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.

TYPES
There are main two types of chemical analysis.
1. Qualitative (identification)
2. Quantitative (estimation)
1. Qualitative
analysis is
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 analytical techniques 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 methods
a) volumetric or titrimetric methods
b) gravimetric methods
c) gasometrical analysis
2.Electrical methods
3.Instrumental methods
4.Biological and microbiological
1. 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 titrations
iii) Precipitation titrations
iv) Oxidation reduction titrations
v) 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 methods
Electrical 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 bioassays.
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 isA = log (I0/I) = Ecl
Where, A = absorbance
I0= intensity of light incident upon sample cell
I = intensity of light leaving sample cell
C = molar concentration of solute
L = 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 partsLight 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.
ii.
iii.

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.
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.
Turn the wavelength control knob to 265nm with blank solution to calibrate the
spectrophotometer.

Preparation of the solution:

i.
ii.
iii.
iv.

Prepare a stock solution of 0.1% salicylic acid by measuring 100mg of

salicylic acid and dissolving in 100mL acetate buffer.
From the above stock solution perform serial dilution containing 0.05%,
0.025%, 0.01%, 0.005%, 0.0025%, and 0.001%.
Label the test tubes from 1 to 6. Then add sufficient amount of acetate buffer
to make the volume up to 10mL.
Then prepare standard blank of 20mL acetate buffer.

Results summarised in following table:

Determination of absorbance
i.
ii.
iii.
iv.

Set blank solution in the cuvette and calibrate at wavelength of 290nm.

Insert cuvette containing the sample
Read and record the absorbance for all the solution
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 + 0.006
Where,
Y= Value from y-axis i.e. the absorbance
X= 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 cisalkenes 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
as high-pressure liquid chromatography), is a technique in analytical chemistry used
to separate the components in a mixture, to identify each component, and to quantify
each component. It relies on pumps to pass a pressurized liquid solvent containing
the sample mixture through a column filled with a solid adsorbent 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. Chromatography can be
described as a mass transfer process involving adsorption. 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 and temperature play 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), dipole
dipole 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
for quantitative analysis of the sample components. A digital microprocessor and
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 composition gradient in the mobile phase.
Various detectors are in common use, such as UV/Vis, photodiode array (PDA) or
based on mass 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) InstrumentationThe 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 solutionThe 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 solution

To 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) Linearity
Accurately 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) Specificity
To 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) Accuracy
The 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

as pharmaceuticals, foods, life sciences, environment, forensics, etc.
Common application areas in pharmaceutical analysis are:

Assay Related Substance Analytical Method Validation

Stability Studies

Compound Identification

Working Standards

Common 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 SPECTROSCOPY

AIM: 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 the Beer-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 given wavelength). 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. The radiation flux without 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)n

Where 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 procedures

Analysis 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 value
2) 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 non
polar), 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 (4
hydroxyphenyl) acetamide. Acetaminophen is both an analgesic and an
antipyretic.
Bayergeneric name is aspirin, chemical name is 2 acetoxybenzoic acid.
Aspirin acts as an analgesic, antipyretic, and an anti inflammatory,
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 (4
isobutylphenyl)propanoic acid. Works mostly as an anti inflammatory 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, silica plates, aspirin solution, watch glass, 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 of FTIR is 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. During FTIR analysis, a spot on the specimen is subjected to a
modulated IR beam. The specimen's transmittance and reflectance of the infrared
rays at different frequencies is translated into an IR absorption plot consisting of
reverse peaks. The resulting FTIR spectral pattern is then analysed and matched
with known signatures of identified materials in the FTIR library.

Working :
To understand the powerfulness and usefulness of FTIR spectrometer, it is essential
to have 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 radiation sources are inert solids that
are heated electrically to promote thermal emission of radiation in the infrared region
of the electromagnetic spectrum. The monochromator is 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.
Detectors are 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 Sample
i. 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 electrophoresis is 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/6r
Where- Electrophoretic mobility
Q-Net charge on the ion
r- Ionic radius of the solute
- Viscosity of the medium
POLY ACRYLAMIDE GEL ELECTROPHORESIS
Polyacrylamide 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 COOand 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 Gels
Gel %

30%
Acrylamide

Water

5x TBE

APS

TEMED

8%

3.2 ml

6.4 ml

2.4 ml

200 l

10 l

10%

4.0 ml

5.6 ml

2.4 ml

200 l

10 l

12%

4.8 ml

4.8 ml

2.4 ml

200 l

10 l

MATERIALS REQUIRED:
1. Mini gel Apparatus (Vertical)
2. D.C power supply with Cord
3. Test tubes
4. Eppendorfs
5. Micropipettes and tips
6. Gel storage bag
Reagents 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 buffer
Preparation of reagents:
1. Acrylamide stock (30%)
Acrylamide
29.2gm
Bisacrylamide
0.8gm
Distilled water
100ml
Dissolve the salts in 100 ml water
2. 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
ml

3. 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
current
14. 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.