Jhala Purvashaba S.

003601202940
“INDUSRIAL VISIT TO MAKSON PHARMACEUTICAL PVT.LTD”
A PROJECT REPORT SUBMITED

TO
DEPARTMENT OF CHEMISTRY
S M T S.J VARMORA BBA & BCA MAHILA COLLEGE
SAURASHTRA UNIVERSITY
WADHWAN CITY: - 3633001, GUJARAT (INDIA)
For the partial fulfilment for the degree of

BACHELOR OF SCIENCE
IN
CHEMISTRY
(SEM-6)
JHALA PURVASHABA SHAKTISNH
ENROLLMENT NO: 003601202940
UNDER THE GUIDANCE OF

MS. BANSI JANI ASHSISH MAKWANA
1
SMT SJ VARMORA MAHILA COLLEGE
003601202940
SMT S. J. VARMORA BBA & BCA MAHILA COLLEGE
WADHWAN CITY SURENDRANAGAR – 363001 GUJARAT
(INDIA)
PHONE NO. (02752)22313
WEBSITE: www.sjvarmora.com
CERTIFICATE
This is to certify that the work in corporate in the project report “MAKSON
PHARMACEUTICAL PVT.LTD” by JHALA PURVASHABA S. (003601202940)
carried out Department of chemistry, Smt. S.J.Varmora Mahila College, Saurashtra
University, comprises the result of independent and the Original work for the partial
fulfilment of the degree of Bachelor of science in chemistry.
Guided By,
MS. Bansi Jani

Assistant Professor
Smt. S.J. Varmora College
Gujarat, India.
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SMT S. J. VARMORA BBA & BCA MAHILA COLLEGE
WADHWAN CITY SURENDRANAGAR – 363001 GUJARAT
(INDIA)
PHONE NO. (02752)22313
WEBSITE: www.sjvarmora.com
DECLARATION
I JHALA PURVASHABA S. Student of Department of Chemistry, S. J. Varmora
BBA & BCA Mahila College , Saurashtra University here by under taken that the work
presented in the project report “MAKSON PHARMACEUTICAL PVT.LTD” comprises
the result. Independent and original work carried out. For the partial fulfilment of the degree
of B.SC in chemistry and not subjected to published / present elsewhere without prior
permission.
JHALA PURVASHABA S.
003601202940
Date:
Place:
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ACKNOWLEDGEMENT
I am very much encouraged in presenting my project work and entitled MAKSON
PHARMACEUTICAL PVT.LTD. It has been my humble and endeavour to fulfil whatever
is expected from the student of BACHELOR OF SCIENCE IN CHEMISTRY. It has an
opportunity that provides me a platform to put practice what I am learned in the by gone
years and give back to the society in my own best way. The successful completion of this
project has been credited many persons. This venture would not have possible without the
help of them, whom I would like to acknowledge for their persistent support.
“You want to do right thing and you want to do it for the right reason but if you
don’t have the right guidance you can never hit the right target.”
I am grateful to the god for the good health and well being that where necessary to
complete this project work. I am very much thankful to the owner and whole staff of
MAKSON PHARMACEUTICAL PVT.LTD. for giving me full co-operation during my
study for industrial visit.
I express my deepest sense of gratitude to principal sir Mr. NAYAN VASIYANI.
I wish to express my esteemed guide Ms. BANSI JANI and co-guide Ashish Makwana of S.J
Varmora BBA & BCA Mahila College Kadava Patidar Trust, Saurastra University Wadhwan
City for providing me with all the necessary facilities for the project work. I am extremely
thankful and indebited to both of them for sharing expertise and sincere and valuable
guidance and encouragement.
At last I am thankful to all my friends. Who helped me in the Preparation of Project
report.
JHALA PURVASHABA S.
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INDEX
CHAPTER- 1 INTRODUCTION
1.1 About the Company
1.2 History of the Company
1.3 Board Direction
1.4 Management Team
1.5 Company Journey
1.6 Mission, Vision, Value
1.7 Contact of Company
CHAPTER-2 EXPERIMENTAL SECTION

2.0 Product List
2.1 Product 1 & 2
2.1.1 General introduction
2.1.2 Raw Material
2.1.3 Requirement
2.1.4 Procedure
2.1.5 Reaction
2.1.6 Properties
2.1.7 Result
CHAPTER-3 CHARACTERISTIC SECTION

CHAPTER-4 APPLICATION SECTION
4.0 Application Section
4.1 Use
4.2 Chemical Use
4.3 Advantage
4.4 Disadvantage
4.5 Product Packaging
4.6 Price
4.7 Label Indentification
4.8 Chemical Area & Marketing Flow Chart
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CHAPTER 1
INTRODUCTION
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1.1 ABOUT THE COMPANY.

Processing line: Makson manufacture complete range of processing machines right from
cooking to forming of candies, toffees, lollipops, depositor candies, jellies & toffees and
many more quality machines at competitive cost.
Depositor line: Makson is the first confectionery machinery manufacturer in India who
builds the complete line for depositing hard candies that are clear and smooth.
Cooking: Makson offers two continues cooking system for hard candies. The coil cooker
and the fine-film cooker, depending on the recipe and final moisture content required.
Both offers vacuum extraction if required and can be fed automatically by Makson’s
mixing and dissolving systems.
Depositing:The cooked candy syrup is deposited at final moisture into rigid moulds,
fitted with spring loaded ejector pins. The moulds them travels the length of the cooling
tunnel and returns upside down to the ejection point, where the pins in each mould
impression is depressed, ejecting the sweets. Air is blown by multiple axial fans in a
contra flow direction to the mould movement for efficient cooling. The volume of air is
controlled by dampers.
Makson manufactures depositing line with various capacity like one row, two rows, three
rows & four rows. The capacity of each row is 250 kg/hr. with respect to size & and
candy shape.
The Makson depositing is fitted with flexible silicon rubber moulds for starchless
depositing of low boiled ingredients such as caramel, toffee etc. It deposits single colour,
two colours and center filled “one shot products like chocolate ‘Éclairs’.
Features:
1. High volume efficiency.
2. Continuous production.Labour saving.
3. Consistent high quality production of hard candy, butter scotch, jelly and milk boiled
candies.
4. Good shape control, low wastage percentage.
5. Clear product.Gives high wrapping efficiency.
6. Option of filled & unfilled, striped and various shapes available.
7. Output up to 1000 kg/hr.
1.2 HISTORY OF THE COMPANY

In 1926
The family owned confectionery business started in 1926, as a small, manually operated,
in-house confectionery products manufacturing unit for the local market.
The joint family worked together at a small-scale. Until later when a young man began to
feel the need to fulfil the market’s demands and manufacture for other companies as
well. This was a major step. And thus, the Makson group was formed.
1972
Later, in 1972, the founder & chairman of the company Mr. Dhanjibhai, who happens to
be a mechanical engineer, visited an exhibition at Pragati Maidan, New Delhi. There, he
came across a Double Twist-wrapping machine and decided to build confectionery
machinery. He started building his own machine just by viewing the image of the
machine in the catalogue and finally succeeded after putting in eight years of hard work.
1981
The chairman of the company Mr. Dhanjibhai was awarded "Self made Industrialist"
award in the year 1981 by the Vice President of India and Ex-Officio Chairman of the
Rajya Sabha Mr. Hidayat Ullah. After this award many other awards followed, such as,
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"The National Award for Import Substitution" in the year 1983 and "Export Award" by
the Government of India. He was also awarded the "LENCO Excellence Award" as "The
Best Industrialist & Philanthropist".
1983
Makson expanded as a contract confectionery manufacturer to renowned companies such
as Nestle, Cadbury's, Unilever, Hindustan Lever Limited, Ranbaxy, Cipla, Himalaya,
Boots Piramal, Knoll Pharma, Coca Cola, Blue Cross Labs, etc. Makson has a dedicated
manufacturing facility for P&G, Johnson & Johnson and GlaxoSmithKline. Johnson &
Johnson awarded Makson the "Contract Manufacturer of the Year Award" for 2
consecutive years, 1995 & 1996.
2004, Makson started manufacturing 100 various types of gumbases, of International
Standard for Chewing and Bubble Gum.
Today
Makson is the leading Confectionery Machine Manufacturer & also the largest Contract
Confectionery Manufacturer in India, having its branches at Bhopal & Hyderabad.
1.3 BOARD DIRECTION
1. Mr. Dhanjibhai Makasana Chairman & Founder of Makson Group & Makson
Foundation Trust
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2.Kalpesh Makasana
Director: Makson Pharmaceuticals (I) Pvt. Ltd, Makson Enginnering Export
Proprietor: Makson Export
Trustee: Makson Foundation Trust
3.Rajendra Patel
Director: Makson Industries Pvt. Ltd.
Managing Director: Makson Healthcare Pvt. Ltd
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4. Mayur Patel
Director : Makson Healthcare Pvt. Ltd (Unit 1 & 2), Makson Industries Pvt. Ltd.
1.4 MISSION
We strive for happier healthier tomorrow. Makson should provide total customer
satisfaction and achieve leadership in chosen market, product and services across the
glove, through excellence in technology, based on world class research and
development. Makson human resources will continue to be the most valuable asset in
this pursuit of leadership and the prime driving forces of hir/hes growth. Makson is
responsible to the society. Makson shall be good corporate citizen and will e driven
height ethical standard in our practices.
VISION
We will make heated efforts for the fulfilment of this objective through our
interaction and interdependence with them. We will work and contribute and grow
together in the spirit of Saha Viryam. This is our resolutions and we resolve so we
pray to the almighty fulfill our mission.
1.5 CONTACT OF THE COMPANY

