CHAPTER 1

INTRODUCTION

1.1 General introduction Analytical chemistry may be defined as the science and art of determining the

composition of materials in terms of the elements or compounds contained in them.

Infact,analytical chemistry is the science of chemical identification and

determination of the composition (atomic,molecular,phase) of substances and materials

and and their chemical structure.

The main object of analytical chemistry is to develop scientifically

substantiated methods that allow the qualitative and quantitative evaluation of materials

with certain accuracy. Analytical chemistry derives it’s principles from various branches

of Science like chemistry,physics microbiology,nuclear science, electronics. This

method provides information about the relative amount of one more of these

components.

1.2 Selection of the most appropriate analytical method

should taken in to account the following factors.

The purpose of the analysis, the required time scale and any cost

constraints.

The level of analyte (s) expected and the detection limit required.

The nature of the sample, the amount available and the necessary sample

preparation procedure

The accuracy required for a quantitative method.

The availability of reference materials , standards chemicals and solvents

instrumentation and special facilities.

Possible interferences with the detection or quantitative measurement

of the analyte and the possible need for sample clean up to avoid

matrix interference.

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1.3 Different methods of analysis

By using this process the components of interest is separated and analysed by using

the following techniques .

(a) Spectral methods :-

The spectral techniques are used to measure electromagnetic radiation

which is either absorbed or emitted by the sample

e.g.- U.V. visible spectroscopy ,I R spectroscopy, N M R,

ESR spectroscopy, flame photometery , fluorimetery.

(b) Electro analytical method:-

Electro analytical methods involved the measurement of current voltage or resistance

as a property of concentration of the component in solution mixture.

eg.:- Potentiometry , conductometry, amprometry.

(c) Chromatographic methods :-

Chromatography is a technique in which chemicals in solutions travel down columns or

over surface by means of liquids or gases and are separated from each other due to

there molecular characteristics.

eg:- Paper chromatography

Thin layer chromatography

High performance thin layer chromatography

High performance liquid chromatography

Gas chromatography

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1.4 ULTRAVIOLET SPECTROPHOTOMETRY

The technique of ultraviolet-visible spectroscopy is one of the most frequently employed

techniques in pharmaceutical analysis.

Molecular absorption in ultraviolet & visible region of spectrum is depend on the

electronic structure of molecules. Absorption of energy as quantized, resulting in the

elevation of electrons from orbital in the ground state to higher orbital in the exited state.

The wavelength range of u.v. radiation starts at the blue end of the visible light

and ends at 2000 Ǻ.

The ultraviolet region is subdivided in to two spectral regions.

(1) The region between 2000Ǻ-4000 Ǻ is known as near as U.V. region.

(2) The region below 2000 Ǻ is called far or vaccume U.V. region.

1.4.1 Principle

Any molecule has either and n or combination of these electrons. These bonding and

nonbonding (n) electrons absorb the characteristics radiation and undergo transition

from ground state to exited state. By the characteristic absorption peaks the nature of

electrons present and hence the molecular structure can be elucidated

Ultraviolet absorption spectra arise from transition of electron or electrons with in a

molecule or an ion from a lower to higher electronic energy level and the ultraviolet

emission spectra arise from the reverse type of transition.

When a molecule absorbs ultraviolet radiation of frequency v sec -1, the electron in that

molecule undergoes transition from a lower to a higher energy level or molecular orbital,

energy difference is given by-

E=h v erg

The actual amount of energy required depends on the difference in energy between the

ground state E0 and excited state E1 of the electrons.

E1 -E0 =hv

We known that total energy of a molecule is equal to the sum of electronic vibrational

and rotational energy. The magnitude of these energies decrease in following order :- E

etec , E vib and E rot.

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The most important characteristic of spectrophotometry are there wide applicability, high

sensitivity, moderate to high sensitivity, good accuracy and convenience. The assay of

an absorbing substance may be quickly carried out by preparing a solvent in a

transparent solvents and measuring its absorbance at a suitable wavelength.

This is governed by Beer-Lambert’s law which states-

A = a b c

Where,

A is the absorbance.

a is the absorbitivity.

b is the path length.

c is the concentration.

