M.SC.

(FINAL) BIOCHEMISTRY

PROJECT REPORT SUBMITTED TO:

GOVT. MAHARANI LAXMIBAI GIRLS PG COLLEGE KILA MAIDAN INDORE (M.P.)

Signature guide:

Signature student:

Name of guide:
PROF. ASHA CHOUDHARY ( H.O.D of Biochemistry Dept.)

Name of student:
MISS RADHA RANI PRAJAPATI

ACKNOWLEDGEMENT
It is a great pleasure to acknowledge a deep sense of gratitude and indebtedness to our H.O.D. (Biochem. Dept.) Prof. Mrs. Asha Choudhary for providing permission for carrying out this project. I give my sincere thanks and gratitude to Mrs. Archana Mehta (Assistant of general manager of Q.C.) of IPCA labs, Indore for providing me all the best facilities during the industrial training in their highly esteemed organization. I am very much thankful to Prof. Mrs. Tasneem Dharwala and prof. Miss Harsha Garg and Prof. Miss Shweta Azmera for their kind help during project preparation. Last not but least I wish to record our thanks to the entire staff of Biochemistry Department for their help and cooperation throughout the project.

Declaration of the student

I Radha Rani Prajapati daughter of Shri Abadh Bihari Prajapati certify that the project report entitled Microbial Analysis Of Drinking Water prepared by me is my personal and an authentic work under the guidance of PROF. Mrs. ASHA CHOUDHARY.

Date: 07/05/2010 Place : Indore

Signature of Student: Name: Class: Roll number: Address: Contact number:

SYNOPSIS

 ABSTRACT INTRODUCTION REVIEW OF LITERATURE AIM AND OBJECTIVE MATERIALS AND METHOD

RESULT AND DISCUSSION

CONCLUSION SUMMARY REFERENCE

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ABSTRACT
In the previous project analysis of antimalarial drug is done which is used to cure malaria with the help of some technique like chromatography, spectroscopy and chemical analysis and now the analysis of antifungal drum is perform with the help of same technique.

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INTRODUCTION

ANALYSIS OF ANTIFUNGAL DRUG ( FLUCANOZOLE )

Fluconazole is a triazole antifungal drug used in the treatment and prevention of superficial and systemic fungal infection. In a bulk powder form, it appears as a white crystalline powder, and it is very slightly soluble in water and soluble in alcohol. It is commonly marketed under the trade name DIFLUCAN or TRICAN.
MODE OF ACTION :-

Like other imidazole and triazole class antifungal, Fluconazole inhibits the fungal cytochrome P450 enzyme 14 alpha demethylase. Mammalian demethylase activity is much less sensitivity to fluconazole than fungal demethylase. This inhibit prevention the conversion of lanosterol to ergo sterol, an essential component of the fungal cytoplasmic membrane, and subsequent accumulation of 14 alpha- methyl sterols. Fluconazole is primarily fungistic, however may be fungicidal against certain organisms in a dose dependent manner. Interestingly, when fluconazole was in development at Pfizer it was decided early in the process to avoid producing any chiral centers in the drug so that subsequent synthesis and purification did not encounter difficulties with enantiomer separation and associated variations in biological effect. Fluconazole is active against the following microorganism:
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Blastomyces dermatidis Candida

Coccidiodes immitis Epidermophyton spp. Histoplasm capsulatum Microsporoum spp.

ADVERSE EFFECT: Common rash, Headache, Dizziness, Nausea, Vomiting, Abdominal pain, Diarrhoea, or elevated liver enzymes. Infrequent anorexia, fatigue, constipation. Rare oliguria, hypokalamia, parasthesia, seizures, alopecia, thrombocytopenia,other blood dyscrasias.

MOLECULAR FORMULA:MOLECULAR WT. :SYNONYMS :-

C13H12N6OF2 306.3

2- (2,4- DIFLUOROPHENYL) 1, 3 bis (1H 1,2,4 triazole- 1- yl) 2 propanol.

