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1. INTRODUCTION
Analytical chemistry may be defined as the Science and art of determining the
composition of materials in terms of the elements or compounds contained.
Analytical method is a specific application of a technique to solve an analytical
problem. The use of instrumentation is an exciting and fascinating part of chemical
analysis that interacts with all areas of chemistry and with many other areas of pure and
applied science. Analytical instrumentation plays an important role in the production and
evaluation of new products and in the protection of consumers and the environment. This
instrumentation provides the lower detection limits required to assure safe foods, drugs,
water and air. The manufacture of materials, whose composition must be known
precisely, is to be monitored by analytical instruments.1
Typical Instrumental Techniques:
The methods of estimation of drugs are divided into physical, chemical,
physicochemical and biological ones of them, physical and physicochemical methods are
used mostly. Physical methods of analysis involve the studying of the physical properties
of a substance. They include determination of the solubility, transparency or degree of
turbidity, color density or specific gravity (for liquids), moisture content, melting,
freezing and boiling points. Physicochemical methods are used to study the physical
phenomenon that occurs as a result of chemical reactions. Among the physicochemical
methods are optical refractometry, polarimetry, emission and fluorescent methods of
analysis, photometry including photocolorimetry, spectrophotometry, nephelometry and
turbidometry, electrochemical (potentiometry, amperometry, coulometer, polarography)
and chromatography (column, paper, thin layer, gas, high performance liquid) methods
are generally preferable. Methods involving nuclear reactions such as nuclear magnetic
resonance (NMR) and paramagnetic resonance (PMR) are becoming more popular. The
combination of mass spectroscopy with gas chromatography is one of the most powerful
tools available. The chemical methods include the gravimetric and volumetric
procedures, which are based on complex formation, acid-base, precipitation and redox
reactions. Titrations in non-aqueous media and complexometric have been widely used in
pharmaceutical analysis whenever the existing amounts are in milligram level and the
interferences are negligible. The methods (HPLC, GLC, NMR and Mass Spectroscopy)
of choice for assay involve sophisticated equipment that are very costly and pose
problems of maintenance. Hence they are not in the reach of most laboratories and smallscale industries, which produce bulk drugs and pharmaceutical formulations. These
analytical techniques can be classified as follows.2

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A) Spectrometric Techniques

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 Techniques
Gas Chromatography
High performance Liquid Chromatography
Thin Layer Chromatography
C) Miscellaneous Techniques
Kinetic Techniques
Mass Spectrometry
Thermal Analysis
D) Hyphenated Techniques
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)
The visible spectrophotometric (or colorimetric) methods which fall in the
wavelength region 400-800 nm and fluorimetric methods (may fall in UV & Visible
regions) are very simple, cheap and easy to carry out estimations of drugs in bulk form
and their formulations. The limitations of many colorimetric or fluorimetric methods of
analysis lie in the chemical reactions upon which the procedures are based rather than the
instruments available. Many of the reactions involve color or fluorescence of a particular
drug are quite selective or can be rendered selective through the introduction of masking
agents, control of PH, use of solvent extraction technique, adjustment of oxidation states
or by prior removal of interfering ingredients with the aid of chromatographic separation .
1. This is preferably followed by general methodology for UV-Visible and HPLC
method developments.
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2. Followed by literature of drugs used in Analysis

1.1. CHROMATOGRAPHY3:
Chromatography is a family of analytical chemistry techniques for the separation
of mixtures. It involves passing the sample, a mixture which contains the analyte, in the
"mobile phase", often in a stream of solvent, through the "stationary phase." The
stationary phase retards the passage of the components of the sample. When components
pass through the system at different rates they become separated in time, like runners in a
marathon. Ideally, each component has a characteristic time of passage through the
system. This is called its "retention time."
A chromatograph takes a chemical mixture carried by liquid or gas and separates
it into its component parts as a result of differential distributions of the solutes as they
flow around or over a stationary liquid or solid phase. Various techniques for the
separation of complex mixtures rely on the differential affinities of substances for a gas
or liquid mobile medium and for a stationary adsorbing medium through which they pass;
such as paper, gelatin, or magnesium silicate gel.
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.
1.2 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY4-7:
HPLC is a modern technique, it is a much more reliable and reproducible method
for the standardization of both single and compound formulation. HPLC is a separation
technique based on a stationary phase and a liquid mobile phase. Separations are
achieved by partition, adsorption or ion exchange process, depending upon the size of
stationary phase used.
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.
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Types of Modes in HPLC

