INDEX

 COMPANY PROFILE
 DEPARTMENT OF COMPANY
 QUALITY CONTROL DEPARTMENT
1. INTRODUCTION
2. INSTRUMENT USED IN QUALITY
CONTROL TEST
 Analytical Balance
 Autoclave
 Centrifuge
 Conductivity meter
 Desintegrator
 Dissolution tester
 HPLC
 IR Spectroscopy
 Ph meter
 Sonicator
 Tablet hardness tester
 Viscometer
Unichem Laboratories was founded in 1944 by Padma Bhushan Amrut V Mody, a
pioneer in the Indian pharmaceuticals business.
The initial public offering of Unichem Laboratories Limited was in 1963, after its
registration in 1962,[4] and the company was consequently listed on BSE Limited and
NSE Limited.
Unichem Laboratories Limited is currently active in finished formulations, APIs, contract
manufacturing, custom synthesis, etc. Their R & D site at Goa has expertise in product
development, process chemistry and developing complex APIs for global market.

Vision
So far our vision is to be a leading Pharmaceutical Company in the global Pharma
market, to continually create value and bring pride to the community at large.

Mission
Our goal is to ensure that no patient should be denied access to high quality and
affordable medication and support. We wish to create innovations to meet the
demands of the patients using technology and uniqueness. Our clientele consists
of many prolific Government Institutions and Foreign Firms.

Vision
our firm encourage honesty, perfection and dedication in their work and we
promise their growth and development at fullest we also emphasize respect for
each other

General Information
INTERNATIONAL ACCREDITATIONS

Backed by a highly capable and motivated team of over 3100 people,

Unichem is headquartered in Mumbai, India, and has six drug manufacturing
locations across the country.
In keeping with its commitment to benchmark quality standards, several of
the company’s facilities have been accredited by reputed international
organisations such as the US FDA, UK MHRA, ANVISA (Brazil), COFEPRIS
(Mexico), PMDA (Japan), EUGMP, EDQM (Europe), WHO (Geneva) and TGA
(Australia).

 RESEARCH &
DEVELOPMENT
THE NEXT FRONTIER

The Company has taken several important strides in the critical area of Research and
Development (R&D). It has expanded its R&D facility in Goa to spearhead research in
Novel Drug Delivery Systems (NDDS) and chemically synthesize non-infringing routes
for the manufacture of medical products directed at regulated markets. In its effort to
drive innovation, Unichem has established a proprietary formulation development centre
in Goa to focus on the development of generic formulations comprising Immediate
Release as well as NDDS and ANDAs exclusively for the US market. The facility has
also been equipped to handle the development of formulations for New Chemical
Entities (NCEs). Further, the company has also established a Biotech facility at Goa to
invent, design, develop and commercialise Biotech and Biosimilar products.
With a robust business model, seamless processes and innovative research, Unichem
is positioned to take on challenges of the future while remaining firmly rooted in a solid
foundation of values.

Type of products manufactured at the site

The unit has a numbers of product permissions for tablets (Uncoated/ Film
Coated/ Sugar Coated/ Enteric Coated Tablets), and Liquid Orals in the following
therapeutic range:

Type of products manufactured at the site

 Arkamin H  Unistar
 Izra L  Vizylac DT
 Lezyncet D  Zilsar
 Losar Beta H  Zisper plus
 Momoz F  Zulu AT
 Pregaba M  Zyncet (syrup,tablet)
 Trilosar
 Trilosar HS

Area Statement of Site

Plant Area: 83,070 sq.m.

Covered Area: 28,217 sq.m.

Quality Management System: Quality Policy:
The firm has very clear and recorded quality policy which states that: “We, at
Unichem laboratories limited, aim to provide satisfaction to our customers in
domestic/ international market by fully meeting their requirements in terms of
Quality, Cost and Delivery. We have created a company committed to quality by
bringing about involvement of all employees at all levels for continuous
improvement in Quality.”

Quality control resources

The quality control laboratories have following facilities.

 Stability Testing Facilities.

 Instrumental Analysis laboratories.
 In-process Quality Control facilities.
 Chemical Analytical laboratories.
 Control samples storage facilities.
 Microbiology Lab
 Packing Materials Testing Facility
 QUALITY CONTROL

INTRODUCTION:
The Q.C. Dept is the heart of any industry; the quality controldepartment
deals with regular and periodic checking of raw materials, finished products,
packed goods. The tests carried out include assays, related substance detection,
average weight, moisture contents etc. Various Analyses of Quality Control are:

1)Raw material
. 2)Finished product
.3 ) S t a b i l i t y .
4)Method validation

. Following are some of the important functions of Quality Control Department:

1)To see that the product produced in company should be of consistent [Link] should be free
from contaminants and also be free from environmental vectors
2)To maintain consistency in obtaining yield of the product, this is
d o n e b y strictly obeying the Standard Operating Procedures (SOP) and
monitoring the reactions from time to time.

3)To responsibly monitor the quality of the raw materials, the reactions going in the reactors and
the finished goods produced.

4)To check the documents from stores, manufacturing, packing depart

m e n t before release of product.

To check whether the new products prepared by R&D are as per

validation protocol.

 Quality Control Equipments

 Analytical Balance  High Performance Liquid

 Autoclave Chromatography
 Centrifuge  Infrared Spectroscopy
 Conductivity meter  pH meter
 Disintegrator  Sonicator
 Dissolution Tester  Tablet hardnes tester
 Viscometer

ANALYTICAL INSTRUMRNT USED IN QUALITY CONTROL

 Analytical balance
To use the analytical balance effectively, the analyst must have a thorough
knowledge of the construction, design, and operation of the balance.
Furthermore, the correct use and interpretation of measurements made with the
balance is dependent on an understanding of the absolute precision with which
samples can be weighed.

Working principle
The quickest way to understand the principle of how electronic balances work, is
to first understand how they are constructed. There are two basic types of
electronic balance designs.

