”

Carried out at
IOL CHEMICAL AND PHARMACEUTICALS LIMITED
BARNALA, PUNJAB
Submitted in Partial Fulfillment of the requirement for the award of degree of

(Commercial Methods of Chemical Analysis)

Submitted by

Under the supervision of

Dr. Jaspal Singh
(Associate Professor, Department of Chemistry)

DEPARTMENT OF CHEMISTRY
GURUKULA KANGRI
VISHWAVIDYALAYA
HARIDWAR-249404 U.K. (INDIA)

2023- 2024
 CERTIFICATE

This is to certify that work incorporated in this project entitled

“INSTRUMENTATION AND CHEMICAL ANALYSIS OF ACTIVE PHARMACEUTICAL
INGREDIENT IBUPROFEN” has been carried out by
Anant Dubey s/o Mr.Surjeet Dubey (M.Sc. Chemistry IV semester) during 05-01-
2024 to 30-03-2024 at “IOL CHEMICAL AND PHARMACEUTICALS LIMITED,
BARNALA,PUNJAB” under my supervision for the fulfillment of the award of
Master Degree in Chemistry (Commercial Methods of Chemical Analysis ) from
Gurukula Kangri Vishwavidyalaya, Haridwar.

DATE: Dr. Jaspal Singh.

(Assistant professor)
 CANDIDATE DECLARATION

I hereby declare that the work being presented in the project report entitled
“INSTRUMENTATION AND CHEMICAL ANALYSIS OF ACTIVE
PHARMACEUTICAL INGREDIENT (API): IBUPROFEN” is an authentic record work
carried out by Anant Dubey during 05-01-24 to 30-03-24 at IOL Chemicals &
Pharmaceuticals limited, Barnala, Punjab, under the guidance of Dr. Jaspal Singh
Assistant Professor, Department of Chemistry, Gurukula Kangri Vishwavidyalaya,
Haridwar. It is being submitted for partial fulfill the requirement for the award of
the degree of Master of Science in Chemistry (Commercial Methods of Chemical
Analysis).

DATE: Anant Dubey

 ACKNOWLEDGEMENT
IOL Chemicals & Pharmaceuticals are a global renowned pharmaceutical
organization. It provides a well training program to the trainees. Working in IOL
Chemicals & Pharmaceuticals was a great experience for me.
It is matter of great pleasure and happiness to make and submit this project
report. During the course of completion of this project work, many persons have
assisted me and offered their support.
I want to express my thanks to Dr.R.K. Shukla, H.O.D., Department of Chemistry,
Gurukula Kangri Vishwavidyalaya, Haridwar, for his suggestions given during the
period of entire study.
I express my profound sense gratitude to Dr. Jaspal Singh, Associate Professor,
Gurukula Kangri Vishwavidyalaya, Haridwar for his valuable suggestions, advice and
encouragement from time to time.
I am grateful to Dr. UTTAM BAHADUR SINGH (Vice President, QC) Mr. Kushinder
Singh Chauhan (SGM,QC) Mr. RAJESH VERMA (AGM, QC), Mr. JEET PAL
SINGH (Manager, QC) Mr. DHANANJAY SINGH (Deputy Manager, QC) for
providing the facilities which were required for this work.
It is my privilege to acknowledge Mr. BASANT SINGH (Head H.R.) for providing
me the opportunity for this training.
Finally, I express my regards to my parents who inspired me throughout my
studies and completion.

Thanking you

Anant Dubey
 Contents

CHAPTER CHAPTER TITLE PAGE

NO. NO.

1 INTRODUCTION
1.1 ABOUT COMPANY
1.2 IBUPROFEN
1.2.1 HISTORY OF IBUPROFEN
1.2.2 MECHANISM OF IBUPROFEN
1.2.3 SIDE EFFECTS

2 ANALYSIS
2.1 INSTRUMENT USED

3 RESULTS
4 CONCLUSION
5 REFERENCES
INTRODUCTION

COMPANY PROFILE
IOL Chemical and Pharmaceutical is an API based pharmaceutical company with substantive
manufacturing capacities. These API portfolio covers various therepeutic categories such as
pain management ,anti diabetic,anti hypertensive and anti convulsant. IOL Chemicals and
Pharmaceuticals Limited (IOLCP) is a leading organic chemicals manufacturer and supplier. We
are Indian manufacturer of industrial chemicals & bulk drugs for over two decades. By
pursuing & implementing the highest standards of excellence in our
operations, we have nurtured our capabilities. IOLCP is the backword integrated company
producing all intermediates and key starting materials of ibuprofen. By delivering consistent
results have earned the admiration of customers and stakeholders alike.

1. We have built up our expertise responding to diverse customer requirements Environmental

policies.

2. Our products cater to the key industrial sectors of Textiles, Pharmaceutical

& Packaging
3. Sufficient teamwork & strong associations have guided us to success.
4. Accolades for our have come from the highest levels of power.
Through an unwavering focus on Quality, Commitment & Delivery, we have charted our way to
success in our operations and have won the admiration of our customers. Our success is built
on the strong pillars of innovation, quality, & dedicated customer service. By incorporating
these & other business strengths, we have boosted our capabilities to maintain the leading
edge in the industry & earn the loyalty of our customers.

A GLANCE ABOUT COMPANY:

● Company has been established on 29th September, 1986 as public limited company.

● World’s largest producer of IBUPROFEN.

● Among the largest producers of Ethyl Acetate in India.
● Second largest producer of Iso Butyl Benzene in World.
● USFDA approved Ibuprofen facility.
 ● Totally integrated plant, producing all key stating material (KSM) of Ibuprofen
in GMP environment.
● 17-Megawatt power generation plant for captive consumption.

Infrastructure
IOL Chemicals and Pharmaceuticals Limited is a major Indian manufacturer of active

pharmaceutical ingredient, organic chemicals and intermediates. IOLCP is founded in 1986, has

grown from a single product company to a major global player with a diversified multi product

facility and quality professionals. IOLCP’s success is based on its strengths in chemistry,

excellent facilities and absolutely dependable quality achieved with the help of highly qualified,

competent & committed men power. The company has built-up long-term relationships with

its business associates in India and worldwide by supplying quality products on schedule and

its commitment to ethical. The facility compiles fully with CGMA standards.