Based on modes of separation:
Normal Phase Chromatography
Reverse Phase Chromatography
Based on principle of separation:

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

Based on elution technique:

Isocratic separation
Gradient separation
Partition chromatography
Partition chromatography was the first kind of chromatography that
chemists developed. The partition coefficient principle has been applied in paper
chromatography, thin layer chromatography, gas phase and liquid-liquid applications.
The 1952 Nobel Prize in chemistry was earned by Archer John Porter Martin and Richard
Laurence Millington Synge for their development of the technique, which was used for
their separation of amino acids. Partition chromatography uses a retained solvent, on the
surface or within the grains or fibers of an "inert" solid supporting matrix as with paper
chromatography; or takes advantage of some additional coulombic and or hydrogen
donor interaction with the solid support. Molecules equilibrate (partition) between a
liquid stationary phase and the eluent. Known as Hydrophilic Interaction
Chromatography (HILIC) in HPLC, this method separates analytes based on polar
differences. HILIC most often uses a bonded polar stationary phase and a non-polar,
water miscible, mobile phase. Partition HPLC has been used historically on unbonded
silica or alumina supports. Each works effectively for separating analytes by relative
polar differences, however, HILIC has the advantage of separating acidic, basic and
neutral solutes in a single chromatogram.

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The polar analytes diffuse into a stationary water layer associated with the polar
stationary phase and are thus retained. Retention strengths increase with increased analyte
polarity, and the interaction between the polar analyte and the polar stationary phase
(relative to the mobile phase) increases the elution time. The interaction strength depends
on the functional groups in the analyte molecule which promote partitioning but can also
include coulombic (electrostatic) interaction and hydrogen donor capability. Use of more
polar solvents in the mobile phase will decrease the retention time of the analytes,
whereas more hydrophobic solvents tend to increase retention times.
Normal phase chromatography
Also known as Normal phase HPLC (NP-HPLC), or adsorption
chromatography, this method separates analytes based on adsorption to a stationary
surface chemistry and by polarity. It was one of the first kinds of HPLC that chemists
developed. NP-HPLC uses a polar stationary phase and a non-polar, non-aqueous mobile
phase, and works effectively for separating analytes readily soluble in non-polar solvents.
The analyte associates with and is retained by the polar stationary phase. Adsorption
strengths increase with increased analyte polarity, and the interaction between the polar
analyte and the polar stationary phase (relative to the mobile phase) increases the elution
time. The interaction strength depends not only on the functional groups in the analyte
molecule, but also on steric factors. The effect of sterics on interaction strength allows
this method to resolve (separate) structural isomers.
The use of more polar solvents in the mobile phase will decrease the retention
time of the analytes, whereas more hydrophobic solvents tend to increase retention times.
Very polar solvents in a mixture tend to deactivate the stationary phase by creating a
stationary bound water layer on the stationary phase surface. This behavior is somewhat
peculiar to normal phase because it is most purely an adsorptive mechanism (the
interactions are with a hard surface rather than a soft layer on a surface).
Displacement Chromatography
The basic principle of displacement chromatography is: A molecule
with a high affinity for the chromatography matrix (the displacer) will compete
effectively for binding sites, and thus displace all molecules with lesser affinities. There
are distinct differences between displacement and elution chromatography. In elution
mode, substances typically emerge from a column in narrow, Gaussian peaks. Wide
separation of peaks, preferably to baseline, is desired in order to achieve maximum
purification. The speed at which any component of a mixture travels down the column in
elution mode depends on many factors. But for two substances to travel at different