Makson Group of Companies
Rajkot Highway, Surendranagar Gujarat, INDIA, Pincode -363020
Mobile no: +91-2752-289999
Email: makson@makson-group.com
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CHAPTER 2
EXPERIMENTAL SECTION
2.0 PRODUCT LIST
A. Hajmola Maha Candy
B. Nutrine Maha Lato
2.1(A) HAJMOLA MAHA CANDY
2.1.1 GENERAL INTRODUCTION
About Dabur Hajmola Maha Candy Albela Aam and Chulbuli Imli Mix Dabur
Hajmola Maha Candy contains mixed candies of both the flavours Aam and Imli. The
Candy's Khatta Meetha taste is available in two different fun-filled flavours - Albela
Aam and Chulbuli Imli as ingredients. It can help in improving digestion and soothing
other common stomach ailments.
2.1.2 RAW MATERIAL
Amra
Imli
Shunthi
Pippali
Samudra Lavana
Sauvarchala Lavana
Maricha
Pudina
Dhanyaka
Sharkara (Sugar)
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2.1.3 REQUIREMENT
A. Tray feeding
B. Dumping tray-starch filling-stamp moulds
C. Depositor
D. Tray outlet
2.1.4 PROCEDURE
Chocolate manufacturers must follow a set of guidelinesand quality criteria
if they are to produce products that maintain the consumers’ loyalty to their
products. Before processing, the quality of beans is evaluated using two
different methods. With the first technique, the beans are assessed for the
following indicators:
1. Degree of fermentation
2. Moisture content (maximum 6%)
3. Number of defects
4. Number of broken beans
5. Bean count (number per 100 g)
6. Degree of mouldiness
7. Flavour profile
8. Colour
9. Fat content (minimum 52%)
10. Fat quality relating to percentage of free fatty acids (asoleic acid)
11. Shell content (10–12%)
12. Uniformity of bean size
13. Insect and rodent in the second technique is evaluated based on the size
of the beans using either bean count (Number of beans per 100 g) or the
weight in grams of 100 beans. On the international cocoa Market,
different bean sizes attract different prices. Beans with smaller sizes usually
contain proportionately lower amount of nibs, higher shell content, lower
fat content and attract lesser prices. Typically, beans from Asian origin
have higher shell content than beans from West Africa.
2.1.5 REACTION
Chocolate’s antioxidant potential may have a range of health benefits. The
higher the cocoa content, as in dark chocolate, the more benefits there are.
Dark chocolate may also contain less fat and sugar, but it is important to check
the label.
Eating chocolate may have the following benefits:
1. Lowering cholesterol levels
2. Preventing cognitive decline
3. Reducing the risk of cardiovascular problems
2.1.6 PROPERTIES
A. Chatpata tablets, fun to have at any time of the day.
B. Improves digestion for kids and adults.
C. Useful in cases of indigestion, loss of appetite & flatulence.
D. Great way to enjoy while staying healthy.
E. Hajmolas Chatpata richness is good for a change of taste.
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2.1.7 RESULT
Brand Hajmola maha candy

Product Type Chocolate
Product Model Maha candy
Ingredients Type Vegetarian
Allergens Contains Soy, Wheat and Barley
Dimension (LxWxH) 21.5 x 11.5 x 7 cm
Weight 2875g
Warranty As Per Manufacturer Terms and Condition
Other Details
Dimension (CM) - L x B x H
Unit of Measurement
Weight 2.575 kg
Generic Type Chocolates
2.2 (B)NUTRINE MAHA LACTO
2.2.1 GENERAL INTRODUCTION

While other candy brands are downsizing due to cost pressures, Nutrine
Mahalacto has increased its size by 20 per cent by adding more milk and
caramel into the candy. And to communicate a larger sized candy to its
consumers, Nutrine Mahalacto didn’t take the usual offer route.The candy
brand from Godrej Hershey Ltd has come out with a new TVC that brings
alive the brand as a Mahalacto boy who loves milk and has milk on the sly. He
is a naughty and fun character who is adept at pole vaulting. He once
accidentally falls into a bowl of milk and to avoid drowning drinks off the
entire bowl of milk through a straw. Commenting on the the launch of
Mahalacto, Mahesh Kanchan, VP & Marketing Head of Godrej Hershey Ltd,
said, “In today’s time of high inflation, when all players have taken down the
size of their candies and chocolates, we have done exactly the opposite by
adding more milk and caramel to our Nutrine Mahalacto and have made it 20
per cent bigger and tastier. The creative trigger was ‘bigger is better & tastier’
when it comes to a candy. Consumers love Mahalacto for its rich milky taste
and we had to accentuate the fact that we have added more of what they love.
The simple big idea was to bring Mahalacto alive as a character and make this
little character perform and speak to the consumers.”The creative duo of Priya
Pardiwalla, VP & Senior Creative Director, and Steve Mathias, AVP & Senior
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Creative Director at JWT India, has co-written this film. Pardiwalla said, “For
us, this was a great opportunity to create a strong property for Mahalacto,
which goes beyond just the film. We literally decided to bring the candy to
life.
2.2.2 RAW MATERIAL

A. Sugar,
B. Liquid glucose,
C. Full cram condensed milk,
D. Milk fat,
E. Malt extract,
F. Wheat
G. Salt and emulsifier.
2.2.3 REQUIREMENT
A. Tray feeding
B. Dumping tray-starch filling-stamp moulds
C. Depositor
D. Tray outlet
2.2.4 PROCEDURE
Chocolate manufacturing processes generally share common features such

as:
(1) Mixing
(2) Refining
(3) Conching of chocolate paste
(4) Tempering and depositing
(5) Moulding and demoulding
The outcome sought is smooth textures of products considered desirable
in modern confectionery and elimination of oral perceptions of grittiness.
Mixing
Mixing of ingredients during chocolate manufacture is a fundamental
operation employed using time–temperature combinations in a continuous
or batch mixers to obtain constant formulation consistency. In batch
mixing, chocolate containing cocoa liquor, sugar, cocoa butter, milk fat
and milk powder (depending on product category) is thoroughly mixed
normally for 12–15 minutes at 40– 50◦C. Continuous mixing is usually
used by large chocolate manufacturers such as Nestl´e and Cadbury using
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well-known automated kneaders, producing somewhat tough texture and

plastic consistency.
Refining
Refining of chocolate is important to the production of smooth texture
that is desirable in modern chocolate confectionery. Mixtures of sugar
and cocoa liquor (and milk solids depending on the type of chocolate)
at an overall fat content of 8–24% are refined to particle size of less than 30
μm normally using a combination of two- and five-roll refiners. Final
particle size critically influences the rheological and sensory properties.
Conching
Conching is regarded as the endpoint or final operation in the manufacture of
bulk chocolate, whether milk or dark product.
Tempering
Uncontrolled crystallization of the chocolate is called Tempering. It is the final
process.
2.2.5 PROPERTIES
a) Chocolate is believed to contain high levels of antioxidants.
b) Some studies have suggested chocolate could lower cholesterol levels
and prevent memory decline.
c) Chocolate contains a large number of calories.
d) People who are seeking to lose or maintain weight should eat chocolate
only in moderation.
2.2.6 REACTION
 Chocolate receives a lot of bad press because of its high fat and sugar
content. Its consumption has been associated with acne, obesity, high
blood pressure, coronary artery disease, and diabetes.
 However, according to a reviewTrusted Source of chocolate’s health
effects published in the Netherlands Journal of Medicine, it’s not all bad
news.
 The authors point to the discovery that cocoa, the key ingredient in
chocolate, contains biologically active phenolic compounds.
 This has changed people’s views on chocolate, and it has stimulated
research into how it might impact aging, and conditions such as
oxidative stress, blood pressure regulation, and atherosclerosis.
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2.2.7 RESULT
Yummier than yummy, so creamy,get ready for Maha fun and Maha flavour.
This is a 100% vegetarian product.
Brand Nutrine
Product Type Chocolate
Product Model Maha Lacto
Ingredients Type Vegetarian
Allergens Contains Soy,Wheat and Barley
Dimension (LxWxH) 21.5 x 11.5 x 7 cm
Weight 2875g
Warranty As Per Manufacturer Terms and Condition
Other Details
Dimension (CM) - L x B x H
Unit of Measurement
Weight 2.875 kg
Generic Type Chocolates
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CHAPTER 3
CHARACTERISTICS
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
INTRODUCTION
Chromatography – is an analytical technique whereby a sample is s an analytical
technique whereby a sample is separated into its individual components separated into its
individual components. During separation the sample components are distributed between a
stationary phase and a mobile phase. After separation the components can be quantified and
even identified.
High Performance Liquid Chromatography (HPLC) is a process of separating

