Chapter 1

Introduction

INTRODUCTION

1.1 PHARMACEUTICAL ANALYSIS:

Pharmaceutical analysis1 is a branch of Analytical chemistry that involves a
series of process for identification, determination, quantification and purification of a
substance and also separation of the components of the solution or a mixture, or
determination of structure of chemical compounds. The substance may be a single
compound or a mixture of compounds and it may be in any of the dosage form. The
drug substance used as pharmaceuticals are from any of the source like animals,
plants, microorganisms, minerals and various synthetic products.

1.1.1 TYPES OF ANALYSIS:

There are main two types of chemical analysis.

A) Qualitative (identification)
B) Quantitative (estimation)

A) Qualitative analysis2:
 Qualitative inorganic analysis seeks to establish the presence of a given
element or inorganic compound in a sample.
 Qualitative organic analysis seeks to establish the presence of a given
functional group or organic compound in a sample.

B) Quantitative analysis3:

 Quantitative analysis seeks to establish the amount of a given element or

compound in a sample.

1.1.2 METHODS OF DETECTING ANALYTES4:

A) Physical means

 Mass
 Color
 Refractive index
 Thermal conductivity

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B) With electromagnetic radiation

 Absorption
 Emission
 Scattering

C) By an electric charge

 Electro chemistry
 Mass Spectrometry

D) Traditional analytical techniques:

 Titration
 Gravimetry

1.1.3 INSTRUMENTAL TECHNIQUES:

A) Spectrometric Techniques5:

 Atomic Spectrometry (Emission and Absorption)

 Electron Spin Resonance Spectroscopy
 Fluorescence and phosphorescence Spectro photometry
 Infrared Spectro photometry
 Nuclear Magnetic Resonance Spectroscopy
 Radiochemical Techniques including activation analysis
 Raman Spectroscopy
 Ultraviolet and visible Spectro photometry
 X-Ray Spectroscopy

B) Chromatographic Techniques6-8:

 Gas Chromatography
 High performance Liquid Chromatography
 Thin Layer Chromatography

C) Miscellaneous Techniques9-11:

 Kinetic Techniques
 Mass Spectrometry
 Thermal Analysis

D) Hyphenated Techniques12-15:

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 GC-MS (Gas Chromatography – Mass Spectrometry)
 ICP-MS (Inductivity Coupled Plasma- Mass Spectrometry)
 GC-IR (Gas Chromatography – Infrared Spectroscopy)
 MS-MS (Mass Spectrometry – Mass Spectrometry)

1.2 SPECTROSCOPY:

Spectroscopy16-18 is the branch of science dealing with the study of interaction

between Electro Magnetic Radiation and matter. It involves the measurement and
interpretation of Electro Magnetic Radiation (EMR) absorbed or emitted when the
molecule or atoms or ions of a sample move from one energy state to another energy
state. This change may be from ground state to excited state or excited state to ground
state. At ground state, the energy of a molecule is the sum of rotational, vibrational
and electronic energy. In other words, Spectroscopy measures the changes in
rotational, vibrational or electronic energies.

It is a most powerful tool available for the study of atomic and molecular
structures and is used in the analyses of wide range of samples. Optical spectroscopy
includes the region on electromagnetic spectrum between 100 Å0 and 400μm.

The regions of electromagnetic spectrum are

Table No.1.1: Regions of electromagnetic spectrum

Region Wavelength
Far (or 10-200 nm
vacuum)ultraviolet

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Near ultraviolet 200-400 nm
Visible 400-800 nm
Near infrared 0.75- 2.5 μm
Mid infrared 2.5-50 μm
Far infrared 50-1000 μm

1.2.1 ULTRAVIOLET-VISIBLE SPECTROPHOTOMETRY:

UV-Visible Spectrophotometry19-20 is one of the most frequently employed

technique in pharmaceutical analysis. It involves measuring the amount of ultraviolet
or visible radiation absorbed by a substance in solution. Instrument which measure the
ratio, or function of ratio, of the intensity of two beams of light in the UV- visible
region are called Ultraviolet-Visible Spectrophotometers.

Principle involved in Ultraviolet Spectrophotometry:

Ultraviolet Spectrophotometry is concerned with the study of absorption of

UV radiation which ranges from 200 nm to 400 nm. Any molecule has n, π or σ or a
combination of these electrons. These bonding (σ and π) and non bonding (n)
electrons absorb the characteristic radiation and undergoes transition from ground
state to excited state.

The types of electrons present in any molecule may be conveniently classified as:

1. ‘σ’ electrons: These are the ones present in saturated compounds. Such
electrons do not absorb near UV, but absorb vacuum UV radiation (<200nm)
2. ‘π’ electrons: These electrons are present in unsaturated compounds
(double bonded and triple bonded compounds).
3. ‘n’ electrons: These are non bonded electrons which are not involved in any
bonding lone pair of electrons like in S, O, N and Halogens (X).

Any molecule has either n, π, σ or a combination of these electrons. These

bonding (σ and π) and non- bonding electrons absorb the characteristic radiation and
undergoes transition from ground state to excited state.

By the characteristic absorption peaks, the natures of the electrons present and
hence the molecule structure can be elucidated.

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Principle involved in Visible Spectrophotometry (Colorimetry):

Colorimetry is concerned with the study of absorption of visible radiation

whose wavelength ranges from 400 nm to 800 nm. Any coloured substance will
absorb radiation in this wavelength region.

In the absorption curve, the wavelength at which maximum absorption of

radiation takes place is called as λmax. This λmax is characteristic or unique for every
coloured substance and this is a qualitative aspect, useful in identifying the substance.

Laws governing absorption of radiation:

The two laws related to the absorption of radiation are:

1. Beer’s law
2. Lambert’s law
Beer’s law: It states that “the intensity of a beam of monochromatic light decreases
exponentially with increase in the concentration of absorbing species arithmetically.”
Lambert’s law: It states that “the rate of decrease of intensity with the thickness of
the medium is directly proportional to the intensity of incident light.”

Mathematical equation for Beer- Lambert’s law: A= Σ ct

Where A = Absorption or optical density or extinction co-efficient

Σ = Molecular extinction coefficient

c = Concentration of drug

t = Path length

Σ can also be expressed as follows: Σ = E 1% 1cm x Molecular weight/10

E 1% 1cm means the absorbance of 1% w/v solution, using a path length of 1cm.

Limitations of Beer-Lambert’s Law:

The Beer-Lambert law is rigorously obsessed provided a single species gives

rise to the observed absorption. However the law may not obsessed when,

1. Different forms of the absorbing molecules are in equilibrium.

2. Solute and solvent from association complexes.

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3. There is a thermal equilibrium between ground electronic state and a low lying
excited state.
4. The compounds are charged by irradiation (fluorescent compounds).

1.3 CHROMATOGRAPHY:
Chromatography21-23 is a branch of analytical chemistry; this technique is
employed for the separation of mixture into individual components using a stationary
phase and a mobile phase. Russian botanist Michael Tswett invented chromatography
as a separation technique. He described in detail the separation of pigments, the
colored substances by filtration through column, followed by developments with pure
solvents. The first paper of Tswett, was published in 1903, contains a study of more
than 100 absorbents used in conjunction with several different solvents.
Analytical chromatography is used to determine the identity and concentration
of molecules in a mixture. Preparative chromatography is used to purify larger
quantities of a molecular species. Most of the following refers to analytical
chromatography.