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speeds, and thereby be resolved, there must be substantial differences in some interaction
between the biomolecules and the chromatography matrix. Operating parameters are
adjusted to maximize the effect of this difference. In many cases, baseline separation of
the peaks can be achieved only with gradient elution and low column loadings. Thus, two
drawbacks to elution mode chromatography, especially at the preparative scale, are
operational complexity, due to gradient solvent pumping, and low throughput, due to low
column loadings. Displacement chromatography has advantages over elution
chromatography in that components are resolved into consecutive zones of pure
substances rather than peaks. Because the process takes advantage of the nonlinearity
of the isotherms, a larger column feed can be separated on a given column with the
purified components recovered at significantly higher concentrations.
Reverse Phase Chromatography (RPC)
Reversed phase HPLC (RP-HPLC or RPC) has a non-polar stationary phase and
an aqueous, moderately polar mobile phase. 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. 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. RPC is so commonly used that it is often
incorrectly referred to as "HPLC" without further specification. The pharmaceutical
industry regularly employs RPC to qualify drugs before their release.
RPC operates on the principle of hydrophobic forces, which originate from the
high symmetry in the dipolar water structure and play the most important role in all
processes in life science. RPC is allowing the measurement of these interactive forces.
The binding of the analyte to the stationary phase is proportional to the contact surface
area around the non-polar segment of the analyte molecule upon association with the
ligand in the aqueous eluent. This solvophobic effect is dominated by the force of water
for "cavity-reduction" around the analyte and the C 18-chain versus the complex of both.
The energy released in this process is proportional to the surface tension of the eluent
(water: 7.3 10-6 J/cm, methanol: 2.2 10-6 J/cm) and to the hydrophobic surface of the
analyte and the ligand respectively. The retention can be decreased by adding a less polar
solvent (methanol, acetonitrile) into the mobile phase to reduce the surface tension of
water. Gradient elution uses this effect by automatically reducing the polarity and the
surface tension of the aqueous mobile phase during the course of the analysis.

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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 noninteracting 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 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.
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. SEC 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. 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.

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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.
This form of chromatography is widely used in the following applications: water
purification, preconcentration of trace components, ligand-exchange chromatography,
ion-exchange chromatography of proteins, high-pH anion-exchange chromatography of
carbohydrates and oligosaccharides, and others.
Bioaffinity chromatography
This chromatographic process relies on the property of biologically active
substances to form stable, specific, and reversible complexes. The formation of these
complexes involves the participation of common molecular forces such as the Vander
Waals interaction, electrostatic interaction, dipole-dipole interaction, hydrophobic
interaction, and the hydrogen bond. An efficient, biospecific bond is formed by a
simultaneous and concerted action of several of these forces in the complementary
binding sites.
Aqueous Normal Phase Chromatography
Aqueous normal phase chromatography (ANP) is a chromatographic technique
which encompasses the mobile phase region between reversed-phase chromatography
(RP) and organic normal phase chromatography (ONP). This technique is used to achieve
unique selectivity for hydrophilic compounds, showing normal phase elution using
reverse-phase solvents.

1.3 HPLC INSTRUMENTATION7-15:

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Figure 1: HPLC instrumentation

a) Mobile phase reservoir
In HPLC the mobile phase can be an aqueous-organic mixture or buffer solution
or a mixture of organic solvents.
The mobile phase is pumped under pressure from one or several reservoirs and
flows through the column at a constant rate. With micro particulate packing, there is a
high -pressure drop across a chromatography column. Eluting power of the mobile phase
is determined by its overall polarity, the polarity of the stationary phase and the nature of
the sample components. For normal phase separations eluting power increases with
increasing polarity of solvent but for reversed phase separations, eluting power decreases
with increasing solvent polarity. Optimum separating conditions can be achieved by
making use of mixture of two solvents. Some other properties of the solvents, which need
to be considered for a successful separation, are boiling point, viscosity, detector
compatibility, flammability and toxicity.
b) Solvent degassing system
The constituents of the mobile phase should be degassed and filtered before use.
Several methods are employed to remove the dissolved gases in the mobile phase. They
include heating and stirring, vacuum degassing with an aspirator, filtration through 0.45
filters, vacuum degassing with an air-soluble membrane, helium purging, ultra-sonication
or purging or combination of these methods. HPLC systems are also provided an online