1. Electromagnetic balancing type

2. Electrical resistance wire type (load cell type)

These are based on completely different principles, but what they both have in
common is that neither directly measures mass. They measure the force that acts
downward on the pan. This force is converted to an electrical signal and displayed
on a digital display. As a means of measuring force, the electromagnetic balance
method utilizes the electromagnetic force generated from a magnet and coil,
whereas the electrical resistance wire method utilizes the change in resistance
value of a strain gauge attached to a piece of metal that bends in response to a
force. The mass is displayed because the reference standards for mass are
weights, which are placed on a pan to inform the electronic balance that a given
force is equivalent to a given number of grams, which is used for conversion.
Consequently, electronic balances that do not perform this conversion accurately
cannot display accurate mass values.

 Autoclave
An autoclave is a device to sterilize equipment and supplies by subjecting them to
high pressure saturated steam at 121 °C or more, typically for 15-20 minutes. In
its most basic form the autoclave is a pressure cooker. Water is heated in a
pressurized environment to create steam. Using pressure makes it possible to
heat to higher temperatures with less energy. Autoclaves are usually made of
steel and have various configurations for removing air prior to pressurization.
Downward displacement autoclaves use gravity to remove air

. Some common temperatures at which autoclaves operate are: 115 degrees C/10
p.s.i., 121 degrees C/15 p.s.i., and 134 degrees C/30 p.s.i. (p.s.i.=pounds per
square inch). The temperature, pressure and time of operation depend on the
degree of sterilization needed. An autoclave using standard settings can kill most
bacteria, spores, viruses and fungi (all models of Osworld Autoclaves).

 Centrifuge

Centrifuge is applying centrifugal force to separate the useful component in

mixtures of liquids and solids or liquids and liquids. Centrifuge is mainly used to
separate solids from liquids in suspension or separate two liquids with different
density and non-homogenous liquids, for example, separate cream form milk; and
also it can be used to remove liquids existed in solids, such as special speeding
tubular centrifuges can separate the mixed gas content with different density,
depending different density and particle size of solid particles in the liquid and
different characteristics of the subsiding speed centrifuge, the sedimentation
centrifuge also can classified solids according to different density and particle size.
Centrifuge is widely used in chemical, oil, food, pharmaceutical, beneficiation,
coal, water treatment and shipping etc. Part Centrifuge has a drum rotating its
axle called bowl, generally drived by motor. Suspension or emulsion is introduced
to the bowl and rotate with bowl with the same speed, eject separately under the
centrifugal [Link],high separation speed, high separation ratio. The
principle of centrifuge is divided to centrifugal filtering and centrifugal
sedimentation. Centrifugal filtering is made suspension become filtrate under the
centrifugal force and the centrifugal sedimentation is applied different density to
separate suspension and emulsion and realize liquid-solid or liquid- liquid
separation.

 Conductivity meter

Conductivity measurement is an extremely widespread and useful method,

especially for quality control purposes. Surveillance of feed water purity, control
of drinking water and process water quality, estimation of the total number of
ions in a solution or direct measurement of components in process solutions can
all be performed using conductivity measurements. The high reliability, sensitivity
and relatively low cost of conductivity instrumentation makes it a potential
primary parameter of any good monitoring program. Some applications are
measured in units of resistivity, the inverse of conductivity. Other applications
require the measurement of total dissolved solids (TDS), which is related to
conductivity by a factor dependent upon the level and type of ions present.
Conductivity measurements cover a wide range of solution conductivity from pure
water at less than 1x10-7 S/cm to values of greater than 1 S/cm for concentrated
solutions. In general, the measurement of conductivity is a rapid and inexpensive
way of determining the ionic strength of a solution. However, it is a nonspecific
technique, unable to distinguish between different types of ions, giving instead a
reading that is proportional to the combined effect of all the ions present.

Working A typical conductivity meter applies an alternating current (I) at an

optimal frequency1) to two active electrodes and measures the potential (V).
Both the current and the potential are used to calculate the conductance (I/V).
The conductivity meter then uses the conductance and cell constant to display
the conductivity. Conductivity2) = cell constant x conductance

Note: the current source is adjusted so that the measured potential (V) is equal to
the reference potential (Er) (approximately ± 200 mV).

 Disintegrator
An orally administered drug must disintegrate to attain good absorption of its
active substance. The first step toward dissolution is usually the break-up of the
tablet; a process described as disintegration. The disintegration test results in a
time necessary to disintegrate a group of tablets into small particles under
standard conditions. The disintegration test is a valuable tool in quality control
environments. The test is used for batch release and trending of lot-to-lot
variations during manufacturing of tablets. The disintegration test determines
whether tablets or capsules disintegrate within the prescribed time when placed
in a liquid medium in the experimental conditions prescribed below.

Disintegration is considered to be achieved when:

a) no residue remains on the screen, or b) if there is a residue, it consists of a soft

mass having no palpably firm, unmoistened core, or c) only fragments of
coating (tablets) or only fragments of shell (capsules) remain on the screen; if
a disc has been used (capsules), fragments of shell may adhere to the lower
surface of the disc.

Operation The operation of disintegrator is quite simple, with a electronic

Resistor-Capacitor (RC) circuit controlling the timer settings and a temperature
sensor (for example, AD590) constantly monitoring the water bath temperature
and completing a feedback circuit. The purpose of the feedback circuit is
maintenance of the temperature within the prescribed range. There are two
stepper motors which is electronically connected to the overall circuitry and
controls the vertical movement of the basket rack assembly during device
operation. A Stepper Motor or a step motor is a brushless, synchronous motor
which divides a full rotation into a number of steps. Unlike a brushless DC motor
which rotates continuously when a fixed DC voltage is applied to it, a step motor
rotates in discrete step angles. The Stepper Motors therefore are manufactured
with steps per revolution of 12, 24, 72, 144, 180, and 200, resulting in stepping
angles of 30, 15, 5, 2.5, 2, and 1.8 degrees per step.
  Dissolution Test

Tablets or capsules taken orally remain one of the most effective means of
treatment available. The effectiveness of such dosage forms relies on the drug
dissolving in the fluids of the gastrointestinal tract prior to absorption into the
systemic circulation. The rate of dissolution of the tablet or capsule is therefore
crucial. One of the problems facing the pharmaceutical industry is to optimize the
amount of drug available to the body, i.e. its bioavailability. Inadequacies in
bioavailability can mean that the treatment is ineffective and at worst potentially
dangerous (toxic overdose). Drug release in the human body can be measured in-
vivo by measuring the plasma or urine concentrations in the subject concerned.
However, there are certain obvious impracticalities involved in employing such
techniques on a routine basis. These difficulties have led to the introduction of
official in-vitro tests which are now rigorously and comprehensively defined in the
respective Pharmacopoeia.