1.3.1 TYPES OF CHROMATOGRAPHY24:

A) Based upon the nature of stationary and mobile phase

 Gas- solid chromatography

 Gas- liquid chromatography
 Solid- liquid chromatography
 Liquid- liquid chromatography

B) Based on the principle of separation

 Adsorption chromatography
 Partition chromatography

C) Based on the modes of chromatography

 Normal phase mode

 Reverse phase mode

D) Miscellaneous

 Ion exchange chromatography

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 Gel permeation chromatography
 Chiral chromatography

1.4 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY:

High Performance Liquid Chromatography25 is the fastest growing analytical

technique for the analysis of drugs. Its simplicity, high specificity and wide range of
sensitivity make its ideal for the analysis of many drugs in both dosage forms and
biological fluids. This technique is based on the same methods of separation as that
of classical column chromatography i.e. adsorption, partition, ion exchange and gel
permeation, but it differs from the column chromatography in the fact that the mobile
phase is passed through the packed column under high pressure.
HPLC is one of the most versatile instruments used in the field of pharmaceutical
analysis. It provides the following features.

 High resolving power

 Speedy separation
 Continuous monitoring of the column effluent
 Accurate quantitative measurement
 Repetitive and reproducible analysis using the same column
 Automation of the analytical procedure and data handling.

1.4.1 TYPES OF HPLC METHODS26:

Based on modes of separation:

 Normal Phase Chromatography

 Reverse Phase Chromatography

Based on principle of separation:

 Adsorption chromatography
 Ion exchange chromatography
 Size exclusion chromatography
 Affinity chromatography
 Chiral phase chromatography

Based on elution technique:

 Isocratic separation
 Gradient separation

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Normal phase chromatography:

In normal phase chromatography, the stationary phase (e.g. silica gel) is polar
in nature and the mobile phase is non- polar (isopropane with hexane) in nature, non-
polar compounds travel faster and are eluted first. This is because less affinity
between solute and stationary phase. Polar compounds are retained for longer time in
the column because more affinity towards stationary phase and takes more time to be
eluted from the column. This is not advantageous in pharmaceutical applications since
most of the drug molecules polar in nature and takes longer time to be eluted and
detected. Hence this technique is not widely used in pharmacy.

Reverse Phase Chromatography:

In reversed phase mode, the stationary phase is non-polar in nature and the
mobile phase is aqueous, moderately polar in nature. One common stationary phase is
silica which has been treated with RMe2SiCl, where R is a straight chain alkyl group
such as C18H37 or C8H17 and most commonly used solvents for mobile phase are water,
methanol, and acetonitrile. With these stationary phases, retention time is longer for
molecules which are more non-polar, while polar molecules elute more readily. An
investigator can increase retention time by adding more water to the mobile phase;
thereby making the affinity of the hydrophobic analyte for the hydrophobic stationary
phase stronger relative to the now more hydrophilic mobile phase. Similarly, an
investigator can decrease retention time by adding more organic solvent to the eluent.
Reverse phase chromatography is so commonly used that it is often incorrectly
referred to as "HPLC" without further specification. The Pharmaceutical industry
regularly employs Reverse phase chromatography to qualify drugs before their
release.

Structural properties of the analyte molecule play an important role in its

retention characteristics. In general, an analyte with a larger hydrophobic surface area
(C-H, C-C, and generally non-polar atomic bonds, such as S-S and others) results in a
longer retention time because it increases the molecule's non-polar surface area, which
is non-interacting with the water structure. On the other hand, polar groups, such as
-OH, -NH2, COO- or -NH3+ reduce retention as they are well integrated into water.
Very large molecules, however, can result in an incomplete interaction between the

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large analyte surface and the ligand's alkyl chains and can have problems entering the
pores of the stationary phase.

Retention time increases with hydrophobic (non-polar) surface area. Branched

chain compounds elute more rapidly than their corresponding linear isomers because
the overall surface area is decreased. Similarly organic compounds with single C-C-
bonds elute later than those with a C=C or C-C-triple bond, as the double or triple
bond is shorter than a single C-C-bond.

Adsorption Chromatography:

The principle of separation in normal phase mode and reverse phase mode is
adsorption; separation of components takes place because of difference in affinity of
compounds towards stationary phase. The component which has more affinity
towards stationary phase travels slower and eluted later, the component which has less
affinity towards stationary phase travels faster and eluted first. Since no two
components have the same affinity towards stationary phase, the components are
separated.

Size Exclusion Chromatography:

Size Exclusion Chromatography (SEC), also known as gel permeation

chromatography or gel filtration chromatography, separates particles on the basis of
size. It is generally a low resolution chromatography and thus it is often reserved for
the final, "polishing" step of purification. It is also useful for determining the tertiary
structure and quaternary structure of purified proteins. It is used primarily for the
analysis of large molecules such as proteins or polysaccharides. SEC works by
trapping these smaller molecules in the pores of a particle. The larger molecules
simply pass by the pores as they are too large to enter the pores. Larger molecules
therefore flow through the column quicker than smaller molecules, that is, the smaller
the molecule, the longer the retention time.

Ion Exchange Chromatography:

In Ion Exchange Chromatography, retention is based on the attraction between solute

ions and charged sites bound to the stationary phase.

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Ions of the same charge are excluded.

Types of ion exchangers include:

 Polystyrene resins – These allow cross linkage which increases the stability of
the chain. Higher cross linkage reduces swerving, which increases the
equilibration time and ultimately improves selectivity.
 Cellulose and dextran ion exchangers (gels) – These possess larger pore sizes
and low charge densities making them suitable for protein separation.
 Controlled-pore glass or porous silica

In general, ion exchangers favor the binding of ions of higher charge and smaller
radius.
An increase in counter ion (with respect to the functional groups in resins)
concentration reduces the retention time. An increase in pH reduces the retention time
in cation exchange while a decrease in pH reduces the retention time in anion
exchange.

Affinity Chromatography:
This is the most selective type of chromatography employed. It utilizes the
specific interaction between one kind of solute molecule and a second molecule that is
immobilized on a stationary phase. For example, the immobilized molecule may be an
antibody to some specific protein. When solute containing a mixture of proteins is
passed by this molecule, only the specific protein is reacted to this antibody, binding it
to the stationary phase. This protein is later extracted by changing the ionic strength or
pH.

Chiral phase chromatography:

Separation of optical isomers can be done by using chiral stationary phases.
Different principles operate for different types of stationary phases and for different
samples. The stationary phases used for this type of chromatography are mostly
chemically bonded silica gel.

Isocratic separation:

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In this technique, the same mobile phase combination is used throughout the process
of separation.

The same polarity or elution strength is maintained throughout the process.

Gradient separation:

In this technique a moblie phase combination of lower polarity or elution

strength is used followed by gradually increasing the polarity or elution strength.