components in a liquid mixture. A liquid sample is injected into a stream of solvent (mobile
phase) flowing through a column packed with a separation medium (stationary phase).
Sample components separate from one another by a process of differential migration as they
flow through the column.
As bands emerge from the column, flow carries them to one or more detectors which
deliver a voltage response as a function of time. This is called a chromatogram. For each
peak, the time at which it emerges identifies the sample constituent with respect to a standard.
The peak’s area represents the quantity.
PRINCIPLE
The separation principle of HPLC is based on the distribution of the analyte (sample)
between a mobile phase and a stationary phase (packing material of the column). Depending
on the chemical structure of the analyte, the molecules are retarded while passing the
stationary phase. The specific intermolecular interactions between the molecules of a sample
and the packing material define their time “on-column”. Hence, different constituents of a
sample are eluted at different times. Thereby, the separation of the sample ingredients is
achieved.
A detection unit (e.g. UV detector) recognizes the analytes after leaving the column. The
signals are converted and recorded by a data management system (computer software) and
then shown in a chromatogram. After passing the detector unit, the mobile phase can be
subjected to additional detector units, a fraction collection unit or to the waste. In general, a
HPLC system contains the following modules: a solvent reservoir, a pump, an injection
valve, a column, a detector unit and a data processing unit (Fig. 1). The solvent (eluent) is
delivered by the pump at high pressure and constant speed through the system. To keep the
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drift and noise of the detector signal as low as possible, a constant and pulseless flow from
the pump is crucial. The analyte (sample) is provided to the eluent by the injection valve.
WORKING
How does high-performance liquid chromatography work? Let’s get into the process that makes
HPLC what it is.
Mobile Phase
In this beginning stage, the solvent, also known as the mobile phase, will remain in a reservoir. That
is, until the high-pressure pump on HPLC machines pushes the solvent through to mix with the liquid
sample in testing. Once the solvent and sample are mixed, they enter the next phase through the
HPLC column.
Stationary Phase
The HPLC column, also known as the stationary phase in the process, is where the separation of the
mixture occurs. The inside of the column contains absorbent particles that help distinguish each
compound during separation.
Compound Measurement
Once the mixture separates the compounds within the column, the machine measures the compounds
by a detector based on their reaction to the solvent. Depending on the compound, lab technicians may
need different types of detectors based on the electrical signal provided. For example, if the
compound results in a fluoresce, they will need a fluorescence detector. Based on the results gathered
by the detector, the lab technician can make an educated decision about their tested sample.
Two HPLC Phases
First, there’s normal phase HPLC, which primarily measures polar compounds by using non-polar
solvents. The key distinction with normal-phase HPLC is that polar compounds tend to remain in the
column longer while non-polar compounds will pass through. The next type is known as reverse-
phase HPLC, the most common method of HPLC. Unlike normal-phase HPLC, the solvent here is
polar instead of non-polar. In reverse-phase HPLC, the non-polar compounds remain in the column
longer while polar compounds pass through faster.
High-performance liquid chromatography is a favoured method in several types of labs because of its
versatility across industries. In general, HPLC separates mixtures using a liquid mobile phase and a
column stationary phase. Within the column the mixture separates because of particles that absorb
certain compounds in its most basic sense. Once separated, a detector measures the compounds based
on their reaction to the solvent, which will become data.
HPLC Applications
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High-performance liquid chromatography is used for various applications across many different
industries that affect our daily lives. The most common applications and industries for HPLC analysis
include:
 Cannabis testing.
 Clinical research.
 Forensic & Toxicology research.
 Pharmaceuticals.
 Environmental testing.
 Food & beverage testing.
 Flavour & fragrance.
HOW DOES HPLC WORK?
ADVANTAGES
 Controls and automates chromatography instrumentation.

 Provides data management, security features, and reporting and instrument validation.
 Powerful and adaptable.
 Increases productivity by managing all the areas of analysis - from sample to instrument,
and from separation to reporting results.
 Affordable.
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DISADVANTAGES
 Cost.
 Complexity.
 Low sensitivity for some compound.
 Irreversibly adsorbed compound not detected.
 Co-elution difficult to detect.
APPLICATION FOR HPLC
HPLC can be used to analyze or separate many different mixtures. It is used as an analytical
tool in research to qualitatively analyze samples, identifying what molecules are present, and
quantitatively to identify how much of a compound is present. This means it can be used in
chemical, biochemical, biological, and medical applications to identify and measure specific
compounds in a laboratory setting. The advent of mobile units has meant that fieldwork can
be carried out as well. For example, field analysis of hemoglobin in Africa to detect sickle
cell anemia.
It is also a useful tool in testing for pesticides, herbicides, fungicides, pharmaceutical

products, illegal food additives, toxins, and other organic contaminants. It can also be used to
detect illegal or performance-enhancing drugs in humans.
With increasing developments in detection and recording equipment and the development of
more sophisticated media columns, the potential uses for HPLC are likely to continue to
grow.
APPARATUS AND REQUIREMENTS
 A Solvent Reservoir.
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 A Pump.
 An Injection.
 A Column.
 A Detector unit.
 A Data Processing unit.
ULTRA PERFORMANCE LIQUID CHROMATOGRAPHY (UPLC)
INTRODUCTION
UPLC is a special version of HPLC which has the advantage of technological strides made in
particle chemistry performance, system optimization, detector design, data processing and
control. These achievements lead to a very significant increase in resolution, sensitivity and
efficiency with faster results and less consumption of solvents which lowers the cost and
make the technology environment friendly.
To achieve the above targets, Waters in 2004, launched and trademarked Ultra Performance
Liquid Chromatography (UPLC) which is based upon small, porous particles (sub 2micron
particles). Van Deemter equation is the principle behind this evolution which correlates the
connection between linear velocity and plate height. The small particles require a high
pressure to work with UPLC i.e., 6000 psi which is typically the upper limit of conventional
HPLCs. It was observed that when the particle size is decreased below 2.5 µm, there is a
remarkable increase in the effectiveness and this effectiveness does not lessen on increasing
the linear speed or rate of flow. The use of speed, particles with a small radius and maximum
number of resolvable peaks (peak capacity) comprehends the efficiency together with
resolution. This method reduces the mobile phase volume consumption by at least 80%
compared to HPLC with a shorter runtime of about 1.5 min. The smaller sized particles
increase the pressure up to 1000 bars or more which can alone increase the retention factor of
the separation. Lower injection volume is required for UPLC which results in higher
efficiency and increase in resolution. The higher column temperature reduces the mobile
phase viscosity resulting in the high diffusion coefficient and flow rate without significant
loss in efficiency and increase in column back pressure.
PRINCIPLE
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The underlying principle of UPLC is based on the van Deemter relationship

which explains the correlation between flow rate and plate height. The van Deemter equation
(i) shows that the flow range with the smaller particles is much greater in comparison with
larger particles for good results.
H a 1/w
(i)
Where H represents height equivalent to the theoretical plate (HETP), A, B & C are the
constants and v is the flow rate (linear velocity) of the carrier gas. The aim is to minimize
HETP to improve column efficiency. The term A does not depend on velocity and indicates
eddy mixing. It is smaller if the columns are filled with small and uniform sized particles.
The term B denotes the tendency of natural diffusion of the particles. At high flow rates, this
effect is smaller, so this term is divided by v. The term C represents the kinetic resistance to
equilibrium during the process of separation. The kinetic resistance is the time lag involved in
moving from the mobile phase to the stationary phase and back again. The higher the flow
rate of the mobile phase, the more a molecule on the packing material inclines to lag behind
molecules in the mobile phase. Thus, this term is inversely proportional to linear velocity.
Consequently, it is likely to enhance the throughput, and without
affecting the chromatographic performance, the separation can be speeded up. The
emergence of UPLC has necessitated the improvement of existing instrumentation facility for
LC, which takes the benefit of the separation performance (by decreasing dead volumes) and
consistent pressures (about 500 to 1000 bars, compared with 170 to 350 bars in HPLC).
Efficiency is proportionate to the length of the column and inversely proportional to the
radius of the particles. Consequently, the column length can be reduced by the similar factor
as the particle radius without affecting the resolution. The use of UPLC has helped in the
detection of drug metabolites and enhancement of the quality of separation spectra.
WORKING
1. UPLC systems operate under very high pressure to accommodate the use of small
particles.
2. This technology allows for faster flow rates while maintaining the separation
efficiency and thus increasing the sample throughput of analysis.
3. As an alternative to wholly porous sub-2-μm particles, 1.7-μm fused-core particles
surrounded by a 0.5-μm porous silica layer with 90 Å pores, have emerged.
4. In a comparative study, substantially lower back pressure was reported when the
fused-core particles were used.
5. This allowed for columns to be coupled in series, which increased the peak efficiency
up to 92,750 plates.
6. Wider-pore fused-core particles have an average pore diameter of 160 Å.
7. The wider-pore particles are particularly useful for increased sample loading and the
rapid separation of peptides using volatile mobile phases.
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HOW DOES UPLC WORK?
INSTRUCTION
 When performing fast analyses, note that a peak of interest can be as narrow as 0.5
seconds.
 Waters recommends a sampling rate of 25 to 50 point across the peak, which provides
good quantitation and peak representation.
 Sampling rates faster than 20 points per peak yields higher baseline noise and filter
constants should be adjusted accordingly.
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ADVANTAGES
 It is more selective and sensitive with high resolution performance and faster resolving
power.
 It also reduces process cycle time and assures end-product quality with reduced cost of
operation and decreased run time.
 It increases sensitivity and provides quick analysis through the use of a novel column
material of very small particle size.
 It decreases the consumption of solvent and increases sample throughput and also
provides real-time analysis in step with manufacturing processes.
DISADVANTAGES
 A major disadvantage of UPLC is the higher back pressures compared to conventional