Tablet Dissolution is a standardized method for measuring the rate of drug

release from a dosage form. The principle function of the dissolution test may be
summarized as follows: Optimization of therapeutic effectiveness during product
development and stability assessment. Routine assessment of production quality
to ensure uniformity between production lots. Assessment of ‘bioequivalence’,
that is to say, production of the same biological availability from discrete batches
of products from one or different manufacturers. Prediction of in-vivo availability,
i.e. bioavailability (where applicable). Although initially developed for oral dosage
forms, the role of the dissolution test has now been extended to drug release
studies on various other forms such as topical and transdermal systems and
suppositories.

 High Performance Liquid Chromatography

The acronym HPLC stands for High Performance Liquid Chromatography.

“Chromatography” is a separation technique, “chromatogram” is the
chromatography result, and “chromatograph” is the chromatography apparatus.
HPLC is a technique for disjointing, determinating, and quantifying each
component in a mixture.

Spectroscopy is used to identify and quantify the mixture, which is separated using
the fundamental concept of column chromatography. Some of the major
components of chromatographs include devices specialised in molecule separation
known columns and high-performance pumps for supplying solvent at a steady
flow rate, among the different technologies created for chromatography. The
system once known as High Performance Liquid Chromatography became known
simply as “LC” as associated technologies got more advanced. Ultra High
Performance Liquid Chromatography (UHPLC), which is capable of high-speed
analysis, is becoming increasingly popular and is being widely used nowadays.

HPLC can only evaluate chemicals that are dissolved in solvents. HPLC separates
chemicals dispersed in a liquid sample, allowing for qualitative and quantitative
examination of which components are present in the sample and how much of each
component is present.

In the 1960s, LC with low-pressure glass columns evolved into high-pressure

chromatography (HPLC) using metal columns. As a result, it is a better version of
TLC. Instead of allowing a solvent to drop through a column under gravity, it is
pushed through at up to 400 atmospheres of pressure.

Principle of HPLC

 A separation column separates the stationary and mobile phases during

purification.
 In a separation column, the stationary phase is a granular substance with
very small porous particles.
 The mobile phase is a solvent or solvent combination that is pushed through
the separation column under high pressure.
 The sample is loaded into the mobile flow regime from the pump to the
separation column using a syringe through a valve with a linked sample
loop, i.e. a tiny tube or capillary made of stainless steel.
 A chromatogram is generated in the HPLC software at the conclusion of this
operation/run.
 The chromatogram allows the various compounds to be identified and
quantified.
 As a result, owing to interactions with the stationary phase, the constituent
components of a mixture migrate through the column at different speeds.
  Individual compounds are identified by an appropriate detector after exiting
the column and transmitted as a signal to the computer’s HPLC software.

Types of HPLC

Normal Phase

This method separates analyses based on their extreme nature. A liquid stationary
stage and a non-polar portable stage are used in NP-HPLC. As a result, polar
specimens are kept on the polar surface of the column pressing for longer than less
polar ones.

Reverse Phase

The stationary stage is hydrophobic, whereas the versatile stage is a polar liquid,
such as water-methanol or acetonitrile mixes. It is based on the hydrophobic
collaboration rule, which states that the more nonpolar the substance, the more it
will be retained.

Size Exclusion

Molecules migrate into pores in a porous media and are segregated based on their
size in comparison to the pore size. Large molecules elute first, followed by
smaller ones.

Ion Exchange

The mobile phase is buffer, and the column packing comprises ionic groups. It is
used to distinguish between anions and cations.

Parts of HPLC
Uses of HPLC

 Purification of water.
 Impurity detection in the pharmaceutical industry.
 Trace components are pre-concentrated.
 Chromatography based on ligand exchange.
 Protein chromatography via ion exchange.
 Carbohydrate and oligosaccharide anion-exchange chromatography at high
pH.

Importance of HPLC in Pharmaceuticals: A Critical Tool for Drug

Development and Quality Control

The significance of HPLC in the pharmaceutical industry is multifaceted and

encompasses various stages of drug development and production. From the initial
stages of drug discovery to the final stages of quality control, HPLC proves to be
an invaluable asset.

Drug Discovery and Development: HPLC plays a crucial role in drug discovery
by facilitating the identification, purification, and characterization of potential drug
candidates. It enables scientists to isolate and quantify specific compounds from
complex mixtures, such as extracts from natural sources or synthetic libraries,
aiding in the selection of promising drug leads.

Drug Formulation and Quality Control: HPLC is an indispensable tool in drug

formulation development, enabling scientists to optimize the formulation of drugs
to ensure consistent and effective delivery. It is also employed in quality control
laboratories to monitor the purity and stability of drugs throughout the
manufacturing process and during their shelf life.

Therapeutic Drug Monitoring: HPLC is utilized in therapeutic drug monitoring

(TDM) to measure drug concentrations in patients’ blood or plasma. This
information is crucial for clinicians to tailor drug regimens and optimize patient
outcomes.

In conclusion, HPLC has emerged as an indispensable tool in the pharmaceutical

industry, revolutionizing drug development and quality control practices. Its
versatility, precision, and sensitivity have made it an essential component of
modern pharmaceutical research and manufacturing, ensuring the safe and
effective delivery of drugs to patients worldwide.