1.4.2 HPLC INSTRUMENTATION27:

Fig No.1.1: Waters HPLC instrument

The essential parts of the High Performance Liquid Chromatography are:

A. Solvent reservoir
B. Mobile phase
C. Pump system
D. Sample Injection System
E. Column
F. Detector
G. Recorder and integrators

RECORD
ER

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Fig No.1.2: Schematic diagram of HPLC

A) SOLVENT RESERVOIR:

A modern HPLC apparatus is equipped with one or more glass or stainless

steel reservoirs. The reservoir is often equipped with an online degasser which
removes the dissolved gasses usually oxygen and nitrogen, which interfere by forming
bubbles. Degasser may consist of vacuum pumping system, distillation system,
system devices for heating, and solvent stirrer.

B) MOBILE PHASE:

The primary objective in the selection of mobile phase is to achieve optimum

separation of all individual impurities and degradants from each other and from
analyte peak. For a given stationary phase, the retention of a given solute depends
directly upon the mobile phase. Sufficient solubility of solute molecules in the mobile
phase must be ensured in order to prevent precipitation.

For the mobile phase, first variable to be decided is whether an organic or

aqueous eluent should be used. With RP-HPLC analysis, either an aqueous eluent or
variety of organic solvents such as methanol or acetonitrile is tried first. If the k
values are too large with an aqueous solvent, then the separation should be attempted
by using mixture, in various proportions. Many simple analyses can be carried out
with isocratic elution using an aqueous eluent to which an organic modifier is added.
If the sample to be analyzed contains a very complex mixtures or mixture of
compounds of diverse structure and retention behaviour, then either a ternary mixture
of solvents can be used isocratically or gradient elution may be necessary.

Initially water alone or very dilute acid, base or buffer solution is tried. If it is
used, the pH and ionic strength of buffer can be varied. The stability of the buffer and
the ability to maintain the desired pH are very important considerations in choosing a
buffer agent. If the separation cannot be achieved or resolution is inadequate, an
organic modifier may be added. Isocratic separations with mixed solvents are

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preferable in the determination of only one or two components in the sample,
especially in developing routine analysis of pure compounds.

In choosing an organic solvent or modifier, the first considerations are

solubility of solutes and compatibility of the solvent with water, methanol and
acetonitrile. Ethanol, dimethyl sulfoxide, dimethyl formamide, tetrahydrofuran and
dioxane also used. More than one organic modifier may be used to achieve the desired
resolution, or water may be used as a modifier in a predominantly organic eluents.

C) PUMPS:

Pumps are required to deliver a constant flow of mobile phase at pressures

ranging from 1 - 550 bar pumps capable of creating pressure up to 8000 psi provide a
wide range of flow rates of mobile phase, typically from 0.01-10ml/min. Low flow
rates (10-100µl/min) are used with micro bore columns, intermediate flow rates (0.5-
2ml/min) are used with conventional analytical HPLC columns, and fast flow rates
are used for preparative or semi preparative columns and for slurry packing
techniques. Mechanical pumps of the reciprocating piston type view a pulsating
supply of mobile phase. A damping device is therefore required to smooth out the
pulses so that excessive noise at high levels of sensitivity or low pressure does not
detract from detection of small quantities of sample. This type of pump is extremely
useful, however in that a constant volume of liquid is delivered, the actual value being
set by adjustment of piston stroke. This means that the pressure shown on a gauge
acts as indicator of working conditions. Thus, if the column becomes partially
blocked, rise in pressure occurs until ultimately the relief wall operates.

Classification of Pumps:

HPLC pump can be classified in to the following groups according to the manner in
which they operate:
 Constant flow rate pump (or) constant displacement pump
i) Reciprocating piston pump
ii) Syringe drive pump
 Constant pressure pump
i) Simple gas displacement pump
ii) Pneumatic amplifier pump

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i) Reciprocating pump:
Reciprocating pumps usually consist of a small chamber in which the solvent
is pumped by the back and forth motion of a motor driven piston. Two check valves
control the flow of solvent. Reciprocating pumps have a disadvantage of producing
pulsed flow, which must be damped as its presence is manifested as base line noise on
the chromatogram. Advantages of this pump include their small internal volume, high
output pressure, ready adaptability to gradient elution, and independent of column
backpressure and viscosity of solvent.

ii) Displacement pump:

Displacement pumps usually consist of large syringe like chambers equipped

with a plunger that is activated by a screw driven mechanism powered by stepping
motor. Displacement pumps also produce a flow that tends to be independent of
viscosity and backpressure. In addition, the output is pulse free. Disadvantages
include limited solvent capacity (250 ml) and considerable inconvenience when
solvents must be changed.

iii) Pneumatic pumps:

In pneumatic pumps, the mobile phase is contained in a collapsible container

housed in a vessel that can be pressurized by a compressor gas. Pumps of this kind
are inexpensive and pulse free. They suffer from limited capacity, pressure output,
dependence of flow rate on solvent viscosity and column backpressure. In addition,
they are not amenable to gradient elution and are limited to pressures less than about
2000 psi.

D) SAMPLE INJECTION SYSTEM:

The limiting factor in the precision of LC measurements lie in reproducibility

with which samples are introduced into the column packing. The earliest and simple
means of sample introduction was syringe injection through a self-sealing
electrometric septum. In stop flow injections, the flow of solvent is stopped

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momentarily, and fitting at column head is removed and the sample is injected directly
into the head of column packing. After replacing the fitting the system is again
pressurized.

Commercial chromatographs use valves for sample injection. With these

devices, sample is first transferred at atmospheric pressure from a syringe into a
sample loop. Turning the valve from load to inject position connects the sample loop
into the high-pressure mobile Phase stream, whereby the contents of the sample loop
are transferred on to the column. In Rheodyne 7125 valve, sample from a micro litre
syringe is loaded into the needle port, filling the sample loop, which is a small piece
of stainless steel tube connected between ports. Any excess goes to waste from
another port. On turning to ‘inject’, the loop contents are flushed on to the column. A
variety of loop volumes is available, commonly 10-50 µl.

E) COLUMN:

Fig No.1.3: Types of Columns

The Column is the heart of HPLC separation processes. The availability of

a stable high performance column is essential in developing a rugged reproducible
method. Most column packings used for HPLC separations make use of silica particle
(SiO2´H2O).

Columns are usually made of polished stainless steel, are between 50 and
300 mm long, and have an internal diameter of between 2 to 50 mm. They are
commonly filled with a stationary phase with a particle size of 5 -20 µm. columns

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with internal diameters of less than 2 mm are often reoffered to as micro bore columns
.ideally, the temperature of the mobile phase and the column should be kept to
constant during an analysis. Most separations’ are performed at an ambient
temperature, but columns may be heated to give better efficiency normally, columns
should not be heated above 600C because of the potential for stationary phase
degradation or changes occurring to the composition of the mobile phase.

The silanol groups on the surface of silica give it a polar character, which is
exploited in adsorption chromatography using non -polar organic eluents. Silica can
be drastically altered by reaction with organo chloro silanes or organo alkoxy silanes
giving Si-O-Si-R linkages with the surface. The attachment of hydrocarbon chain to
silane produces a non-polar surface suitable for reversed phase chromatography where
mixture of water and organic solvents are used as eluents. The most popular material
is octadecyl silica (ODS-Silica), which contains C18 chains, but materials C2, C6, C8
and C22 chains are also available. During the manufacturing, such materials may be
reacted with a small mono functional silane (e.g. trimethyl chloro silane), which
reduce the number of silane groups remaining on the surface (end-capping). There is a
vast range of materials which have intermediate surface polarities arising from the
bonding of silica with other organic compounds which contain phenyl, nitro, amino
and hydroxyl groups. Strong ion exchangers are also available in which sulfonic acid
groups or quaternary ammonium groups are bonded to silica. The useful pH range for
columns is 2 to 8, since siloxane linkages are altered below pH 2 while at pH values
above 8, silica may dissolve.