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REVIEW OF LITERATURE

HK Johansen, PC Gotzsche Jama, in 1999: Problems in the design and reporting of trials of antifungal agents encountered during meta-analysis of fluconazole. M Nunez, JE Peacock Jr, R Chin Jr Chest, in 2000: Pulmonary cryptococcosis in the immunocompetent host. Therapy with oral fluconazole suggests that fluconazole may be an appropriate choice for the treatment. LL Patton, AJ Bonito, DA Shugars in 2001: The effectiveness of antifungal drugs for the prevention and treatment of oropharyngeal candidiasis in HIVpositive patients. EJ Bow, M Laverdiere, N Lussier, C Rotstein, MS Cancer, in 2002: The use of parenteral therapeutic antifungal therapy for severely neutropenic chemotherapy recipients.

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KA Marr, W Leisenring, F Crippa, JT in 2004:

Cyclophosphamide metabolism is affected by azole antifungals hematologylibrary.org

SB Girois, F Chapuis, E Decullier, BGP in 2006: Adverse effects of antifungal therapies in invasive fungal infections.

E Robenshtok, A Gafter-Gvili, E Goldberg, in 2007: Antifungal prophylaxis in cancer patients after chemotherapy or hematopoietic stem-cell transplantation.

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AIM AND OBJECTIVE

AIM: The analysis of antifungal drug fluconazole . OBJECTIVE:

MATERIALS AND METHOD SYNTHETIC STEPS

Raw material required in the synthesis: TDA { alpha[ 1H1,2,4-TRIAZOLE]- 1-Y}2,4DIFLUORO ACETOPHENONE. ATDA {alpha[ 4 AMINO,1,2,3- TRIAZOLE}2,4 DIFLUORO ACETOPHENONE. ACETONITRILE ALUMINIUM CHLORIDE 1,3 DIFLUORO BENZENE ETHYLENE DICHLORIDE SODIUM NITRITE POTASSIUM HYDROXIDE TRIMETHYL SULPHOXIDE DE MINERALISED WATER (7)

1ST intermediate: ATDA Description :- it is off white powder, which on drying gets converted to a pale yellow crystalline powder

pH:

6.5 7.5

This intermediate is tested for its i) Alkali insoluble ii) Related impurity ( by TLC ) 2ND intermediate:- TDA TDA obtained from the dryer is tested for its water content by karl fischer apparatus. After purification TDA is tested for its i) Melting point ii) Solubility iii) Acid insolubility iv)Water content v) Purity by gas chromatography

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CRUDE FLUCONAZOLE Description :- off white to pale yellow crystalline powder. It is tested for : pH color index iron content

related impurity purity or assay by non- aqueous titration crude fluconazole is purified with charcoal and demineralised water to get a filteration which with acetone and is reflux with isopropyl alcohol to give pure fluconazole. FLUCONAZOLE:Fluconazole (pure) is an off white to pale yellow crystalline powder.

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FLOW DIAGRAM OF SYNTHESIS

ATDA EDC, 1,3 DFB ALCL3 , ACETONITRILE TDA

NaNo3, DM WATER FLUCONAZOLE CRUDE DM WATER, KOH TRIMETHYL SULPHOXIDE

CENTRIFUGE FLUCONAZOLE PURE DRYING

MILLING SHIFTING FLUCONAZOLE PURE (FINAL PRODUCT

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ANALYSIS OF FLUCONAZOLE

CHEMICAL ANALYSIS: Involving determination of

Following: Melting point Moisture content pH Specific gravity Solubility Bulk density Loss on drying INSTRUMENTATION SPECTROSCOPY Infra Red Spectroscopy UV/Visible Spectroscopy

CHROMATOGRAPHY Thin Layer Chromatography Gas chromatography H.P.L.C

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DETERMINATION OF MELTING POINT:

DEFINATION: Melting point is the temperature at which liquid and crystalline solid phases of a compound coexist. Melting range or temperature of a substance is defined as those point of temperature within which, the substance begins to coalesce and is completely melted.

PROCEDURE: After connecting the power supply, the stirrer is started to circulate the liquid in the heating vessel so that temperature become uniform. The sample is taken in a capillary tube up to a height of 4 to 6 mm. temperature is maintained at about 10 C below the expected melting point and capillary is inserted in the space given in the Teflon capsule. Temperature is increased slowly and materials is observed carefully, the temperature range between which sample begin to melt and is completely melt as seen by the formation of a definite meniscus is taken as its melting point range. Melting point range of FLUCONAZOLE between 136 to 140 C .