The company has two units: -

API Unit
This Unit is manufacturing IBUPROFEN as per IP / BP / USP / Ph.Eur. / JP/ Micronized grade
with installed Capacity of 10 MTPD.

Chemical Unit
This unit is manufacturing organic chemicals (Glacial Acetic Acid, Ethyl Acetate, Acetic
Anhydride, Chloro acetic Acid, Acetyl Chloride, Isobutyl Benzene). The unit is DCS (Distributed
Control System) based control system. The capacity of plant for Acetic Acid 240 MTPD, Ethyl
Acetate is 150 MTPD; Acetic Anhydride is 60 MTPD, Chloro acetic Acid 20 MTPD, Acetyl Chloride
16 MTPD, Isobutyl benzene 30 MTPD.
Awards & Recognitions

1. KOSHER Certificate: -

The IBUPROFEN is complying the LAWS OF KUSHRUTH and is process and manufactured

under HALACHA REGULATIONS. This is approved by Kosher Inspection service, India.

2. HALAL Certificate: -

IBUPROFEN is registered from the HALAL COMMITTE JAMIAT ULAMA-EMAHARASHTRA.

ISO 9001:2000, ISO 14001:2004 is registered from BSI.

3. ENERGY Awards: -

The IOL Chemicals and Pharmaceuticals Ltd. is awarded from Ministry of Power for Energy

savings in 2005, 2006, and 2007 in Chemical Sector.

4. SAFETY Awards: -

The IOL Chemicals and Pharmaceuticals Ltd. is awarded from Punjab State Safety in 2008
for largest reduction in frequency of accidents in chemical.
5. FINANCE Awards: -

Ministry of Commerce and Industry gives IOL Chemicals and Pharmaceuticals Ltd. as a

status of Star Export House.

ESTABLISHMENT OF VARIOUS PLANTS

1) 1986- Incorporation

2) 1991-Acetic acid plant

3) 1996-Ethyl Acetate Plant

4) 1999-Acetic Anhydride Plant2000-Ibuprofin Plant (Domestic Supplies)

5) 2006-Co-generation plant

6) 2007- Ibuprofen Plant

7) 2009- Isobutyl benzene (IBB), Monochloro acetic acid and acetyl chloride plant.

8) 2013-Multipurpose Plant-Pharma
9) 2012-Ibuprofen Unit-2

10) 2016-Commencement of Unit-3

11) 2017- Commencement of Unit-4 for Metaformin Hydrochloride

12) 2018- Commencement of Unit-5 for Clopidogrel Bisulfate form-II

13) 2019- Commencement of Unit-6 for Pantoprazole Sodium

14) 2020- Commencement of Unit-7&8

15) Commencement of new Plant i.e., FLU

VISION OF THE COMPANY

To be the most admired and valuable company in APIs/bulk drugs, Intermediates and specialty

chemicals globally.

MISSION OF THE COMPANY

To provide top quality prodcts in APIs, Intermediates and specialty chemicals through

continuous innovation and cutting-edge technology, with due regards to safetyand

environment.

QUALITY POLICY
The IOLCP is committed to be the centre of excellence in providing cutting edge products for

the chemical & pharmaceutical sector.

 ANALYSIS

ASSAY OF ALDEHYDE: -
Take sample accurately 1.0 gm to 1.5 gm in a conical flask and then take another conical flask
and add 2-3 drops of methyl orange and neutralize with 1N HCl and weigh accurately Hydroxyl
Ammonium Hydrochloride same as the sample and add 2-3 drops of Methyl Orange and to
neutralize add 1N NaOH then mix the solution in first conical flask and put it for 30 minutes
then titrate it with NaOH until the color is changes from pink to yellow.

ASSAY = (B.R. × 19.0 × NORMALITY OF NaOH)

Weight of the sample

Aim to obtain 35% HCL from the byproduct of Ibuprofen: -

PROCEDURE: - Take a 250 ml conical flask and add 50ml water and then add 2 ml of sample
and note the weight of the sample. Now add 2 drop of phenolphthalein indicator and titrate
with 1N NaOH and note the end point.

STRENGTH = B.R x N x 36.5 x W x 100

Ws x V x 1000

N=Normality of NaOH
W=Weight of the sample taken in gm.
Ws=Total weight of the sample.
V=Volume of the sample
 POLY ALUMINUM CHLORIDE (PAC): -
During the production of isobutyl aceto phenone (IBAP) some amount of by product is also

formed in the quencher unit of the IBAP plant which is nothing but poly aluminum chloride

(PAC). In quencher, quenching of the reaction mass is actually takes place which results in

formation of two layers, one is organic layer and the other is poly aluminum chloride layer.

Earlier this PAC from IBAP plant will be transferred to some other companies at low rate

because of lot of impurities present in it.

As poly aluminum chloride has a lot of applications in industry as well as in daily use. Therefore,
in order to sell that PAC at higher rate, plans are made to increase the concentration of poly
aluminum chloride by establishing a separate unit for it. Therefore, poly aluminum chloride
from IBAP plant is transferred to poly aluminum plant of the company where concentration of
poly aluminum chloride can be increased according to the sale specification set by the

authorities. As PAC is the result of hydrolysis of aluminum chloride which is an exothermic

reaction too. Therefore, concentration can be increased by input more aluminum chloride into
the poly aluminum chloride which we are getting from the IBAP plant. One of the major cause
of impurities in the poly aluminum is the presence of solvent tri chloro ethene in it due its water
solubility.

SOLUTION A:- Take sample 2-2.5 g of the sample in 250 ml of volumetric and make it with
water.

BLANK: -Take a conical of 250 ml and add 50 ml of EDTA and 2 ml of 1:12 nitric acid and now

A) boil it and then cool it and now add 10 ml of 2M SODIUM ACCETATE and 3 drop of xylenol orange
indicator and titrate it with zinc chloride and note down the and point reading

B) ALUMINA TEST: - Take a 250ml conical flask add 50 ml of EDTA and now add 2ml of
diluted 1:12 nitric acid and 10 ml of sample solution A. and boil it and cool then add 2M
sodium acetate and 3 drop of xylenol orange indicator. And now titrate it with 0.01M zinc
chloride.
 .