In HPLC, generally two types of columns are used, normal phase columns and
reversed phase columns. Using normal phase chromatography, particularly of non
-polar and moderately polar drugs can make excellent separation. It was originally
believed that separation of compounds in mixture takes place slowly by differential
adsorption on stationary silica phase. However, it now seems that partition plays an
important role, with the compounds interacting with the polar silanol groups on the
silica or with bound water molecules.

Normal phase involves the passage of a relatively non-polar mobile phase over
a polar stationary phase; reversed phase chromatography is carried out using a polar
stationary phase. A range of stationary phases (C 18, C8, -NH2, -CN, -phenyl etc.) is

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available and very selective separations can be achieved. The pH of the mobile phase
can be adjusted to suppress the ionization of the drug and thereby increase the
retention on the column. For highly ionized drugs ion-pair chromatography is used.

F) DETECTORS:

The function of the detector in HPLC is to monitor the mobile phase emerging
from the column. The output of the detector is an electrical signal that is proportional
to some property of the mobile phase and/or the solutes

LC detectors are basically of two types.

Bulk property detectors respond to mobile phase bulk property such as refractive
index, dielectric constant or density.

Solute property detectors respond to some property of solutes, such as UV absorbing,

fluorescence, diffusion current, which are not possessed by the mobile phase.

Most common HPLC detectors:

 UV-Visible Absorbance Detector (UV-VIS)

 Photo Diode Array Detector (PDA)
 Fluorescence Detector
 Electro Chemical Detector (ECD)
 Refractive Index Detector (RI)
 Mass Detectors (MS)
 Conductometric Detector
 Chiral Detector (Polarimetric and Circular dichrosim)
 Evaporative Light Scattering Detector (ELSD)
 Radio Chemical Detector

UV Absorbing Detector:

The general applicability and ease of operation of UV-Visible absorbance

detectors have made it most popular in LC. It measures the absorbance of the
monochromatic light by the solute according to the well known Beer-Lambert’s law.

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I = I0 10 –act

Where, ‘I’ is the intensity of the transmitted light, ‘Io’ is intensity in the
absence of the analyte. ‘a’ is molar absorptivity of analyte at the wavelength used, ‘t’
is the length of the optical cell used and ‘c’ is the concentration of the Analyte in the
cell.
Table No.1.2: UV cut-off wavelengths for solvents

SOLVENT λ min (nm) SOLVENT λ min (nm)

Acetone 330 Dimethylformamide 270
Acetonitrile 190 Ethanol 205
Chloroform 240 Ethlyacetate 260
Cyclohexane 200 n-Hexane 190

Photo Diode Array Detector:

In Photo Diode Array detector, the single detecting of UV detector has been
replaced by an array of solid state detecting elements (photo-diodes). These detectors
typically have large numbers of diodes in array (256, 512, 1024). In PDA,
polychromatic radiation, after passing through the sample, is dispersed by a fixed
grating and then falls on to an array of photodiodes. Each diode measures a narrow
band of wavelengths in the spectrum, thus the PDA has parallel data acquisition, all
points in the spectrum being measured simultaneously. The spectrum of each peak in
the chromatogram can be stored and subsequently compared with standard spectra,
which facilitates the identification of peaks.

This system is superior to other detection systems as:

There are no moving parts to wear out, wavelength-resetting errors are

reduced and the instrument is likely to require less maintenance than does a
conventional spectrophotometer. The ability to make multiwavelength measurements
and the speed of data acquisition mean that various signal-averaging techniques can
be used to reduce noise and improve sensitivity.

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Fluorescence Detector:

Many compounds are capable of absorbing UV radiation and subsequently

emitting radiation of a longer wavelength, either instantly (fluorescence) or after a
time delay (Phosphorescence).Such compounds are suitable for fluorescent detection.
Radiation from a xenon or deuterium source is focused on the flow cell. The simplest
detectors employ a mercury excitation source and one or more filters to isolate the
band of emitted radiation. An interchangeable filter allows different excitation
wavelengths to be used. The fluorescent radiation is emitted by the sample in all
directions, but is usually measured at 90º to the incident beam. FD for HPLC is
similar in design to the fluorometers or spectrofluorometer.

Refractive Index Detector:

RI detectors are bulk property detectors in which the solvent passes through
one half of the cell on its way to the column. A glass plate mounted at an angle such
that bending of the incident beam occurs if the two solutions differ in refractive index

separates the two compartments. The resulting displacement of the beam with respect
to the photosensitive surface of the detectors causes variation in the output signal.

Evaporative Light Scattering Detector (ELSD):

Evaporative Light Scattering Detection is the preferred concentration detection

method for Liquid Chromatography .Their principal requirement is that the analyte
should be less volatile than the mobile phase. An ELSD cannot detect highly volatile
analytes. In this detector, the column effluent is passed into a nebuliser where, it is
converted into fine mist by a flow of nitrogen. The fine droplets are then carried
through a controlled temperature drift tube where evaporation of mobile phase occurs
leading to the formation of fine particles of the analyte. The cloud of analyte particles
then passes through a laser beam. The scattered radiation is detected at right angle to
the flow by a silicon photo diode.

The major advantage of this type of detector is its response is reported same
for all non-volatile solutes. In addition, it is significantly more sensitive than the RID
and UVD with detection limits stated to be 5 ng / 25 µL. It is stable during gradient
elutions, it is not susceptible to ambient temperature changes, and it does not produce

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negative peaks. It responds to the relative mass of the analyte. The ELSD does not
respond to the mobile phase disruption seen as solvent front peaks in the void volume
with UV and RI detectors.

Electro Chemical Detector:

Electro Chemical Detection (ECD) is based on the transfer of electrons

between either oxidisable / reducible molecule in the solution and a solid conductor.
ECD measure either the conductance of the eluent or the current associated with the
oxidation or reduction of solutes. To be capable of detection using the first method
the solutes must be ionic and using the second method the solutes must be relatively
easy to oxidize or reduce. The areas of application of electrochemical detection are
not large, but the compounds for which it does apply, represent some of the most
important drug, pollutant and natural product classes. For these, the specificity, and
sensitivity make it very useful for monitoring these compounds in complex matrices
such as body fluids and natural products. Sensitivities for compounds such as phenol,
catechol amines, nitrosamines, and organic acids are in the picomole (nanogram)
range. An inherent advantage of ECD is that the measured quantity is already electric
signal this makes the instrument relatively simple.

Conductivity Detectors:

The conductivity detectors are used for the detection of inorganic or organic
ions, usually after separation by ion exchange chromatography. These detectors
oxidize or reduce only a small quantity of the solute, so the currents observed are very
small (nanoamps). An aqueous buffer is used as the mobile phase and the
conductivity changes due to the elution of sample ions have been measured on a high
background level. Such currents are not too difficult to measure using modern
amplifiers and the detector has a high sensitivity. The electrical conductivity of the
column effluent can be monitored by measuring the current when an a.c. voltage is
applied between two electrodes in a flow cell. Conductivity detection is
predominantly applied in ion chromatography.

