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INTRODUCTION is essential to many aspects of human en
INTRODUCTION
1, INTRODUCTION
1.1 Analytical Chemistry’
‘Analytical chemistry is a scientific discipline used to study the chemical composition,
structure and behaviour of matter. The purposes of chemical analysis are together and
interpret chemical information that will be of value to society in a wide range of contexts.
Quality control in manufacturing industries, the monitoring of clinical and environmental
samples, the assaying of geological specimens, and the support of fundamental and applied
research are the principal applications. Analytical chemistry involves the application of a
range of techniques and methodologies to obtain and assess qualitative, quantitative and
structural information on the nature of matter.
Qualitative analysis is the identification of elements, species and/or compounds
present in sample.
<> Quantitative analysis is the determination of the absolute or relative amounts of
elements, species or compounds present in sample.
Structural analysis is the determination of the spatial arrangement of atoms in an element or
molecule or the identification of characteristic groups of atoms (functional groups). An
‘element, species or compound that is the subject of analysis is known as analyte. The
remainder of the material or sample of which the analyte(s) form(s) a part is known as the
matrix.
‘The gathering and interpretation of qualitative,
.deavour, both terrestrial and extra-terrestrials. The
throughout the world and the
quantitative and structural information
maintenance of an improvement in the quality of li
"management of resources heavily on the information provided by chemical analysis.
Manufacturing industries use analytical data to monitor the quality of raw materials,
intermediates and finished products. Progress and research in many areas is dependent on
establishing the chemical composition of man-made or natural materials, and the monitoring
the environment is of ever increasing importance. Studies of biological
of toxie substance:
and other complex systems are supported by the collection of large amounts of analytical
s and situations that include not
data. Analytical data are required in a wide range of discip
ist chemistry and most ater sciences, from biology'to zoology, butte ats, such as painting
‘and sculpture, and archaeology. Space exploration and clinical diagnosis are two quite
desperate areas in which analytical data is vital. Important areas of application include the
BNPCW Page 1 INTRODUCTION
Lene
Quality control (QC) in many manufacturing industries, the chemical composition of raw
materials, intermediates and finished products needs to be monitored to ensure satisfactory
quality and consistency. Virtually all consumer products from automobiles to clothing,
pharmaceuticals and foodstuffs, electrical goods, sports equipment and horticultural products
rely, in part, on chemical analysis.
The food, pharmaceutical and water industries in
particular have stringent requirements backed by legislation for major components and
permitted levels of impurities or contaminants. The electronic industry needs analyses at
ultra-trace levels (parts per billion) in relation to the manufacture of semi-conductor
materials. Automated, computer-controlled procedures for process-stream analysis are
‘employed in some industries.
Monitoring and control of pollutants: The presence of toxic heavy metals (e.g., lead,
icals (e.
and vehicle exhaust gases (oxides of carbon,
cadmium and mercury), organic cher
polychlorinated biphenyls and detergents)
ygen and sulphur, and hydrocarbons) in the
environment are health hazards that need to be monitored by sensitive and accurate methods
of analysis, and remedial action taken. Major sources of pollution are gaseous, solid and
liquid wastes that are discharged or dumped from industrial sites, and vehicle exhaust gases.
Clinical and biological studies: The levels of important nutrients, including trace metals
(e.g. sodium, potassium, calcium and zinc), naturally produced chemicals, such as
cholesterol, sugars and urea, and administered drugs in the body fluids of patients undergoing
is often a crucial factor and
hospital treatment require monitoring. Speed of analy:
automated procedures have been designed for such analyses.
Geotogical assays: The commercial value of ores and minerals are determined by the levels
of particular metals, which must be accurately established. Highly accurate and reliable
analytical procedures must be used for this purpose, and referee laboratories are sometimes
employed where disputes arise.
Fundamental and applied research: The chemical composition and structure of materials
used in or developed during research programs in numerous disciplines can be of
significance, Where new drugs or materials with potential commercial value are synthesized,
a complete chemical characterization maybe required involving considerable analytical work
Combinatorial chemistry is an approach used in pharmaceutical research that generates very
large numbers of new compounds requiring confirmation of identity and structure
‘Analytical techniques: There are numerous chemical or physico-chemical processes that can
bee used to provide analytical information. The processes are related to a wide range of atomic
and molecular properties and phenomena that enable elements and compounds to be detected
Department of Pharmaceutical Analysis BNPCW Page 2 INTRODUCTION
ce Ua REnnnmnmnemmraeed
and/or quantitatively mensured under controlled conditions, ‘The underlying, processes define
the various analytical techniques, The more important of these are listed in [Link].1
together with their suitability for qualitative, quantitative or structural analysis and the levels
of analyte(s) in a sample that can be measured, Atomle, molecular spectrometry and
chromatography, which together comprise the largest and most widely used groups of
techniques, can be further subdivided according to their physico-chemical basi
Spectrometric techniques may involve either the emission or absorption of electromagnetic
radiation over a very wide range of energies, and can provide qualitative, quantitative and
structural information for analytes from ma
\jor components of a sample down to ultra-trace
levels. The most
portant atomic and molecular spectrometric techniques and their principal
tions are listed in Table,No.2.
Chromatographic techniques provide the means of separating the components of mixtures
and simultaneous qualitative and quantitative analysis, as required. ‘The linking of
chromatographic and spectrometric techniques, called hyphenation, provides a powerful
means of separating and identifying unknown compounds.
Electrophoresis's another separation technique with similarities to chromatography that is
Particularly useful for this parathion of charged species. The principal separation techniques
and their applications are listed in [Link].3.
Analytical methods
An analytical method consists of a detailed, stepwise list of instructions to be
followed in the qualitative, quantitative or structural analysis of a sample for one or more
analytes and using a specified technique. It will include a summary and lists of chemicals and
reagents to be used, laboratory apparatus and glassware, and appropriate instrumentation. The
quality and sources of chemicals, including solvents, and the required performance
characteristics of instruments will also be specified as will the procedure for obtaining a
representative sample of the material to be analyzed, This is of crucial importance in
obtaining meaningful results. ‘The preparation or pre-treatment of the sample will be followed
by any necessary standardization of reagents and/or calibration of instruments under specified
Conditions. Qualitative tests for the analyte(s) or quantitative measurements under the same
Conditions as those used for standards complete the practical part of the method.
Leper reese sey
Department of Pharmaceutical Analysis, BNPCW Page 3 INTRODUCTION
The remaining steps will be concerned with data processing, computational methods
for quantitative analysis and the formatting of the analytical report. The statistical assessment
of quantitative data is vital in establishing the reliability and value of the data, and the use of
various statistical parameters and tests is widespread. Many standard analytical methods
have been published as papers in analytical journals and other scientific literature, and in
textbook form.
Collections by trades associations representing, for example, the cosmetics, food, iron
and steel, pharmaceutical, polymer plastics and paint, and water industries are available
standards organizations and statutory authorities, instrument manufacturer’s applications
notes, the Royal Society of Chemistry and the US Environmental Protection Agency are also
valuable sources of standard methods. Often, laboratories will develop their own in-house
‘methods or adapt existing ones for specific purposes.
Method development forms a significant part of the work of most analytical laboratories,
‘and method validation and periodic revalidation is a necessity. Selection of the most
appropriate analytical method should take into account the following factors:
‘The purpose of the analysis, the required time scale and any cost constraints;
*
‘The level of Analyte(s) expected and the detection limit required;
‘The nature of the sample, the amount available and the necessary sample preparation
*
procedure;
+
‘The accuracy required for a quantitative analysis;
‘The availability of reference materials, standards, chemicals and solvents,
*
instrumentation and any special facilities;
Possible interference with the detection or quantitative measurement of the analyte(s)
and the possible need for sample clean-up to avoid matrix imerference;
methods may be selective for a small number of
*
The degree of selectivity available —
*
analytes or specific for only one.
+ Quality control and safety factors.