HPLC which decreases the life of the columns.
 Increasing the column temperature reduces the back pressure problem in UPLC.
 Moreover, the particles of less than 2 μm are mostly non-regenerable and, therefore, have
a narrow use.
APPLICATIONS
Determination of Pesticides in Groundwater
UPLC coupled with triple quadrupole tandem mass spectrometry (UPLC-MS/MS) can be
utilized to determine the trace level pesticides in groundwater in less time and speedy
manner. The technique has enhanced the analysis speed, sensitivity, and resolution.
Improved Resolving Power in Peptide Maps
Peptide mapping is an essential technique for the characterization of proteins. Due to

exceptionally reduced instrument and column dispersion, the analyzes of tryptic digest of
phosphorylase by UPLC technology provides significantly improved resolution, peak
capacity, and sensitivity compared to HPLC, allowing the detailed characterization of the
protein.
Rapid Dose Formulation Analysis
Nowadays, the use of UPLC together with UV and MS detection has been widely utilized in
pharmaceutical applications. Several commercial drug formulations were used as models to
study the efficiency of separations with the change of flow rate. The efficiency was judged on
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the parameters of resolution, theoretical plates, column ruggedness, retention time, and peak
area. For example, mefenamic acid and chloramphenicol separation was studied in
dimethylacetamide/ polyethylene glycol-200 vehicle.
Analysis of Traditional Chinese Medicines (TCM)
The identification and quantification of components of TCM by chromatographic analysis is

one of the major challenges. TCM is a complex matrix in which all the constituents play a
specific role for the overall efficacy. Therefore, the analysis of all the constituents is
synchronously necessary for the quality control. The new technique UPLC is used for the
quality control of the TCM.
Multi-Residue Analysis of Pharmaceuticals in Waste Water
The water used in the pharmaceutical companies is found to have the traces of various
cholesterol-lowering statin agents, anti-ulcer agents, antibiotics, beta-blockers, analgesics,
anti-inflammatory agents, lipid regulating agents, psychiatric drugs, and histamine H2
receptor antagonists. UPLC coupled with Q-TOF-MS is used to confirm and screen these
drugs in the samples of waste water treatment plant.
Identification of Metabolites
The identification and detection of all the possible metabolites of the candidate drugs for the
discovery of new chemical entities is a very important step. For the identification of the
metabolites, a high sample throughput is required to be maintained by the analysts to provide
quick results to the medicinal chemists. UPLC-MS/MS is helpful in biomarker discovery as it
meets tough analytical requirements and provides sensitivity, mass accuracy, dynamic
range, and resolution.
In Manufacturing / Quality Assurance (QA) / Quality Control (QC)
Identification, quantification, purification, efficacy and safety are key parameters to be

evaluated during manufacturing of a drug product and pharmaceutical dosage form. Material
stability is also observed as a component of QA and QC. UPLC is used as an important tool
in QA/QC laboratories for the quantitative and extremely regulated analysis.
Impurity Profiling
Impurity profiling should be efficient for consistent detection and separation of all the
impurities present in the active compound. The drug development and formulation process
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demand accurate measurement/testing of low-level impurities present with the active

pharmaceutical ingredients or the excipients or the raw materials used in the preparation of
the final product. Thus, the presence of excipients in the sample makes the profiling difficult
and with HPLC method, it takes longer time for analysis to achieve sufficient
resolution. Thus, the combination of UPLC with mass spectrometry has been useful for the
documentation of drug and endogenous metabolites in the final product.
APPARATUS AND REQUIRMENTS
A. Sample Injection.
B. UPLC Columns.
C. Detectors.
A. A. Sample Injection
In UPLC, sample introduction is critical. Conventional injection valves, either automated or

manual, are not designed and hardened to work at extreme pressure. To protect the column
from extreme pressure fluctuations, the injection process must be relatively pulsefree and the
swept volume of the device also needs to be minimal to reduce potential band spreading. A
fast injection cycle time is needed to fully capitalize on the speed afforded by UPLC, which
in turn requires a high sample capacity. Low volume injections with minimal carryover are
also required to increase sensitivity. There are also direct injection approaches for biological
samples.
B. B. UPLC Columns
Resolution is increased in a 1.7μm particle packed column because efficiency is better.

Separation of the components of a sample requires a bonded phase that provides both
retention and selectivity. Four bonded phases are available for UPLC separations:
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C. C. Detectors
The detectors are use in UPLC analysis is UV/Visible detector. Detection of analytes is
conventionally based on absorbance that is concentration sensitivity detectors. In UPLC the
flow cell volume would have to be reduced to maintain concentration and signal. Based on
Beer’s Law, smaller volume conventional flow cells would also reduce the path length upon
which the signal strength depends. A reduction in cross-section means the light path is
reduced, and transmission drops with increasing noise. Therefore, if a conventional HPLC
flow cell were used, UPLC sensitivity would be compromised. The ACQUITY Tunable
UV/Visible detector cell consists of a light guided flow cell equivalent to an optical fiber.
Light is efficiently transferred down the flow cell in an internal reflectance mode that still
maintains a 10mm flow cell path length with a volume of only 500mL. Tubing and
connections in the system are efficiently routed to maintain low dispersion and to take
advantage of leak detectors that interact with the software to alert the user to potential
problems.
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GAS CHROMATOGRAPHY (GC)
INTRODUCTION
Gas chromatography (GC) is a common type of chromatography used in analytical
chemistry for separating and analyzing compounds that can be vaporized without
decomposition. Typical uses of GC include testing the purity of a particular substance, or
separating the different components of a mixture. In preparative chromatography, GC can be
used to prepare pure compounds from a mixture.
Gas chromatography is also sometimes known as vapour-phase chromatography (VPC),