G) RECORDERS AND INTEGRATORS:

Recorders are used to record the responses obtained from detectors after
amplification, if necessary. They record the baseline and all the peaks obtained, with

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respect to time. Retention time for all peaks can be found out from such recordings,
but the area of individual peaks cannot be known.

Integrators are improved version of recorders with some data processing

capabilities. They can record the individual peaks with retention time, height and
width of the peaks, peak area, percentage of area etc. Integrators provide more
information on peaks than recorders. Now a day’s computers and printers are used for
recording and processing the obtained data and for controlling several operations.

1.4.3 METHODS OF QUANTITATIVE ANALYSIS28 :

The sample or solute is analyzed quantitatively in HPLC by either peak

height or peak area measurements. Peak areas are proportional to the amount of the
material eluting from the column as long as the solvent flows at constant rate. Peak
heights are proportional to the amount of the material only when peak widths are
constant and are strongly affected by the sample injection techniques. They are five
principles evaluation methods for quantifying the solute.

a) Calibration by Standards

Calibration curves for each component are prepared from pure standards, using
identical injection volumes of operating conditions for standards and samples. The
concentration of solute is read from its curve if the curve is linear
X=K x Area
Where,
X=concentration.
K=proportionality constant (slope of the curve).
In this evaluation method, only the area of the peaks of interest is measured.
Relative response factors must be considered when converting areas to volume and
when the response of the given detector differs for each molecular type of
compounds.

b) Internal Standard Method:

In this technique, a non quantity of internal standard is chromatographed and

area versus concentration is ascertained. Then a quantity of internal standard is added

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to the raw sample prior to any sample pre treatment or separation operations. The
peak area of the standard in the sample run is compared with the peak are when the
standard is run separately. This ratio serves as correction factor for variation in sample
size, for losses in any preliminary pretreatment operations, or for incomplete elution
of the sample. The material selected for the internal standard must be completely
resolved from adjacent sample components and should not interfere with the sample
components and never be present in samples.

Area of sample
Area ratio =
Area of internal standard
Area of sample
Sample concentration = x concentration of standard
Area of internal standard

c) Area Normalization:
This technique is often used for the sample having identical components. It is
used to evaluate the absolute purity of the sample. The procedure is to total up the
areas under all peaks and then calculates the percentage of the total area that is
contributed by the compound of interest. For this method the entire sample must be
eluted, all components must be separated and each peak must be completely resolved.

d) Standard Addition method:

If only few samples are to be chromatographed, it is possible to employ the

method of standard edition (s).the chromatogram of the unknown is recorded, then a
known amount of analyte (s) is added and the chromatogram is repeated using same
reagents, instruments and other conditions. From the increase in the peak area (or
peak height), the original concentration can be computed by interpolation.

If an instrumental reading(area/height) ‘Rx’ is obtained, from a sample of

unknown ‘x’ and a reading ‘Rt’ is obtained from the sample to which a known
concentration ‘a’ of analyte has been added, then ‘x’ can be calculated from

x Rx
__________ = ________

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x+a Rt
A correction for dilution must be made if the amount of standard added,
changes the total sample volume significantly. It is always advisable to check the
result by adding at least one other standard.

1.5 ANALYTICAL METHOD DEVELOPMENT29-30:

The philosophy of method development is based on several considerations.

There exist today a good practical understanding of the chromatographic separation
and how it varies with sample and with experimental conditions. In most cases, a
desired separation can be achieved easily with only a few experiments. In other cases
a considerable experiments may be needed. A good method development strategy
should require only as many experimental runs as are necessary to achieve the desired
final result.

Ideally every experiment will contribute to the end result, so that there are no
wasted runs. Usually, this requires that the result of each chromatographic run be
assessed before proceeding with the next experiment. Sometimes the chemical
structures of the sample components are known, other times this is not the case.
Methods are developed for new products when no official methods are available.
Alternate methods for existing (non-pharmacopoeia) products are developed to reduce
the cost and time for better precision and ruggedness. Trial runs are conducted,
method is optimized and validated. When alternate method proposed is intended to
replace the existing procedure comparative laboratory data including merit/demerits
are made available.

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Fig No.1.4: Flow chart for HPLC method development

1.5.1 DATA TO BE COLLECTED BEFORE STARTING A METHOD

DEVELOPMENT:

Nature of the sample:

Before beginning method development, we need to review what is known

about the sample. Important information’s about the sample composition and
properties that should be collected are as follows;

a) Number of compounds present

b) Chemical structure (functionality) of the compounds

c) Molecular weight of the compounds

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d) pKa values of the compounds

e) UV-spectra of the compounds

f) Concentration range of the compounds in the sample of

interest

g) Sample solubility

Table No.1.3: Mode of chromatography based on nature of sample

Sample Requirements

Neutral & ionic Reverse-phase chromatography (isocratic, gradient, ion-pair,

NARP, normal phase)

Inorganic-ions Ion chromatography

Isomers Normal phase HPLC/reverse phase HPLC using cyclodextrin-

silica columns

Enantiomers Separation by protein-based column by varying the type &

conc of organic mobile phase modifier, pH, ionic strength,
temp.
E.g.: OVM column

Biologicals Several factors make sample of this kind “special” :molecular

conformation, polar functionality & wide range of
hydrophobicity

Macro-molecules Big molecules require column packing with large pores( >10
nm diameter); moreover biological sample requires special
conditions

Separation goals:

The goals of the HPLC separation need to be specified clearly. Some related questions
that should be asked at the beginning of method development include;

a) Is the primary goal quantitative analysis, the detection of an (undesired)

substance, the characterization of unknown sample components, or the isolation
of the purified material?

b) Is it necessary to resolve all sample components?

c) If quantitative analysis is required, what level of accuracy and precision are

required?

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d) For how many sample matrices should the method be designed?

e) How many samples are analyzed at one time?

f) What HPLC equipment and operator skills are present in the laboratory that
will use the final method?

Table No.1.4: Separation goal and comments

GOAL COMMENT
Resolution(Rs) Greater than 1.5

Separation time 5-10 minutes for routine work

Quantization < 2% for assays; <5% for less demanding analyses;

<15% for trace analyses

Pressure Less than 150 bar is desirable; less than 200 is usually
essential

Peak height Narrow peaks are desired for higher signal to noise
ratio

Solvent Minimum mobile phase use per run

consumption

Sample pre-treatment and detection:

Samples come in various forms.

 Solutions ready for injection

 Solutions that require dilution, buffering, addition of an internal standard or

other volumetric manipulation

 Solids that must first be dissolved or extracted

 Samples that require sample pre-treatment to remove interferences or protect

the column or equipment from damage

Before the first sample is injected during HPLC-method development, we

must be reasonably sure that the detector selected will sense all sample components of
interest. Variable wavelength UV-visible detectors normally are the first choice,
because of their convenience and applicability fir most samples. When UV-response

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of the sample is inadequate, other detectors are available, such as diode-array detector,
fluorescence detector, electro-chemical detector, etc.