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DETERMINATION OF MOISTURE/ WATER CONTENT BY KARL FISCHEER APPARATUS:

Principle: This is based on stiochemtric titration where 1 mole of iodine reacts with 1 mole of water.

Volumetric Karl Fischer Titrator Model KF-21

Reagent : Karl Fischer reagent is prepared by the action of SO2and I2 solution in a mixture of pyridine and methanol. Procedure: Before using this apparatus, its factor has to be determined every day. Because the moisture content of the air changes every day. For this dehydrated methanol is taken titration vessel and sufficient Karl Fischer reagent is added to give end point. Formula for Factor Determination : Factor = Weight of Water X 1000 Burette reading (final initial) ( 13 )

Now to determine the water content of the given sample, 1 gm of sample is added to the titration vessel, and sufficient amount of Karl Fischer reagent is runned and reading is noted at the end point. Moisture content = Burette reading x factor x 100 (Solid) Weight of sample (Gms) x 1000 Moisture content = (Liquid) Burette reading x factor x 100 Weight of sample (ml) x density x 1000

Ex.

Sample : FLUCONAZOLE Reagent factor : 5.61 mg/ml Burette reading : 1.35 ml Weight of sample : 1.0317 gms. 1.35 x 5.61 x 100 1.0317 x 1000 0.7347%W/W.

Moisture Content = =

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DETERMINATION OF pH:
pH: it is defined as the negative logarithm of hydrogen ion concentration. pH = -Log [H+]

Procedure : 3 gm of FLUCONAZOLE dissolve in 30 ml water in a 100 ml beaker and tested the pH by pH meter.

Before taking pH doing calibrate the pH meter and after this observed pH by deep the electrode in solution and observed pH of fluconazole. Limit of pH: 6.5 7.5
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DETERMINATION OF SPECIFIC FGRAVITY BY PYCNOMETER : Specific Gravity = Weight of sample Weight of water

For this measurement the procedure is as follows: Take weight of empty pycnometer = A Weight of pycnometer with water = B Weight of dry pycnometer with sample = C So weight of sample = C A Weight of water = B A Ex. Sample: fluconazole Weight of empty pycnometer (A) = 34.1177gm Weight of pycnometer with water (B) = 58.4175gm Weight of water = (B - A) = 58.4175 34.1177 = 24.2998gm Weight of dry pycnometer with sample (C) = 58.7279gm So weight of sample = 24.6102gm

Specific gravity

= 24.6102 24.2998 = 1.01277w/w

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DETERMINATION OF SOLUBILITY: Solubility is tested for the purity of drug substance. Ex :- FLUCONAZOLE Solubility Methanol - freely soluble Acetone - soluble Isopropanol sparingly soluble Chloroform - sparingly soluble Water slightly soluble Toluene - very slightly soluble Terms used in solubility : Descriptive Term Very soluble Freely soluble Soluble Sparingly soluble Approx solubility in parts by vol. of Solvent for 1 part by wt. of solute. Less than 1 From 1 10 1 30 30 100

Slightly soluble Very slightly soluble Practically insoluble

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100 1000 1000 10,000 10,000 and over

DETERMINATION OF BULK DENSITY Definition : Bulk density of sample is the value of the density in gm/ml i.e. expressed in mass/volume, under specified condition. The bulk density of a powder depends primarily on other particle size distribution, particle shape and tendency of particle to adhere to one another. The particle may pack in a way as to leave large gap between their surface, resulting in a light powder and powder of low density. The smaller particle may shift between the large ones to form a heavy powder or one of high bulk density. Procedure : 10gm of sample is taken in a 50 ml bulk density cylinder, and covered with a cork. The volume of this sample in cylinder is noted down. This cylinder is set into the clamp of bulk density apparatus. Note : Wt. of sample taken and number of tapping differs according to the sample. Untapped Bulk Density = Wt. of sample before tapping Volume of sample

Ex. FLUCONAZOLE Wt. of sample = 9.3969 Volume of sample = 33ml

Untapped bulk density

= 9.3969 33l = .28gm/ml

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After tapping Tapped Bulk density = Wt. of sample After Tapping Volume of sample Wt. = 9.3969 Volume = 18ml Tapped Bulk density = 9.3969 18 = .52gm/ml Bulk Density is found for those drugs which can be packed in the form of capsules.

