High Performance Liquid Chromatography (HPLC)

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Mr. Sanket P. Shinde
Assistant Professor
Pune-Maharashtra.

INTRODUCTION
• HPLC stands for “High-performance liquid chromatography”
(sometimes referred to as High-pressure liquid chromatography).
• High performance liquid chromatography is a powerful tool in analysis,
it yields high performance and high speed compared to traditional
columns chromatography because of the forcibly pumped mobile phase.
• HPLC is a chromatographic technique that can separate a mixture of
compounds.
• It is used in biochemistry and analytical chemistry to identify, quantify
and purify the individual components of a mixture.
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Mr. S. P. Shinde

• Chromatography : Physical method in which separation of
components takes place between two phases-a stationary phase and
a mobile phase
• Stationary phase : The substance on which adsorption of the
analyte (the substance to be separated during chromatography) takes
place. It can be a solid, a gel, or a solid liquid combination
• Mobile phase : Solvent which carries the analyte (a liquid or a gas)
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Mr. S. P. Shinde

The principle involved in HPLC can be either adsorption or partition.
➢ Interaction of sample with both stationary phase and mobile phase.
➢ The analytes are injected into the flow of mobile phase.
➢ The outlet of the column is connected to a detector where the eluted
substances are detected.
➢ The separation is achieved in the column packed with stationary
phase material of low particle size
➢ The reliable flow rate of the mobile phase with appropriate pressure is
applied.
➢ The sample mixture is interacted between the stationary phase and
mobile phase.
➢ The interaction between these phases results in the separation.
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Mr. S. P. Shinde

o When a mixture of components are introduced into the column . Various
chemical and/or physical interactions take place between the sample
molecules and the particles of the column packing.
o They travel according to their relative affinities towards the stationary
phase. The component which has more affinity towards the adsorbent,
travels slower.
o The component which has less affinity towards the stationary phase
travels faster.
o Since no two components have the same affinity towards the stationary
phase, the components are separated.
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Mr. S. P. Shinde

The theory behind the liquid chromatography can be explained under
two major categories.
1. Plate theory
2. Rate theory
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Mr. S. P. Shinde

1. Plate Theory:
o The basis for the plate theory is the 'theoretical plates'. The separation
efficiency of a column can be expressed in terms of number of
theoretical plates.
o A theoretical plate represents a single equilibrium step occur during
separation process.
o It is considered as a discrete continuous horizontal layer in a column,
in which the equilibrium of solute takes place between mobile phase
and stationary phase.
o The greater the number of theoretical plates/equilibrium step, the
greater the resolving power of the column.
The number of theoretical plates is denoted by ‘N’. The plate height 'H' is
expressed by the length of column 'L' divided by 'N'.
H = N
L
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Mr. S. P. Shinde

HEIGHT EQUIVALENT OF A THEORETICAL PLATE (HETP)
o A theoretical plate is an imaginary or hypothetical unit of a column
where distribution of solute between stationary phase and mobile
phase has attained equilibrium. It can also be called as a functional
unit of the column.
o The height of one theoretical plate is referred to as the ‘Height
Equivalent of a Theoretical Plate‘
o A theoretical plate can be of any height, which describes the
efficiency of separation. If HETP is less, the column is more
efficient. If HETP is more, the column is less efficient.
o HETP = length of the column/ no. of theoretical plates
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Mr. S. P. Shinde

2. Rate Theory:
o This theory explains the effect of parameters that affects the width
of an elution band.
o The broadening of peak or band is the effect of several sources
such as the mobile phase velocity, diffusivity, migration pattern
which are in turn depends upon the particle size, shape and
uniformity of packing.
o The path length that solvent moves from the top of the column and
time to reach the bottom of column also affects the width of the
band.
o The theory has been well explained by the Van Deemter equation.
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Mr. S. P. Shinde

Van Deemter Equation:
Van Deemter equation is given below.
A = Eddy's diffusion
B = Longitudinal diffusion
C = Mass transfer
μ = Linear gas velocity
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Mr. S. P. Shinde

Eddy's diffusion :
Consider a column packed with adsorbent. Depending upon the column
packing, the solute molecules reach the bottom in different manner. The
path length of solutes A, B and C is different. C will have longer path
length than B and A. The net effect leads to band broadening. This process
is called as Eddy's diffusion.
How to minimize :
The Eddy's diffusion can minimized by using smaller particles of uniform
size, uniform packing and smaller diameter column.
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Mr. S. P. Shinde

Longitudinal or molecular diffusion :
The term 'B' in Van deemter equation represents the longitudinal or
molecular diffusion. This type of diffusion occurs due to the concentration
gradients within the column. The molecules tend to migrate from
concentrated zone to the diluted zone. This resulted in band broadening.
This is more in case of gas used as mobile phase than liquid.
How to minimize :
As the flow rate is the major factor for longitudinal diffusion, changing the
type or flow rate of carrier gas can be done. Denser gases can be used with
high flow rate.
For example carbon dioxide or nitrogen can be used instead of hydrogen or
helium.
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Mr. S. P. Shinde