1.5.2 PREFERRED EXPERIMENTAL CONDITIONS FOR THE INITIAL

HPLC METHOD DEVELOPMENT

Table No.1.5: Conditions for initial HPLC development

Separation Variables Preferred initial Choice

Column dimension(length, ID) 15*0.46 cm

Particle size of column 5 µm

Stationary phase C8 or C18

Mobile phase(solvent A & B) Buffer-Acetonitrile

% of B 80-100 %

Buffer 25 mM potassium phosphate, pH: 2-3

Additive(amine-modifier, ion-pair Don’t use initially

reagent)

Flow-rate 1.5-2.0 mL /min

Temperature 35-45 degree centigrade

Sample volume Less than 25 micro litre

Sample weight Less than 100 micro gram

1.5.3 PERFORMANCE CALCULATIONS:

Calculating the following values (which can be including in a custom report)

used to access overall system performance.

1. Relative retention

2. Theoretical plates

3. Capacity factor

4. Resolution

5. Peak asymmetry

6. Plates per meter

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The parameters used to calculate these system performance values for the
separation of two chromatographic components. (Note: Where the terms W and t both
appear in the same equation they must be expressed in the same units).

Relative retention (Selectivity):

 = (t2 - ta) / (t2 - ta)

Theoretical plates:

n = 16 (t / W) 2

Capacity factor:

K' = (t2 / ta) – 1

Resolution:

R = 2 (t2 - t1) / (W2 + W1)

Peak asymmetry:

T = W0.05 / 2f

Plates per meter:

N=n/L

HETP = L/n Where,

 = Relative retention.

t1 = Retention time of the first peak measured from point of injection.

t2 = Retention time of the second peak measured from point of injection.

ta = Retention time of an inert peak not retained by the column, measured

from point of injection.

n = Theoretical plates.

t = Retention time of the component.

W = Width of the base of the component peak using tangent method.

K' = Capacity factor.

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R = Resolution between a peak of interest (peak 2) and the peak preceding it

(Peak 1).

f = Distance from the peak maximum to the leading edge of the peak.

N = Plates per meter

L = Column length, in meters

T = Peak asymmetry, or tailing factor.

W0.05 = Distance from the leading edge to the tailing edge of the peak, that can

be measured at a point 5 % of the peak height from the baseline.

W1, W2 = Width of the base of component peak 1, 2

1.5.4 METHOD OPTIMIZATION:

During the optimization stage, the initial sets of conditions that have evolved
from the first stages of development are improved or maximized in terms of
resolution and peak shape, plate counts asymmetry, capacity, elution time,
detection limits, limit of Quantization, and overall ability to quantify the
specific analyte of interest.
Optimization of a method can follow either of two general approaches:

1. Manual and 2. Computer driven

The manual approach involves varying one experimental variable at a time,
while holding all others constant, and recording changes in response .The variables
might include flow rates, mobile or stationary phase composition, temperature,
detection wavelength, and pH this univariate approach to system optimization is slow,
time consuming and potentially expensive. However, it may provide a much better
understanding of the principles and theory involved and of interactions of the
variables.

In the second approach, computer driven automated methods development,

efficiency is optimized while experimental input is minimized. Computer driven
automated approaches can be applied to many applications .In addition, they are

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capable of significantly reducing the time, energy and cost of virtually all-
instrumental methods development.

The various parameter s that include to be optimized during method

development

a) Mode of separation
b) Selection of stationary phase
c) Selection of mobile phase
d) Selection of detector
e) Column temperature

a) Selection of mode of separation:

In reverse phase mode, the mobile phase is comparatively more polar than the
stationary phase. For the separation of polar or moderately polar compounds, the most
preferred mode is reverse phase. The nature of the analyte is the primary factor in the
selection of the mode of separation. A second factor is the nature of the matrix.

b) Selection of stationary phase / column:

Selection of the column is the first and the most important step in method
development. The appropriate choice of separation column includes three different
approaches:

1. Selection of separation system.

2. The particle size and the nature of the column packing.
3. The physical parameters of the column i.e. the length and the diameter.
Some of the important parameters considered while selecting chromatographic
columns are:

 Length and diameter of the column.

 Packing material.
 Shape of the particles.
 Size of the particles.
 % of Carbon loading.
 Pore volume.

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 Surface area.
 End capping.
The column is selected depending on the nature of the solute and the
information about the analyte. Reversed phase mode of chromatography facilitates a
wide range of columns like dimethyl silane (C2), butylsilane (C4), octylsilane (C8),
octadecylslane (C18), base deactivated silane (C18) BDS phenyl, cyanopropyl (CN),
nitro, amino etc. C18 was chosen for this study since it is most retentive one. The
sample manipulation becomes easier with this type of column.

Generally longer columns provide better separation due to higher theoretical

plate numbers. As the particle size decreases the surface area available for coating
increases. Columns with 5µm particle size give the best compromise of efficiency,
reproducibility and reliability. In this case; the column selected had a particle size of 5
µm and an internal diameter of 4.6 mm.

Peak shape is equally important in method development. Columns that provide

symmetrical peaks are always preferred while peaks with poor asymmetry can result
in,

 In accurate plate number and resolution measurement

 Imprecise quantitation
 Degraded and undetected minor bands in the peak tail
 Poor retention reproducibility
A useful and practical measurement of peak shape is peak asymmetry factor
and peak tailing factor. Peak asymmetry is measured at 10% of full peak height and
peak tailing factor at 5%. Reproducibility of retention times and capacity factor is
important for developing a rugged and repeatable method.
A column which gives separation of all the impurities and degradants from
each other and from Analyte peak and which is rugged for variation in mobile phase
shall be selected.

c) Selection of mobile phase:

The primary objective in selection and optimization of mobile phase is to

achieve optimum separation of all the individual impurities and degradants from each
other and from analyte peak. In liquid chromatography, the solute retention is

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governed by the solute distribution factor, which reflects the different interactions of
the solute– stationary phase, solute – mobile phase and the mobile phase – stationary
phase .For a given stationary phase, the retention of the given solute depends directly
upon the mobile phase, the nature and the composition of which has to be judiciously
selected in order to get appropriate and required solute retention. The mobile has to be
adapted in terms of elution strength (solute retention) and solvent selectivity (solute
separation) Solvent polarity is the key word in chromatographic separations since a
polar mobile phase will give rise to low solute retention in normal phase and high
solute retention in reverse phase LC. The selectivity will be particularly altered if the
buffer pH is close to the pKa of the analytes; the solvent strength is a measure of its
ability to pull analyte from the column. It is generally controlled by the concentration
of the solvent with the highest strength. The following are the parameters, which shall
be taken into consideration while selecting and optimizing the mobile phase.

 Buffer.
 pH of the buffer.
 Mobile phase composition.

 Buffer, if any and its strength:

Buffer and its strength play an important role in deciding the peak symmetries
and separations. Some of the most, commonly employed buffers are:
 Phosphate buffers prepared using salts like KH2PO4, K2HPO4, NaH2PO4 and
Na2HPO4 etc.
 Phosphoric acid buffers prepared using H3PO4.
 Acetate buffers – Ammonium acetate, Sodium acetate, etc.
 Acetic acid buffers prepared using CH3COOH.
The retention times also depend on the molar strengths of the buffer – Molar
strength is increasingly proportional to retention times. The strength of the buffer can
be increased, if necessary, to achieve the required separations.
The solvent strength is a measure of its ability to pull analytes from the
column. It is generally controlled by the concentration of the solvent with the highest
strength.

 pH of the buffer:

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pH plays an important role in achieving the chromatographic separations as it
controls the elution properties by controlling the ionization characteristics.
Experiments were con ducted using buffers having different pH to obtain the required
separations. It is important to maintain the pH of the mobile phase in the range of 2.0
to 8.0 as most columns does not withstand to the pH which are outside this range.
This is due to the fact that the siloxane linkage area cleaved below pH 2.0, while pH
valued above 8.0 silica may dissolve.