Principles of HPLC in Pharmaceuticals

Components of HPLC System

A high-performance liquid chromatography (HPLC) system consists of several key

components that work together to achieve effective separation and analysis of
compounds. These components include:

1.
1. Solvent Delivery System (Pump): The heart of the HPLC system,
the pump delivers the mobile phase, a liquid solvent that carries the
sample through the column. It provides a constant flow of solvent at
high pressure, ensuring consistent and reproducible separation.
HPLC solvent delivery system (pump)

2.
2. Sample Injector: The sample injector introduces a precise volume of
the sample solution into the mobile phase stream. It ensures accurate
and reproducible sample introduction, minimizing errors and
maintaining the integrity of the sample.

HPLC sample injector

 3.
3. Chromatographic Column: The column is the separation chamber
where the sample components interact with the stationary phase. It is
typically a long, narrow tube packed with small, porous particles
coated with a stationary phase material. The stationary phase
selectively interacts with different sample components, allowing them
to separate as they pass through the column.

HPLC chromatographic column

4. Detector: The detector measures the concentration of each sample

component as it elutes from the column. It generates a signal that is
proportional to the concentration of the component, providing
quantitative information about the sample. Various detectors exist,
each suited for different types of analytes.
 5. Data Recorder and Processor: The data recorder receives the signal
from the detector and converts it into a digital format. It records and
processes the data, generating chromatograms that display the elution
time and response of each sample component.

HPLC data recorder and processor

Types of HPLC Techniques

HPLC encompasses a variety of techniques that differ in the type of stationary

phase and the mode of interaction between the sample components and the
stationary phase. Some common HPLC techniques include:

1. Reverse-Phase HPLC (RP-HPLC): In RP-HPLC, the stationary phase is

hydrophobic, while the mobile phase is hydrophilic. This technique is
widely used for separating nonpolar compounds, such as organic molecules
and drugs.
2. Normal-Phase HPLC (NP-HPLC): In NP-HPLC, the stationary phase is
polar, while the mobile phase is nonpolar. This technique is suitable for
separating polar compounds, such as carbohydrates and peptides.
3. Ion-Exchange HPLC (IEX-HPLC): In IEX-HPLC, the stationary phase is
charged, and the separation is based on the electrostatic interaction between
the sample components and the stationary phase. IEX-HPLC is particularly
useful for separating ions, such as proteins and inorganic compounds.
4. Size-Exclusion Chromatography (SEC): In SEC, the stationary phase
consists of porous particles of varying sizes. Separation is based on the size
of the sample components, with smaller molecules eluting first and larger
molecules eluting later. SEC is often used to determine the molecular weight
distribution of polymers and proteins.
Applications in Drug Development

High-performance liquid chromatography (HPLC) plays a pivotal role in various

stages of drug development, from the initial identification of potential drug
candidates to the optimization of drug formulations. Its versatility, precision, and
sensitivity make it an indispensable tool in the pharmaceutical industry.

Drug Discovery

In the early stages of drug discovery, HPLC is employed to:

1. Identify and isolate potential drug candidates: HPLC facilitates the

screening of large libraries of compounds, enabling scientists to identify and
isolate promising drug leads from complex mixtures, such as extracts from
natural sources or synthetic libraries.
2. Purify and characterize potential drug candidates: HPLC provides a
robust method to purify and characterize potential drug candidates, ensuring
their purity and confirming their identity.
3. Evaluate drug potency and selectivity: HPLC can be used to measure the
concentration of a drug substance or its metabolites in biological fluids,
allowing scientists to assess drug potency and selectivity.
4. Determine drug stability: HPLC can be used to monitor drug stability
under various conditions, such as temperature and pH, to ensure its shelf life
and efficacy.

Formulation Development

HPLC plays a crucial role in the development and optimization of drug

formulations. Its applications include:

1. Excipient selection and compatibility: HPLC can be used to evaluate the

compatibility of various excipients with the drug substance, ensuring that
they do not interact or degrade the drug.
2. Dissolution studies: HPLC is used to assess the dissolution rate of drug

Quality Control and Assurance

High-performance liquid chromatography (HPLC) is an essential tool in quality
control and assurance (QC/QA) practices within the pharmaceutical industry. Its
ability to separate, identify, and quantify compounds with high precision and
sensitivity makes it invaluable for ensuring the purity, stability, and consistency of
drug products.

Purity Testing

HPLC plays a critical role in purity testing of drug substances and products. It
enables the detection and identification of impurities that may arise from the
synthesis, formulation, or storage of drugs. The presence of impurities can have
significant adverse effects on the safety, efficacy, and stability of drugs.

1. Detection and quantification of impurities: HPLC can detect and quantify

impurities present at very low concentrations, typically in the parts per
million (ppm) or parts per billion (ppb) range. This sensitivity ensures that
even small amounts of impurities are identified and controlled.
2. Identification of impurities: HPLC can provide information about the
structure and identity of impurities. This information is crucial for
understanding the potential impact of impurities on drug safety and efficacy.
3. Monitoring impurity levels during drug manufacturing: HPLC can be
used to monitor impurity levels throughout the drug manufacturing process,
ensuring that impurities are controlled within acceptable limits.

Stability Testing

HPLC is an essential tool for assessing the stability of pharmaceuticals over time.
Drug stability studies are crucial for ensuring the shelf life of drugs and
maintaining their safety and efficacy throughout their storage and distribution.

1. Monitoring drug degradation: HPLC can be used to monitor the

degradation of drugs over time and under various conditions, such as
temperature, humidity, and light exposure.
2. Identification of degradation products: HPLC can identify and
characterize degradation products that may form during drug storage.
Understanding the degradation profile of a drug is crucial for establishing
appropriate storage conditions and shelf life.
3. Assessing the impact of formulation factors on stability: HPLC can be
used to evaluate the impact of formulation factors, such as excipients and
packaging, on drug stability.
Conclusion

High-performance liquid chromatography (HPLC) has revolutionized the

pharmaceutical industry, providing scientists with a powerful tool for drug
discovery, formulation development, and quality control. Its versatility, precision,
and sensitivity have made it an indispensable asset in ensuring the safety, efficacy,
and consistency of drugs, ultimately contributing to improved patient care and
outcomes..