BNPCW Pose
Department of Pharmaceutical Analysis INTRODUCTION
ES
TableNo.1. Analytical techniques and principal applications
Principal areas of
Technique Property Measured
application
Weight of pure analyte or | Quantitative for major
Gravimetry
compound of known as stoichiometry | or minor components
‘Volume of standard reagent solution | Quantitative for major
Titrimetry
reacting with the analyte or minor Component
Qualitative,
Wavelength and intensity of | quantitative or
Atomic molecular a ‘ ;
electromagnetic radiation emitted/ | structural or for major
spectrometry
absorbed by the analyte down to trace level
‘components
Qualitative or structural
Mass for major down to trace
Mass of analyte or fragments of it
spectrometry level components
isotope ratios
Qualitative and)
Chromatography |
4 Various physicochemical properties | quantitative separations
an
of separated analytes of mixtures at major to
electrophoresis
trace levels
Characterization of
Chemical/physical changes in the | single or mixed
Thermal analysis, oe
analyte when heated or cooled major/minor
compounds
Qualitative and
Electrochemical | Electrical properties of the analyte in | quantitative for major
analysis, solution to trace devel
‘components
aie Qualitative and
Radiochemical | Characteristic ionizing nuclear . |
‘yt quantitative at major to
i i i by the analyte
analysis radiation emitted by the analy as
L
———_
Department of Pharmaceutical Analysis BNPCW Page S INTRODUCTION
See Ee
[Link].2. Spectrometric Techniques and Principal Applications
Technique Basis Principal applications
‘Atomic emission after | Determination of metals
Plasma emission aoe
excitation in high | and some —_non-metals
spectrometry A
temperature gas plasma | mainly at trace levels
Flame emission 5 i
Atomic emission after | Determination of alkali
spectrometry a =
flame excitation and alkaline earth metals
‘Atomic absorption after | Determination of trace
Atomic absorption
atomization by flame or | metals and some _non-
spectrometry
electro thermal means _ | metals
‘Atomic fluorescence | Atomic fluorescence | Determination of mercury
spectrometry emission after flame | and hydrides of non-metals
excitation at trace levels
‘Atomic or atomic | Determination of major
X-ray emission | fluorescence and minor —_ elemental
spectrometry emission after | components of
excitation by electrons | metallurgical and
or radiation ‘geological samples
Monitoring of radioactive
‘spectrometry y-ray emission after .
" elements in environmental
nuclear excitation
samples
Ultraviolet/visible Electronic molecular | Quantitative determination
spectrometry absorption in solution | of unsaturated organic
Vibrational molecular | Identification of organic,
Infrared spectrometry
absorption compounds
"| Tdentifieation and
Nuclear magnetic | Nuclear absorption
structural analysis of
resonance spectrometry (change of spin states) .
organic compounds
i Tonization and | Identification and
lass spectrometi y
2 ” fragmentation of | structural analysis of
molecules ‘organic compounds
TS
Department of Pharmaceutical Analysis BNPCW Page 6 INTRODUCTION
‘[Link].3. Separation techniques and principal applications
Technique Basis Principal applications
Differential rates of migration
of analytes through a a ;
‘Thin-layer Qualitative analysis of.
stationary phase by movement | ~
chromatography ane | mixtures.
of a liquid or gaseous mobile
phase
‘Quantitative and qualitative
Gas chromatography determination of volatile
compounds
High-performance Quantitative and qualitative
liquid a determination of non-
chromatography volatile compounds
Differential rates of migration | Quantitative and qualitative
Electrophoresis of analytes through a buffered | determination of ionic
medium
compounds
1.2 Chromatography ?
1.2.1 Introduction
The chromatography was discovered by Russian Chemist and botanist
Micheal Tswett (1872-1919) who first used the term chromatography (colour writing
derived from Greek for colour — Chroma , and write —
raphein) to describe
is work on the
to bands on a column of chalk and other material
such as polysaccharides, sucrose and insulin.
separation of coloured plant pigments
“| Chromatography is a method in which the components of a mixture are separated
on an adsorbent column in a flowing system".
‘The adsorbent material, or stationary phase, first described by Russian scientist
named Tswett in 1906, has taken many forms over the years, including paper, thin layers of
solids attached to glass plates, immobi
ed
ids, gels, and solid particles packed in
columns. The flowing component of the system, or mobile phase, is either a liquid or a gas.
Concurrent with development of the different adsorbent materials has been the development
eel
Department of Pharmaceutical Analysis BNPCW Page 7 INTRODUCTION
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of methods more specific to particular classes of analytes. In general, however, the trend
development of chromatography has been toward faster, more efficient.
“In his early papers of Tswett (1906) stated that chromatography is a method in which
the component of a mixture are separated on an adsorbent column in a flowing system.
Chromatography has progressed considerably from Tswett’s time and now includes a number
of variations on the basic separation process”.
“Chromatography is a physical method of separation in which the component to be
separated are distributed between two phases of which in stationary while other moves in a
definite direction (IUPAC)”
1.2.2. Chromatographic Process‘
Chromatographic separations are based on a forced transport of the liquid (mobile
phase) carrying the analyte mixture through the porous media and the differences in the
interactions at analytes with the surface of this porous media resulting in different migration
times for a mixture components. In the above definition the presence of two different phases
is stated and consequently there is an interface between them. One of these phases provides
the analyte transport and is usually referred to as the mobile phase, and the other phase is
immobile and is typically referred to as the stationary phase.
A mixture of components, usually called analytes, are dispersed in the mobile phase at
the molecular level allowing for their uniform transport and interactions with the mobile and
stationary phases. High surface area of the interface between mobile and stationary phases is
essential for space discrimination of different components in the mixture. Analyte molecules
undergo multiple phase transitions between mobile phase and adsorbent surface. Average
residence time of the molecule on the stationary phase surface is dependent on the interaction
energy.
For different molecules with very small interaction energy difference the presence of
significant surface is critical since the higher the number of phase transitions that analyte
molecules undergo while moving through the chromatographic column, the higher the
difference in their retention. The nature of the stationary and the mobile phases, together with
the mode of the transport through the column, is the basis for the classification of
chromatographic methods.
—_—————
Department of Pharmaceutical Analysis, BNPCW Page 8 INTRODUCTION
EE
1.2.3. Types of Chromatography
‘The mobile phase could be either a liquid o a gas, and accordingly we can subdivide
chromatography into Liquid Chromatography (LC) or Gas Chromatography (GC). Apart
from these methods, there are two other modes that use a liquid mobile phase, but the nature
of its transport through the porous stationary phase is in the form of either (a) capillary
forces, as in planar chromatography (also called Thin-Layer Chromatography, TLC), or (b)
electro osmotic flow, as in the case of Capillary Electro Chromatography (CEC).
[Link].1. Showing flow chart for classification of chromatography‘
CHROMATOGRAPHY
ae
Gas [Supercritical] [Liquid] [Planar] [cec]
Gas-Solid] [Gas-Liquid np] [Rp] [iex] [sec
Capillary GC
[Link].4. Principles and classification of chromatography
Principal
- Stationary Mobile
Technique Format | sorption
Phase Phase
mechanism
Partition
Paper
(adsorption,
chromatography Paper (cellulose) | Liquid | Planar | |
ion-exchange,
(Pc) .
exclusion)
Silica, cellulose,
° Adsorption
‘Thin-layer ion-exchange, " i
. (partition, ion-
chromatography resin, Liquid | Planar
. exchange,
(TLC) controlled porosity
exclusion)
solid
Gas chromatography (GC)
Gas-liquid Liquid Gas | Column | Partition
i
Department of Pharmaceutical Analysis BNPCW Page 9 INTRODUCTION
ee
chromatography
(GLC)
Gas-solia
chromatography _| Solid Gas | Column | Adsorption
(Gscy
Liquid Chromatography (LC)
High Performance
Modified
Liq Solid or bonded- -
Liquid | Column | partition
Chromatography —_| phase
(adsorption)
(HPLC)
Size-Exclusion
Controlled porosity :
‘Chromatography id Liquid | Column | Exclusion:
soli
(SEQ)
Ton-Exchange
Chromatography
Ion-exchange resin
(IEC), Ton Column | Ion-exchange
or bonded-phase
Chromatography
ic
Chiral ° ~
Solid chiral Selective
Chromatography Liquid | Column
Selector adsorption
(cc)
———————
Department of Pharmaceutical Analysis BNPCW Page 10 INTRODUCTION
ES
1.3 METHODS IN CHROMATOGRAPHY!
1, According to nature of stationary and mobile phase
> Solid- Liquid chromatography
> Liqui
> Gas- Solid chromatography
|-Liquid chromatography
> Gas -Liquid chromatography
2. According to principle of separation
A. Adsorption chromatography
> Gas Solid chromatography
> Thin layer chromatography
> Column chromatography
> High performance liquid chromatography
>
Affinity phase chromatography
v
Hydrophobic Interaction chromatography (HIC)
B. Partition chromatography
> Gas liquid chromatography
> Paper partition chromatography
> Column parti
mn chromatography
Based on modes of chromatography
> Normal phase chromatography
> Reversed phase chromatography
4, Other types of chromatography
y
Size exclusion chromatography (SEC)
.