 Mobile phase composition:

Most chromatographic separations can be achieved by choosing the optimum

mobile phase composition. This is due to that fact that fairly large amount of
selectivity can be achieved by choosing the qualitative and quantitative composition
of aqueous and organic portions. Most widely used solvents in reverse phase
chromatography are Methanol and Acetonitrile. Experiments were conducted with
mobile phases having buffers with different pH and different organic phases to check
for the best separations between the impurities. A mobile phase which gives
separation of all the impurities and degradants from each other and from Analyte peak
and which is rugged for variation of both aqueous and organic phase by at least ±0.2%
of the selected mobile phase composition.

d) Selection of detector:

The detector was chosen depending upon some characteristic property of the
analyte like UV absorbance, fluorescence, conductance, oxidation, reduction etc.
characteristics that are to be fulfilled by a detector to be used in HPLC determination
are,
 High sensitivity, facilitating trace analysis
 Negligible baseline noise. To facilitate lower detection
 Large linear dynamic range
 Low dead volume
 Non destructive to sample
 Inexpensive to purchase and operate

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Pharmaceutical ingredients do not all absorb UV light equally, so that
selection of detection wavelength is important. An understanding of the UV light
absorptive properties of the organic impurities and the active pharmaceutical
ingredient is very helpful.

For the greatest sensitivity λmax should be used. UV wavelengths below

200nm should be avoided because detector noise increases in this region. Higher
wavelengths give greater selectivity.

e) Column Temperature:

An increase in column temperature by 1 degree centigrade will usually

decrease retention (k) by 1 to 2 % .For a large increase in temperature, it may be
necessary to reduce % of B to maintain k value between 0.5 to 20 .Many examples
have been reported where changes in temperature results in useful changes in band
spacing.

Until recently temperature has not been used widely for controlling band
spacing, because of the following reasons:

 HPLC systems is often not equipped with column thermostat

 HPLC columns are not stable at higher temperature, particularly for a
mobile phase of pH below 3 or above 6.
 Solvent viscosity and vapor pressure depend strongly on temperature.
1.6 ANALYTICAL METHOD VALIDATION:

VALIDATION:
Validation is defined as follows by different agencies:

Food and Drug administration (FDA): Establishing documentation evidence, which

provides a high degree of assurance that specific process, will consistently produce a
product meeting its predetermined specification and quality attributes.

World Health Organization (WHO): Action of providing that any procedure

process, equipment, material, activity, or system actually leads to the expected results.

European Committee (EC): Action of providing in accordance with the principles

of good manufacturing practice, that any procedure, process, equipment material,

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activity or system actually lead to the expected results. In brief validation is a key
process for effective Quality Assurance.

Objective of Validation31:

The primary objective of validation is to form a basis for written procedures

for production and process control which are designed to assure that the drug products
have the identity, strength, quality and purity they purport or are represented to
process. Quality, safety and efficacy must be designed and built into the products.
Each step of the manufacturing process must be controlled to maximize the
probability that the finished product meets all quality and design specifications.

Analytical method validation32:

Method validation can be defined as (ICH) “Establishing documented

evidence, which provides a high degree of assurance that a specific activity will
consistently produce a desired result or product meeting its predetermined
specifications and quality characteristics”.

Method needs to be validated or revalidated

 Before their introduction into routine use
 Whenever the conditions changes for which the method has been validated ,
e.g., Instrument with different characteristics
 Whenever the method is changed, and the change is outside the original scope
of the method.
Purpose of Validation:
1. Enable the scientists to communicate scientifically and effectively on technical
matter.
2. Setting the standards of evaluation procedures for checking compliance and taking
remedial action.
3. Economic: Reduction in cost associated with process sampling and testing.
4. As quality of the product cannot always be assured by routine quality control
because of testing of statistically insignificant number of samples.
5. Retrospective validation is useful for trend comparison of results compliance to
cGMP/cGLP.
6. Closure interaction with Pharmacopoeial forum to address analytical problems.

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7. International Pharmacopoeial harmonization particularly in respect of impurities
determination and their limits.

Steps followed for validation procedures:

1. Proposed protocols or parameters for validations are established.
2. Experimental studies are conducted.
3. Analytical results are evaluated.
4. Statistical evaluation is carried out.
5. Report is prepared documenting all the results.

1.6.1 VALIDATION PARAMETERS33:

1. Accuracy:
Accuracy is the closeness of test results obtained by that method to the true
value. Accuracy can also be described as the closeness of agreement between the
value that is adopted, either as a conventional, true or accepted reference value, and
the value found.
In case of assay of a drug substance accuracy may be determined by
application of the analytical method to an analyte of known purity (e.g. reference
standard) or by comparison of the results of the method with those of a second well
characterized method, the accuracy of which has been stated or defined.
Accuracy is calculated as the percentage of recovery by the assay of the known
added amount of analyte in the sample, or as the difference between the mean and the
accepted true value, together with confidence intervals.
The ICH documents recommended that accuracy should be assessed using a
Minimum of nine determinations over a minimum of three concentrations levels,
Covering the specified range (i.e., three concentrations and three replicates of each
Concentration).

2. Precision:
Precision is the degree of agreement among individual test results when the
method is applied repeatedly to multiple samplings of a homogenous sample.
Precision of an analytical method is usually expressed as the standard deviation or
relative standard deviation (coefficient of variation) of a series of measurements. The
measured standard deviation can be subdivided into 3 categories: repeatability,

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intermediate precision and reproducibility. Precision is determined by assaying a
sufficient number of aliquots of a homogenous sample to be able to calculate
statistically valid estimates of standard deviation or relative standard deviation
(coefficient of variation).
 The repeatability of a test procedure is assessed by carrying out complete
separate determinations on the separate samples of the same homogeneous
batch of the material and this will provide a measure of the precision of the
procedure under normal laboratory operating conditions

 Intermediate precision is a term that has been defined by ICH (4) as the long-
term variability of the measurement process. It is determined by comparing the
results of a method run within a single laboratory over a number of weeks. A
method’s intermediate precision may reflect discrepancies in results obtained

1. From different operators,

2. From inconsistent working practice (thoroughness) of the same operator,
3. From different instruments,
4. With standards and reagents from different suppliers,
5. With columns from different batches or
6. A combination of these.
The objective of intermediate precision validation is to verify that in the same
laboratory the method will provide the same results once the development phase is
over.
 Reproducibility, as defined by the ICH, represents the precision obtained
between different laboratories. The objective is to verify that the method will
provide the same results in different laboratories. The reproducibility of an
analytical method is determined by analyzing aliquots from homogeneous lots
in different laboratories with different analysts, and by using operational and
environmental conditions that may differ from, but are still within, the
specified parameters of the method (inter laboratory tests).