Biopharmaceutical Applications

Biopharmaceuticals, a class of drugs derived from biological sources, such as

proteins, antibodies, and peptides, have revolutionized medicine. HPLC has
emerged as an essential tool for the analysis and characterization of these large
molecules.

1. Protein purification and characterization: HPLC can be used to purify

proteins from complex mixtures, enabling the isolation of pure proteins for
further study or therapeutic use.
2. Antibody analysis: HPLC is employed to characterize antibodies,
determining their purity, molecular weight, and glycosylation patterns.
3. Protein-ligand interactions: HPLC can be used to study protein-ligand
interactions, providing insights into drug binding and activity.
4. Peptide analysis: HPLC can be used to analyze peptides, a class of small
proteins with therapeutic potential.

HPLC techniques for biopharmaceutical applications include:

1. Size-exclusion chromatography (SEC): SEC separates proteins based on

their molecular weight, providing information about protein size
distribution.
2. Reversed-phase HPLC (RP-HPLC): RP-HPLC separates proteins based
on their hydrophobic properties.
3. Ion-exchange chromatography (IEX-HPLC): IEX-HPLC separates
proteins based on their charge.
4. Hydrophobic interaction chromatography (HIC): HIC separates proteins
based on their hydrophobic properties at high salt concentrations.
5. Affinity chromatography: Affinity chromatography separates proteins
based on their specific binding to a ligand immobilized on the stationary
phase.
  Infrared Spectroscopy

Infrared spectroscopy has been a workhorse technique for materials analysis in

the laboratory for over seventy years. An infrared spectrum represents a
fingerprint of a sample with absorption peaks which correspond to the
frequencies of vibrations between the bonds of the atoms making up the
material. Because each different material is a unique combination of atoms, no
two compounds produce the exact same infrared spectrum. Therefore, infrared
spectroscopy can result in a positive identification (qualitative analysis) of every
different kind of material. In addition, the size of the peaks in the spectrum is a
direct indication of the amount of material present. With modern software
algorithms, infrared is an excellent tool for quantitative analysis. In infrared
spectroscopy, IR radiation is passed through a sample. Some of the infrared
radiation is absorbed by the sample and some of it is passed through
(transmitted). The resulting spectrum represents the molecular absorption and
transmission, creating a molecular fingerprint of the sample. Like a fingerprint no
two unique molecular structures produce the same infrared spectrum. This makes
infrared spectroscopy useful for several types of analysis. Fourier Transform
Infrared Spectroscopy (FTIR) Fourier Transform Infrared (FT-IR) spectrometry was
developed in order to overcome the limitations encountered with dispersive
instruments. The main difficulty was the slow scanning process. A method for
measuring all of the infrared frequencies simultaneously, rather than individually,
was needed. A solution was developed which employed a very simple optical
device called an interferometer. The interferometer produces a unique type of
signal which has all of the infrared frequencies “encoded” into it. The signal can
be measured very quickly, usually on the order of one second or so. Thus, the
time element per sample is reduced to a matter of a few seconds rather than
several minutes. Most interferometers employ a beam splitter which takes the
incoming infrared beam and divides it into two optical beams. One beam reflects
off of a flat mirror which is fixed in place. The other beam reflects off of a flat
mirror which is on a mechanism which allows this mirror to move a very short
distance (typically a few millimeters) away from the beam splitter. The two beams
reflect off of their respective mirrors and are recombined when they meet back at
the beam splitter. Because the path that one beam travels is a fixed length and
the other is constantly changing as its mirror moves, the signal which exits the
interferometer is the result of these two beams “interfering” with each other. The
resulting signal is called an interferogram which has the unique property that
every data point (a function of the moving mirror position) which makes up the
signal has information about every infrared frequency which comes from the
source. This means that as the interferogram is measured, all frequencies are
being measured simultaneously. Thus, the use of the interferometer results in
extremely fast measurements. Because the analyst requires a frequency spectrum
(a plot of the intensity at each individual frequency) in order to make an
identification, the measured interferogram signal can not be interpreted directly.
A means of “decoding” the individual frequencies is required. This can be
accomplished via a well-known mathematical technique called the Fourier
transformation. This transformation is performed by the computer which then
presents the user with the desired spectral information for analysis.

FTIR has a numerous practical applications such as it can identify unknown

materials, can determine the quality or consistency of a sample or can determine
the amount of components in a mixture. Instrumentation The normal
instrumental process is as follows:

1. The Source: Infrared energy is emitted from a glowing black-body source. This
beam passes through an aperture which controls the amount of energy presented
to the sample (and, ultimately, to the detector).

2. The Interferometer: The beam enters the interferometer where the “spectral
encoding” takes place. The resulting interferogram signal then exits the
interferometer.

3. The Sample: The beam enters the sample compartment where it is transmitted
through or reflected off of the surface of the sample, depending on the type of
analysis being accomplished. This is where specific frequencies of energy, which
are uniquely characteristic of the sample, are absorbed.
 4. The Detector: The beam finally passes to the detector for final measurement.
The detectors used are specially designed to measure the special interferogram
signal.

5. The Computer: The measured signal is digitized and sent to the computer
where the Fourier transformation takes place. The final infrared spectrum is then
presented to the user for interpretation and any further manipulation.

Because there needs to be a relative scale for the absorption intensity, a

background spectrum must also be measured. This is normally a measurement
with no sample in the beam. This can be compared to the measurement with the
sample in the beam to determine the “percent transmittance.” This technique
results in a spectrum which has all of the instrumental characteristics removed.
Thus, all spectral features which are present are strictly due to the sample. A
single background measurement can be used for many sample measurements
because this spectrum is characteristic of the instrument itself.