Gel permeation chromatography
¥
Gel chromatography
a
Gel Filtration
”
Gel permeation chromatography
Y
Ion exchange chromatography
¥
Chiral chromatography
1, Adsorption chromatography
Chromatography in which separation is based mainly on difference between the
adsorption affinities of the sample components for the surface of an active solid. The analyte
interact with solid stationary surface and are displaced with eluent for active sites on surface.
RS
Department of Pharmaceutical Analysis BNPCW Page 11 INTRODUCTION
SS
2. Partition chromatography
This method results from a thermodynamic distribution of analytes between two liquid
phases. On the basis of relative polarities of stationary and mobile phase, partition
‘chromatography can be divided in to normal phase and reverse phase chromatography. In
normal phase chromatography, the stationary phase bed is strongly polar in nature (e.g. Silica
gel) and the mobile phase is non-polar (such as n-hexane or tetrahydrofuran). Polar sample
are thus retained on polar surface of the column packing longer than polar material while in
reverse phase chromatography, the stationary bed is non-polar (hydrophobic in nature, while
the mobil
phase is polar liquid, such as mixture of water and methanol or acetonitrile. Here
the more non polar the material is, the longer it will retain.
3. Size-exclusion chromatography
This involves a solid stationary phase with controlled pore size. Solids are separated
according to molecular size, with the large molecule unable to enter the pores eluted first.
4. Ton- exchange chromatography
Involves a solid stationary phase with anionic or cationic groups on the surface to
separation, HPLC and HPTLC methods have widely been exploited in pharmaceutical
analysis because of its simplicity, precision, accuracy and reproducibility of result.
5. Solid-Phase Extraction [SPE]
‘A sample preparation technique that uses LC principles to isolate, enriches, and/or
purifies analytes from a complex matrix applied to a miniature chromatographic bed. Offline
SPE is done with larger particles in individual plastic cartridges or in micro-elution plate
wells, using low positive pressure or vacuum to assist flow. Online SPE is done with smaller
particles in miniature HPLC columns using higher pressures and a valve to switch the SPE
column online with the primary HPLC column, or offline to waste, as appropriate. SPE
methods use step gradients to accomplish bed conditioning, sample loading, washing, and
elution steps. The goal is to remove matrix interferences and to isolate the analyte in a
solution, and at a concentration, suitable for subsequent analysis,
13.1. High Performance Liquid Chromatography (HPLC) *
The acronym HPLC, coined by the Late Prof. Csaba Horvath for his 1970
Pittconpaper, originally indicated the fact that high pressure was used to generate the flow
required for liquid chromatography in packed columns. In the beginning, pumps only had a
pressure capability of 500 psi (35 bars}. This was called high pressure liquid
chromatography, or HPLC. The early 1970s saw a tremendous leap in technology. These new
HPLC instruments could develop up to 6,000 psi [400 bars] of pressure, and incorporated
Department of Pharmaceutical Analysis BNPCW Page 12 INTRODUCTION
aS
improved injectors, detectors, and columns. With continued advances in performance during
this time [smaller particles, even higher pressure], the acronym HPLC remained the same, but
the name was changed to
performance liquid chromatography.
High Performance Liquid Chromatography is now one of the most powerful tools in
analytical chemistry. It has the ability to separate, identify, and quantitative the compounds
that are present in any sample that can be dissolved in a liquid. Today, compounds in trace
concentrations as low as parts per trillion (ppt) may casily be identified. HPLC can be, and
has been, applied to just about any sample, such as pharmaceuticals, food, nutraceuticals,
cosmetics, environmental matrices, forensic samples, and industrial chemicals.
[Link].2. High-Performance Liquid Chromatography [HPLC] System
1 = eluent reservoir
2 = filter
3 = high pressure pump
with pulse dampener
4= pressure gauge
5
10 = recorder (integrator, PC ete.)
ample injection valve with
syringe
1.3.2. Types of HPLC techniques”
1. Based on modes of separation
> Normal phase chromatography
> Reversed phase chromatography
2. Based on principal of separation
v
Adsorption chromatography
v
Partition chromatography
Ton exchange chromatography
Ton pair chromatography
Size exclusion or Gel permeation chromatography
vvvyv
Affinity chromatography
————
Department of Pharmaceutical Analysis BNPCW Page 13 INTRODUCTION
Leen ee eee eo!
> Chiral phase chromatography
3. Based on elution technique
> Isocratic elution
> Gradient elution
4. Based on scale of operation
> Analytical HPLC
> Preparative HPLC
5. Based on types of analysis
> Qualitative analysis
> Quantitative analysis
1.3.3. Normal phase chromatography
Normal phase HPLC (NP-HPLC) was the first kind of HPLC chemistry used, and separate
analytes, based on polarity. This method uses a polar stationary phase and a non-polar mobile
phase, and is used when the analyte is fairly polar in nature. The polar analyte associates with
and is retained by the polar stationary phase. Absorption strengths increase with increase in
analyte polarity, and the interaction between the polar analyte and the polar stationary phase
increases the elution time. The interaction strength not only depends on the functional groups
in the analyte molecule, but also on stearic factors and structural isomers is often resolved
from one another. Use of more polar solvents in the mobile phase will decrease the retention
time of the analyte while more hydrophobic solvents tend to increase retention times.
Particularly polar solvents in a mixture tend to deactivate the column by occupying the
stationary phase surface.
[Link].3. Normal-Phase Chromatography
‘Stationary Phase is Polar (Sica)
Mobie Pnaso
—+ 1s Non-Polar
(Hoxane)
1.3.4, Reversed phase chromatography (RPC)
Reversed phase HPLC (RP-HPLC) consists of a non-polar stationary phase and an
aqueous, moderately polar mobile phase. One common stationary phase is siliea which has
been treated with RMe2SiCI, where R is a straight chain alkyl group such as C\sH37 or CsHy7.
‘The retention time is therefore longer for molecules which are more non-polar in nature,
allowing polar molecules to elute more readily. Retention Time (R) is it
reased by the
addition of polar solvent to the mobile phase and decreased by the addition of more
Department of Pharmaceutical Analysis BNPCW Page 14 INTRODUCTION
Neen ee eee ee ee EEE
hydrophobic solvent, The pharmaceutical industry regularly employs RPC to qualify drugs
before their release.
RPC operates on the principle of hydrophobic interactions, which result from repulsive
forces between a polar eluent, the relatively non-polar analyte, and the non-polar stationary
phase. The binding of the analyte to the st
jonary phase is proportional to the contact surface
area around the non-polar segment of the analyte molecule upon. association with the ligand
in the aqueous eluent, The energy released in
process is proportional to the surface
tension of the eluent (water: 73 erg/em, methan
: 22 erp/em*) and to the hydrophobic
surface of the analyte and the ligand respectively. The retention can be decreased by adding
less-polar solvent (MeOH, ACN) into the mobile phase to reduce the surface tension of
water. Gradient elution uses this effect by automatically changing the polarity of the mobile
phase during the course of the analysis.
[Link].4. Reversed-Phase Chromatography
Stationary Phase is Non-Polar (Cu)
sag Sa, Mobile Phase
4, ~~ ped
sore
‘WHY Do They Separate? Se
13.5. Isocratic flow and gradient elution
[Link].5. High-Pressure-Isocratic System
sempte
infection port
al
Dsoneser? | [Deusseer2 pres
High pressure pump
A separation in which the mobile phase composition remains constant throughout the
procedure is termed isocratic (meaning constant composition). The word was coined by
Csaba Horvath, who was one of the pioneers of HPLC. The mobile phase composition does
not have to remain constant. A separation in which the mobile phase composition is changed
during the separation process is des:
bed as a gradient elution, One example is a gradient
<7 Ts
Department of Pharmaceutical Analysis BNPCW Page 15, INTRODUCTION
starting at 10% methanol and ending at 90% methanol after 20 minutes. The two components
of the mobile phase are typically termed "A" and "B"; A is the "weak" solvent which allows
the solute to elute only slowly, while B is the "strong" solvent which rapidly elutes the
solutes from the column. In reverse-phase chromatography, solvent A is often water or an
aqueous buffer, while B is an organic solvent miscible with water, such as acetonitrile,
methanol, THF, or isopropanol.