ALUMINA CONTENT = {(B.R-T.V)x 2.549 x Normality of Zncl2}

2 x Weight of Sample in gm

C) CHLORIDE TEST: -Take 25 ml of solution A in a 250 conical flask and 10 ml very diluted
nitric acid and mixed indicator .and now titrate it with 0.01 M mercuric nitrate and note the end
point.

Chloride Content = B.R x N x 35.5 x 100

W x 1000
BR= burette reading
N= normality of mercuric nitrate

W= weight of sample taken

D) SPECIFIC GRAVITY: -
We take a pycnometer and weigh it as empty then weigh with water and then weigh with
sample at the same temperature.
Calculation=
{wt. of the sample with pycnometer −wt. of empty pycnometer}

{(wt. of the water with pycnometer) −wt. of the pycnometer}

ASSAY OF SODIUM SULPHATE (Na2SO4): -

First we prepare 12% of BaCl2 solution and then we take 2 gm of sample in 250 ml of conical

flask and add 25 ml of Water and 2 ml of 36% conc. HCl and put it on hot plate at 1000C. for

40 minutes until the boiling is started then cool for small time and check precipitation by adding

a drop of BaCl2. After sometime again check precipitation until precipitation is stopped., then

take weight of empty Sintered Glass Crucible. Filter through this crucible using Vacuum pump
and dry at 1500C and weigh it..

% Assay = (0.608XWeight loss of sample X 100)

Weight of initial sample
 TEST BY TLC

Reagents: -

 n- Hexane (AR grade)

 1, 4-dioxane (AR grade)
 Methanol (AR grade)
 Triethylamine (AR grade)
Preparation of diluents: - Prepare a suitable quantity of a mixture of methanol and water in the ratio of
1:1 mix well.

Preparation of standard solution: -

Accurately weigh and transfer about 50 mg of Ibuprofen working standard/USP Ibuprofen RS to

a 100 ml volumetric flask. Dissolve in diluents and make up the volume with diluents.

Preparation of sample solution: -

Determine the average weight of tablets and crush them to a fine powder. Accurately weigh
and transfer powder equivalent to about 50 mg of Ibuprofen to a 100 ml of volumetric flask.
Add about 60 ml of diluents and sonicate for about 30 min. Make up the volume with diluents
and mix.Filter through 0.45 µm nylon membrane.

Result: - The principal spot obtained with sample solution is found to be concordant in position and size
to that obtained with standard solution.

LOSS ON DRYING (LOD)

Procedure: - Dry clean glass-stoppered, shallow weighing bottle at 105 + 2o C for 30 minutes.
Cool to room temperature in desiccators and take the weight accurately (W 1 g). Crush not less
than four tablets into fine powder and transfer accurately about 1 gram of sample into weighing
bottle by gentle sidewise shaking, distribute the sample as evenly as practicable to a depth of 5
mm. Take the weight accurately (W2 g). Place the loaded bottle in the oven, removing the
stopper and placing it also in the oven. Dry at temperature 105 + 2o C for three hours under
vacuum and open the oven, close the bottle promptly. Place it in desiccators; allow cooling to
room temperature and taking the weight accurately (W3 g).
 Formula used: -

(W₃-W1)
% loss on drying =
(W₂ – W1)

Weight of empty L.O.D bottle (W1) = 44.3509 g

Weight of sample taken = 1.0000 g

Weight of L.O.D bottle with sample taken (W2) = 45.3509 g

Weight of L.O.D bottle with sample after drying (W₃) = 45.3541 g

Calculation: -

% loss on drying = (45.3541 – 45.3509) X 100

(45.3509 – 44.3509)
= 0.0032 X 100

= 0.32%

Result: - The loss on drying of Ibuprofen is found to be 0.32 %

IDENTIFICATION

(A). By HPLC

The retention time of the major peak in the chromatogram of the sample solution corresponds
to that in the chromatogram of the standard solution, as obtained in the assay.

Result: -The retention time of the major peak in the chromatogram of the sample solution corresponds to that in
the chromatogram of the standard solution, as obtained in the assay.

BY UV
Preparation of standard solution: -

Accurately weigh and transfer about 10 mg of ibuprofen working standard to a 100 ml

 volumetric flask. Add about 50 ml of water and sonicate to dissolve. Make up the volume with
water and mix. Dilute 10 ml of this solution to 100 ml with water and mix. Filter through 0.45
µm nylon membrane filter.

Preparation of sample solution: -

5gm fine powder. Accurately weigh and transfer sample equivalent to about 10 mg of ibuprofen
to a 100 ml volumetric flask. Add about 50 ml of water and sonicate for 10-15 min with
intermittent shaking. Make up the volume with water and mix. Dilute 10 ml of this solution to
100 ml with water & mix filter through 0.45 µm nylon membrane filters.
Procedure: -

scan the std. and sample solution between 200 & 350 nm & record the spectra.
The ultra - violet adsorption spectrum of the standard solution and sample. Exhibit maxima at thesame
wavelength
Result: - The ultraviolet absorption spectrum of the standard and sample solutions exhibit maxima at the
ASSAY (BY HPLC)
Reagents: -
Tri ethylamine (HPLC grade)
Ortho phosphoric acid (AR
grade)

Acetonitrile (HPLC grade)

Preparation of buffer (pH 3.5): -

Add about 1 ml of Tri ethylamine in 1000 ml of water. Adjust the pH to 3.5+0.05 with dilute ortho
phosphoric acid. Filter through 0.45 µm nylon membrane filter.

Preparation of mobile phase: - Prepare a suitable quantity of mixture of buffer (pH 3.5) and acetonitrile in

the ratio of 70:30. Mixwell and degas.

Preparation of diluents: -

Prepare a suitable quantity of a mixture of water and acetonitrile in the ratio of 80:20. Mix well.

Preparation of standard solution: -

 Accurately weigh and transfer about 50 mg of Ibuprofen working standard to 50 ml volumetric
flask. Add about 20 ml diluents and sonicate to dissolve. Make up the volume with diluents and
mix. Dilute 5 ml of this solution to 100 ml with diluents and mix. Filter through 0.45 µm nylon
membrane filter.

Preparation of sample solution: - Transfer 10 intact tablets to a 100 ml volumetric flask. Add
about 50 ml of diluents and sonicate for about 20 minutes with intermittent shaking. Allow the
sample to attain room temperature and make the volume with diluents and mix. Dilute 5ml of
this solution to 100 ml with diluents and mix. Filter through 0.45 µm nylon membrane filter.