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DETERMINATION OF LOSS ON DRYING Loss on drying test: Loss on drying is the loss in weight in percent weight / weight resulting from water and volatile matter of any kind that can be driven of under special conditions. Procedure: A glass stoppered weighing bottle that has been dried for 30 min. under the same condition to be employed in the determination is weighed. 1 gm sample is taken in the bottle covered properly and again weighed. This bottle is placed in the drying chamber (oven) for 4 hrs at 105, After drying bottle is taken out, brought to room temperature and weighed accurately. Calculation: Wt. of bottle Wt. of sample + bottle Wt. of sample Wt. of sample + Bottle After drying % of LOD = W1 gm = W2 gm = (W2 W1) gm. = W3 gm. = (W2 W3) gms. X 100 (W2 W1) gms.

Ex. Sample: FLUCUNAZOLE Limit at 105 for 3hrs NMT .50%w/w

Wt. of Bottle = 45.9165gm Wt. of sample + Bottle = 46.9885 gm Wt. of sample = 1.0685 gm Wt of sample + Bottle = 46.9845gm. After drying % of LOD = ( 46.9885- 46.9845) x 100 ( 46.9885- 45.9165) = 0.046%

INSTRUMENTATION SPECTROSCOPY
Various instrument are used in carrying out different types of analysis such as infra red spectroscopy, uv spectroscopy,

UV SPECTROSCOPY
Principle: The absorption in UV OR Visible region occurs due to excitation of electrons. This transition requires presence of unsaturated functional groups. It is based on the principle of BEER LAMBERT LAW. BEER LAMBERT LAW Beer Law : According to this law the absorbance is directly proportional to the concentration. A C Lambert Law : According to this law the absorbance is directly proportional to the length of the tube. A L

Then according to the Beer Lambert law we can say that A C. L A = e. C. L. Where, A e C L = Absorbance = Molar absorptivity = Concentration in moles per liter = Path length.
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Main parts of spectrophotometer :

Power supply (b) Source (c) Wavelength selector (d) Sample compartment (e) Radiant power detector (f) Meter recorder of computer readout.
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Power supply:
A constant power supply is required for spectrophotometer because the fluctuation in power supply disturbs the output radiation of lamp.

Source : i) Visible source :

A tungsten lamp provides 15% of its output in the visible region ( between 325 nm to 900 ) and provides a reliable and stable source.
ii)

UV source : For measurement in the UV region ( between 200 nm to 325 nm ) electric discharge source are used in excitation of the gaseous molecule is caused by the passage of electron through the gas. Hydrogen lamp is

commonly used and has output rising rapidly below 375 nm. Quartz source is another option which absorbs below 200 nm the use of deuterium in place of hydrogen lamp result in radiant power several times greater than those from the hydrogen lamp but the spectral range is basically the same. iii) Wavelength selector: - A filter is used for the selection of Particular wavelength region. Generally prism and gratings are used as dispersing elements.
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Sample Compartment : The sample holder is designed carefully so that positioning of the sample in the light path will always be the same. The sample cell should be exactly perpendicular to the light path to minimize reflection losses. Detector : The detector that converts photons signal into electrical signal is called as photoelectric detectors, one such inexpensive and rugged detector is barrier layer cell. Working: The incoming beam from the light source has been sharply defined by the reference slit. It is dispersed into its component wavelength by prism or a grafting. Then a small portion of spectrum is selected with the exit slit whose size controls the amount of measured radiant power and control the band pass. Ex: Analysis of FLUCONAZOLE Procedure: 0.1 gm of sample is dissolved in 100ml water, its 1ml is taken and diluted to 10% solution. This sample is examined at below given wavelength to give specified absorbance. For Fluconazole product, the absorbance should fall in the given range of wavelengths.