1.3.6. Working Principle of HPLC *
The components of a basic High-Performance Liquid Chromatography [HPLC]
system are shown in the simple diagram in figure 5. A reservoir holds the solvent {called the
mobile phase, because it moves]. A high-pressure pump [solvent delivery system or solvent
manager] is used to generate and meter a specified flow rate of mobile phase, typically
millilitres per minute. An injector is able to introduce [inject] the sample into the
continuously flowing mobile phase stream that carries the sample into the HPLC column.
‘The column contains the chromatographic packing material needed to effect the
separation. This packing material is called the stationary phase because it
is held in place by
the column hardware. A detector is needed to see the separated compound bands as they elute
from the HPLC column. The mobile phase exits the detector and can be sent to waste, or
collected, as desired. When the mobile phase contains a separated compound band, HPLC
provides the ability to collect this fraction of the elute containing that purified compound for
further study. This is called preparative chromatography.
‘The detector is wired to the computer data station, the HPLC system component that
records the electrical signal needed to generate the chromatogram on its display and to
identify and quantitative the concentration of the sample constituents, Since sample
compound characteristics can be very different, several types of detectors have been
developed. For example, if a compound can absorb Ultra
ht, a UV-absorbance
detector is used. If the compound does not have either of these characteristics, a more
universal type of detector is used, such as an Evaporative-Light-Scattering Detector [ELSD].
‘The most powerful approach is the use multiple detectors in series. For example, a UV and/or
ELSD detector may be used in combination with a Mass Spectrometer [MS] to analyze the
results of the chromatographic separation. This provides, from a single injection, more
Department of Pharmaceutical Analysis, BNPCW Page 16 INTRODUCTION
eS
comprehensive information about an analyte. The practice of coupling a mass spectrometer to
an HPLC system is called LC/MS.
1.4 Components of HPLC”
+ Solvent
“Solvent Delivery System (Pumps)
+ Injector
+ Column
~~ Detectors
% Recorder (Data Collection)
[Link].6. Schematic of a modular HPLC instrument
(Als
‘Solvent rack
(mobile phase) a
Degasser 2
Analytical pump 3
Injector
(autosampler) 4
Thermostatted 5
column compartment
UV Detector 6
—— a7. nT ST
Department of Pharmaceutical Analysis BNPCW Page 17 INTRODUCTION
el
[Link].7. Schematic of the dual-headed reciprocating pump
Working cylinder Equilieygtion cylinder
B
Proportoning
TMobile phase] Hoptan.
Exit valve
Pulse damper
Exit valve
Towards inj
and column
Outer
np
Solvents (maximum 4)
‘The most simplified way of explaining the cycle of operation, without taking into
account the compressibility of the solvents, is as follows. From the moment when the outlet
valve of cylinder a closes and its entrance valve open, the piston in A, moving backwards,
sucks the eluent through the inlet check valve and the chamber fills. Meanwhile cylinder B is
‘open and its piston moves forward to force the mobile phase towards the injector and the
column. The volume displaced by piston B is half of that available in the chamber of piston
A. With chamber A full, the entrance valve of a closes and the corresponding outlet valve
opens. Piston a now advances and pushes out the contents of the chamber. Half of this
volume is expelled directly towards the column, the other half serves to fill cylinder B as
piston B retracts. A pulse absorber is located between the two cylinders (diagram courtesy of
Agilent Technologies).
1.4.1. Solvent
Compatible with the instrument (pumps, seals, fitings, detector ete).
Compatible with the stationary phase.
“Readily available (often use liters/day) and of adequate purity,
+ Spectroscopic and trace-composition usually.
Not too compressible (causes pump/flow problems),
Free of gases (which cause compressibility problems).
eee
‘The polarity index is a measure of the relative polarity of a solvent, It is used for
identifying suitable mobile phase solvents.
Department of Pharmaceutical Analysis BNPCW Page 18 INTRODUCTION
ES
+ Increasing eluent strength or polarity index values mean increasing solvent
polarity, and
‘ The analyte(s) and samples must be mobile phase and stationary phase
compatible.
1.4.2, Pumps
+ It drives the mobile phase from the reservoir to the column.
Because of the small particles used in modern HPLC, modem pumps need to
operate reliably and precisely at pressures of 10,000 p.s.i. or at least 6,000 p.s.i.
Ps
To operate at these pressures and remain sensibly inert to the wide variety of
solvents used, HPLC pumps usually have sapphire pistons, stainless steel
cylinders and return valves fitted with sapphire balls and stainless steel seats.
For analytical purposes HPLC pumps should have flow rates that range from 0 to
10 ml/min., but for preparative HPLC, flow rates in excess of 100 ml/min may be
required.
The level of constancy is required because most HPLC detectors are flow
sensitive and errors in quantization will result from changes in flow rate.
TYPES
Syringe pumps
Reciprocating pumps
oe
Pneumatic pumps
Other errors are added to this due to capillary action and the small dimensions/cavities inside
the injector.
1.4.3. Injectors
4 There are three types of injectors, they are
1, Septum injectors
2. Stop flow injectors
3, Rheodyne injectors
‘There are two modes of sample injection in LC, they are
1. Load position
2. Inject position
mmr 77 EE ST
Department of Pharmaceutical Analysis, BNPCW Page 19 INTRODUCTION
[Link].8, Injectors port used in HPLC.
we
Y
=
thy? e
as
tot |
coum | coun For adsorption chromatography
4 Adsorbents such as silica or alumina are available as micro porous forms.
+ Pellicular systems generally have a high efficiency but low sample capacity
and so micro porous supports are preferred.
4 Spherical shape gives good efficiency and flow properties.
Disadvantages
‘ Developing solvent may gradually wash off the liquid Phase with repeated
use.
+ To overcome this problem bonded phases have been developed.
+ Ex. silica, silicone polymer etc.,
Stationary phase
Polar (normal phase): Silica, alumina, Cyano, amino or diol terminations on the bonded
phase.
Non-polar (reversed phase): C18 to about C8 terminations on the bonded phase, Phenyl and
Cyano terminations on the bonded phase.
> Mixtures of functional groups can be used.
Packed particles in a column require:
Frits at the ends of the column to keep the particles in,
“ Filtering of samples to prevent clogging with debris
“High pressure pumps and check-valves
+ Often a Guard Column to protect the analytical column
1.4.5. General HPLC column care”
The correct use of an HPLC column is extremely important for the life time of a
column and therefore for the benefit of your HPLC analysis. The following lines will give us
some guid
for the use, cleaning and storage of HPLC columns. These guidelines depend
‘on the one hand on the nature of the chromatographic support (silica, polymers or others) and
on the other hand on the surface chemistry of the corresponding stationary phase.
Silica based columns
General guidelines
Silica is the ideal support for HPLC columns. It offers a large mechanical stability,
excellent physicochemical surface properties, a wide range of bonding chemistries and is
compatible with a broad range of organic solvents. However, the following points are
extremely important to know when working with silica based HPLC columns.
TT
Department of Pharmaceutical Analysis, ‘BNPCW Page 22 INTRODUCTION
ee EEE EEEIDEEESREIRDEEEEEEEREEEnEEnpnnpeenememmemmenee sees a aoe ead
pi! stability
In general, HPLC columns are stable wi
in a pH range of 2 to 8. If you are
measuring a pH value, the measurement must be done in the aqueous media before mixing
the eluent with organic solvents. Modern HPLC columns can be used outside that pH range.