Note: - Standard and sample solutions are stable for at least 24 h at 50C.
Chromatographic parameters: -

Column: - Hypersil BDS C-18, 5 micro m (250 mm X 4.6

mm)Column oven temperature: - 400C

Flow rate: - 1.0 ml/min

Injection volume: - 20 micro

lit

Detector: - UV at 230 nm Runtime: - 12 min

Evaluation of system suitability: -

The system is suitability for analysis if and only if;

The tailing factor for ibuprofen peak is not more than 2.0.

The column efficiency determined from the IBUPROFN peak is not more than 2.0 %.

Make adjustments, if necessary, to meet system suitability parameters.

Procedure: -
Inject 20 micro liters of diluents, standard (5 times), sample solution (twice) into the
chromatograph and record the chromatograms.
 Note: - 1) The retention time of ibuprofen peak is about 7 min to 9 min.

2) To meet the retention time flow rate can be adjusted to + 50%.

Calculations: -

AT DS P 1
ibuprofen = ------ X ------ X ------ X ------ (% of

m/m) AS DT 100 C1

Where
AT = Average area counts of Ibuprofen

Peak in the chromatograms of sample solution.

AS = Average area counts of Ibuprofen

Peak in the chromatograms of standard solution as obtained under system suitability

DS = Dilution factor for standard solution.

DT = Dilution factor for sample solution.

P = Percent potency of Ibuprofen
Working standard, on as is basis.
C1 = Label claim of ibuprofen, in mg.

Note: – For 10 mg: Each 280 mg of powder contain 10 mg of ibuprofen

Result: - The assay of ibuprofen by HPLC is 95.12%.

INSTRUMENTS
USED
HPLC (HIGH PERFORMANCE LIQUIDCHROMATOGRAPHY)

SYSTEM SPECIFICATION: -

a) Integrated solvent and sample management to ensure consistent system-to-

system performance and high reproducibility.

b) Flexibility for analyzing a wide variety of sample type, under many different
conditions, with ease.

c) Wide range of detection capabilities.

d) Wide range of columns, sample preparation devices, and accessories.

 HPLC BASIC INSTRUMENTATION WATERS 2695
INTRODUCTION
High-performance liquid chromatography (sometimes referred to as high-pressure
liquid chromatography), HPLC, is a form of liquid chromatography to separate
compounds that aredissolved in solution.
HPLC instrument consists of a reservoir of mobile phase, a pump, an injector, a
separation column, and a detector. Compounds are separated by injecting a plug of
sample mixture onto the column. The different components in the mixture pass
through the column at differentrates due to differences in their partitioning
behavior between the mobile liquid phase and thestationary phase. When the
mobile phase has passed through the column it enters into thedetector that
detects the different molecules as they have pass through it. A signal goes from the
detector to a primer that presents the separation graphically.

PRINCIPLE
HPLC is based on the principle of separation. The separation depends on the relative affinity of
Compounds towards stationary and mobile phase. The compounds under the influence of
mobile phase (driven by capillary action) travel over the surface of stationary phase. During this
movement the compounds with higher affinity to stationary phase travel slowly while the others
travel faster. Thus, separation of components in the mixture is achieved.

TYPES OF HPLC

BASED ON MODE OF SEPARATION: -

A. Normal phase chromatography: - Also known Normal phase HPLC (NP-HPLC),
this method separates analytes based on polarity. NP-HPLC uses a polar
stationary phase and a non- polar mobile phase. The polar analyte interacted
with and is retained by the polar stationary phase. Adsorption strengths
increase with increased analyte polarity, and the interaction between the
polar analyte and the polar stationary phase increases the elution time.
 B. Reversed phase chromatography: - Reversed phase HPLC (RP-HPLC or RPC) has a
nonpolar stationary phase and an aqueous, moderately polar mobile phase. RPC
operates on the principle of hydrophobic interactions, which result from repulsive
forces between a polar eluent, the relatively non-polar analyte, and the non-polar

C. stationary phase: - The binding of the analyte to the stationary phase is proportional to
the contact surface area around the non-polar segment of the analyte molecule upon
association with the ligand in the aqueous eluent.

A. Polar: –polar bonds and non –polar –non polar bonds have more affinity than
Polar non polar bonds Reverse phase chromatography is more commonly used as
drugs are usually hydrophilic

D. Size exclusion chromatography: - Size exclusion chromatography (SEC), also called as

gel permeation chromatography or gel filtration chromatography mainly separates

particles on the basis of size. It is also useful for determining the tertiary structure and

quaternary structure of proteins and amino acids. This technique is widely used for

the molecular weight determination of polysaccharides.

E. Ion exchange chromatography: - In Ion-exchange chromatography, retention is based on

the attraction between solute ions and charged sites bound to the stationary phase. Ions

of the same charge are excluded. This form of chromatography is widely used in

purifying water, Ligand-exchange chromatography, Ion-exchange chromatography of

proteins, High-pH anion-exchange chromatography of carbohydrates and

oligosaccharides, etc.

BASED ON ELUTION TECHNIQUE

1. Isocratic elution

● A separation in which the mobile phase composition remains constant

throughout the procedure is termed isocratic elution.
 ● In isocratic elution, peak width increases with retention time linearly with the number
of theoretical plates. This leads to the disadvantage that late eluting peaks get very flat
and broad.

● Best for simple separations

● Often used in quality control applications that support and are in close proximity to a

manufacturing process.

2. Gradient elution

● A separation in which the mobile phase composition is changed during the

separation process is described as a gradient.

A. Gradient elution decreases the retention of the later – eluting components

so that they elute faster, giving narrower peaks. this also improves the peak
shapeand the peak height Best for the analysis of complex samples

B. Often used in method development for unknown mixtures

C. Linear gradients are most popular

BASED ON SCALE OF OPERATION: -

a) Analytical HPLC: - No recovery of individual components of substance.

b) Preparative HPLC: - Individual components of substances can be recovered.

BASED ON TYPE OF ANALYSIS

a) Qualitative analysis
● Analysis of a substance in order to ascertain the nature of its chemical constituents
● We can separate individual components but cannot asses the quantity in this analysis

b) Quantitative analysis
● Determination the amounts and proportions of its chemical constituents.