FTIR v/s IR The original infrared instruments were of the dispersive type. These
instruments separated the individual frequencies of energy emitted from the
infrared source. This was accomplished by the use of a prism or grating. An
infrared prism works exactly the same as a visible prism which separates visible
light into its colors (frequencies). A grating is a more modern dispersive element
which better separates the frequencies of infrared energy. The detector measures
the amount of energy at each frequency which has passed through the sample.
This results in a spectrum which is a plot of intensity vs. frequency. Fourier
transform infrared spectroscopy is preferred over dispersive or filter methods of
infrared spectral analysis for several reasons:

• It is a non-destructive technique

• It provides a precise measurement method which requires no external

calibration

• It can increase speed, collecting a scan every second

• It can increase sensitivity – one second scans can be co-added together to ratio
out random noise

• It has greater optical throughput

• It is mechanically simple with only one moving part

pH meter

An acidic solution has far more positively charged hydrogen ions in it than an
alkaline one, so it has greater potential to produce an electric current in a certain
situation—in other words, it's a bit like a battery that can produce a greater
voltage. A pH meter takes advantage of this and works like a voltmeter: it
measures the voltage (electrical potential) produced by the solution whose acidity
we're interested in, compares it with the voltage of a known solution, and uses
the difference in voltage (the "potential difference") between them to deduce the
difference in pH. Instrumentation: A typical pH meter has two basic components:
the meter itself, which can be a moving-coil meter (one with a pointer that moves
against a scale) or a digital meter (one with a numeric display), and either one or
two probes that you insert into the solution you're testing. To make electricity
flow through something, you have to create a complete electrical circuit; so, to
make electricity flow through the test solution, you have to put two electrodes
(electrical terminals) into it. If your pH meter has two probes (like the one in the
photo at the top of this article), each one is a separate electrode; if you have only
one probe, both of the two electrodes are built inside it for simplicity and
convenience. The electrodes aren't like normal electrodes (simple pieces of metal
wire); each one is a mini chemical set in its own right. The electrode that does the
most important job, which is called the glass electrode, has a silver-based
electrical wire suspended in a solution of potassium chloride, contained inside a
thin bulb (or membrane) made from a special glass containing metal salts
(typically compounds of sodium and calcium). The other electrode is called the
reference electrode and has a potassium chloride wire suspended in a solution of
potassium chloride.
Working The potassium chloride inside the glass electrode (shown here colored
orange) is a neutral solution with a pH of 7, so it contains a certain amount of
hydrogen ions (H+ ). Suppose the unknown solution you're testing (blue) is much
more acidic, so it contains a lot more hydrogen ions. What the glass electrode
does is to measure the difference in pH between the orange solution and the blue
solution by measuring the difference in the voltages their hydrogen ions produce.
Since we know the pH of the orange solution (7), we can figure out the pH of the
blue solution. When we dip the two electrodes into the blue test solution, some
of the hydrogen ions move toward the outer surface of the glass electrode and
replace some of the metal ions inside it, while some of the metal ions move from
the glass electrode into the blue solution. This ion- swapping process is called ion
exchange, and it's the key to how a glass electrode works. Ion- swapping also
takes place on the inside surface of the glass electrode from the orange solution.
The two solutions on either side of the glass have different acidity, so a different
amount of ion- swapping takes place on the two sides of the glass. This creates a
different degree of hydrogen-ion activity on the two surfaces of the glass, which
means a different amount of electrical charge

 Refractometer

A refractometer is a laboratory or field device for the measurement of an index of

refraction (refractometry). The index of refraction is calculated from Snell's law
and can be calculated from the composition of the material using the Gladstone–
Dale relation.

Working Principle When light enters from a medium with a lower refractive index
as for example air into a medium with a higher refractive index as for example
water it thus changes its speed. This has as a consequence that a beam of light
changes its angle when it passes from one medium with a refractive index n1 to
another medium with a refractive index [Link] ratio of the sines of the two
angles is equivalent to the opposite ratio of the refractive indices of the two
media. This mathematical relationship is known as Snell's law. The refractive
index depends on the temperature of the media: The higher the temperature of a
media, the higher the speed of light in the media and the lower its refractive
index. In all other media, however, the speed of light depends as well on its
wavelength: The shorter the wavelength of the light, the higher its speed. This
frequency dependency of the refractive index is known as dispersion and causes a
prism or a rainbow (where the light travels from air through water) to divide
white light into its constituent spectral colors.

The refractive index has thus always to be stated together with the wavelength of
the light used for the measurement and the temperature of the media. The
refractive index is normally measured at a temperature of 20°C using light with
the wavelength of the sodium D line (589.29 nm) and is therefore expressed as
nD20. In digital refractometers the light

(1) travels from a prism

(2) with a high refractive index (normally glass or artificial sapphire) into the
sample

(3). If the angle of incidence exceeds a certain value, the light is reflected at the
prism/sample boundary (see 'total internal reflection' in the chapter above). The
reflected light is detected by a CCD (or CMOS) sensor

(4): The lower the refractive index of the sample being measured, the smaller the
critical angle and the bigger the illuminated surface of the sensor.

Sonicator

Sonication is the act of applying sound energy to agitate particles in a sample, for
various purposes. Ultrasonic frequencies (>20 kHz) are usually used, leading to
the process also being known as ultra sonication or ultra-sonication. In the
laboratory, it is usually applied using an ultrasonic bath or an ultrasonic probe,
colloquially known as a sonicator. Sonication can be used to speed dissolution, by
breaking intermolecular interactions. It is especially useful when it is not possible
to stir the sample, as with NMR tubes. It may also be used to provide the energy
for certain chemical reactions to proceed. Sonication can be used to remove
dissolved gases from liquids (degassing) by sonicating the liquid while it is under a
vacuum. This is an alternative to the freeze-pump-thaw and sparging methods.
Working principle High frequency electrical energy is converted into ultrasound
waves by means of ultrasonic transducers, which are bonded to the base of a
Stainless Steel Water Tank. These high frequency sound waves create in the liquid
countless, microscopic vacuum bubbles, which rapidly expand and collapse. This
phenomenon is called cavitation. These bubbles act like miniature high speed
brushes, driving the liquid into all the openings and minute recesses of the object
immersed in the liquid. Intense scrubbing by the process of cavitation cleans away
all the dirt and soil from the object immersed and the object comes out perfectly
cleaned. Intricate objects can be cleaned with either complete or little
dismantling.