or gas–liquid partition chromatography (GLPC). These alternative names, as well as their
respective abbreviations, are frequently used in scientific literature.
Gas chromatography is the process of separating compounds in a mixture by injecting a
gaseous or liquid sample into a mobile phase, typically called the carrier gas, and passing the
gas through a stationary phase. The mobile phase is usually an inert gas or an unreactive gas
such as helium, argon, nitrogen or hydrogen. The stationary phase is a microscopic layer
of viscous liquid on a surface of solid particles on an inert solid support inside a piece of
glass or metal tubing called a column. The surface of the solid particles may also act as the
stationary phase in some columns. The glass or metal column through which the gas phase
passes is located in an oven where the temperature of the gas can be controlled and the eluent
coming off the column is monitored by a computerized detector.
PRINCIPLE
A gas chromatograph is made of a narrow tube, known as the column, through which the
vaporized sample passes, carried along by a continuous flow of inert or nonreactive gas.
Components of the sample pass through the column at different rates, depending on their
chemical and physical properties and the resulting interactions with the column lining or
filling, called the stationary phase. The column is typically enclosed within a temperature
controlled oven. As the chemicals exit the end of the column, they are detected and identified
electronically.
WORKING
A Gas Chromatograph is comprised of a heated inlet port, an oven, an analytical column, and
a detector. Let’s walk through the process:
Step 1: Sample Prep
Samples are generally dissolved or diluted in a solvent and then injected onto the inlet port.
Some samples such as essential oils are not diluted. Other methods of sample preparation,
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include sample introduction techniques such as thermal desorption and headspace. These are
easy techniques for there is typically no or minimal sample preparation.
Step 2: Vaporization
This is as simple as it sounds. The liquid sample is vaporized in the hot inlet and becomes a
gas.
Step 3: Separation
Here an inert gas such as helium carries the sample through the column. Different substances
in the sample interact differently with the column’s stationary phase, depending on their
chemistry. This causes them to travel through the column at different speeds, thus separating
them.
Step 4: Detection
The separated compounds then leave the column one after the other and enter a detector, such
as a mass spectrometer (MS). The use of a MS detector is helpful. Because of the huge
amount of organic compounds, at times, compounds may elute at the same time. A MS helps
identify what the compound is and separates them by mass. The time taken for a compound
to travel through the column is called its retention time.
Step 5: Chromatogram
The GC produces a graph called a chromatogram, which shows peaks: the size of a peak
indicates the amount of each component reaching the detector. The number of peaks shows
different compounds present in the sample. The position of each peak shows the retention
time for each compound.
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HOW DOES GC WORK?
INSTRUCTION
1. Check gas filters, carrier gas supply.
If your carrier gas supply employs oxygen and water filters, check the indicators on these
filters to ensure they are not exhausted and replace if necessary.
2. Place correct nut and ferrule on the column and cut column end.
Once the nut and ferrule have been placed on the column, a 10 cm section of the column
needs to be removed. Hold the column between your index finger and thumb and scribe
across the surface of the column with cutting tool. This will leave a scratch in the polyimide
surface. Apply a slight pressure to either side of scratch mark. The column will snap and
should leave a clean square end. If a clean break is not achieved, repeat the scribing process
making sure you are further into the column than the initial scribe.
3. Install column into the GC inlet.
As the GC system vary, consult your GC installation manual for the correct distance a
column should be inserted into GC inlet. Once inserted, fingers tighten the nut. Using a
wrench, tighten by about another ½ turn. You should not be able to pull the column out of
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ferrule. If movement is felt, tighten until secure. If this cannot be achieved check the correct
ferrule has been used.
4. Turn on carrier gas and check flow.
Turn on the carrier gas and adjust the column pressure to the desired value. If the pressure for
the column has not been pre-determined, adjust flow rate temporarily. Cut the column end
according to the procedure described in step 2. Check column flow by dipping the column
end into a small vial containing a solvent. A stream of bubbles should be observed. If not,
check for possible leaks in the GC inlet.
5. Install column into detector.
Place nut and ferrule on the column. Cut the column end again to ensure no ferrule material is
deposited in the column end. As all instruments require the column end to be located at
different positions in the detector, consult your GC installation manual. Determine the signal
is stable and is not subject to the sharp movements. This would indicate a problem with the
column position. The baseline should stabilize in a uniform manner.
6. Check the leaks.
Once the column is installed and preliminary gas flow applied, check for leaks. For non GC-
MS application you only need to check the injector system. Use an electronic leak detector if
possible. To check for leaks at GC-MS interface, use a stream of argon. If a leak is present,
an argon signal will be detected on the MS system.
7. Set the carrier gas flow rate.
Set the carrier gas flow rate according to method requirements. Set the spit flow, septum
purge flow, and any other applicable gas rates.
ADVANTAGES
 The major advantage of gas chromatography is its high sensitivity, resolution, and
separation ability, which allows it to separate a wide range of volatile compounds.
 It can be upgraded to a mass spectrometer (MS), which is used to determine the mass-
to-charge ratio of ions.
 It comes with a variety of detectors and injectors that can be used for various
pharmaceuticals as well as other applications.
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 Gas chromatography can analyze a sample much faster than other chromatographic
techniques.
 It is a robust method of separation that gives the superior signal-to-noise ratio.
 It only takes a very small amount of sample to inject, and its detectors are extremely
sensitive, allowing it to detect extremely low concentrations.
 As per the requirement of the molecule, there are different types of GC columns are
available in many diameters and lengths.
 Gas chromatography is easy, automated, and has quick analysis of data which gives
comparatively high precision, accuracy, and reproducible results.
 Operational parameters such as flow rate, temperature, and pressure, etc. are easy to
change even during chromatographic runs.
DISADVANTAGES
 The major disadvantage of GC is that only volatile and thermally stable compounds
can be separated using gas chromatography.
 Detectors which are used in the GC are destructive, except for MS.
 Selectivity in HPLC or TLC is also better as a mobile phase can be easily changed. In
GC, you can just modify the temperature of the column and oven, but you cannot change the
mobile phase as it has a constant flow of carrier gas (helium, nitrogen).
 Since hydrogen gas, which is used for flame, is highly flammable, care must be taken
when using it.
 It is impossible to recover individual sample components.
APPLICATION
Manufacturing
Manufacturing is another area where gas chromatography is useful. Pharmaceutical
companies, in particular, need to know that their products contain the exact amount of
medicine required without any inconsistencies or impurities.
Gas chromatography can do exactly that, identifying and quantifying each component that
makes up a particular product. It’s also used in the chemical industry to do the same for
solvents and emulsifiers. In both cases, it makes sure production can be scaled up without the
risk of unsafe or deficient products.
Pollution monitoring
Pollution is one of the most talked-about topics over the past decade, and there’s no signs of
that changing. One of the most immediate issues is the emission of pollutants into the air we
breathe. Most commonly attributed to cars and industrial facilities, these emissions have been
linked to breathing difficulties, fatal illnesses like cancer, and even birth defects.
Gas chromatography can be used to monitor pollution levels in the air. In turn, that gives
researchers a better insight into where pollution is worse, how it changes throughout the day
and year, and how it can be combatted in the long run.
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Prevention
Another pollution-related application of gas chromatography is preventing it. Volatile organic
compounds are released by a number of products, from carpets and furniture to paint and
cleaning solutions.
Gas chromatography can be used to identify and quantify specific chemicals that are released
into the air by these products. That gives manufacturers a better chance of removing them
altogether, or at the very least advising consumers on practices like ventilation to reduce their
impact.
Drug testing
Because gas chromatography can identify specific components within a substance, it can also
be used to detect drugs from samples of bodily fluids. This is sometimes used for forensic
purposes, testing whether someone has consumed drugs or ingested poison in the hours
leading up to their death. However, it can also be used in legal cases and by sporting bodies
to test for illegal or prohibited drug usage.
APPARATUS AND REQUIREMENTS

1. A sample injector,
2. Flow controller,
3. GC column,
4. Column oven,
5. A detector,
6. A computer for data acquisition and analysis.
MASS SPECTROMETRY
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INTRODUCTION
Mass is an intrinsic property of a body. It was traditionally believed to be related to
the quantity of matter in a physical body, until the discovery of the atom and particle physics.
It was found that different atoms and different elementary particles, theoretically with the
same amount of matter, have nonetheless different masses. Mass in modern physics has
multiple definitions which are conceptually distinct, but physically equivalent. Mass can be
experimentally defined as a measure of the body's inertia, meaning the resistance
to acceleration (change of velocity) when a net force is applied. The object's mass also
determines the strength of its gravitational attraction to other bodies.
The SI base unit of mass is the kilogram (kg). In physics, mass is not the same as weight,
even though mass is often determined by measuring the object's weight using a spring scale,
rather than balance scale comparing it directly with known masses. An object on the Moon
would weigh less than it does on Earth because of the lower gravity, but it would still have
the same mass. This is because weight is a force, while mass is the property that (along with
gravity) determines the strength of this force.
PRINCIPLE
The basic principle of mass spectrometry (MS) is to generate ions from either inorganic or
organic compounds by any suitable method, to separate these ions by their mass-to-charge
ratio (m/z) and to detect them qualitatively and quantitatively by their respective m/z and
abundance. The analyte may be ionized thermally, by electric fields or by impacting energetic
electrons, ions or photons. The ions can be single ionized atoms, clusters, molecules or their
fragments or associates. Ion separation is effected by static or dynamic electric or magnetic
fields. Although this definition of mass spectrometry dates back to 1968 (Kienitz, 1968), it is
still valid. One should add that ion separation by m/z can also be effected in field-free
regions, provided the ions possess a well-defined kinetic energy at the entrance of the flight
path.
It follows directly from this definition that atoms or molecules need to carry an electric
charge, i.e., they need to be transformed into ions, for MS to work. The electric charge acts
like a handle that allows to grab these atoms or molecules. In contrast to neutrals, ions can be
accelerated and decelerated, can be shot into defined orbits or other flight paths, and can
finally be collected and detected. The “race tracks” of these ions can be determined by
application of electric and/or magnetic fields. While the Coulombic force is exerted on ions
in electric fields, the Lorentz force influences ions moving with a component orthogonal to
the magnetic field.
WORKING
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In a regular mass spectrometer, we initially have the material to be analyzed, but we need it
to be ionized to pass through the spectrometer with enough energy. Thus, the sample is
bombarded by electrons to ionize it.
This ionized beam is now passed through a series of electric or magnetic fields depending on
the type of the sample and its properties. The ions are deflected by the field through which
they are passed through in such a way that the ions with the same mass to signal ratio will
follow the same path to the detector.
These charged and deflected ions are now incident onto a detector which is capable of
distinguishing the charged particles falling on it. Based on the mass spectrum produced by
the charged ions, we can identify the atoms or molecules constituting the sample by
comparing them with known masses or through a characteristic fragmentation pattern.
HOW DOES MASS SPECTOMETRY WORK?
ADVANTAGES
 A big advantage of mass spectrometry is that it is incredibly sensitive (parts per million)
over many other techniques.
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 It is an excellent tool for identifying or confirming the presence of unknown components