The new bonding chemistries allow use down to pH-1 for some stationary phases. However,
please check vendor’s product information before using silica based column outside the pH
range of 2 to 8. However, best lifetimes are obtained between pH 2.0 and pH 6.8. Stationary
phases based on ultra-pure silica gel can also be used at higher pH ranges, up to pH 11,
depending on the chemical nature of the modifier used in the mobile phase. Large bases (like
Pyrolidine) are not able to attack the surface of the silica and therefore can be used at higher
pH values. If you are working at pH values above 8 with small bases as modifier (like
Ammonia), we highly recommend using stationary phases based on Polymers or
dioxide.
irconium
Mechanical stal
ty
Stationary phases based on silica are mechanically very stable. The packed columns
show no pressure limit and can be used at more than 40 Mpa (6000 psi) without any problem.
However, please avoid pressure shocks on the column. Pressure shocks lead to channelling in
the column, which results in peak sj
g in the corresponding chromatogram.
Mobile phases (Eluents)
Silica based stationary phases are compatible with all organic solvents in the above
mentioned pH range, Please use the highest quality solvents available (HPLC grade). Also,
please filter all prepared buffer through a 0.5m filter before using them in your HPLC
system. The use of non pure solvents in HPLC causes irreversible adsorption of impurities on.
the column head,
These impurities block adsorption sites, change the selectivity of the column and lead
to peak split
ies cause so called “Ghost
ing in the chromatogram. In gradient elution, imput
Peaks”, Ghost peaks are peaks that always appear in the same position on the chromatogram.
Their origin is not the sample, but the impurities from the solvents or solvent additives.
Therefore, it is highly recommended to run a gradient jection in the beginning of
each method to determine the ghost peaks. To avoid irreversible adsorption at the column
head, you should always use a pre-column. The use of a pre-column increases the life time of
column dramatically. In addition, a pre-column can filter solid parts stemming from pump
————
Department of Pharmaceutical Analys! BNPCW Page 23 INTRODUCTION
ET
seals or injection rotors. An alternative to a pre-column is an in-line filter. These filters are
attached directly to the column. These filters get rid of solid parts in the eluent but will not
avoid irreversible adsorption of organic impurities.
Proper storage of HPLC columns
For short term storage, i.e. overnight, columns can be stored in the eluent used in last
analysis.
For middle term storage, i
. 2 days or over the weekend, columns should be flushed with
pure water to prevent microbial growth.
For long term storage
Silica based columns should be stored in an aprotic solvent.
+ The water content should not be higher than 50%. The best storing solvent is
acetonitrile,
Caution: Please make sure that all buffers are washed out of the column before
flushing with Acetonitrile. Buffer salts are mainly not soluble in Acetonitrile and can
block the capillaries and the column,
Factors affecting column effi
ney
“Dimensions of column
“Particle size of adsorbent
+ Nature of solvent
+ Temperature of column
> Pressure
+ Packing of column
Equilibration time
The equilibration time of a column depends on the column dimensions. In general, a
column is equilibrated after flushing with 20 column volumes. The equilibration time
for the most important column dimensions is summarized in the following Table,
No.5.
Shorter equilibration times are possible if you simply increase the flow rate. It is no
*
problem to do that if no chromatography is done. However, 20 column volumes are
necessary to ensure a 100% equilibration.
ary
a
Department of Pharmaceutical Analysis, BNPCW Page 24 INTRODUCTION
a CS
[Link].5. Equilibration times
Equilibration |
Column | Column volume | Flow rate
time
dimension {ml]* {ml/min}
[min]
250% 4.6mm 291 1.00 38
150 x 4.6 mm 174 1.00 35
100 x 4.6 mm 116 1.00 2B
50x 4.6mm 0.58 1.00 12
250x 4.0 mm 2.20 1.00 44
125x 4.0mm 1.10 1.00 22
2502.0 mm 0.55 0.25 44
150x 2.0 mm 033 028 26
50x 2.0mm O1L 0.25 9
Regeneration of a column
+ Irreversible adsorption of impurities stemming from the matrix on the column head
‘can cause changes in selectivity or peak splitting. Often those “dirty columns” can be
regenerated by applying the following protocols.
Regeneration of RP packing’s
RP- packing’s are C18, C8, and C4, Cl, C30, CN or Phenyl! stationary phases.
Flush the column with 20 column volumes water
ees
Flush the column with 20 column volumes acetonitrile
°
Flush the column with 5 column volumes isopropanol
Flush the column with 20 column volumes heptane
“> Flush the column with 5 column volumes isopropanol
Regeneration of NP (Normal Phase) packing’s
> NP-packing's are Silica, Diol, Nitro and Amino stationary phases.
> Flush the column with 20 column volumes Heptane
> Flush the column with 5 column volumes Isopropanol
% Flush the column with 20 column volumes Acetonitrile
> Flush the column with 20 column volumes Water
Tr EE TD
Department of Pharmaceutical Analysis BNPCW Page 25 INTRODUCTION
SS
> Flush the column with 20 column volumes Acetonitrile
> Flush the column with 5 column volumes Isopropanol
> Flush the column with 20 column volumes Heptane
Regeneration of Ion Exchange Pickings
Ion exchange packing’s are Anion or Cation exchangers (WCX, SCX, WAX and
SAX) Flush the column with 20 column volumes of the same eluent, but double the buffer
concentration. Follow the regeneration protocol for RP packing’s (see above) Flush with 20
column volumes of water equilibrate the column now to the original conditions.
Polymer based columns
Polymer based stationary phases show higher pH stability but lower mechanical
stability, compared to silica based columns. Also, polymer’ based packing’s are not
compatible with all organic solvents. They swell or shrink in some organic solvents.
Unfortunately, the pressure stability and solvent compatibility are different for the different
nature of polymers and from manufacturer to manufacturer. Therefore, no general rules for
the column care of polymer based materials can be given. Always read the instructions for the
use of those columns. In case of doubt please contact the corresponding manufacturer.
1.4.6 Detectors of HPLC '°*
‘A chromatography detector is a device used liquid chromatography (LC) to visualize
components of the mixture being eluted off the chromatography column.
‘There are two general types of detectors: destructive and non-destructive.
“The destructive detectors perform continuous transformation of the column effluent
(burning, evaporation or mixing with reagents) with subsequent measurement of
some physical property of the resulting material (plasma, aerosol or reaction
mixture).
“ The non-destructive detectors are directly measuring some property of the column
effluent (for example UV absorption) and thus affords for the further analyte
recovery.
‘The detector for an HPLC is the component that emits a response due to the eluting sample
compound and subsequently signals a peak on the chromatogram. It is positioned
immediately posterior to the stationary phase in order to detect the compounds as they elute
from the column,
———— 1777 a ToSTo
Department of Pharmaceutical Analysis BNPCW Page 26 INTRODUCTION
TT
‘An ideal LC detector should have the following properties
+ Low drift and noise level (particularly crucial in trace analysis).
High sensitivity.
4 Fast response.
‘Wide linear dynamic range (this simplifies quantitation).
4 Low dead volume (minimal peak broadening).
‘There are many types of detectors that can be used with HPLC. Some of the more common
detectors include:
4 Refractive Index (RI)
+ Ultra-Violet (UV)
“Fluorescent
+ Radiochemical
+ Electrochemical
4 Near-Infra Red (Near-IR)
+ Mass Spectrometry (MS)
4 Nuclear Magnetic Resonance (NMR)
ight Scattering (LS)
1. Refractive Index (RI) detectors
> Measure the ability of sample molecules to bend or refraced light. This property for
each molecule or compound is called its refractive index.
> For most RI detectors, light proceeds through a bi-modular flow-cell to a photo
detector.
> Detection occurs when the light is bent due to samples eluting from the column and
this is read as a disparity between the two channels.
‘Types of RI detector
Two types of RI are available, they are:
> Deflection type (most popular)
> Refle
Deflection type RI detector
The reason it’s called deflection type is because deflection is created in a rectangular sample
J type (measure changes in % reflected light at glass-iquid interface)
cell by separating the compartment into two parts with a diagonal glass divider.
Operating Principles
Y Light from the source is focused onto the sample cell, which consist of sample and
the reference chambe
Page 27
Department of Pharmaceutical Analysis BNPCW ‘ag INTRODUCTION
Sd
¥ After deflection from the mirror, light is diverted through an optical. zero
adjustment (beam splitter) into the detector, which actually consist of two photo-
cells, Pl and P2.
Y When a solute elutes off the column the RI of the sample compartment
¥ This causes a change in the amount of deflected light, which in turn changes the
relative amount falling on Pl and P2.