● Quantity of the impurity and individual components can be assessed.

A FLOW SCHEME FOR HPLC
MAIN COMPONENTS OF HPLC: -
I. Solvent Reservoir
II. Pump
III. Injection Port
IV. Column
V. Detector Data Acquisition System

I. SOLVENT RESERVIOR: -Solvent Reservoir is used to store Mobile-Phase. Scott Duran

bottles are commonly used as solvent reservoirs. The solvent reservoir must be made
of inert material such as glass and must be smooth so as to avoid growth of
microorganisms on its walls. It can be transparent or can be amber colored. A
graduated bottle gives a rough estimate of mobile-phase volume in the bottle. Solvent
reservoirs are placed above HPLC system (at higher level) in a tray. They should
never be kept directly above the system as any spillage of solvent on the system may
damage electronic parts of HPLC.

II. PUMP: -
The HPLC Pump is very important component of the system. The Pump delivers the
constant flow of the Mobile Phase or phases so that the separation of the components

of the mixture occurs in a reasonable time. There are two types of Pumping systems: -
Isocratic and Gradient.

III. INJECTION PORT: -

 The sample introduction device such as Injector to introduce the sample in a flow of
mobile phase at high pressure. It is not possible to use direct syringe Injection on

column like GC as the inlet pressure in LC is too high. The value injection through fixed

or variable loop is a common way of introducing the sample. The Rheodyne valve is the

mostly used device. The loop can be partially or fully filled. There are both the types of

injectors available. The advantage of partial filling is the possibility of using small

amount of sample, when there is scarcity of sample. The precision of the injection is

1 % RSD and carryover < 1%.

IV. COLUMNS AND RELATED STUFF: -

The HPLC column holds the stationary phase for separating the components of the

sample. The columns are usually made up of SS-316 grade steel. Apart from columns,

the material of construction of tubing and fittings, plumbing and connections are also

very critical. Apart from resistivity to corrosion, connections and plumbing should have a

very low dead volume.

DETECTORS: -Detectors detect various compounds as they elute out from column. The

detector give response in terms of a mili volt signal that is then processed by the

computer (integrator) to give us a chromatogram. Basically, detector consists of a flow

cell through which the mobile phase and resolved sample moves. Optics shine

through the detector cell and variation in optical properties are detected. A ultra violet

or UV detector detects absorbance of UV light by chromophores in the analyte

compound. A refractive index detector will sense variation in refractive index of mobile

phase stream passing through flow-cell as the sample/analyte mixed mobile phase

enters the detector. Similarly, Fluorescence detector checks for Fluorescence.

PARAMETER USED IN HPLC

1. Retention time (RT): - Retention time is the difference in time between the point of injection and
appearance of peak maxima. Retention time is the required for 50 % of a component to be eluted
from a column. Retention time is measured in minutes or seconds. Retention time is also proportional
to the distance moved on chart paper which can be measured in cm or mm.

2. Retention volume (VR)-: -Retention volume is the volume of mobile required to elute 50
%of the component from the column. It is the product of retention time and flow rate.

a. Retention volume = retention time X flow rate.

3. Baseline: -The portion of the chromatogram recording the detector response

when only the mobilephase emerges from the column.

4. Chromatogram: -
A graphical or other presentation of detector response or other quantity used as
 a measure of the concentration of the analyte in the effluent versus effluent volume ortime.
5. Chromatography: -
A dynamic physicochemical method of separation in which the components to be separated are
distributed between two phases, one of which is stationary (the stationary phase) while the
other (the mobile phase) moves relative to the stationary phase.it is also define as physical
method in which separation of components takesplace between two phase a stationary phase
and a mobile phase.
6. Detector: - A device that indicates a change in the composition of the eluent by measuring
physical or chemical properties [e.g.U.V/visible light absorbance, differential refractive index,
fluorescence, or conductivity]. If the detector’s response is linear with respect to sample
concentration, then, by suitable calibration with standards, the amount of a component may be
quantitated. Often, it may be beneficial to use two different types ofdetectors in series. In this
way, more corroboratory or specific information may be obtained about the sample analytes.
Some detectors [e.g., electrochemical, mass spectrometric] are destructive; i.e., they effect a
chemical change in the sample components. If a detector of this type is paired with a non-
destructive detector, it is usually placed second in the flow path.

7. Eluate:-
The portion of the eluent that emerges from the column outlet containing analytes in
solution. In analytical HPLC, the eluate is examined by the detector for the
concentration or mass of analytes therein. In preparative HPLC, the eluate is collected
continuously in aliquots at uniform time or volume intervals, or discontinuously only
when a detector indicates the presence of a peak of interest. These fractions are
subsequently processed to obtain purified compounds.

8. Eluent :- The mobile phase.

9. Stationary phase: - the substance on which adsorption of analyte (the substance on
to be separate during chromatography) takes place. It can be a solid, a gel, or a solid
liquid combination.

10. Mobile phase: -solvent which carries the analyte (a liquid or a gas)
11. Elute: -To chromatograph by elution chromatography. The process of elution may be stopped
while all the sample components are still on the chromatographic bed [planar thin-layeror paper
 chromatography] or continued until the components have left the chromatographic bed [column
chromatography].
12. Flow Rate The volume of mobile phase passing through the column in unit time.

13. Gradient: -
The change over time in the relative concentrations of two [or more] miscible solvent
components that form a mobile phase of increasing elution strength. A step gradient is
typically used in solid-phase extraction; in each step, the eluent composition is changed
abruptly from a weaker mobile phase to a stronger mobile phase. It is even possible, by
drying the SPE sorbent bed in between steps, to change from one solvent to another
immiscible solvent.
14. Injector (Auto sampler /Sample Manager): -
A mechanism for accurately and precisely introducing a discrete, predetermined
volume of a sample solution into the flowing mobile phase stream. The injector can be a
simple manual device, or a sophisticated auto sampler that can be programmed for
unattended injections of many samples from an array of individual vials or wells in a
predetermined sequence. Sample compartments in these systems may even be.

APPLICATION
The information that can be obtained using HPLC includes identification, quantification, and
resolution of a compound. Preparative HPLC refers to the process of isolation and purification of
compounds. This differs from analytical HPLC, where the focus is to obtain information about
thesample compound.