Validation of reproducibility is important if the method is to be used in different

laboratories.

 Differences in room temperature and humidity

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 Operators with different experience and thoroughness
 Equipment with different characteristics, e.g. delay volume of an HPLC
system
 Variations in material and instrument conditions, e.g. in HPLC, mobile phases
composition, pH, flow rate of mobile phase
 Variation in experimental details not specified by the method
 Equipment and consumables of different ages
 Columns from different suppliers or different batches
 Solvents, reagents and other material with varying quality

3. Specificity:

ICH documents defines specificity as the ability to assess unequivocally the

analyte in the presence of compounds that may be expected to present, such as
impurities, degradation products and matrix components.

4. Limit Of Detection:

Lowest amount of analyte in a sample that can be detected, but not necessarily
quantities as an exact value, under the stated experimental conditions.

The detection limit is usually expressed as the concentration of analyte (e.g.,

percentage parts per million) in the sample.

Limit of Detection = 3.3 σ/ S

Where,
σ = the standard deviation of the response,
S = the slope of the calibration curve.
The slope S may be estimated from the calibration curve of the analyte.
Determination of limit of detection:

For instrumental and non-instrumental methods detection limit is generally

determined by the analysis of samples with known concentration of analyte and by
establishing the minimum level at which the analyte can be reliably detected.

5. Limit Of Quantitation:

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It is the lowest amount of analyte in a sample that can be determined with
acceptable precision and accuracy under the stated experimental conditions.
Quantitation limit is expressed as the concentration of analyte (e.g. percentage, parts
per billion) in the sample.

Determination of limit of quantitation:

For instrumental and non-instrumental methods, the quantitation limit is
generally determined by the analysis of samples with known concentration of analyte
and by establishing the minimum level at which the analyte can be determined with
acceptable accuracy and precision.
Limit of Quantitation = 10 σ/ S
Where,
σ = the standard deviation of the response,
S = the slope of the calibration curve.
The slope S may be estimated from the calibration curve of the analyte.

6. Linearity and Range:

Linearity of an analytical method is its ability to produce results that are
directly proportional to the concentration of analyte in samples.

The range of the procedure is an expression of the lowest and highest levels
of analyte that have been demonstrated to be determinable with acceptable precision,
accuracy and linearity.

Determination of linearity and range

These characteristics are determined by application of the procedure to a series
of samples having analyte concentration spanning the claimed range of the procedure.
When the relationship between response and concentration is not linear,
standardization may be providing by means of a calibration curve. ICH recommends
that for the establishment of linearity a minimum of 5 concentrations normally used.

7. Robustness
Robustness of an analytical method is measure of its capacity to remain
unaffectedly small but deliberate variations in method parameters and provides an
indication of its reliability during normal usage.

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Testing varying some or all condition:
-Column temperature
-pH of buffer in mobile phase
-Reagents and flow rate

8. Ruggedness
Degree of reproducibility of test results obtained by the analysis of the same
samples under a variety of conditions, such as different laboratories, different
analysts, different instruments etc., normally expressed as the lack of influence on test
results of operational and environmental variables of the analytical method.
Ruggedness is a measure of reproducibility of test results under the variation in
condition normally expected from laboratory to laboratory and from analyst to
analyst.
Determination of ruggedness
By analysis of aliquots from homogenous lots in different laboratories, by
different analysts, using operational and environmental conditions that may differ but
are still within the specified parameters of the assay. Degree of reproducibility of test
results is then determined as a function of the assay variables.

1.6.2 SYSTEM SUITABILITY34-36:

System suitability experiments can be defined as tests to ensure that the

method can generate results of acceptable accuracy and precision. The requirements
for system suitability are usually developed after method development and validation
have been completed. (or) the USP (2000) defines parameters that can be used to
determine system suitability prior to analysis.

The parameters that are affected by the changes in chromatographic conditions are,
 Capacity factor (k’),
 Peak asymmetry / tailing factor (As)
 Column efficiency (N) and
 Selectivity (α)

Capacity factor (k'):

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The capacity factor is a measure of the degree of retention of an analyte
relative to an unrestrained peak, where t R is the retention time for the sample peak and
t0 is the retention time for an unrestrained peak.
k' = (t R- t0) / t 0
Recommendations:
The peak should be well-resolved from other peaks and the void volume.
Generally the value of k' is > 2.

Tailing factor (T):

A measure of the symmetry of a peak, given by the following equation where
W0.05 is the peak width at 5% height and f is the distance from peak front to apex
point at 5% height. Ideally, peaks should be Gaussian in shape or totally symmetrical.
The accuracy of Quantitation decreases with increase in peak tailing because
of the difficulties encountered by the integrator in determining where/when the peak
ends and hence the calculation of the area under the peak. Integrator variables are
preset by the analyst for optimum calculation of the area for the peak of interest.

Recommendations:
T should be ≤ 2.

Theoretical plate number / Efficiency (N):

A measure of peak band spreading determined by various methods, some of
which are sensitive to peak asymmetry.
N = 16 (tR / W) 2 = L / H

Theoretical plate number is a measure of column efficiency, t Theoretical plate

number is a measure of column efficiency, that is, how many peaks can be located per
unit run-time of the chromatogram, where tR is the retention time for the sample peak
and W is the peak width.

N is fairly constant for each peak on a chromatogram with a fixed set of

operating conditions. H, or HETP, the height equivalent of a theoretical plate,
measures the column efficiency per unit length (L) of the column. Parameters which
can affect N or H include Peak position, particle size in column, flow-rate of mobile

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phase, column temperature, viscosity of mobile phase, and molecular weight of the
analyte

Recommendations:
The theoretical plate number depends on elution time but in general should be
> 2000.

Resolution (Rs):

Ability of a column to separate chromatographic peaks, Resolution can be

improved by increasing column length, decreasing particle size, increasing
temperature, changing the eluent or stationary phase. It can also be expressed in terms
of the separation of the apex of two peaks divided by the tangential width average of
the peaks. For reliable Quantitation, well-separated peaks are essential for
Quantitation.

Recommendations:
Rs should be > 2 between the peak of interest and the closest potential
interfering peak (impurity, excipient, degradation product, internal standard, etc.) are
desirable.

1.6.3 STATISTICAL ANALYSIS37:

Statistical procedures and representative calculations are as follows:

For Accuracy:

 (x  x )
2
i

Standard deviation () = n 1

Where,
x = sample,
xi = mean value of samples,
n = number of samples.

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The consistency and suitability of the developed method are substantiated
through the statistical analysis like standard deviation, relative standard deviation and
theoretical plates per meter.

Relative Standard Deviation = σ/xi × 100

Where,
σ=standard deviation,
xi =mean.

Sandell’s sensitivity (µg/cm2/0.001 absorbance units) = C/A×0.001

Where,
C= concentration of drug,
A= Absorbance of drug.

According to USP system suitability are an integral part of chromatographic

methods. These tests verify that the resolution and reproducibility of the system are
adequate for the analysis to be performed. One consequence of the evaluation of
robustness and ruggedness should be that a series of system suitability parameters is
established to ensure that the validity of the analytical method is maintained whenever
used. System suitability tests are based on the concept that the equipment, electronics,
analytical operations and samples constitute an integral system that can be evaluated
as a whole.

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Department of Pharmaceutical Analysis TPCP