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degassing system, which continuously removes the dissolved gases from the mobile
phase.
c) Gradient elution devices
HPLC columns may be run isocratically i.e., with constant eluent or they may be
run in the gradient elution mode in which the mobile phase composition varies during the
run. Gradient elution is a means of overcoming the problem of dealing with a complex
mixture of solutes.
d) Pump
The most important component of HPLC in solvent delivery system is the pump,
because its performance directly effects the retention time, reproducibility and detector
sensitivity. Among the several solvent delivery systems (direct gas pressure, pneumatic
intensifier, reciprocating etc.), the reciprocating pump with twin or triple pistons is
widely used, as this system gives less baseline noise, good flow rate, reproducibility etc.
e) Sample introduction systems
Two means for analyte introduction on the column are injection into a flowing
stream and a stop flow injection. These techniques use a syringe or an injection valve.
Automatic injector is a microprocessor-controlled version of a manual universal injector.
Usually, up to 100 samples can be loaded in to the auto injector tray. The system
parameters such as flow rates, gradient, run time, volume to be injected, etc., are chosen,
stored in memory and sequentially executed on consecutive injections.
f) Characteristics of columns and column packings
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 (SiO2H2O).
It consists of a network of siloxane linkages (Si-O-Si) in a rigid three-dimensional
structure containing inter connecting pores. Thus wide ranges of commercial products are
available with surface areas ranging from 100 to 800 m/g and particle sizes from 3 to 50
m.
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

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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 (C18, C8, -NH2, -CN, -phenyl etc.) is
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.
g) Detectors
The purpose of the detector is to monitor the eluent coming out of the column.
Generally two types of detectors are used in HPLC bulk property and solute property
detectors.
a Bulk property detectors: These detectors are based on differential measurement of
a property, which is common to both the sample and the mobile phase. Examples
of such detectors are refractive index, conductivity and dielectric constant
detectors.
b Solute property detectors: Solute property detectors respond to a physical property
of the solute, which is not exhibited by the pure mobile phase.
These detectors measure a property, which is specific to the sample, either with or
without the removal of the mobile phase prior to the detection. Solute property detectors
which do not require the removal of the mobile phase before detection include
spectrophotometric (UV and UV-Vis) detector, fluorescence detectors, polarographic,
electro-chemical and radioactivity detectors, whilst the moving wire flame ionization
detector and electron capture detector, UV-Vis and fluorescent detectors are suitable for
gradient elution, because many solvents used in HPLC do not absorb to any significant
extent.

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h) Derivatization
In HPLC derivatization is used to enhance the sensitivity and selectivity of
detection when available detectors are not satisfactory for the un-derivatized compounds.
Both ultra violet absorbing and fluorescence derivatives have been widely used, for the
above said purpose. Ultra violet derivatization reagents include N-succinimidyl p-nitro
phenyl acetate, phenyl hydrazine and 3, 5-dinitro benzyl chloride, while fluorescent
derivatives can be formed with reagents such as dansyl chloride. 4-bromomethyl-7methoxy-coumarin and fluorescamine. Derivative formation can be carried out before the
sample is injected onto the column or by online chemical reactions between the column
outlet and the detector.
i) Gradient elution
Gradient elution or solvent programming is the change of solvent composition
during a separation in which strength increases from the beginning to the end of the
separation. It is well suited to the analysis of samples of unknown complexity since good
resolution is automatically provided for a wide range of sample polarities.
There are two types of gradient systems: low- pressure gradient mixtures and
high-pressure gradient mixtures. In the former the solvents are mixed at atmosphere
pressure and then pumped into the column, where as in the later, solvents are pumped
into a mixing chamber at high pressure before going into the column.
1.4 GENERAL METHODOLOGY FOR THE DEVELOPMENT OF NEW HPLC
METHODS16-25:
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
1. Careful sampling and sample preparation.
2. Precise sample injection.
3. Appropriate choice of the column.
4. Choice of the operating conditions to obtain the adequate resolution of the mixture.
5. Reliable performance of the recording and data handling systems.
6. Suitable integration/peak height measurement technique.
7. The mode of calculation best suited for the purpose