1.4.2 Instrumentation

(a) Source of light: - The best source of light is the one which is more stable, more

intense and which gives range of spectrum from 180-360 nm (up to 400 nm). The

different sources available are: -

1. Tungston lamp..

2. Hydrogen discharge lamp

3. Deuterium lamp.

4. Xenon discharge lamp.

5. Mercury arc.

The details are as follow:-

(i) Tungston lamp:- The various radiation sources are as follows:

The two most common radiation sources are tungsten lamps and

hydrogen discharge lamps. The tungsten lamp is similar in' its functioning to an electric

light bulb. It is tungsten. Filament heated electrically to white heat. It has two

shortcomings. The intensity of radiation at short wavelengths (<350 nm) is small.

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Furthermore, to maintain a constant intensity, the electrical current to the lamp must be

carefully controlled. However, the lamps are generally stabled, robust, and easy to use.

Typically, the emission intensity varies with wavelength.

(ii) Hydrogen discharge lamps:-

In these lamps, hydrogen gas is stored under relatively high pressure. When an

electric discharge is passed through the lamp, excited hydrogen molecules will be

produced which emit UV radiations. The high pressure in the hydrogen lamps causes

the hydrogen to emit a continuum rather than a simple hydrogen spectrum.

Hydrogen lamps cover the range 3500-1200 A. These lamps are stable, robust and

widely used.

The hydrogen discharge lamp consists of hydrogen gas under relatively

high pressure through which there is an electrical discharge. The hydrogen molecules

are excited electrically and emit UV radiation. The high pressure brings about many

collisions between the hydrogen molecules, resulting in pressure broadening. This

causes the hydrogen to emit a continuum (broad band) rather than a simple hydrogen

line spectrum. The lamps are , stable, robust, and widely used. If deuterium (D2) is used

instead of hydrogen, the emission intensity is increased by as much as a factor of 3 at

the short-wavelength end of the UV range. Deuterium lamps are more expensive than

hydrogen lamps but are used when higher intensity is required.

(iii) Deuterium lamps:-

If deuterium is used in place of hydrogen, the intensity of radiation emitted is 3 to

5 times the intensity of a hydrogen lamp of comparable design and wattage.

Deuterium lamp is more expensive than hydrogen lamp. But, it is used when high

intensity is required.

(iv) Xenon discharge lamps:-

In these lamps, xenon gas is stored under pressure in the range of 10-30

atmospheres. The xenon lamp possesses two tungsten electrodes separated by about

8 mm. When applying a low voltage forms an intense arc, the ultraviolet light is

produced.

The intensity of ultraviolet radiation produced by xenon discharge lamp is

much greater than that of hydrogen lamp.

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(v) Mercury arc:-

In this, the mercury vapour is under high pressure, and the excitation of mercury

atoms is done by electric discharge. The mercury are, a standard source for much

ultraviolet work, is generally not suitable for continuous spectral studies because of the

presence of sharp lines or bands.

(b) Monochromators:-Filters and prism monochromators are not used because of low resolution. On the other

hand grating provides a band pass of 0.4 to 2nm. Hence they are more widely used,

especially in expensive spectrophotometers .the mirrors, gratings etc are made up of

quartz, since glass absorbs UV radiations from 200-300 nm. mirrors are front surfaced

to prevent absorption of radiation .

e.g.: - Prism.Gratings

(c) Sample cells:-

The design of sample cell used are similar to that used in colorimetry except that it is

made up of quartz. Quartz cells only must be used UV spectroscopy since glass cells

will absorb UV radiation. The path length of the cell are 10nm or 1cm.

(d) Solvents:-

Solvent plays an important role in UV spectra. Hence the solvent for a sample is

selected in such a way that the solvent neither absorbs in the region of measurement

not affects the absorption of the sample.

(e) Detectors:-

Although any one of the detectors used in calorimetric can be used, photomultipliter

tubes are mainly used, since the cost of such UV spectrophotometers are high and

more accurate measurements are to be made.

E.g.: - (1) Barrier layer cell or photo voltaic cell.

(2) Photo tubes (or) photo emissive cell.

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(3) Photo multiplier tubes (PMT).

1.4.3 APPLICATION

(1) Qualitative Analysis:-

(a) Detection of impurities: To limit the presence of impurities, we can use

UV spectrophotometer measurements additional peaks can be due to

impurities in the sample and can be compared with that of standard raw

material. Also by absorbance measurement as specific wavelength the

impurities can be detected.

(b) Structure elucidation of organic compound

(c) Structure analysis of organic compound

(2) Quantitative Analysis:-

(a) Using values can we find out the quantity of drug molecule?