in a sample.
DISADVANTAGES
 The disadvantages of mass spec are that identifying hydrocarbons that produce similar
ions is not very good and it is not able to separate optical and geometric isomers.
 The disadvantages are offset by combining MS with other methods, for example gas
chromatography.
 One disadvantage of mass spectrometry is over hydrocarbons.
 The method fails to distinguish between hydrocarbons producing similarly fragmented
ions.
APPLICATION
Mass spectrometry is an efficient method to elucidate the chemical composition of a sample

or molecule. More recently, it has been used to classify biological products, in particular
proteins and protein complexes, in a number of species. Usually, mass spectrometers can be
used to classify unknown substances by molecular weight measurement, to measure known
compounds, and to determine the structure and chemical properties of molecules.
 Due to its capability to distinguish between substances, Mass spectrometry is used to

determine unknown substances.
 To identify the isotopes of a substance.
 In analytical laboratories that study the chemical, physical and biological properties of
substances. It is favored over several other analytical techniques as it has less
background interference since it is performed in a vacuum
 Ionizer
 Accelerator
 Deflector
 Detector
1. Ionizer – The bombarding of the sample is done by the electrons. These electrons
move between cathode and anode. When the sample passes through the electron
stream between the cathode and anode, electrons with high energy knock
electrons out of the sample and form ions.
2. Accelerator – The ions placed between a set of charged parallel plates get
attracted to one plate and repel from the other plate. The acceleration speed can
be controlled by adjusting the charge on the plates.
3. Deflector – Magnetic field deflects ions based on its charge and mass. If an ion is
heavy or has two or more positive charges, then it is least deflected. If an ion is
light or has one positive charge, then it is deflected the most.
4. Detector – The ions with correct charge and mass move to the detector. the ratio
of mass to charge is analyzed through the ion that hits the detector.
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INFRA-RED (IR) SPECTROSCOPY
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INTRODUCTION
Infrared spectroscopy (IR spectroscopy) is the spectroscopy that deals with the infrared
region of the electromagnetic spectrum, that is light with a longer wavelength and lower
frequency than visible light. It covers a range of techniques, mostly based on absorption
spectroscopy. As with all spectroscopic techniques, it can be used to identify and study
chemicals. A common laboratory instrument that uses this technique is a Fourier transform
infrared (FTIR) spectrometer.
The infrared portion of the electromagnetic spectrum is usually divided into three regions; the
near-, mid- and far- infrared, named for their relation to the visible spectrum. The higher
energy near-IR, approximately 14000-4000 cm-1 (0.8-2.5μm wavelength) can excite overtone
or harmonic vibrations. The mid-infrared, approximately 4000-400 cm-1 (2.5-25μm) may be
used to study the fundamental vibrations and associated rotational vibrational structure. The
far-infrared, approximately 400-10 cm-1 (25-1000μm), lying adjacent to the microwave
region, has low energy and may be used for rotational spectroscopy. The names and
classifications of these subregions are conventions, and are only loosely based on the relative
molecular or electromagnetic properties.
PRINCIPLE
The IR spectroscopy theory utilizes the concept that molecules tend to absorb specific
frequencies of light that are characteristic of the corresponding structure of the molecules.
The energies are reliant on the shape of the molecular surfaces, the associated vibronic
coupling, and the mass corresponding to the atoms.
For instance, the molecule can absorb the energy contained in the incident light and the result
is a faster rotation or a more pronounced vibration.
WORKING
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The infrared radiation is split into two beams. One beam passes through the sample cell and
the other passes through the reference cell. The reference cell (like a control group) is used to
measure the effect of the material of the sample cell, the solvent and the conditions of the
atmosphere so that these effects can be discounted from information retrieved by the sample
cell .The difference in transmittance (transmitted radiation) between the sample and the
reference cell is because of the Absorption of certain frequencies by the molecules of the
sample. These absorbtions then result in changes in the vibrational energy of the molecule
under examination. This is picked up by the detector.
HOW DOES IR SPECTROSCOPY WORKS?
INSRUCTION
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1. Clean the machine before use.
2. Use a cotton swab to clean the needle tip and reservoir of the machine with methanol.
3. Dry using a Kim wipe.
4. Contamination of results may occur if the machine is not properly cleaned.
5. Please be courteous and also clean the machine after use.
6. Wait a few minutes to ensure the machine is completely dry before proceeding.
ADVANTAGE
1. Qualitative and quantitative analysis: One of the key advantages of Infrared

spectroscopy is without destroying the sample it can provide qualitative and quantitative
chemical information.
2. Sample Preparation: The major advantage of infrared spectroscopy is that the sample
does not need any particular preparation.
3. Sensitive and Time-saving technique: IR spectroscopy is very sensitive, hence it
required minimum sample quantity to scan the sample spectrum and it takes a few
seconds to scan a whole range of IR.
4. It's versatility: Solid, liquid, gases and semisolid samples can be analyzed by the IR
spectroscopy.
5. Easy for interpretation: The Peak intensities, peak positions, peak widths, shapes, and
functional groups provide all helpful information.
DISADVANTAGE
1. Disadvantages include sometimes difficult handling procedures and maintenance of

the sample cells.
2. There are no infrared spectra in atoms or monatomic ions, hence it cannot analyses.
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3. To use infrared spectroscopy is that it requires very sensitive and properly tuned
devices.
4. The sample having aqueous solutions and complex mixtures are complicated to
analyze by infrared spectroscopy.
APPLICATION
Infrared spectroscopy is widely used in industry as well as in research. It is a simple and

reliable technique for measurement, quality control and dynamic measurement. It is also
employed in forensic analysis in civil and criminal analysis.
Some of the major applications of IR spectroscopy are as follows:
1. Identification of functional group and structure elucidation
Entire IR region is divided into group frequency region and fingerprint region. Range of
group frequency is 4000-1500 cm-1 while that of finger print region is 1500-400 cm-1.
In group frequency region, the peaks corresponding to different functional groups can be
observed. According to corresponding peaks, functional group can be determined.
Each atom of the molecule is connected by bond and each bond requires different IR region
so characteristic peaks are observed. This region of IR spectrum is called as finger print
region of the molecule. It can be determined by characteristic peaks.
2. Identification of substances
IR spectroscopy is used to establish whether a given sample of an organic substance is
identical with another or not. This is because large number of absorption bands is observed in
the IR spectra of organic molecules and the probability that any two compounds will produce
identical spectra is almost zero. So if two compounds have identical IR spectra then both of
them must be samples of the same substances.
IR spectra of two enantiomeric compound are identical. So IR spectroscopy fails to
distinguish between enantiomers.
For example, an IR spectrum of benzaldehyde is observed as follows.
C-H stretching of aromatic ring- 3080 cm-1
C-H stretching of aldehyde- 2860 cm-1 and 2775 cm-1
C=O stretching of an aromatic aldehyde- 1700 cm-1
C=C stretching of an aromatic ring- 1595 cm-1
C-H bending- 745 cm-1 and 685 cm-1
No other compound then benzaldehyde produces same IR spectra as shown above.
3. Studying the progress of the reaction
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Progress of chemical reaction can be determined by examining the small portion of the
reaction mixure withdrawn from time to time. The rate of disappearance of a characteristic
absorption band of the reactant group and/or the rate of appearance of the characteristic
absorption band of the product group due to formation of product is observed.
4. Detection of impurities
IR spectrum of the test sample to be determined is compared with the standard compound. If
any additional peaks are observed in the IR spectrum, then it is due to impurities present in
the compound.
5. Quantitative analysis
The quantity of the substance can be determined either in pure form or as a mixure of two or
more compounds. In this, characteristic peak corresponding to the drug substance is chosen
and log I0/It of peaks for standard and test sample is compared. This is called base line
technique to determine the quantity of the substance.
1. A source of continuous infrared radiation and