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8. Validation of the developed method.

1) 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.
Number of compounds present
Chemical structures (functionality of compounds)
Molecular weights of compounds
pKa values of compounds
UV spectra of compounds
Concentration range of compounds in samples of interest
Sample solubility
2) Separation goals
The goals of HPLC separation need to be specified clearly include
The use of HPLC to isolate purified sample components for spectral
identification or quantitative analysis
It may be necessary to separate all degradants or impurities from a product for
reliable content assay or not
In quantitative analysis, the required levels of accuracy and precision should be
known (a precision of 1 to 2% is usually achievable)
Whether a single HPLC procedure is sufficient for raw material or one or more
for mutations and/or different procedures are desired for formulations
When the number of samples for analysis at one time is greater than 10, a run
time of less than 20 minutes often will be important.
3) Sample preparation
Samples come in various forms
Solutions ready for injections
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 pretreatment to remove interferences and/or protect the
column or equipment from damage.
Most samples for HPLC analysis require weighing and or volumetric dilution
before injection. Best results are often obtained when the composition of the sample
solvent is close to that of the mobile phase since this minimizes baseline upset and other
problems. Some samples require a partial separation (pretreatment) prior to HPLC,
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because of need to remove interferences, concentrate sample analytes or eliminate

column killers. In many cases the development of an adequate sample pretreatment
can be challenging than achieving a good HPLC separation. The detector selected should
sense all sample components of interest. Variable-wavelength ultraviolet (UV) detectors
normally are the first choice, because of their convenience and applicability for most
samples. For this reason, information on the UV spectra can be an important aid for
method development. When the UV response of the sample is inadequate, other detectors
are available (fluorescence, electrochemical etc) or the sample can be derivatized for
enhanced detection.
4) Precise sample injection
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 detectors normally are the first choice, because of their
convenience and applicability for most samples. Information on the UV spectra can be an
important aid for method development.
5) Choice of the Column
The selection of the column in HPLC is somewhat similar to the selection of
columns in G.C, 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 and size of the ions generated by the sample molecules and the
nature of ionizable group on the stationary phase.
Table 1: Selection of columns based on the method is briefly summarized in the table
below
Method/ Description/Columns
Reverse-phase HPLC

Preferred Method

Uses water- organic mobile phase

Columns: C18 (ODS), C8, phenyl,
trimethylsilyl (TMS), and cyano.

First choice for most samples, especially neutral

or non-ionized compounds that dissolve in
water-organic mixtures

Ion-pair HPLC
Uses water-organic mobile
phase,
a buffer to control pH, and an
ion-pair reagent

Acceptable choice for ionic or ionisable

compounds, especially bases or cations.

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Columns: C18, C8, Cyano

Normal-phase HPLC
Uses mixtures of organic solvents as
mobile phase Columns: cyano, diol,
amino, silica

Good second choice when reverse-phase or ionpair HPLC is ineffective; first choice for
lipophilic samples that do not dissolve well in
water-organic mixtures; first choice for
mixtures of isomers and for preparative HPLC

6) Choice of the operating conditions to obtain the adequate resolution of the

mixture
If HPLC is chosen for the separation, the next step is to
classify the sample as regular or special. Regular samples mean typical mixtures of small
molecules (<2000Da) 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 bases
usually require the addition of a buffer to the mobile phase. For basic or cationic samples,
less acidic reverse phase columns are recommended.