Disadvantages of RI detector
a) Not-suitable for gradient elution, changes in solvent composition change RI. Therefore,
baseline shifts and S/N are affected.
) Require careful control of the column and detector temperature.
c) Moderate sensitivity10° to 10""g, not useful for trace analysis.
2, Fluorescence detector
‘Measure the ability of a compound to absorb then re-emit light at given wavelengths.
* Each compound has a characteristic fluorescence.
[Link].9. Fluorescence detector
Fluorescence Detectors
Bee
4 ‘The excitation source passes through the flow- The sample compound or molecule is ionized, it is passed through a mass analyzer
and the ion current is detected.
> There are various methods for ionization:
A) Electron Impact (EI) An electron current or beam created under high electric potential is
used to ionize the sample migrating off the column,
B) Chemical Ionization A less aggressive method which utilizes ionized gas to remove
electrons from the compounds eluting from the column,
C) Fast Atom Bombardment (FAB) Xenon atoms are propelled at high speed in order to
ionize the eluents from the column.
Ithas detection limit of 10° to 107° g/ml.
5, Light-seattering (LS) detectors
When a source emits a parallel beam of light which strikes particles in solution, some light is
reflected, absorbed, transmitted or scattered.
Two forms of LS detection may be used to measure the later occurrences,
A) Nephelometry: This is defined as the measurement of light scattered by a particulate
solution, This method enables the detection of the portion of light scattered at a multitude of
Department of Pharmaceutical Analysis BNPCW Page 29 INTRODUCTION
angles. The sensitivity depends on the absence of background light or scatters since the
Detection occurs at the black or null background,
B) Turbidimetry: This is defined as the measure of the reduction of light transmitted due to
particles in solution. It measures the light scatter as a decrease in the light that is transmitted
through the particulate solution, Therefore, it quantifies the residual light transmitted.
Sensitivity of this method depends on the sensitivity of the machine employed, which can
range from a simple spectrophotometer to a sophisticated discrete.
6. Nuclear magnetic resonance (NMR) detectors
Certain nuclei with odd- numbered masses, including H and 13C, spin about an axis
in a random fashion.
are aligned either
+ However, when placed between poles of a strong magnet, the 5
parallel or anti-parallel to the magnetic field, with the parallel orientation favored
since it is slightly lower in energy.
‘> The nuclei are then irradiated with electromagnetic radiation which is absorbed and
places the parallel nuclei into a higher energy state; consequently they are now in
"resonance" with the radiation.
+ Each H or C will produce different spectra depending on their location and adjacent
molecules or elements in the compound, because all nuclei in molecules are
surrounded by electron clouds which change the encompassing magnetic field and
thereby alter the absorption frequency.
7. Near-Infrared detectors
> Operates by scanning compounds in a spectrum from 700 to 1100 nm.
Stretching and bending vibrations of particular chemical bonds in each molecule are
3
detected at certain wavelengths.
‘This is a method which offers several advantages
—
+ Speed (sometimes less than I second)
«> Simplicity of preparation of sample
<> Multiple analyses from single spectrum
+ Non-consumption of the sample
aan BNPCW Page 30
Department of Pharmaceutical Analysis INTRODUCTION
8, Ultra-Violet (UV) detector
It is divided in to three types they are fixed wavelength, variable wavelength, and
diode array detectors.
[Link].10. Ultra-Violet (UV) Detector
Sompie Catt or Flypocell
oa [Ce time.
why,
eS
ture
TeronColoan “isesrs
It measures the ability of a sample to absorb light. This can be accomplished at one or several
wavelengths
> Fixed wavelength measures at one wavelength, usually 254 nm
> Variable wavelength measures at one wavelength at a time, but can detect over a
wide range of wavelengths
> Diode Array measures a spectrum of wavelengths simultaneously
> UV detectors have a sensitivity to approximately 10° or 10° gmv/ml.
A. Fixed wavelength detectors
a) It is the most common and inexpensive detector. The use of suitable 2 is
determined by the nature of the light source used.
b) Deuterium lamp can be used over a range of wavelength (covers a continuum of
wavelengths), hence covering most of the UV spectral region.
B. Variable wavelength detectors
Provides detection of eluted peak at any selected intensity.
a) It is less sensitive than fixed wavelength but the detection wavelength can be
varied.
b) Deuterium source is mostly used because it provides continuum source. This can be
combined with a suitable monochromator in dual beam mode.
C. Diode Array detectors (DAD)
> A diode array consists of a number of photosensitive diodes placed side by side and
insulated from one another in the form of a multi-layer sandwich.
> The common use of a diode array is to monitor light that has passed through a liquid
sensor cell as in a multi-wavelength liquid chromatography detector.
Department of Pharmaceutical Analysis BNPCW Page 31 INTRODUCTION
Se
> There are two major advantages of diode array detection:
1. In the first, it allows for the best wavelength to be selected for actual analysis. This
is particularly important when no information is available on molar absorptivities at
different wavelengths.
2. The second major advantage is related to the problem of peak purity. Often, the
peak shape in itself does not reveal that it actually corresponds to two (or even more)
components.
1.5. Chromatographic parameters *
1, Retention time (R))
.. Efficiency (N)
. Retention volume (V,)
2
3
4, Resolution factor (R.)
5, Column Efficiency (N)
6, HETP (High Equivalent Theoretical Plates)
7. Capacity factor (mass distribution ratio, Dx)
8. Symmetry factor (A,)
9, Tailing Factor (T)
Retention time
Retention time is the difference in time between the points of injection and eluted
from a column, Retention time is measured in minutes or seconds. Retention time is also
proportional to the distance moved on a chart paper, which can be measured in em or mm.
Retention volume
‘The distance of the peak maxima from the injection point expressed in time units is
called retention time (ta), and it serves as an identifier for the given analyte on that particular
system, Retention time is probably the most widely used descriptor ofthe analyte behaviour,
and itis the most easily measurable parameter. However, even though it is easily measurable,
's the least universal parameter. Analyte retention time is dependent on the mobile phase
flow rate; the faster the flow rate, the smaller the analyte retention time, It is also dependent
tion time and the mobile-phase flow
on the flow rate stability, The product of the analyte rete
rate is the retention volume (Va):
Retention volume (x) = Retention time (¢4) x flow rate
BNPCW Page 32
Department of Pharmaceutical Analysis: INTRODUCTION
HETP (High Equivalent Theoretical Plates)
A theoretical plate can be of any height, which decides the efficiency of separation. If
HETP is less the column is more efficient. If HETP is more, the column is less efficient. The
height equivalent to a theoretical plate (HETP) is given by
HETP=Length of column (LYN
Where
N= plates per meter
RT= retention time of the components.
W = width of the base of the component peak using tangent method.
L= column length in meters
Capacity factor (mass distribution ratio, Da)
This factor determines the retention of a solute and can be calculated from the
chromatogram using the following formula:
Where,
tn= retention time of the solute
t¢= retention time of an unretained component
‘A low Dm value indicates that the peak elutes close to the solvent front, which may
‘compromise selectivity. A minimum Dp, value of | is recommended for the peak of interest.
Void volume
‘The volume of the liquid phase in the column is called “void volume” (w). Several
other names are also used in the chromatographic literature: “dead volume,” “hold-up
volume,” and sometimes “retention volume of non retained component.” In this book we will
be using term “void volume.”
ve
te=t®, = "B
BNPCW Page 33 INTRODUCTION
Void time can be interpreted as part of the total analyte retention time that the analyte
actually spends in the mobile phase moving through the column, and for the rest of the
retention time the analyte sits on the stationary phase surface.
Retention factor
The ratio of the reduced retention volume to the void volume is a widely used
dimensionless parameter called retention factor, k.
VAL ta-bo
Vo to
Retention factor (sometimes called capacity factor) is a very convenient
chromatographic descriptor since it is dimensionless and independent on the mobile phase
flow rate and column dimensions. Ideally if the retention of the same analyte was measured
‘on two instruments equipped with columns of different dimensions with the same type of
stationary phase and the same mobile phase, theoretically the retention factors of that analyte
‘on both systems should be identical.