Applications Laboratory: for glassware, filter cleaning & HPLC mobile phase,
degassing Industrial: semi-conductors, electronic components, precious parts &
mechanisms Medical: dental & surgical instruments Optical: glasses, glasses
frames, lenses Jewelry: for all kinds of jewelry, precious stones, etc. Removes:
dust, oils, grease, polishing compounds, waxes, stains, soils, and any other.

 Hardness Tester

Tablet hardness testing, is a laboratory technique used by the pharmaceutical

industry to test the breaking point and structural integrity of a tablet "under
conditions of storage, transportation, and handling before usage" The breaking
point of a tablet is based on its shape. It is similar to friability testing, but they are
not the same thing. There are 2 main processes to test tablet hardness:
compression testing and 3 point bend testing. For compression testing, the
analyst generally aligns the tablet in a repeatable way, and the tablet is squeezed
by 2 jaws. The first machines continually applied force with a spring and screw
thread until the tablet started to break. When the tablet fractured, the hardness
was read with a sliding scale.

UV Visible Spectrometer
 What is UV-Vis spectroscopy?

UV-Vis 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. Since this spectroscopy technique relies
on the use of light, let’s first consider the properties of light.

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.1 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-Vis
spectroscopy to analyze or identify different substances by locating the specific
wavelengths corresponding to maximum absorbance (see the Applications of UV-
Vis spectroscopy section).

How does a UV-Vis spectrophotometer work?

Whilst there are many variations on the UV-Vis spectrophotometer, to gain a better
understanding of how an UV-Vis spectrophotometer works, let us consider the
main components, depicted in Figure 1.
Figure 1: A simplified schematic of the main components in a UV-Vis
spectrophotometer.

Light source

As a light-based technique, a steady source able to emit light across a wide range
of 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,2 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
[Link] 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 [Link] holographic diffraction gratings tend to provide
significantly better quality measurements.

 Absorption filters - Absorption filters are commonly made of colored 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.

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

 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)2
and UVB (280-315 nm)2 but allowing some UVA (315-400 nm)2 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 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, photodiodes and charge-coupled
devices (CCDs) are two of the most common detectors based on semiconductor
technology.

After the electric current is generated from whichever detector was used, the signal
is then recognized and output to a computer or screen. Figures 2 and 3 show some
simplified example schematic diagrams of UV-Vis spectrophotometer
arrangements.

Figure 2: Schematic diagram of a cuvette-based UV-Vis spectroscopy system.

Figure 3: Schematic diagram of a cuvette-free UV-Vis spectroscopy system.

The absorbance (A) is equal to the logarithm of a fraction involving the intensity of
light before passing through the sample (Io) divided by the intensity of light after
passing through the sample (I). The fraction I divided by Io is also called
transmittance (T), which expresses how much light has passed through a sample.
However, Beer–Lambert's law is often applied to obtain the concentration of the
sample (c) after measuring the absorbance (A) when the molar absorptivity (ε) and
the path length (L) are known. Typically, ε is expressed with units of L mol-1 cm-1,
L has units of cm, and c is expressed with units of mol L-1. As a consequence, A
has no units.

Sometimes AU is used to indicate arbitrary units or absorbance units but this has
been strongly discouraged.

Beer–Lambert's law is especially useful for obtaining the concentration of a

substance if a linear relationship exists using a measured set of standard solutions
containing the same substance. Equation 1 shows the mathematical relationships
between absorbance, Beer–Lambert's law, the light intensities measured in the
instrument, and transmittance.

Equation 1: A set of equations showing the relationships between absorbance A,

Beer–Lambert's law, the light intensities measured in the instrument, and
transmittance.

The term optical density (OD) is sometimes incorrectly used interchangeably with
absorbance. OD and absorbance both measure the amount of light intensity lost in
an optical component, but OD takes into consideration loss from light scattering
whereas absorbance does not. If very little light scattering is present in a
measurement, then OD may be approximated directly using absorbance and Beer–
Lambert's law may be used.

Knowing the experimental conditions during measurements is important. Cuvettes

designed for a 1 cm path length are standard and are most common. Sometimes,
very little sample is available for examination and shorter path lengths as small as
1 mm are necessary. Where quantitation is required, absorbance values should be
kept below 1, within the dynamic range of the instrument. This is because an
absorbance of 1 implies that the sample absorbed 90% of the incoming light, or
equivalently stated as 10% of the incoming light was transmitted through the
sample. With such little light reaching the detector, some UV-Vis
spectrophotometers are not sensitive enough to quantify small amounts of light
reliably. Two simple possible solutions to this problem are to either dilute the
sample or decrease the path length.

As mentioned above, recording a baseline spectrum using a “blank” reference

solution is essential. If the instrument was absolutely perfect in every way, the
baseline would have zero absorbance for every wavelength examined. In a real
situation, however, the baseline spectrum will usually have some very small
positive and negative absorbance values. For best practice, these small absorbance
values are often automatically subtracted from the sample absorbance values for
each wavelength of light by the software to obtain the true absorbance values. 1

Depending on the purpose of the analysis, the construction of a calibration curve

may be desirable. Building a calibration curve requires some data analysis and
extra work but it is very useful to determine the concentration of a particular
substance accurately in a sample based on absorbance measurements. There are
however, numerous circumstances in which a calibration curve is not necessary
including OD measurements for bacterial culturing, taking absorbance ratios at
specific wavelengths for assessing the purity of nucleic acids or identifying certain
pharmaceuticals.