I. Chemical Separations It is based on the fact that certain compounds have different
migration rates given a particular column and mobile phase, the extent or degree of
separation is mostly determined by the choice of stationary phase and mobile phase.
II. Purification: - Purification is defined as the process of separating or extracting the
target compound from a mixture of compounds or contaminants. Each compound
showed a characteristic peak under certain chromatographic conditions. The migration
of the compounds and contaminants through the column need to differ enough so that
the pure desired compound can be collected or extracted without incurring any other
undesired compound.
III. Identification Generally assay of compounds are carried using HPLC. The parameters of
this assay should be such that a clean peak of the known sample is observed from the
chromatograph. The identifying peak should have a reasonable retention time and

should be well separated from extraneous peaks at the detection levels which the assay
will be performed. Other applications of HPLC: Other applications of HPLC includes
pharmaceutical applications: -
• Tablet dissolution study of pharmaceutical dosages form.
• Shelf-life determinations of pharmaceutical products
• Identification of active ingredients of dosage forms
• Pharmaceutical quality control

IV. Environmental applications

• Detection of phenolic compounds in Drinking Water
V. Identification of diphenhydramine in sedimented samples

• Bio-monitoring ofpollutant

VI. Forensics
• Quantification of the drug in biological samples.
• Identification of anabolic steroids in serum, urine, sweat, and hair
• Forensic analysis of textile dyes.
• Determination of cocaine and metabolites in blood

VII. Clinical
• Quantification of ions in human urine Analysis of antibiotics in blood plasma.
• Estimation of bilirubin and bilivirdin in blood plasma in case of hepatic disorders.
• Detection of endogenous neuropeptides in extracellular fluids of brain.

VIII. Food and Flavor

• Ensuring the quality of soft drink and drinking water.

 • Analysis of beer.
• Sugar analysis in fruit juices.
• Analysis of polycyclic compounds in vegetables.
• Trace analysis of military high explosives in agricultural crops.
(1) Assay of compound: - The total substances that are analyzed is present in

percentage.Formula used:
(2) a) As is basis: -
(Area of sample peak X wt. of standard X dilution of standard) X Potency of standard
(area of standard peak X wt. of sample X dilution of sample)
b) As dried basis: -
(area of sample peak X wt. of standard X dilution of standard) X Potency of standard
(area of standard peak X wt. of sample X dilution of sample)X(100-LOD)
c) As moisture basis: -
(area of sample peak X wt. of standard X dilution of standard) X Potency of standard
(area of standard peak X wt. of sample X dilution of sample)X(100-water content)

(3) Purity of compounds: -

It explains about the how much the sample is pure. it is routine analysis.

(4) Related substances (RS) in compounds: -

How many impurities are present in the sample shows the related substances in
which some are known impurities and some are unknown impurities. Because of
these impurities have lies nearly of substance sample wavelength.

(5) Reaction monitoring: -

It is the intermediate analysis of samples that is helpful for next step of reactions thatmeans it monitor the reactions
according to their analysis
AN OVERVIEW OF SOFTWARE OF WATER’S ALLIANCE

Fig. EMPOWER PRO

Fig. In hardware the programs that are in working and also the system
setup/display.

Fig. How the peak is integrated an overview

AN OVERVIEW OF HPLC OF AG
ILENTINFINITY MODEL 1260

Fig. HPLC of Agilent model infinity 1260

Fig. open lab software for Agilent HPLC

Fig. software of Agilent infinity model 1260
Differences between HPLC and GC

• HPLC is a more popular technique with broad range of applications and GC is used
on morespecific occasions.

S. SPECIFICATIONS GC HPLC
NO.

1 COLUMN CAPPILARY TYPE SILICA COLUMN

2 COLUMN LENGTH 25 TO 30 METER 15-25 CM

3 DETECTORS FID, TCD etc. PDA, UV VIS, RI etc.

4 PRSSURE LESS PRESSURE REQUIRED HIGH PRESSURE

REQUIRED (4000-6000 bar

5 TEMPERATURE HIGH TEMPERATURE LESS PRESSURE

REQUIRED (200-250 REQUIRED (30-50
DEGREE CELCIUS) DEGREE CELCIUS)

6 TYPES OF VOLATILE NON-VOLATILE

COMPOUNDS
DETECTED

7 MOBILE PHASE GAS LIQUID

 pH METER

SPECIFICATION: -

Model no: S220/S210 K Seven Compact pH/Ion

pH range: -2.000 to 20.000 pH resolution: User
definable: 0.001/0.01/0.1 pH relative accuracy:
+/-0.002 MV range: -2.000 to 2000.0
MV-resolution: User-definable:0.1/1

MV-relative accuracy: +/- 0.2

Temperature range: -30.0 to 130

Temperature accuracy 0c: +/- 0.1

Display: TFT Color 4.3 inch

Power supply: Ext power supply 9-12 V/10

Conc. range: 1.00 E-9 to 9.99E+9 Conc.

Accuracy: +/-0.5 %
INTRODUCTION ( pH – Potential Of Hydrogen)

The –ve logarithm of H ion conc. Is called pH. A pH meter consist two electrode one as glass
electrode and other is refrence electrode. A pH meter is an electronic instrument used to
measure the pH (acidity or basicity) of a liquid (though special probes are sometimes used to
measure the pH of semisolid substances). A glass electrode connected to an electronic meter
that measures and displays the pH reading.

pH = -log[H+]

INSTRUMENTATION

The pH probe measures pH as the activity of hydrogen ions surrounding a thin wall
glass bulb at its tip. The probe produces a small voltage (about 0.06 volt per pH unit) that is
measured and displayed as pH units by the meter.

The meter the meter circuit is no more than a voltmeter that displays measurements in pH units
instead of volts. The input impedance of the meter must be very high because of the high
resistance approximately 20 to 100m ohm of the glass electrode probes typically used with pH
meters. The circuit of a sample pH meter usually consists of operational amplifiers in an
inverting configuration, with a total voltage again of about – 17. The inverting amplifier
converts the small voltage produced by the probe (+ 0.05 volt/pH) into Ph units, which are then
offset by seven volts to give a reading on the pH scale.
 KARL FISHER AUTOTITRATOR

INTRODUCTION

Karl fisher titration is widely used method for determining the micro amount of water in a
variety of products. Since its invention by the German petroleum chemist Karl Fischer in the
1930’s, the iodometric titration method that bears his name has become an increasingly
popular analytical technique for quantifying water in a variety of industries. During this time,
Karl Fischer titration has evolved from an esoteric novelty to a widely used instrumental method
employed in research and development, production, and quality control.