Table 2: Preferred experimental conditions for the initial HPLC separation

Separation Variable

Preferred Initial Choice

Column

Dimensions (length, ID)

Particle size
Stationary phase
Mobile phase
Solvents A and B

15 X 0.46 cm
5 m
C8 or C18
Buffer- acetonitrile

%B

80-100%

Buffer (compound,
pH, concentration)

25mM potassium phosphate,

2.0<pH<3.0

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Additives (e.g., amine

modifiers, ion- pair reagents)

Do not use initially

Flow rate

1.5-2.0 mL/min

Temperature

35-45oC
Sample Size

Volume

< 25L

Polar solvent
a. 3.5 m particles are an alternative, using a 7.5 cm column.
b. For an initial isocratic run; an initial gradient run is preferred.
c. No buffer required for neutral samples; for pH <2.5, pH-stable columns are
recommended.
d. Smaller values required for smaller-volume columns (e.g., 7.5 x 0.46-cm, 3.5-m
column).
If typical reverse-phase conditions provided cause is inadequate sample retention,
suggesting the use of either ion-pair or normal phase HPLC. Alternatively, the sample
may be strongly retained with 100% ACN as mobile phase suggesting the use of nonaqueous reverse phase chromatography or normal phase HPLC.
7) Getting Started On Method Development
One approach is to use an isocratic mobile phase of some average solvent strength
(50%) organic solvent. A better alternative is to use a very strong mobile phase first (80100%) 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.
Table 3: Goals that are to be achieved in method development
Goal

Comment

Resolution

Precise and rugged quantitative analysis requires that Rs be

greater than 1.5.

Separation time

<5-10 min is desirable for routine procedures.

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2% (1 SD) for assays; 5% for less-demanding analyses
15% for trace analyses.

Pressure

<150 bar is desirable, <200 bar is usually essential (new

column assumed).

Peak height

Narrow peaks are desirable for large signal/noise ratios.

Solvent consumption

Minimum mobile-phase use per run is desirable.

Roughly in order of decreasing importance but may vary with analysis requirements.
Separation or resolution is a primary requirement in quantitative HPLC. The
resolution (Rs) value should be maximum (Rs > 1.5) 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 Rs=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 separation of the compounds of
interest (qualitative analysis). In such cases only enough 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).
Conditions for the final HPLC method should be selected so that the operating
pressure with a new column does not exceed 170 bar (2500 psi) and an upper pressure
limit below 2000 psi is desirable. There are two reasons for this pressure limit, despite the
fact that most HPLC equipment can be operated at much higher pressures. First, during
the life of a column, the backpressure may rise by a factor of as much as 2 due to the
gradual plugging of the column by particulate matter. Second, at lower pressures (<170
bars, pumps, sample valves and especially auto samplers operate much better, seals last
longer, columns tend to plug less and system reliability is significantly improved. For
these reasons, a target pressure of less than 50 % of the maximum capability of the pump
is desirable. When dealing with more challenging samples or if the goals of separation are

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particularly stringent, a large number of method development runs may be required to

achieve acceptable separation.
8) Repeatable separation
As the experimental runs described above are being carried out, it is important to
confirm that each 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 mobile phase and
temperature.
Usually column equilibration is achieved after passage of 10 to 20 column
volumes of the new mobile phase through the column. However, this should be
confirmed by repeating the experiment under the same conditions. When constant
retention times are observed in two such back-to-back repeat experiments ( 0.5% or
better), it can be assumed that the column is equilibrated and the experiments are
repeatable.
Completing the HPLC method development
The final procedure should meet all the goals that were defined at the beginning
of method development. The method should also be robust in routine operation and
usable by all laboratories and personnel for which it is intended.
Parameters of HPLC method
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 validations have been
completed.
The criteria selected will be based on the actual performance of the method as
determined during its validation. For example, if sample retention times form part of the
system suitability criteria, their variation (SD) can be determined during validation.
System suitability might then require that retention times fall within a 3 SD
range during routine performance of the method.
The USP (2000) defines parameters that can be used to determine system
suitability prior to analysis. These parameters include plate number (N), tailing factor, k
and or, resolution (Rs) and relative standard deviation (RSD) of peak height or peak
area for respective injections.
Retention Time (Rt)
The time between the injection point and the maximum detector response for
correspondent compound.