Wavelengths 342 nm 329nm 256nm 235nm 220nm

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Absorbance 0.36 - 0.39 0.32 - 0.35 0.30 - 0.33 0.35 - 0.39 0.60 - 0.66

INFRARED SPECTROPHOTOMETER
Introduction : I.R. region of spectrum is spread from a wavelength of 0.75 300 micro( or wave no. from 13000 33 cm). this region is subdivided into following.
a)

Near infrared region (0.75 2.5)/ (142904000cm) b)

c)

Middle infrared region ( 25 15micro m)/ 4000 666 cm) Far infrared region (14.3- 50micro m)/(700 200 cm)

I.R spectrophotometer: it is used for recording the spectra in the infrared region. It consists of an optical system capable of providing the monochromatic light in the region of 0.8micro to 50 micro and means of measuring the quotient of the intensity of the transmitted light and the incident light. Now a days Fourier transform Infrared spectrophotometer are replacing the conventional dispersive instruments

Principle: since atom in a molecule are not hold rigidly so when I.R. radiation are passed through the sample. The vibrational and

rotational energies of the molecule are increased. The energy required for exciting emission is atleast for rotaton, higher for vibrational and still higher for electronic transitions. If excitation energy is kept so small that it produces transition from one rotational quantum number to another with in a given vibrational
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level, the spectra than produced is called rotational spectrum such a spectra is formed in far infra- red regions. If the excitation energy can cause transition from are vibrational quantum level to another within a given electronic level, emission are observed corresponding to the change in vibrational quantum numbers. Any change in vibrational level also involves change in rotational levels do the spectrum produced is called vibrational rotational spectrum. This type of spectrum appears in near infrared region. Radiation source: Nernst glower Glower source Mercury arc Sample preparation: solid sample can be identified in two ways: a. Disc: in this method about 1gm of the sample is saturated with 300mg of dry powdered KBr . after 150mg of this powder taken in a set and put under pressure so as to get thin transparent disc of approx. 13mm in diameter. b. Null: in this sample is saturated with liquid paraffin to give a creamy paste is null which is compressed between 2 suitable plates and IR spectra is taken. Procedure :- 0.1 gm of sample is dissolved in 10ml water and approx. 1 2 ml dilute NaOH is added and washed with 40ml of methylene dichloride. This solution is poured over anhydrous

sodium sulphate in a funnel to separate the organic layer. This organic layer is evaporated to dryness on hot plate and then liquid paraffin is added.
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CHROMATOGRAPHIC TECHNIQUE
THIN LAYER CHROMATOGRAPHY
Introduction :-Thin layer chromatography is a type of adsorption chromatography where the adsorbent is thin layer if solids like sillicagel, alumina or cellulose. It can be applied directly to the plate or can be bonded to the plate by means of plaster of paris. It can be used for determining the related impurities present in the sample. Thin layer chromatography is a chromatography technique used to separate mixtures. Thin layer chromatography is performed on a sheet of glass ,plastic or aluminium foil, which is coated with a thin layer of adsorbent material, usually silica gel, aluminium oxide or cellulose. This layer of adsorbent is known as the stationary phase. After the sample has been applied on the plate , a solvent or solvent mixture( known as the mobile phase) is drawn up the plate via capillary action. Because different analysis ascend the TLC plate at different rates, separation is achieved. TLC find many application including: Assaying the radiochemical purity of radio pharmaceuticals Determining of the pigment a plant contains

Detection of pesticides or insecticides in food Analyzing the dye composition of fibers in forensics Identifying compounds present in a given substance Monitoring organic reaction
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TESTING OF FLUCONAZOLE Test solution: Dissolve 50mg of the substance to be examined in methanol and dilute to 5ml with the same solvent. Reference solution A : Dissolve 50mg of fluconazole in methanol and dilute to 5ml with the same solvent. Reference solution B : Dilute 50 mg of econazole and 50mg of fluconazole in methanol and dilute to 5ml with the same solvent. Plate Coating TLC silica gel F 254R Development Acetic acid, water, andmethylisobutylketone (25:25:50) Mobile phase concentrated NH2R, methanol and methylene chloride(1:40:60) v/v/v. Detection apply separately to the plate 10 microl of each solution. Develop over apath of 10cm. allow the plate to dry in air. Examine under the UV light .