In UV-Vis spectroscopy, the wavelength corresponding to the maximum

absorbance of the target substance is chosen for analysis. This choice ensures
maximum sensitivity because the largest response is obtained for a certain analyte
concentration.1 An example of a UV Vis absorption spectrum of Food Green 3 and
a corresponding calibration curve using standard solutions are provided in Figure
5. Note that two maximum absorbance peaks are present in the Food Green 3 dye,
a smaller maximum absorbance peak at 435 nm and a more intense maximum
absorbance peak at 619 nm. To gain maximum sensitivity when calculating an
unknown concentration of Food Green 3, the maximum absorbance peak at 619 nm
was used for analysis. Standard solutions across a range of known concentrations
were prepared by diluting a stock solution, taking absorbance measurements and
then plotting these on a graph of absorbance versus concentration to build a
numerical relation between concentration and absorbance. A calibration curve was
created using a least squares linear regression equation. The closer the data points
are to a straight line, the better the fit. The y intercept in the straight line equation
was set to zero to indicate no absorbance when no dye was present.

For data analysis, the graph of absorbance versus concentration can indicate how
sensitive the system is when building a calibration curve. When a linear least
squares regression equation is used, the slope from the line of best fit indicates
sensitivity. If the slope is steeper, the sensitivity is higher. Sensitivity is the ability
to differentiate between the small differences in the sample concentration. From
Beer–Lambert's Law, the sensitivity can be partially indicated by the molar
absorptivity ε. Knowing the ε values beforehand, if available, can help to determine
the concentrations of the samples required, particularly where samples are limited
or expensive.

For reliability and best practice, UV-Vis spectroscopy experiments and readings
should be repeated. When repeating the examination of a sample, in general, a
minimum of three replicate trials is common, but many more replicates are
required in certain fields of work. A calculated quantity, such as the concentration
of an unknown sample, is usually reported as an average with a standard deviation.
Reproducible results are essential to ensure precise, high quality measurements.
Standard deviation, relative standard deviation, or the coefficient of variation help
to determine how precise the system and measurements are. A low deviation or
variation indicates a higher level of precision and reliability.

Strengths and limitations of UV-Vis spectroscopy

No single technique is perfect and UV-Vis spectroscopy is no exception. The

technique does, however, have a few main strengths listed below that make it
popular.

 The technique is non-destructive, allowing the sample to be reused or

proceed to further processing or analyses.
 Measurements can be made quickly, allowing easy integration into
experimental protocols.
 Instruments are easy to use, requiring little user training prior to use.
 Data analysis generally requires minimal processing, again meaning little
user training is required.
 The instrument is generally inexpensive to acquire and operate, making it
accessible for many laboratories.

Although the strengths of this technique seem overwhelming, there are also certain
weaknesses:

 Stray light - In a real instrument, wavelength selectors are not perfect and a
small amount of light from a wide wavelength range may still be transmitted
from the light source,1 possibly causing serious measurement errors.9 Stray
light may also come from the environment or a loosely fitted compartment
in the instrument.1

 Light scattering - Light scattering is often caused by suspended solids in

liquid samples, which may cause serious measurement errors. The presence
of bubbles in the cuvette or sample will scatter light, resulting in
irreproducible results.

 Interference from multiple absorbing species - A sample may, for

example, have multiple types of the green pigment chlorophyll. The
different chlorophylls will have overlapping spectra when examined together
in the same sample. For a proper quantitative analysis, each chemical species
should be separated from the sample and examined individually.

 Geometrical considerations - Misaligned positioning of any one of the

instrument's components, especially the cuvette holding the sample, may
yield irreproducible and inaccurate results. Therefore, it is important that
 every component in the instrument is aligned in the same orientation and is
placed in the same position for every measurement. Some basic user training
is therefore generally recommended to avoid misuse.

Applications of UV-Vis spectroscopy

UV-Vis has found itself applied to many uses and situations including but not
limited to:

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 . 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.

What does UV What causes UV absorbance at this

absorbance at wavelength?
this wavelength
Wavelength
indicate the
used in
presence of?
absorbance
analysis

in nanometers
230 Protein Protein shape
 260 DNA and RNA Adenine, guanine, cytosine, thymine,
uracil
280 Protein Mostly tryptophan and tyrosine

Table 1: Summary of useful UV absorbance when determining 260/280 and

260/230 absorbance ratios.

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. For example, benzocyanine, a local
anesthetic, 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 mililiter 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.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,for which UV light can facilitate quantification. Certain classes of colored
substances, such as anthrocyanin 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 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
  FRIABILITY TEST:

FRIABILITY is the phenomenon where the surface of the tablet is damage or

shown a site of damage due to mechanical shock.”

• PURPOSE: To evaluate the ability of the tablets to withstand the breakage

during the transportation and handling.

• Friability testing is a method, which is employed to determine physical strength

of uncoated tablets upon exposure to mechanical shock and attrition. In simple
words, friability test tells how much mechanical stress tablets are able to withstand
during their manufacturing, distribution and handling by the customer.

• It means Surface Erosion by certain mechanical shock and lost of material from
intact tablet.

Equipment used in this test:

Roche friabilator tester is most commonly used for determining % friability of

tablet.
Procedure:
[Link] 20 tablets and take initial weight of it and put it into friabilator.
[Link] rotate the drum at 25 rpm per min or 100 rpm for 4 mins.
[Link] this,tablet gets dropped on plastic from 6 inches, it will pass through
mechanical shocks.
[Link] 4 minutes,calculate final weight of tablets and that of % friability. %
Friability can be calculated by following formula-

% Friability = W1 – W2/W1 × 100

Where, W1 = weight of tablets before testing W2 = weight of tablets after testing.

Standards for friability:

% Friability should be upto 0.5 to 1% for all standard compressed tablets.

• For effervescent,chewable,WHISKERING,orodispersible tablets % friability
should be greater than
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