[Link].11. showing retention factor
Resolution
‘The distance between the peak maxima reflects the selectivity of the system. The
preater the distance, the higher the selectivity. The width of the chromatographic peak
reflects the system band broadening and thus efficiency. Resolution, R, is defined as the ratio
of the distance between two peaks to the average width of these peaks (at baseline), and this
descriptor encompasses both the efficiency and selectivity.
For the resolution of a so-called “critical pair” of analytes (two analytes in the mixture that
have minimal distance between them compared to all other analytes in the
fraa trea
Wr +
Department of Pharmaceutical Analysis BNPCW Page 34
R=2 INTRODUCTION
NES
icure, if they have relatively high retention factors (k_> 5) that their peak wid ean be
assumed as equal, reduces to
Peak width could be expressed from equation
Ate
VN
If we select the retention of the second analyte for the calculation of the peak width, then
applying equation in to below equation.
“
pateactea YN
tre 4
Relatively simple algebraic conversion will bring us to so-called Master Resolution Equation
Efficiency (N)
The efficiency is the measure of the chromatographic band broadening and the number of the
theoretical plates (\) in the column and is usually calculated using the following equation
Where ta is the analyte retention time and w is the peak width measured in time units as the
distance between the intersections of the tangents to the peak inflection points with the
baseline, as shown in [Link].12, Column efficiency is mainly dependent on the kinetic
factors of the chromatographic system such as molecular diffusion, mass-flow dynamics,
Properties of the column packing bed, flow rate, and so on, The smaller the particles and the
‘more uniform their packing in the column, the higher the efficiency. The faster the flow rate,
the less time analyte molecules have for diffusive band-broadening. At the same time, the
faster the flow rate, the further analyte molecules are from the thermodynamic equilibrium
TS
Department of Pharmaceutical Analysis BNPCW Page 35
a INTRODUCTION
swith the stationary phase. This shows that there should be an optimum flow rate that allows
achievement of an optimum efficiency for a given column,
[Link].12. Optimum Efficiency
Retention time
Efficiency and selectivity are complementary chromatographic descriptors. A column with
high efficiency can generate narrow chromatographic zones and allows the separation of
analytes with low selectivity.
[Link].13. Efficiency and Selectivity,
cersancny
system to discriminate different analytes is
Selectivity The ability of the chromatograph
called selectivity. Selectivity is determined as the ratio of the retention factors of two
analytes, or the ratio of the reduced retention times.
try at
e 3
Department of Pharmaceutical Analysis BNPCW Page 36 INTRODUCTION
‘roiling factor (T) the Tailing factor T, a measure of peak symmetry is unity for perfectly
ymmetical peaks and its value increases as tailing becomes more pronounced. In some
cases, values less than 1 maybe observed. As peak asymmetry increases integration and
hence precision becomes less reliable,
‘T= Woas /2f
Where,
Woos = width of peak at 5% height
f= Distance from the peak maximum to the leading edge of the peak, the distance
being measured at a point 5% of the peak height from the baseline Limit: <2
1.5. Analytical Method Development'™"”
‘A good method development strategy should require only as many experimental runs
as are necessary to achieve the desired final result. Finally method development should be as
simple as possible and it should allow the use of sophisticated tools such as computer
modeling. The important factors, which are to be taken into account to obtain reliable
quantitative analysis, are
ms
Careful sampling and sample preparation.
¥
Appropriate choice of the column.
¥
Choice of the operating conditions to obtain the adequate resolution of the mixture.
Reliable performance of the recording and data handling systems.
v
Suitable integration/peak height measurement technique.
7
‘The mode of calculation best suited for the purpose.
7
Validation of the developed method.
Careful sampling and sample preparation’?
Before beginning method development it is need to review what is known about the
sample in order to define the goals of separation, The sample related information that is
important is summarized in following.
‘The sample related summarized relation
> Number of compounds present, chemical structures
> Molecular weights of compounds
> pKa values of compounds, UV spectra of compounds
> Concentration range of compounds in samples of interest
Department of Pharmaceutical Analysis BNPCW Page 37 5 anthonerion
> Sample solubility
se chemical composition of the sample can pro
le valuable clues for the best choice of
| conditions for an HPLC separation,
inital ‘
‘Table No. 6: Table showing special samples and customized conditions
Sample Requirements
Tnorganie ions Detection is primary problem; use fon chromatography.
‘Some isomers can be separated by reversed-phase HPLC and are
isn then classified as regular samples better separations of isomers
are obtained using either (1) normal-phase HPLC or (2) reversed-
phase separations with cyclodextrin-silica columns.
‘These compounds require “chiral” conditions for their
separation.
‘Several factors make samples of this kind “special”: molecular
Biologicals conformation, polar functionality, and a wide range of
hydrophobicity.
Choice of the column
‘The selection of the column in HPLC is somewhat similar to the selection of columns
in GC, in the sense that, in the adsorption and partition modes, the separation mechanism is
based on inductive forces, dipole-dipole interactions and hydrogen bond formation. In case of
ion-exchange chromatography, the separation is based on the differences in the charge, size
of the ions generated by the sample molecules and the nature of ionisable group on the
stationary phase, In the ease of size-exclusion chromatography the selection of the column is
based on the molecular weight and size of the sample components.
Choice of the operating conditions to obtain the adequate resolution of the mixture
Most of the drugs come under the category of regular samples. Regular samples mean
typical mixtures of small molecules that can be separated using more or less standardized
starting conditions, Regular samples can be further classified as neutral or ionic, Samples
classified as ionic include acids, bases, amphoteric compounds and organic salts. If the
Sample is neutral buffers or additives are generally not required in the mobile phase, Acids or
or cationic
bases usually require the addition of a buffer to the mobile phase. For ba
Pa
Department of Pharmaceutical Analysis BNBCW, ae INTRODUCTION
sanples, less acidic reverse phase columns are recommended. Based on recommendations of
the conditions, the first exploratory run is carried and then improved systematically. On the
tosis of the initial exploratory run isocratic or gradient elution can be selected as most
suitable. Ir typical reverse-phase conditions provided inadequate sample retention it suggests
the use of either ion-pair or normal phase HPLC. Alternatively the sample may be strongly
retained with 100% acetonitrile as mobile phase suggesting the use of non-aqueous reverse
phase eromatography ot normal phase HPLC,
Getting started on method development
‘One approach is to use an isocratic mobile phase of some average organic solvent
strength (50%). A better alternative is to use a very strong mobile phase first (80-10%) then
reduce %B as necessary. The initial separation with 100% B results in rapid elution of the
‘entire sample but few groups will separate. Decreasing the solvent strength shows the rapid
separation of all components with a much longer run time, with a broadening of latter bands
and reduced retention sensitivity. Goals that are to be achieved in method development are
briefly summarized.
Separation or resolution is a primary requirement in quantitative HPLC. The
resolution (R,) value should be maximum (R>2) favors maximum precision. Resolution
usually degrades during the life of the column and can vary from day to day with minor
fluctuations in separation conditions. Therefore values of R=2 or greater should be the goal
during method development for sample mixtures. Such resolution will favor both improved
assay precision and greater method ruggedness. Some HPLC assays do not require base line
ive analysis). In such cases only enough
separation of the compounds of interest (qual
separation of individual components is required to provide characteristic retention times for
peak identification, The time required for a separation (runtime = retention time for base
band) should be as short as possible and the total time spent on method development is
reasonable (runtimes 5 to 10 minutes are desirable).
Repeatable separation
As the experimental runs described above are being carried out, itis important to confirm that
cach chromatogram can be repeated. When we change conditions (mobile phase, column, and
temperature) between method development experiments, enough time must elapse for the
column to come into equilibrium with the new mo!
BNPCW Page 39
Department of Pharmaceutical Analysis
phase and temperature. INTRODUCTION
Usually column equilibration is achieved after passage of 10 to 20 volumes of the new
sobile phase through the column. However this should be confirmed by repeating the
daperiment under the sane conditions. When constant retention times are observed in two
soch back-to-back repeat experiments (+ 0.5% or beter), it can be assumed thatthe column is
equirated and the experiments are repeatable
Optimization of HPLC method
During the optimization stage, the initial sets of conditions that have evolved from the
fast stages of development are improved or maximized in terms of resolution and peak shape,
plate counts, asymmetry, capacity factor, elution time, detection limits, limit of quantitation
and overall ability to quantify the specific analyte of interest.