Performance the principle spot in the chromatogram obtained with the test solution is similar in the position and size to the principle spot in the chromatogram obtained with reference solution A. this is not valid unless reference solution B shows two clearly separated spots.
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GAS CHROMATOGRAPHY
Introduction :- The distinguish features of gas chromatography are a gaseous mobile phase and a solid or immobilized liquid stationary phase in packed or capillary column. In the packed column, the liquid phase is deposited on finally divided inert solid support, such as di- atomaceous earth, porous polymer or graphotype carbon. The solid phase is an active adsorbent such as alumina, silica or carbon. Principle :- when a gas or vapor comes in contact with an adsorbent, certain amount of gas is adsorbed on the solid phase and when it comes in contact with a liquid , a fixed amount of gas is dissolved in the liquid that is Henrys law or partition is obtained.

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Instrumentation and working: - Gas chromatography consists of a carrier gas source, an injection port, column, and detector and recording device. The injection port , column, and detector are temperature controlled. The typical gas is helium, nitrogen or hydrogen, depending on the column and detector in use. The gas is supplied from a high pressure cylinder or high purity gas generator and passes through suitable pressure reducing valves and flow meter to the injection port and column. Compound to be chromatograhed either in solution or as gases are injected into the gas stream at the injection port. Depending upon the configuration of the apparatus, the test mixture may be injected directly into the column or be vaporized in the injection port mixed into the following carrier gas prior to entering the column. Once in the column, compounds in the test mixture are separated by virtue of differences in their capacity factors, which in turn depends vapor pressure and degree of interaction with the stationary. The capacity factor, which governs resolutions, retention time, column efficiencies of components of test mixture, is also temperature dependent. The use of temperature programmable column ovens takes advantages of this dependence to achieve efficient separation of compounds differing widely in vapor pressure. As resolved compound emerge separately from the column, they pass through a differential detectors, which responds to amount of each compound present. The type of detector to be used depends upon the nature of the compound to be analyzed and is specified in the individual monograph. Detector is heated to prevent condensation of the eluting compounds. Detector output is recorded as a function of time, producing a chromatograph which consists of a series peaks on a time axis. Each peak represents a compound in the vaporized test mixture, although some peaks may overlay.
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The elution time is characterized of a individual compound and the instrument responses, measured as peak area of peak height, is a function of the amount present. Injector: sample injection device range from simple syringes to fully programmable automatic injectors. Column: capillary column which are usually made of fused silica gel are typically 0.2 to 0.53mm in internal diameter and 5 to 60 m in length. The liquid or stationary phase, which is sometimes chemically bonded to the inner surface, is 0.1 to 1.0 micrometer thick, although stationary phases may be upto 5 micron thick. Column, made of glass or metal, are 1 to 3 m in lengthand diameter 2 to 4 mm. Detector : Flame ionization detector (FID) are used for most pharmaceutical analysis, with lesser use made of thermal conductivity detector (TCD), electron capture detector(ECD), nitrogen phosphorous detector( NPD),and Mass spectrometric detector(MSD). FID has a wide linear range and is sensitivity to most organic compounds. Limit for fluconazole: NLT( not less than) 98% GC Condition : Column Oven temp. FID temp. INJ. Temp N flow rate Injection vol. Runtime (minimum) Required base
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10%SEV- 30 280 c for 5 min. 280 c 200 c 20ml/min 1 micro litre 20 min. NLT 98%

HIGH PRESSURE LIQUID CHROMATOGRAPHY (HPLC)

Between 1967 and 1969, Kirkland and huber describe the first high pressure liquid chromatography(HPLC). They proposed high pressure systems capable of operating at pressure upto 2.07* 10 Nm (3000psi). in HPLC, small diameter columns ( 1 3 mm) with support particle size in the region of 30 micro m are used and the eluent is pumped through the column at a high flow rate. It has been found that separation by HPLC may be effected about 100 times faster than by the use of conventional liquid chromatography. HPLC is particularly suitable to the analysis of those compounds which are not readily handled by GLC. In HPLC, eluent from the solvent resvoir is filtered, pressurized and pumped through the chromatographic column. A mixture of solutes injected at the top of the column is separated into components on traveling down the column and the individual solutes are monitored by the detector and recorded as peaks on a chart recorder.
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Related substance by HPLC in FLUCONAZOLE Individual impurity: NMT 0.30% Total impurity: NMT 0.50%