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Retention Volume (Vr)

tR x eluent flow rate
Theoretical Plates
The column performance can be defined in terms of column plate number n is
defined by
n = 5.54 (tR/W)2
Where tR is the retention time of the peak and
W is the width of the peak at half peak height.
HETP
Another way to express efficiency of column is by calculating height equivalents
of theoretical plates (HETP).
h = L/n
Where h = HETP; L = Length of column; n = number of theoretical plates. Lower
the HETP, higher is the efficiency of the column, i.e., higher the theoretical plates more
efficient the column is.
Resolution
The resolution of two adjacent peaks can be calculated by using the formula
Rs = 1.18(t2-t1) / W0.5, 1 + W0.5, 2
Where t1 and t2 are retention times of the adjacent peaks and
W0.5, 1 and W0.5, 2 are the width of the peaks at half height.
Rs = 2.0 or greater is a desirable target for method development.
Retention Factor
The retention factor k is given by the equation
k = (tR t0)/ t0
Where tR is the band retention time and t0 is the column dead time.
Peak Symmetry
The peak symmetry can be represented in terms of peak asymmetry factor and
peak tailing factor which can be calculated by using the following formulae
Peak asymmetry factor = B/A
Where B is the distance at 50% peak height between leading edge to the
perpendicular drawn from the peak maxima and A is the width of the peak at half the
peak height.
Capacity Factor / Retention Factor: k1 (K Prime)

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Where T(R) equals the retention time of the peak in minutes and T(0) is the retention
time of an unretained peak. *K Prime must be > 1.00.
k1 = T(R) - T (0) / T (0)
Peak Tailing Factor

According to USP (2000) Peak tailing factor can be calculated by using the
formula
T = W0.05 / 2f

Where W0.05 is the width of the peak at 5 % height and f is the distance from the
peak maximum to the leading edge of the peak, the distance being measured at a point 50
% of the peak height from the base line.
1.5 Method validation26-30:
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 validation is an integral part of the method development; it is the process
by which a method is tested by the developer or user for reliability, accuracy and
preciseness of its intended purpose and demonstrating that analytical procedures are
suitable for their intended use that they support the identity, quality, purity, and potency
of the drug substances and drug products Data thus generated become part of the methods
validation package submitted to Center for Drug Evaluation and Research (CDER).
Simply, method validation is the process of proving that an analytical method is
acceptable for its intended purpose.
Methods should be reproducible when used by other analysts, on other equivalent
equipment, on other days or locations, and throughout the life of the drug product. Data
that are generated for acceptance, release, stability, or pharmacokinetic will only be
trustworthy if the methods used to generate the data are reliable. The process of
validation and method design also should be clearly in the development cycle before
important data are generated. Validation should be on going in the form of re-validation
with method changes.
Though many types of HPLC techniques are available, the most commonly used
method, the reversed-phase HPLC with UV detection, is selected to illustrate the
parameters for validation. The criteria for the validation of this technique can be
extrapolated to other detection methods and chromatographic techniques. For acceptance,
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release or stability testing, accuracy should be optimized since the need to show deviation
from the actual or true value is of the greatest concern.
All the variables of the method should be considered, including sampling
procedure, sample preparation, chromatographic separation, and detection and data
evaluation. For chromatographic methods used in analytical applications there is more
consistency in validation practice with key analytical parameters including
(1) Accuracy
(2) Precision
(3) Linearity
(4) Limit of Detection
(5) Limit of Quantification
(6) Robustness
(7)System suitability
(8)Specificity/selectivity
1. Accuracy
The accuracy of a measurement is defined as the closeness of the measured value
to the true value. In a method with high accuracy, a sample (whose true value is
known) is analyzed and the measured value is identical to the true value. Typically,
Accuracy is represented and determined by recovery studies, but there are three ways to
determine accuracy
comparison to a reference standard
recovery of the analyte spiked into blank matrix, or
Standard addition of the analyte.
2. Precision
Precision can be defined as the degree of agreement among individual test
results when the procedure is applied repeatedly to multiple samplings of a homogenous
sample. A more comprehensive definition proposed by the International Conference on
Harmonization (ICH) divides precision into three types:
Repeatability
Intermediate precision and
Reproducibility