2. A sensitive infrared transducer, or detector.
ULTRA-VIOLET (UV) SPECTROSCOPY
INTRODUCTION
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UV spectroscopy is an analytical technique that measures the amount of discrete wavelengths

of UV or visible light that are absorbed by or transmitted through a sample in comparison to a
reference or blank sample. This property is influenced by the sample composition, potentially
providing information on what is in the sample and at what concentration.
Light has a certain amount of energy which is inversely proportional to its wavelength. Thus,
shorter wavelengths of light carry more energy and longer wavelengths carry less energy. A
specific amount of energy is needed to promote electrons in a substance to a higher energy
state which we can detect as absorption. Electrons in different bonding environments in a
substance require a different specific amount of energy to promote the electrons to a higher
energy state. This is why the absorption of light occurs for different wavelengths in different
substances. Humans are able to see a spectrum of visible light, from approximately 380 nm,
which we see as violet, to 780 nm, which we see as red. UV light has wavelengths shorter
than that of visible light to approximately 100 nm. Therefore, light can be described by its
wavelength, which can be useful in UV spectroscopy to analyze or identify different
substances by locating the specific wavelengths corresponding to maximum absorbance (see
the Applications of UV spectroscopy section).
PRINCIPLE
The Principle of UV-Visible Spectroscopy is based on the absorption of ultraviolet light or

visible light by chemical compounds, which results in the production of distinct
spectra. Spectroscopy is based on the interaction between light and matter. When the matter
absorbs the light, it undergoes excitation and de-excitation, resulting in the production of a
spectrum.
When matter absorbs ultraviolet radiation, the electrons present in it undergo excitation. This
causes them to jump from a ground state (an energy state with a relatively small amount of
energy associated with it) to an excited state (an energy state with a relatively large amount of
energy associated with it). It is important to note that the difference in the energies of the
ground state and the excited state of the electron is always equal to the amount of ultraviolet
radiation or visible radiation absorbed by it.
WORKING
Light source
As a light-based technique, a steady source able to emit light across a wide range of
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wavelengths is essential. A single xenon lamp is commonly used as a high intensity light
source for both UV and visible ranges. Xenon lamps are, however, associated with higher
costs and are less stable in comparison to tungsten and halogen lamps.
For instruments employing two lamps, a tungsten or halogen lamp is commonly used for
visible light, whilst a deuterium lamp is the common source of UV light. 2 As two different
light sources are needed to scan both the UV and visible wavelengths, the light source in the
instrument must switch during measurement. In practice, this switchover typically occurs
during the scan between 300 and 350 nm where the light emission is similar from both light
sources and the transition can be made more smoothly.
Wavelength selection
In the next step, certain wavelengths of light suited to the sample type and analyte for
detection must be selected for sample examination from the broad wavelengths emitted by
the light source. Available methods for this include:

 Monochromators - A monochromator separates light into a narrow band of

wavelengths. It is most often based on diffraction gratings that can be rotated to
choose incoming and reflected angles to select the desired wavelength of light. 1,2 The
diffraction grating's groove frequency is often measured as the number of grooves per
mm. A higher groove frequency provides a better optical resolution but a narrower
usable wavelength range. A lower groove frequency provides a larger usable
wavelength range but a worse optical resolution. 300 to 2000 grooves per mm is
usable for UV-Vis spectroscopy purposes but a minimum of 1200 grooves per mm is
typical. The quality of the spectroscopic measurements is sensitive to physical
imperfections in the diffraction grating and in the optical setup. As a consequence,
ruled diffraction gratings tend to have more defects than blazed holographic
diffraction gratings.3 Blazed holographic diffraction gratings tend to provide
significantly better quality measurements.3

 Absorption filters - Absorption filters are commonly made of coloured glass or

plastic designed to absorb particular wavelengths of light.

 Interference filters - Also called dichroic filters, these commonly used filters are
made of many layers of dielectric material where interference occurs between the thin
layers of materials. These filters can be used to eliminate undesirable wavelengths by
destructive interference, thus acting as a wavelength selector. 1,2

 Cut off filters – Cut off filters allow light either below (short pass) or above (long
pass) a certain wavelength to pass through. These are commonly implemented using
interference-filters.

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 Band pass filters –Band pass filters allow a range of wavelengths to pass through that
can be implemented by combining short pass and long pass filters together.

Monochromators are most commonly used for this process due to their versatility. However,
filters are often used together with monochromators to narrow the wavelengths of light
selected further for more precise measurements and to improve the signal-to-noise ratio.
Sample analysis
Whichever wavelength selector is used in the spectrophotometer, the light then passes
through a sample. For all analyses, measuring a reference sample, often referred to as the
"blank sample", such as a cuvette filled with a similar solvent used to prepare the sample, is
imperative. If an aqueous buffered solution containing the sample is used for measurements,
then the aqueous buffered solution without the substance of interest is used as the reference.
When examining bacterial cultures, the sterile culture media would be used as the reference.
The reference sample signal is then later used automatically by the instrument to help obtain
the true absorbance values of the analytes.
It is important to be aware of the materials and conditions used in UV-Vis spectroscopy

experiments. For example, the majority of plastic cuvettes are inappropriate for UV
absorption studies because plastic generally absorbs UV light. Glass can act as a filter, often
absorbing the majority of UVC (100-280 nm) and UVB (280-315 nm) but allowing some
UVA (315-400 nm) to pass through. Therefore, quartz sample holders are required for UV
examination because quartz is transparent to the majority of UV light. Air may also be
thought of as a filter because wavelengths of light shorter than about 200 nm are absorbed by
molecular oxygen in the air. A special and more expensive setup is required for
measurements with wavelengths shorter than 200 nm, usually involving an optical system
filled with pure argon gas. Cuvette-free systems are also available that enable the analysis of
very small sample volumes, for example in DNA or RNA analyses.
Detection
After the light has passed through the sample, a detector is used to convert the light into a
readable electronic signal. Generally, detectors are based on photoelectric coatings or
semiconductors.
A photoelectric coating ejects negatively charged electrons when exposed to light. When

electrons are ejected, an electric current proportional to the light intensity is generated. A
photomultiplier tube (PMT) is one of the more common detectors used in UV-Vis
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spectroscopy. A PMT is based on the photoelectric effect to initially eject electrons upon
exposure to light, followed by sequential multiplication of the ejected electrons to generate a
larger electric current. PMT detectors are especially useful for detecting very low levels of
light.
When semiconductors are exposed to light, an electric current proportional to the light

intensity can pass through. More specifically, photodiodes6 and charge-coupled devices
(CCDs)7 are two of the most common detectors based on semiconductor technology.2,5
After the electric current is generated from whichever detector was used, the signal is then
recognized and output to a computer or screen.
HOW DOES UV SPECTROSCOPY WORKS?
Fig.1
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Fig.2
ADVANTAGE
 The biggest advantage for chemists and astronomers who use UV-VIS spectrometers is
the accuracy of the device.
 Even small UV-VIS spectrometers can give extremely accurate readings, which is
crucial when you are preparing chemical solutions or recording the movement of
celestial bodies.
 UV-VIS spectrometers are easy to use.
 Most UV-VIS spectrometers used in astronomy attach to telescopes.
 Most of the ones used in chemistry are comparable in size to electron microscopes and
require the same basic skills to use. Because they are simple to operate, there is little
chance of a UV-VIS spectrometer being used improperly.
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DISADVANTAGE
 The main disadvantage of using a UV-VIS spectrometer is the time it takes to prepare to
use one.
 With UV-VIS spectrometers, setup is key. You must clear the area of any outside light,
electronic noise, or other outside contaminants that could interfere with the
spectrometer's reading.
 If the space has been properly prepared ahead of time, UV-VIS spectrometers are
simple to use and give accurate results.
 However, if the space has not been properly prepared, even a small bit of outside light
or vibration from a small electronic device could interfere with the results you are
hoping to achieve in using a UV-VIS spectrometer.
APPLICATION
DNA and RNA analysis
Quickly verifying the purity and concentration of RNA and DNA is one particularly
widespread application. A summary of the wavelengths used in their analysis and what they
indicate are given in Table 1. When preparing DNA or RNA samples, for example for
downstream applications such as sequencing, it is often important to verify that there is no
contamination of one with the other, or with protein or chemicals carried over from the
isolation process.
The 260 nm/280 nm absorbance (260/280) ratio is useful for revealing possible
contamination in nucleic acid samples, summarized in Table 2. Pure DNA typically has a
260/280 ratio of 1.8, while the ratio for pure RNA is usually 2.0. Pure DNA has a lower
260/280 ratio than RNA because thymine, which is replaced by uracil in RNA, has a lower
260/280 ratio than uracil. Samples contaminated with proteins will lower the 260/280 ratio
due to higher absorbance at 280 nm.
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Pharmaceutical analysis
One of the most common uses of UV-Vis spectroscopy is in the pharmaceuticals industry. In

particular, processing UV-Vis spectra using mathematical derivatives allows overlapping
absorbance peaks in the original spectra to be resolved to identify individual pharmaceutical
compounds.12,17 For example, benzocaine, a local aesthetic, and chlortetracycline, an
antibiotic, can be identified simultaneously in commercial veterinary powder formulations by
applying the first mathematical derivative to the absorbance spectra. Simultaneous
quantification of both substances was possible on a microgram per millilitre concentration
range by building a calibration function for each compound.
Bacterial culture
UV-Vis spectroscopy is often used in bacterial culturing. OD measurements are routinely and
quickly taken using a wavelength of 600 nm to estimate the cell concentration and to track
growth.18 600 nm is commonly used and preferred due to the optical properties of bacterial
culture media in which they are grown and to avoid damaging the cells in cases where they
are required for continued experimentation.
Beverage analysis
The identification of particular compounds in drinks is another common application of UV-