Test solution: dissolve 250 mg of the substance to be examined in 5ml of methanol and dilute to 10ml with the mobile phase. Reference solution A: dissolve 250mg of fluconazole WS in 5ml of methanol and dilute to 10 ml with the mobile phase. Dilute 1.0 ml of test solution to 100ml with the mobile phase. Dilute 1.0 ml of this solution to 1.0 ml with the mobile phase. Reference solution B :dissolve 250 mg of fluconazole in 5 ml of 5ml of methanol and dilute to 10 ml with the mobile phase. Dissolve 5mg of benzimidazole in 4ml of the reference sol. (A) and dilute to 10ml with the mobile phase. Dilute 1ml of this solution to 10ml with the mobile phase. Column : Material: stainless steel Packing: octadecylsilyl silica gel for chromatography (5 micro m) Size: L= 0.15m , o = 4.6mm i.e. kromasil C18 or its equivalent Tetra methyl ammonium bromide: Dissolve 7.7 gm tetramethyl ammonium bromide in 1000ml of water. Sodium acetate: Dissolve 1.36gm sodium acetate in 1000ml of water.
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Elution: Acetonitrile, water and a mixture of equal volumes of tetramethyl ammonium bromide and sodium acetate ( adjust to pH 5.0 with acetic acid ) ( 170:80:750) Filter and mobile phase purge with helium while in use if on line degasser is not available. Column temperature: Ambident

Flow rate: 1.0ml/min Detection: spectrophotometric at 261nm Chromatography: injection vol. 20micro l Needle wash: water: acetonitrile ( 50:50v/v) After the system has calibrated, inject mobile phase as a blank run one or two times to set the base line. Inject 20 micro l of the reference solution (b), reference solution(a), and the test solution. Continue the chromatography for more than 6 times the retention of the principle peak in the chromatogram obtained with test solution. Sensitivity: The height of the principle peak in the chromatogram obtained with reference sol(b) is at least 50% of the full scale of the recorder. Performance: resolution of NLT 6.0 between the peaks due to benzimidazole and fluconazole. Injection sequence set up : 1. mobile phase has blank till base line is stable 2. reference sol. (b) for system suitability 3. mobile phase as blank 4. reference sol.(a) dilute Fluconazole replicate for calculation of impurities in test sample. 5. test sample 6. reference sol. (b) for system suitability
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Calculations: Calculate the individual of known and unknown impurities obtained from the chromatogram of test solution w.r.t to area of

Fluconazole peak obtained from reference sol.(a) as per formula given below: % of known/ unknown impurity in active = Rimp x Wtstd x Vol test x Rflu Wt test Vol std Dil test x P Dil std

Where, Rimp = area of each individual impurity peak present in the chromatogram of test solution Rflu = mean peak area of fluconazole obtained from the reference solution (a) P = % purity of fluconazole WS Disregard any peak obtained in the blank For information SubstanceApprox retention time RRTImpurity A5.3min0.5Impurity B4.3min0.41Fluconazole10.4 min1.0 Limit: Individual impurity: NMT 0.30% Total impurity : NMT 0.50%
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RESULT AND DISCUSSION

In quality control all the analysis have their particular importance like: Moisture content : Normal value 1 % But the change in this will affect packaging and wt. of product. Melting point : - For purity 136 to 140 C . TLC : - For impurity Bulk density : - Required for capsule filling and tablet filling. Untapped bulk density: 0.28gm/ml Tapped bulk density: 0.52gm/ml pH : - change in pH will affect GIT. LOD:- 0.046% In this way all the analysis explained in the project. We can say that the quality control is necessary for the quality test of product. If the product is not pure then it may have harmful effect on our body.
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CONCLUSION

After performing all the chemical analysis, spectroscopy and chromatography technique we conclude that the sample or a drug is pure and it not contain any impurity. So from the following analysis the sample is pass and then it is used to make a ANTIFUNGAL Drug for the treatment of fungal infection and preventing those disease caused by this drug.

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PERSONALITY DEVELOPMENT

SYNOPSIS: -

1.

GROUP DISCUSSION Concept. Types. Understanding group task.

