plates on the top of gel mixture.5. Allow the gel to get polymerized for at least 20 min.6. When the polymerization of the gel gets over, remove the comb and the lower spacer strip carefully.7. Remove excess vaseline from the bottom of the gel by wiping with a piece of tissue paper.8. Remove the comb carefully and clean the wells using filter paper.9. Fill the lower reservoir and upper reservoir of the electrophoresis apparatus with the required volume of tank buffer.10. Fix the gel plate to the electrophoresis tank carefully with appropriate clips and clamps.11. Load the protein samples in the wells and equalize with the level electrophoresis buffer in the upper chamber by filling with electrophoresis buffer.12. Raise the level of buffer in the upper reservoir.13. Connect the electrodes to the power pack and the switch on the current and observe for the formation of bubbles which indicates the passage of current14. Keep 15-20mA/75-100V for separating gel.15. Turn off the power supply when the tracking dye reached the bottom of the gel and transfer the gel to the staining solution for 1-2 hours.16. Later transfer the gel to destaining solution until the clear bands are visible.

RESULT:PAGE was successfully performed and the purified fluorescent protein was extracted.