The popularity of the Karl Fischer titration is due to several critical advantages that it holds

over other methods of quantifying water, including: -

• High accuracy and precision

• Selectivity for water

• Small sample quantities required

 • Easy sample preparation
• Short analysis duration

• Nearly unlimited measuring range (1 ppm to 100 %) Suitability for analyzing solids,
liquids, and gases.

• Independence of presence of other volatiles.

• Suitability for automation.

PRINCIPLE: -

The fundamental principle behind the Karl Fischer titration is based on the Bunsen
reaction between iodine and Sulphur dioxide in an aqueous medium shown below:

I2 + SO2 + 2H2O 2HI + H2SO4

Karl Fischer discovered that this reaction could be modified to be used for the determination of
water in a non – aqueous system containing an excess of Sulphur dioxide. He used a primary
alcohol (methanol) as the solvent, and a base (pyridine) as the buffering agent. So, the reaction
changed into:

py.I2 + py.SO2 + H2O + py 2py.HI + py.SO3

py = pyridine

BASIC INGREDIENTS OF KARL FISCHER REAGENT: -

Iodine I2

Sulfur dioxide SO2

Buffer Imidazole or pyridine

Solvent Methanol

MAIN PARTS OF KARL FISCHER AUTOTITRATOR: -

● Titration vessel.
● Two Pt electrodes.
● Magnetic stirrer.
 ● Piston (motor) burette for titrant and solvent component.
● Reservoir for titrant component.
● Burette tip of titrant and solvent burette.
● Stirring bar.
● Wash bottle.
● Display.
● Electrometric indication circuit.

PROCEDURE: -

Preparation for titration before the titration being carried out, some important things should be
considered. They are pH of the sample solution, standardization of Karl Fischer reagent and the
pre – treatment of titrant.

Select a proper pH range: Karl Fischer titration is sensitive to the pH and the rate of the reaction
depends on the pH value of the solvent or working medium. When pH is between 5 and 8, the
titration proceeds normally. However, when the pH is lower than 5, the titration

speed is very slow, on the other hand, when the pH is higher than 8, titration rate is fast, but
only due to an interfering etherification side reaction which produce water, resulting in a
vanishing end point. Thus, the optimal pH range for the Karl Fischer reaction is from 5 to 8, and
highly acidic or basic samples need to be buffered to bring the overall pH into that range.
Standardization of Karl Fischer reagent: Karl Fischer reagent decompose on standing. Because
decomposition is particularly rapid immediately after preparation, it is common practice to
prepare the reagent a day or two before it is to be used. All glassware must be carefully dried
before use, and the standard solution must be stored out of contact of air and must be
standardized against a standard solution of water in methanol or solid crystalline sodium titrate
dehydrate. Standardization is basically done in two steps as follows primary standardization of
Karl Fischer reagent. Secondary standardization of Karl Fischer reagent

Critical areas: -

• Ethanol HCL
• Ketone group

• Acidic sample
 MOISTURE ANALYSER

Instrument specification: -

Model no: - HE73

Memory: - 2.0

Repeatability: - 0.15% (2g sample) ,0.05%

(10g sample).Capacity: - 71g

Temperature range: - 50oC to 200oC.

Weight: - 4.1 kg

Certification /compliance: - CE ISO 9001 ISO 14001.

Display type: - Backlit

LCD. Readability: - 1

mg ,0.01% MC
 INTRODUCTION

Before presenting the various moisture measurements, it is important to define moisture

content. Moisture content is normally expressed as a percentage by weight of either total

product (wet basis) or dry product (dry basis).

Wet Basis Moisture Content: -
M = 100 x (Wet Weight – Dry Weight) / Wet Weight Dry Basis
Moisture Content: -
M = 100 x (Wet Weight – Dry Weight) / Dry Weight

From the above equations, wet basis moisture content cannot exceed 100%. Dry basis moisture
may exceed 100% and is a non-linear function. Moisture content may be determined by
numerous techniques. These may be divided into two major categories, primary and secondary
measurement. Primary Moisture techniques involve direct chemical determination of water
content, usually by extracting the moisture from the product.
All primary methods are destructive and time consuming. Primary methods are performed
offline, but are usually very accurate. Small sample size may not adequately represent bulk
product. The most common primary method is weight loss, in which a sample is weighed, dried
until no further weight loss and then re-weighed. Other methods include Karl-Fischer Titration.
The accuracy of all off-line primary methods is dependent upon laboratory instrument accuracy
and the skill of lab personnel. Since off-line methods require taking a product sample from the
process, the sampling method must provide consistent product samples for testing.
Secondary Moisture techniques measure a property of the variable (moisture) rather than the
variable directly. All continuous moisture analyzers utilize secondary measurement principles
and must be calibrated against a primary reference technique. They have the advantage of
continuous or rapid sampling measurement and may be used for real-time process monitoring
and control.

Calculation: -
 % MC = (W2-W3)/(W2-W1) X 100
Where

MC = Moisture Content

W1 = weight of Empty LOD Bottle (gm)

W2 = weight of Empty LOD Bottle +Sample (gm)

W3 = Total weight of after Drying (gm)

LOSS ON DRYING: -
Principle: - LOD stands for loss on drying. Most standard methods are LOD methods. LOD is a
method that determines the moisture content of a sample upon heating. The loss of weight is
interpreted as loss of moisture of the sample. when all the moisture is out of the sample the
weight of the sample no longer changes. The moisture content of a sample is then calculated by
comparing the initial sample weight to the dried or final sample weight.

Calculation same as of moisture analyzer only difference is we use LOD bottles in this case. And
put it in vacuum oven for 2 Hours.

M = 100 x (Wet Weight – Dry Weight) / Wet Weight

BASIC OF IBUPROFEN
 HISTORY OF IBUPROFEN

Ibuprofen was discovered as a form of prop ionic acid in 1961, how its name came is unknown.
It was made and discovered at the Boots Companying the 1960s and finally patented in 1961.
The Boots Company is now known as Boots UK Limited, a company known for leading the
development of pharmaceuticals.