(b)Single standard or direct comparison method.

(c)Calibration curve method .

(3) Determination of molecular weight

(4) Determination of dissociation constant of acids and bases

(5) Chemical kinetics

(6) Identification of unknown compound.

(7) Detection of poly nuclear hydrocarbons.

(8)Determination of conjugation of geometrical isomerism.

(9) Identification of the compound in different isomerism.

(10) Detection of functional groups.

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Photomultip

Lamp

M 1

Prism

Attenuators Sample

Reference

Source

Monochro- -

mator

Beam

splitter

Sample chamber

Detector

Rotating Sector

DOUBLE BEAM

- ULTRAVIOLET SPECROPHOTOMETER

Photomultiplier

(11) Dissociation in conjugate and non-conjugate

DRUG PROFILE

Drug name:- DICLOFENAC SODIUM

International-Brand-Name :- FENAC PLUS,

DICLOWIN PLUS

Structure :-

Systematic-(IUPAC) name:--2-(2-(2,6-dichlorophenylamino)phenyl)acetic acid

Molecular MASS :- 296.148 g/mol

Empirical formula: - C14H11Cl2N O

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Dose:- Oral: Adults: 50 mg twice daily

Therapeutic category:- NSAID’S

Reported use:- PAIN KILLER

DRUG PROFILE

Drug name:- PARACETAMOL

International-Brand-Name :- DOLO,

DOLOPAR,

CALPOL,

METOPAR

Structure :-

Systematic-(IUPAC)name:--- N-(4-hydroxyphenyl)acetamide

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Molecular MASS :- 151.169 g/mol

Empirical formula: - C8H9N O 2 

Dose:- Oral: Adults: 500 mg twice daily

Therapeutic category:- NSAID’S

Reported use:- ANTIPYRETIC

Description of drug:-

These are the drug used in the treatment of fever pain respectievely.these have

novel mode of action that differentiates it from non-steroidal anti-inflamm Clinical trials

have shown tha Pracetamol & diclofenac sodium is highly effective in relieving the

symptoms of fever and pain. antiflammatory drugs (NSAIDs) and other conventional

forms of drug therapy.

Solubility :-

Paracetmol:- It is soluble in hydrochloric acid .

Diclofenac sodium:- It is soluble in sodium hydroxide

Storage:-

Store in tightly closed container, and reach out from the children,store

the tablet away from light , at room temperature or in the refrigerator.

Presentation:- FENAC PLUS - Blister of 10 Tablet.

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CHAPTER 2

12

OBJECTIVE

AND PLAN OF WORK

OBJECTIVE

SPECTROPHOTOMETRIC ANALYSIS OF PARACETAMOL &

DICLOFENAC SODIUM FORMULATION.

1) Plan of work

1. Determination of the partition coefficient.

2. λ max determination of Paracetamol.

3. λ max determination of Diclofenac sodium.

4. Preparation of calibration curve of Paracetamol.

5. Preparation of calibration curve of Diclofenac sodium.

6. Preparation of sample of paracetamol.

7. Preparation of sample of Diclofenac sodium.

2) Material & Method

1. Hydrochloric sodium 0.1 N .

2. Sodium hydroxide 0.1 N .

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3. Distilled water (H2O )

4. Paracetamol drug ( Pure form )

5. Diclofenac sodium.

CHAPTER 3

14

EXPERIMENTA

L

3.1 max determination:-

15

The uv spectrum of paracetamol and diclofenac sodium in solvent like methanol,0.1N

HCL ,0.1N NAOH . were recorded for a different concentration 10g ml. The spectrum of

paracetamol and diclofenac sodium was found to have good spectrum pattern and

maximum absorbance was obtained . Hence 0.1 N NAOH was selected as the solvent .

the maximum absorbance was found to 257 nm for the paracetamol and 252 for

the diclofenac sodium .

Fig.3.1.1 Spectrum of Diclofenac in NaOH

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Fig.3.1.2 Spectrum of Paracetamol in hydrochloric acid

3.2 Preparation of caibration curve of

1) Paracetamol:-

The pure compound of paracetamol 100mg dissolve in 100 ml 0.1N HCL .form the 10ml

take in 100ml volume metric falsk from this 10 ml take in 100 ml volume metric falsk to

make 10 mg/ ml .solution form this we have made different concentration solution line 2 mg

/ml,4 mg /ml,6 mg /ml,8 mg /ml,10 mg /ml.