Vis spectroscopy. Caffeine content must be within certain legal limits, 1,19 for which UV light
can facilitate quantification. Certain classes of colored substances, such as anthocyanin found
in blueberries, raspberries, blackberries, and cherries, are easily identified by matching their
known peak absorbance wavelengths in wine for quality control using UV-Vis absorbance.
Other applications
This technique may also be used in many other industries. For example, measuring a color
index is useful for monitoring transformer oil as a preventative measure to ensure electric
power is being delivered safely. Measuring the absorbance of hemoglobin to determine
hemoglobin concentrations may be used in cancer research. In wastewater treatments, UV-
Vis spectroscopy can be used in kinetic and monitoring studies to ensure certain dyes or dye
by-products have been removed properly by comparing their spectra over time. It also finds
great utility in food authenticity analysis and air quality monitoring.
UV-Vis spectroscopy is also qualitatively useful in some more specialized research. Tracking
changes in the wavelength corresponding to the peak absorbance is useful in examining
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specific structural protein changes and in determining battery composition. Shifts in peak

absorbance wavelengths can also be useful in more modern applications such as
characterization of very small nanoparticles. The applications of this technique are varied and
seemingly endless.
 A light source,
 A wavelength dispersive element,
 Sample and Detector.
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NUCLEAR MAGNETIC RESONANCE (NMR) SPECTROSCOPY
INTRODUCTION
In 1946, NMR was co-discovered by Purcell, Pound and Torrey of Harvard University and
Bloch, Hansen and Packard of Stanford University. The discovery first came about when it
was noticed that magnetic nuclei, such as 1H and 31P (read: proton and Phosphorus 31) were
able to absorb radio frequency energy when placed in a magnetic field of a strength that was
specific to the nucleus. Upon absorption, the nuclei begin to resonate and different atoms
within a molecule resonated at different frequencies. This observation allowed a detailed
analysis of the structure of a molecule. Since then, NMR has been applied to solids, liquids
and gasses, kinetic and structural studies, resulting in 6 Nobel prizes being awarded in the
field of NMR.
PRINCIPLE
 All nuclei are electrically charged and many have spin.

 Transfer of energy is possible from base energy to higher energy levels when an external
magnetic field is applied.
 The transfer of energy occurs at a wavelength that coincides with the radio frequency.
 Also, energy is emitted at the same frequency when the spin comes back to its base level.
 Therefore, by measuring the signal which matches this transfer the processing of the
NMR spectrum for the concerned nucleus is yield.
WORKING
 Place the sample in a magnetic field.

 Excite the nuclei sample into nuclear magnetic resonance with the help of radio waves to
produce NMR signals.
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 These NMR signals are detected with sensitive radio receivers.

 The resonance frequency of an atom in a molecule is changed by the intramolecular
magnetic field surrounding it.
 This gives details of a molecule’s individual functional groups and its electronic structure.
 Nuclear magnetic resonance spectroscopy is a conclusive method of identifying
monomolecular organic compounds.
 This method provides details of the reaction state, structure, chemical environment and
dynamics of a molecule.
HOW DOES NMR SPECTROSCOPY WORKS?
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ADVANTAGE
 NMR lets the user view molecular dynamics in a liquid or solid state while leaving the
samples intact for future observation and testing.
 Users of NMR spectrometers don't need to go through major preparation processes before
viewing their samples.
 NMR allows users to obtain rich structural information from the vibrations of the
molecules in their natural environment while they're still intact.
 NMR spectrometers simplify and speed up the data acquisition and analysis process.
 Users can use the established libraries of NMR spectrometers to identify molecules.
 Users don't need to manipulate samples that may they would otherwise be unable to study
with other approaches.
 Users are better able to identify and quantify molecules.
DISADVANTAGES
 NMR spectroscopy requires expensive equipment.
 Determining the structures for higher molecular weight molecules is a problem.
 NMR is less sensitive than Mass spectroscopy (MS) since it requires a sample amount in
milligrams, whereas it is a picogram from MS.
 Great care must be taken when using ferromagnetic materials in close proximity to NMR
because of the strong magnet, as they could potentially become dangerous for the
machine and user.
 Ionic states of elements cannot be deciphered using NMR.
 Hydrogen atoms in a molecule having similar resonant frequency could be hard to
resolve.
 Only nuclei having magnetic moments could be analyzed.
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APPLICATION
The two major areas where NMR has proven to be of critical importance is in the fields of
medicine and chemistry, with new applications being developed daily
Nuclear magnetic resonance imaging, better known as magnetic resonance imaging (MRI) is
an important medical diagnostic tool used to study the function and structure of the human
body. It provides detailed images of any part of the body, especially soft tissue, in all possible
planes and has been used in the areas of cardiovascular, neurological, musculoskeletal and
oncological imaging. Unlike other alternatives, such as computed tomography (CT), it does
not used ionized radiation and hence is very safe to administer.
In many laboratories today, chemists use nuclear magnetic resonance to determine structures
of important chemical and biological compounds. In NMR spectra, different peaks give
information about different atoms in a molecule according specific chemical environments
and bonding between atoms. The most common isotopes used to detect NMR signals are 1H
and 13C but there are many others, such as 2H, 3He, 15N, 19F, etc., that are also in use.
NMR has also proven to be very useful in other area such as environmental testing, petroleum
industry, process control, earth’s field NMR and magnetometers. Non-destructive testing
saves a lot of money for expensive biological samples and can be used again if more trials
need to be run. The petroleum industry uses NMR equipment to measure porosity of different
rocks and permeability of different underground fluids. Magnetometers are used to measure
the various magnetic fields that are relevant to one’s study.
 Sample holder – It is a glass tube which is 8.5 cm long and 0.3 cm in diameter.
 Magnetic coils – Magnetic coil generates magnetic field whenever current flows through
it
 Permanent magnet – It helps in providing a homogenous magnetic field at 60 – 100
MHZ
 Sweep generator – Modifies the strength of the magnetic field which is already applied.
 Radiofrequency transmitter – It produces a powerful but short pulse of the radio waves.
 Radiofrequency – It helps in detecting receiver radio frequencies.
 RF detector – It helps in determining unabsorbed radio frequencies.
 Recorder – It records the NMR signals which are received by the RF detector.
 Readout system – A computer that records the data.
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003601202940

“INDUSRIAL VISIT TO MAKSON PHARMACEUTICAL PVT.LTD”

A PROJECT REPORT SUBMITED

For the partial fulfilment for the degree of

ENROLLMENT NO: 003601202940

UNDER THE GUIDANCE OF

MS. Bansi Jani

CHAPTER-2 EXPERIMENTAL SECTION

CHAPTER-3 CHARACTERISTIC SECTION

1.1 ABOUT THE COMPANY.

1.2 HISTORY OF THE COMPANY

1.3 BOARD DIRECTION

1.5 CONTACT OF THE COMPANY

2.1.2 RAW MATERIAL

Brand Hajmola maha candy

2.2 (B)NUTRINE MAHA LACTO

2.2.1 GENERAL INTRODUCTION

2.2.2 RAW MATERIAL

Chocolate manufacturing processes generally share common features such

well-known automated kneaders, producing somewhat tough texture and

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)

High Performance Liquid Chromatography (HPLC) is a process of separating

Two HPLC Phases

HOW DOES HPLC WORK?

 Controls and automates chromatography instrumentation.

APPLICATION FOR HPLC

It is also a useful tool in testing for pesticides, herbicides, fungicides, pharmaceutical

APPARATUS AND REQUIREMENTS

ULTRA PERFORMANCE LIQUID CHROMATOGRAPHY (UPLC)

The underlying principle of UPLC is based on the van Deemter relationship

HOW DOES UPLC WORK?

 A major disadvantage of UPLC is the higher back pressures compared to conventional

 Increasing the column temperature reduces the back pressure problem in UPLC.

Determination of Pesticides in Groundwater

Improved Resolving Power in Peptide Maps

Peptide mapping is an essential technique for the characterization of proteins. Due to

Rapid Dose Formulation Analysis

Analysis of Traditional Chinese Medicines (TCM)

The identification and quantification of components of TCM by chromatographic analysis is

Multi-Residue Analysis of Pharmaceuticals in Waste Water

In Manufacturing / Quality Assurance (QA) / Quality Control (QC)

Identification, quantification, purification, efficacy and safety are key parameters to be

demand accurate measurement/testing of low-level impurities present with the active

APPARATUS AND REQUIRMENTS

In UPLC, sample introduction is critical. Conventional injection valves, either automated or

Resolution is increased in a 1.7μm particle packed column because efficiency is better.

GAS CHROMATOGRAPHY (GC)

Gas chromatography is also sometimes known as vapour-phase chromatography (VPC),

HOW DOES GC WORK?

3. Install column into the GC inlet.

4. Turn on carrier gas and check flow.

5. Install column into detector.

6. Check the leaks.

7. Set the carrier gas flow rate.

APPARATUS AND REQUIREMENTS

HOW DOES MASS SPECTOMETRY WORK?

 It is an excellent tool for identifying or confirming the presence of unknown components

Mass spectrometry is an efficient method to elucidate the chemical composition of a sample

 Due to its capability to distinguish between substances, Mass spectrometry is used to

APPARATUS AND REQUIRMENTS

INFRA-RED (IR) SPECTROSCOPY

HOW DOES IR SPECTROSCOPY WORKS?