The organic compound was first discovered by Stewart Adams along with his team: John
Nicholson, Andrew RM Dunlop, Jeffrey Bruce Wilson, and Colin Burrows.
Stewart Adams had dropped out of school at age 16 to work at a pharmacy. The Boots Company
continued to hold the patent until 1985 and the rights to the Ibuprofen market until 1986.
However, they did licensed Ibuprofen to be produced and sold by the two drug companies White
Hall Laboratories and Upjohn. The Whitehall Laboratories sold ibuprofen as Advil while Upjohn
sold it as Nuprin. Ibuprofen was first intended to be used to treat arthritis starting in the U.K.
(1969) and then going to the U.S. (1974). It was approved by the FDA in 1974.
As the industry for Ibuprofen grew many other companies branched off to creating other
versions of Ibuprofen, for instance Sterling Drugs created the product Midol.
In the end Ibuprofen won Stewart Adams an Order of the British Empire (a high honor in the

U.K.) as well as winning the Queen's Award for Technical Achievement for the Boots

Company.

SYNTHESIS OF IBUPROFEN
The synthesis of Ibuprofen was first done by the Boots Company when the drug was first
developed. First, they used Friedel-Crafts acetylation of the compound isobutyl benzene. This
resulted in a reaction between ethyl chloroacetate otherwise known as Darzens reaction. This
gave the Boots Company a Beta-epoxy ester. They then decarboxylated and hydrolyzed it to
become an aldehyde. They took this and reacted it with hydroxylamine which became oxime.
Then this was covered in nitrile and hydrolyzed to Ibuprofen.
SOME DETAILS OF IBUPROFEN

IUPAC NAME
(RS)-2-(4-(2-methylpropyl) phenyl) propanoic acid

Clinical data
Trade names Advil, Brufen, Motrin, Nurofen, etc.

Pharmacokinetic data

Bioavailability 49–73%

Protein binding 99%

Metabolism Hepatic (CYP2C9)

Half-life 1.8–2 h

Renal

Excretion

Identifier
Formula -C13H18O2 Mol. Mass -206.29 g/mo.
Physical properties

Density 1,03 gr/ml g/cm³

Melt. Point 76 °C (169 °F)

Ibuprofen has an antiplatelet effect, though relatively mild and somewhat short-lived compared
with aspirin or prescription antiplatelet drugs. In general, ibuprofen also acts as a
vasoconstrictor. Ibuprofen is a 'core' medicine in the World Health Organization's Model List of
Essential Medicines necessary to meet the minimum medical needs of a basic healthcare
system.

Ibuprofen was derived from propanoic acid by the research arm of Boots Group during the
1960sand patented in 1961. Originally marketed as Burfen, ibuprofen is available under a
variety of popular trademarks, including Motrin, Nurofen, Advil, and Nuprin. Generic
formulations are available as well.

MEDICAL USES: -
Ibuprofen is used primarily for fever, pain, dysmenorrheal and inflammatory diseases such as
rheumatoid arthritis. It is also used for pericarditis and patent ducts arteriosus.

DOSAGE: -

Ibuprofen has a dose-dependent duration of action of around four to eight hours, which is
longer than suggested by its short half-life. The recommended dose varies with body mass and
indication. A dose of 400 mg per dose and 1200 mg per day is considered the maximum amount
for over-the-counter use, although, under medical direction, the maximum amount for adults is
800 mg per dose or 3200 mg per day based on an individual's response and tolerance.

Unlike aspirin, which breaks down in solution, ibuprofen is stable, thus it can be available in
topical gel form, which is absorbed through the skin, and can be used for sports injuries, with
less risk of digestive problems. ibuprofen plasma concentration, time since ingestion, and risk of
developing renal toxicity inoverdose patients has been published.
 Ibuprofen Structure

Ibuprofen 3-D Structure

 RESEARCH DONE WITH IBUPROFEN

Japan has recently discovered Ibuprofen to be used as an anti-acne medication

because it is used as an anti-inflammatory. However, Ibuprofen is also
being used in some studies to cure Alzheimer's disease overtime. Most recently Ibuprofen has
been associated with lowering the risk of Parkinson's disease. By taking an Ibuprofen, a study
atHarvard shows, that 38% have a lower risk of developing Parkinson's disease. But the
researchis still being developed.

SIDE EFFACTS: -

I. Constipation

II. Diarrhea

III. Gas or Bloating

IV. Nervousness

V. Ringing in the ears

RESULTS
S.NO. Test Specifications Results

White to off-white, round, White to off-white, round,

1 Description deposed with ‘J’ on one deposed with ‘J’ on one
side and ‘10’ on the other. side and ‘10’ on the other.

The ultraviolet absorption The ultraviolet absorption

spectrum of the standard spectrum of the standard
2 Identification By U.V.
and sample solution exhibit and sample solution exhibit
maxima at same wavelength. maxima at same wavelength

3 38% to 42% 39.62%

Acid content

Water (%w/w) (By

4 KF Auto titrator) Not more than4% 0.03%

Loss on drying (LOD) The loss of drying (w/w) is lie 0.32%

5
between ≤0.5%

6 Assay (By HPLC) (95.0-105.0%) 95.12%

 Conclusion
I have gained valuable knowledge about the method development in IOL Chemicals and
Pharmaceuticals. The method development section is a very important section of Quality
Control department. The senior chemist sensually works in QC section. Various instruments
analyze some parameters. Thus, observation and calibration of instruments was also a valuable
experience. DEVELOPMENT OF METHOD is done so as to check the purity of raw material by it
with the particular standards available in the marks. In the present work the result of the raw
material Ibuprofen is within limits as per IP/U.S.P. It shows that the raw material can be used
for production and marketing respectively.
 REFERENCES

1. United states pharmacopoeia (USP’NF-33).

2. European pharmacopoeia (EP VOLUME-6).

3. Indian pharmacopoeia edition published by the controller of publication Delhi.

4. Instrumental methods of chemical analysis (VOGEL).

5. Instrumental methods of chemical analysis by G.R. Chatwal.

6. R.J. Hamilton and swell, introductions to HPLC, 2nd editions.

7. STP/SOP Of IOLChemicals & Pharmaceuticals.

8. Wikipedia.