The paracetamol was found to have maximum absorbance 257nm and hence it was

selected for further studies.

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2) Diclofenac sodium :-

Stock solution of diclofenac sodium of 1000mg/ml was prepared by taking 100mg of durg

dissolved and diluted to 100 ml with 0.1N NaOH.the above stock solution was suitably

diluted with NaOH to get concentration ,ranging from 2-10 g/ml and absorbance were

noted at 252nm and the overlain spectra were show in fig.3.2.2.

Table3.1 :- Data represent the absorbance of the different

concentration of paracetamol at 257.0 nm.

S no. Cocentration

Цg /ml

absorbance

1 1 1.008

2 5 1.625

3 10 1.756

4 15 1.757

5 20 1.758

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Table3..2 :- Data represent the absorbance of the different

concentration of diclofenac sodium in 252.0 nm.

S no. Cocentration

Цg /ml

absorbance

1 2 0.789

2 4 1.365

3 6 1.677

4 8 1.712

5 10 1.731

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Fig.3.2.1 Dilution of paracetomol

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Fig.3.2.2 Dilution of diclofenac sodium

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parcetomal standard graph

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1µg/ml 5µg//ml 10µg/ml 15µg/ml 20µg/ml

conc

Ab

s

Series1

Fig.3.2.3 Calibration graph of paracetamol in 257nm

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Dichlofinac sodium standard graph

0

0.2

0.4

0.6

0.8

1

1.2

2µg/ml 4µg/ml 6µg/ml 8µg/ml 10µg/ml

conc

Ab

s

abs

Fig.3.2.4 Calibration graph of diclofenac sodium in 252nm.

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3.3 prepartion estimation in sample solution of fenac plus :-

20 tablet of 250mg of paracetamol and diclofenac sodium and average weight. Was

calculated

1) sample solution of paracetamol:-

Weigh accurately powder sample equivalent to about 100mg of paracetamol carry out

the dry portion with three 25 ml portion of 0.1N HCL filtering through by sinterdred glass

funnel. Dilute the combined filterate to 100ml with acid dilute further appropriately with acid

got final concentration 100mg /ml .

paracetamol sample graph

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2 µg/ml 4µg/ml 6µg/ml 8µg/ml 10µg/ml

conc

abs Series1

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2) sample:-

The entire residue left after the extraction of paracetamol as described above is used for

estimation of diclofenac sodium. Transfer the residue left in the conical flask and the top

of sintered fannel.qualitatively to 100ml volumetric flask with the help of 0.1 N NAOH

make up the volume and carry out the further dilution appropriately with NAOH toget final

concentration of 100 g/ml.

diclofenac sodium sample

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2 µg/ml 4µg/ml 6µg/ml 8µg/ml 10µg/ml

conc

abs Series1

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RESULT AND DISCUSSION:-

On the basis of experimental data.The paracetamol showed maximum absorbance at

252nm in HCl and diclofenac sodium showed maximum absorbance at 257nm in

NaOH.The data of precision given on method is more accurate and an equal but little time

different from the standard value or calibration curve.

CHAPTER 4

26

BIBLIOGRAPHY

4.1 Bibliography

Douglas-A.-Skoog,-Donald M.West, and JamesF. Holler: Fundamentals of analytical

chemistry :Page No564-567 5th edition.

Gurdeep=R.chatwal,-Sham-K.ANAND:Instrumentalmethods_of_chemical

analysis:Page No. 2.149-2.151, 2.160-2.161,2.176-2.178: 5th edition.

27

J.denny-R.C,-Barnesj.d,Thomas M:Textbook of quantitative chemical analysis:Page

No. 278-279:6th edition.

Scientific & research Publication from , Indian Drug manufactures association :Indian

Drugs :Vol.44 : No 1 :January 2007 :Page No 13 .

Scientific & research Publication from , Indian Drug manufactures association :Indian

Drugs :Vol.44 : No 2 : February 2007 :Page No 145 .

Scientific & research Publication from , Indian Drug manufactures association :Indian

Drugs :Vol.44 : No 3 : March 2007 :Page No 209-210 .

Sharma B. K. Instrumental Methods of chemical analysis :Page No.S-46,S-52 :1st

edition.

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