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Repeatability is the precision of a method under the same operating conditions

over a short period of time. Intermediate Precision is the agreement of complete
measurements (including standards) when the same method is applied many times within
the same laboratory. Reproducibility examines the Precision between laboratories and is
often determined in collaborative studies or method transfer experiments.
3. Linearity and Range
The Linearity of a method is a measure of how well a Calibration plot of response
against concentration approximates a straight line. Linearity can be assessed by
performing single measurements at several analyte concentrations. The data are then
processed using a linear least-squares regression. In the resulting plot slope, intercept,
and Correlation coefficient provide the desired information on Linearity.
The range of an analytical method is the interval between the upper and lower
levels of the analyte including these levels that have been demonstrated to be determined
with precision accuracy and linearity.

Fig 2. Definitions for Linearity, Range, LOQ, LOD

4. Limit of Detection
These limits are normally applied to related substances in the drug substance or
drug product. Specifications on these limits are submitted with the regulatory impurities
method relating to release and stability of both drug substance and drug product.

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Limit of detection is the lowest concentration of analyte in a sample that can be detected,
but not necessarily Quantified, under the stated experimental conditions. With UV
detectors, it is difficult to assure the detection precision of low-level compounds due to
potential gradual loss of sensitivity of detector lamps with age, or noise level variation by
detector manufacturer.
A crude method to evaluate the feasibility of the extraneous peak detection is to use
the percentage claimed for detection limit from the area counts of the analyte. Several
approaches for determining the detection limit are possible, depending on whether the
procedure is a non-instrumental or instrumental.

Based on Visual Evaluation

Based on Signal-to-Noise
Based on the Standard Deviation (SD) of the Response and the Slope

The Limit of detection limit (LOD) may be expressed as:

LOD = 3.3 / S
Where, = 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.
5. Limit of Quantification
Limit of quantification is the lowest concentration of analyte in a sample that can
be determined with acceptable precision and accuracy under the stated experimental
conditions. Several approaches for determining the quantification limit are possible,
depending on whether the procedure is a non-instrumental or instrumental.
Based on Visual Evaluation
Based on Signal-to-Noise Approach
Based on the SD of the response and the slope
The Limit of quantification (LOQ) may be expressed as:

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LOQ = 10 / S
Where, = the standard deviation of the response
S = the slope of the Calibration curve
6. Robustness
The concept of robustness of an analytical procedure has been defined by the ICH
as a measure of its capacity to remain unaffected by small, but deliberate variations in
method parameters. A good practice is to vary, important parameters in the method,
systematically and measure their effect on separation.
7. System suitability
Prior to the analysis of samples of each day, the operator must establish that the
HPLC system and procedure are capable of providing data of acceptable quality. This is
accomplished with system suitability experiments, which 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.
8. Specificity / Selectivity
The terms selectivity and specificity are often used interchangeably. Term specific
generally refers to a method that produces a response for a single analyte only while the
term selective refers to a method which provides responses for a number of chemical
entities that may or may not be distinguished from each other. If the response is
distinguished from all other responses, the method is said to be selective. Since there are
very few methods that respond to only one analyte, the term selectivity is usually more
appropriate. The analyte should have no interference from other extraneous components
and be well resolved from them. A representative HPLC Chromatogram or profile should
be generated and submitted to show that the extraneous peaks either by addition of
known compounds or samples from stress testing are baseline resolved from the parent
analyte.

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