‘The various parameters that include to be optimized during method development are
> Selection of mode of separation.
> Selection of stationary phase.
> Selection of mobile phase.
> Selection of detector.
Selection of mode of separation
the mobile phase is comparatively more polar than the
In reverse phase mode,
+ moderately polar compounds the most
stationary phase. For the separation of polar 0
preferred mode is reverse phase. The nature of the analyte is the primary factor in the
selection of the mode of separation. A useful and practical measurement of peak shape is
0% of full
1g factor. Peak asymmetry is measured at 1
'5%, Reproducibility of retention times and capacity
peak asymmetry factor and peak tai
peak height and peak tailing factor at
factor is important for developing a rugged and repeatable method.
Buffers and buffer capacity
Buffer and its strength play an important role in deciding the peak symmetries and
josphate buffers.
separations. Some of the most commonly employed buffers are ph
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Department of Pharmaceutical Analysis INTRODUCTION
Table No. 7: Table showing buffers and buffer capacity
ae PK, =| Maximum —_| UV Cut-off
(25°C) | Buffer Range | (nm)
ue 03 |- 210 (0.1%)
Phosphate, H2POs pK, 21 113.1 <200
Phosphate, pK2 HPO. 72 |62-82 <200
Phosphate, pKs PO.” 123 113-133 <200
Citrate, pK CsHsO (COzH)2 (CO: | 3.1 2141 230
Citrate, pK2 CsHs0 (COpH): (CO), |4.7- | 3.75.7 230
Citrate, PKs C3Hs0 (CO2-)3 64 | 44-64 230
Carbonate, pK: HCO; 6.1 SATA <200
Carbonate, pK COs fos fos-t13 > 200
Formate 3.8 28-48 210 (10nM)
Acetate 48 3.85.8 210 (10nM.)
Ammonia 93 83-103 200 (100M)
Borate 92 8.2-10.2 NIA
TEA 10.8 | 9.8118 <200
Mobile phase composition
Most chromatographic separations ean be achieved by choosing the optimum mobile
phase composition. This is due to the 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 should be conducted with mol
for the best separations of analyte peak. A
bile phases having buffers with
different pH and different organic phases to check
mobile phase which gives separation of analyte Pe "
at least # 0.2% of the selected mobile phase composition
ak and which is rugged for variation of
both aqueous and organic phase by
should be used, *
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Department of Pharmaceutical Analys! Ee
INTRODUCTION
selection of detector
‘The detector was chosen depending upon some characteristic property of ‘the analyte
ine UV absorbance, florescence, conductance, oxidation, reduction ete. ‘The characteristics
snare to be fulfilled by a detector tobe used in HPLC determination re,
> High sensitivity facilitating trace analysis.
le baseline noise to facilitate lower detection, Low dead volume.
> Negli
Pharmaceutical ingredients do not absorb all UV light equally, so that selection of
ies of
detection wavelength is important. An understanding ofthe UV light absorptive prope
the organic impurities and the active pharmaceutical ingredient is very helpful. For the
greatest sensitivity Anas should be used. Ultra violet wavelengths below 200nm should be
avoided because detector noise increases in this region. Higher wavelengths give greater
selectivity.
1,6 ANALYTICAL METHOD VALIDATION
.n be defined as per ICH “Establishing documented evidence
‘Method validation cai
roduce a
gree of assurance that a specific activity will consistently P
which provides a high de,
acteristics”.
desired result or product meeting its predetermined specifications and quality char
ICH Method validation parameters'*”
hic methods used in analytical applications there is more consistency
For chromatogra
sted sufstenpes are eniamoaly irtsent it vhs phalitcutieal Peel: but
in validation. Rel
those are always within the limits as specified in ICH (Q2B),
> Specificity
Linearity
‘Accuracy
Precision
vvvyv
Limit of Detection
Limit of Quantitation
vv
Robustness
> System suitability
1.5.1 Specificity/Selectivity
Specificity is ability to assess Unt
components that may be expected to be pres
often used interchangeably. According to ICH the term specific genet
that produces a response fora single analyte only ‘wile the term selectivity refers fo a method
equivocally the analyte in the presence of
‘ent, The terms selectivity @
rally refers to & method
and specificity are
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Department of Pharmaceutical Analysis INTRODUCTION
that provides responses for a number of chemical entities that may or may not be
inguished from each other. If the response is
istinguished from all other responses, the
snethod is said to be selective, Since there are very few methods that respond to only one
analyte, the term selectivity is usually more appropriate.
Specificity is the ability of a method to discriminate between the analytes of interest
and other components that are present in the sample. Studies are designed to evaluate the
ies,
degree of interference, if any which can be attributed to other analytes, impu
degradation products, reagent "blanks" and excipients. This provides the analyst with a
degree of certainty that the response observed is due to the single analyte of interest. The
degree of specificity testing varies depending on the method type and the stage of validation.
Specificity should be evaluated continually through the drug development process.
Specificity is sometimes used interchangeably wit the term “selectivity”. The argument over
which term is more correct is one of semantics. Although there is some dissention, the term
“specificity” has been adopted by the regulatory guidance documents and should be used fo
prevent further confusion.
Blank solution to show no interference with any HPLC system.
v
} Placebo to demonstrate the lack of interference from excipients.
Drug substance to show that all significant related substances are resolved from the
v
drug substance.
‘Authentic samples of critical related substances to show that all Known related
3
substances are resolved from each other.
1.5.2 Accuracy
‘The Accuracy of analytical procedure expre
xe value or as an accepted reference value
.sses the closeness of agreement between
the value that is accepted either as a conventional tn
and value found.
‘Accuracy may be inferred once precision,
method should be established from 50% of the
Jinearity and specificity have been
established. Accuracy for the area percent
ICH reporting limit to the nominal concentration of drug substance in the sample solution.
For the high-low and external standard methods, determine accuracy from 50% of the ICH
reporting level to 150% of the proposed shelf life specification of the related substances. In
addition forthe area percent and high-low methods, itis necessary to determine the accuracy
of the related substances and the drug substance:
accuracy of related substances is required. Since the response of the druy
sample solution is not used in the external standard calculation it is mot_necessary 10
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For the external standard method only the
ig substance in the
'BNPCW INTRODUCTION
gerermine accuracy for the drug substance, Typically known amounts of related substances
and the dB substance in placebo are spiked to prepare an accuracy sample of known.
concentration of related substance. According to the ICH accuracy should be determined
using a minimum of nine determinations over a minimum of three concentration levels
covering the range.
1.53 Precision
ICH defines the precision of an analytical procedure as the closeness of agreement
(degree of scatter) between a series of measurements obtained from multiple sampling of the
same homogeneous sample under the prescribed conditions. Precision is the measure of how
close the data values are to each other for a number of measurements under the same
analytical conditions. ICH has defined preci
nn to contain three components: repeatability,
intermediate precision and reproducibility. Ruggedness as defined in USP XXII <1225>,
1990 incorporates the concepts described under the terms “intermediate precision”,
reproducibility" and "repeatability" of this guide,
15.4 Linearity
Linearity of an analytical procedure as its ability (within a given range) to obtain test
results that are directly proportional to the concentration (amount) of analyte in the sample.
Concentration range
‘The concentration range used for linearity should be large enough to encompass the
desired range of the method. A minimum of five concentration ranges should be investigated
and a plot of the detector response vs. the sample concentration should be generated. It is
important that the concentration ranges selected for the linearity study are relatively equally
spaced throughout the range of the method (e.g., 25%, 50%, 75%, 100%, 125% and 150%),
‘and not clustered, as this will provide a skewed estimation of linearity.
Acceptance criteria
Solutions of known concentrations are used to determine the linearity. A. plot of peak
‘ea versus concentration (in percent related substance) is used to demonstrate the linearity.
Authentic samples of related substances with known purity are used to prepare these
Solutions. In most cases, for the linearity of a drug product, spiking the related substance
Authentic sample into excipients is not necessary, as the matrix effect should be investigated
in method accuracy. Visual inspection is the most sensitive method for detecting nonlinearity.
Therefore, the plot has to be linear by visual inspection. In addition, according to ICH
Buidelines, the following results should be reported: slope, correlation coefficient, y-intercept,
enartment of Pharmaceutical Analysl
eee.
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