2. ESSENTIAL QUALITIES FOR SUCESS

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GROUP DISCUSSION
A GD is a methodology used by an organization to gauge whether the candidate has certain personality traits and/or skills that it desires in its members. In this methodology, the group of candidates is given a topic or a situation, given a few minutes to think about the same, and then asked to discuss the it among themselves for 15-20 minutes

Some of the personality traits the GD is trying to gauge may include :

Ability to work in a team Communication skills Reasoning ability Leadership skills Initiative Assertiveness Flexibility Creativity Ability to think on ones feet

Types of GD: - There are two types of GD

a) b)

Topic-based or Case-based.

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Topic based Gds can be classified into three types: Factual topics :- Factual topics are about practical things, which an ordinary person is aware of in his day-to-day life. Typically these are about socio-economic topics. These can be current, i.e. they may have been in the news lately, or could be unbound by time. A factual topic for discussion gives a candidate a chance to prove that he is aware of and sensitive to his environment. E.g. The education policy of India, Tourism in India, State of the aged in the nation. Controversial topics : - Controversial topics are the ones that are argumentative in nature. They are meant to generate controversy.

In GDs where these topics are given for discussion, the noise level is usually high, there may be tempers flying. The idea behind giving a topic like this is to see how much maturity the candidate is displaying by keeping his temper winkin check, by rationally and logically arguing his point of view without getting personal and emotional. E.g. Reservations should be removed, Women make better manager. Abstract topics : - Abstract topics are about intangible things. These topics are not given often for discussion, but their possibility cannot be ruled out. These topics test your lateral thinking and creativity. E.g. A is an alphabet, Twinkle tle little star, The number 10 Case-based Gd: - Another variation is the use of case instead a topic.The case study tries to simulate a real-life situation. Information about the situation will be given to you and you would be asked as a group to resolve the situation. In the case study there are no incorrect answers or perfect solutions. The objective in the case study is to get you to think about the situation from various angles.
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ESSENTIAL QUALITIES OF SUCCESS

There are ten essential quality for success are as follows: 1. Be Optimistic: - Those who expect success tend to succeed, while those who expect failure tend to fail. It therefore makes sense that if you want to succeed in the world of public relations, you need to become an optimist and expect success right from the very beginning.

2. Be Goal-Oriented : - People who know what they want are always more likely to get it. The most successful people in business are those who have clear goals to aim for. So decide right now what your goals are, in terms of income, lifestyle, and so on.. 3. Be Self-Disciplined : -As the old saying goes, the only place success comes before work is in the dictionary. This being the case, start becoming more self-disciplined, both in your business and personal life. Plan your work, then work to your plan. If you do this day in, day out, you will make constant and steady progress towards achieving the goals you have set for yourself. 4. Be Nice : - . The most successful people in business are generally those who are genuinely nice to others. In the world of public relations, as in life, if you are nice to others then the chances are that they will be nice to you, and this can make your job a great deal easier. 5. Be Helpful : - Aim to be genuinely helpful to your clients. Don't make the mistake of doing only what you are paid to do. Instead, go that extra mile. Treat your clients as members of your own family and your helpfulness will automatically increase their loyalty to you as their PR consultant.
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6. Be Honest: - In all your business dealings, be honest. Don't say what you think a potential client wants to hear just to please him or her. Instead present your advice and opinions honestly. 7. Be Studious : - Even if you have completed a training course in public relations (and you should), don't ever think that you know it all. Instead, be a life-long student. Buy books on PR and promotion, and study how other PR consultants operate and learn from them.

8. Be Creative : - The world of public relations is a lot like the world of advertising -- the more creative you are, the more successful you will be. Of course, more often than not you will be using fairly standard techniques such as press releases to promote your client. However, if you can inject some of your own personal creativity into these tried-and-tested methods, you will find they become even more effective as a result, you will become a more successful PR consultant. 9. Be Enthusiastic : - Be passionate about your business and life. Enthusiasm sets you apart as someone who loves what they do rather than as one who merely shows up in order to fulfill an obligation. 10. Be Persistent : - Success won't come overnight, and it won't come easily. It is likely that you, like any other professional, will face obstacles and setbacks as you tread the royal road to success. To get through these rough patches you must become a person who is habitually persistent. Remember, winners never quit and quitters never win. Persist in pursuing your goals and as long as you refuse to throw in the towel you will eventually achieve them.
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