Mass transfer :
o The term 'C' represents the mass transfer. In the column
chromatography, generally an equilibrium of the solute established
between mobile phase and stationary phase.
o The time taken for such equilibrium throughout the column is not
uniform.
o When fresh mobile phase passed through the column, the equilibrium
is not established instantly with the front or starting zone.
o But the solute may moved down the column and these will be
equilibrium in the further zone.
How to minimize :
1. By increasing the temperature so that the solubility of sample in the
stationary liquid phase in GC can be increased.
2. A thin film of stationary liquid phase can be used. This minimize the
diffusion within the phase.
3. In liquid chromatography, small particles, thin stationary films, high
temperature and low viscous mobile phases can be used.
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Efficiency
o The efficiency of a chromatographic peak is a measure of the
dispersion of the analyte band as it travelled through the HPLC system
and column.
o Each plate is the distance over which the sample components achieve
one equilibration between the stationary and mobile phase in the
column. Therefore, the more (‘theoretical’) plates available within a
column, the more equilibrations possible and the better quality the
separation.
Mr. S. P. Shinde

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HPLC can be broadly classified into two major types-
1. Normal phase chromatography- stationary phase is polar
(hydrophilic) and mobile phase is non-polar (hydrophobic).
2. Reverse phase chromatography- stationary phase is non-polar
(hydrophobic) and mobile phase is polar (hydrophilic).
o Polar-Polar bonds and Non Polar-Non Polar bonds have more affinity
than Polar-Non Polar bonds.
o Reverse phase chromatography is more commonly used as drugs are
usually hydrophilic
Mr. S. P. Shinde

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The elution in HPLC can be classified into two categories:
1. Isocratic Elution :
o In this technique the mobile phase composition is fixed constant throughout
the chromatographic procedure.
o For example, if a method consisting of mobile phase as methanol and water
in the ratio of 70:30, the same ratio is maintained for the entire
chromatographic procedure in isocratic method.
2. Gradient Elution :
o In this technique, the composition of mobile phase is changed either
stepwise or continuously as elution proceeds.
o For example, Initially a composition (methanol :water, 70:30) for some
time period (10 min), is maintained then the polarity is modified by
changing the ratio to (80:20) for next 5 min and then to 90:10 for another
5 min.
o Changing of composition to bring out the desirable separation is called as
gradient elution method.
Mr. S. P. Shinde

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The instrument of a HPLC consist of the following components:
1. Mobile phase reservoir
2. Pumping system
3. Sample injection systems
4. Column
5. Detector
6. Data collection device or recorder.
Mr. S. P. Shinde

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o Generally reservoirs made up of glass are used.
o One or two reservoirs of 500 ml capacity or more quantity if required
are used.
o The quantity of the mobile phase to be used in the separation depends
upon the nature of solvent, quantity of mixture to be separated, time
required for the separation, particle size of the solvent type of stationary
phase used and other such factors.
Mr. S. P. Shinde

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Degassing:
It is necessary to remove the dissolved gases present in the mobile phase
solvent.
Different techniques are followed for degassing which are discussed below.
(a) External Vacuum Degassing :
o In this method the solvent in a container is kept in a ultrasonic bath.
o Ultrasonication is done under vacuum using a vacuum pump.
o This process will remove the dissolved gases from the solvents.
Mr. S. P. Shinde

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(b) Helium Sparging :
o In this technique, helium is bubbled into the solvent which will remove
the other dissolved gases.
o Helium is insoluble in the mobile phase so it escapes out without
interfering.
o This can be done online or offline.
Mr. S. P. Shinde

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(c) Online Degassing:
In this method, vacuum pump is equipped with the HPLC and vacuum is
applied on the semi permeable tubes in which the solvents run. The air from
the solvents is removed and goes to the waste collecting container.
(d) Filters :
Other than the above methods filters are also used to remove the dust and
other matters from the solvents. Membrane filters of 45u are usually used.
The mobile phase filtered through these filters using Buchner funnel under
vacuum followed by ultrasonication.
Mr. S. P. Shinde

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o The mobile phase is forced from reservoir to the column using Pumps.
o More resistant to the flow of mobile phases due to the small internal
diameter of the column and lower particle size of the stationary phase.
o Hence high pressure is required to push the mobile phase with uniform
flow rate. This is achieved by the pumps.
The general requirements for an HPLC pump are as follows.
1. It should provide the desirable pressure (Generally upto 6000 psi).
2. It should provide uniform flow rate ranging from 0.1 ml to 10 ml/min. In
case of preparative analysis it should provide more flow rate.
3. It should provide pulse less flow.
4. It should be chemically resistant to all solvents.
5. It should provide reproducibility.
Mr. S. P. Shinde

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Mr. S. P. Shinde
1. Syringe Pumps
o Consists of large syringe like chamber.
o Suitable for small bore column.
o These pumps have a volume between 250 to 500mL

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Mr. S. P. Shinde
2. Reciprocating - Piston Pumps
o The term reciprocating describes any continuously repeated
backwards and forwards motion.
o Widely used type of pump.
o Solvent is sucked during back stroke and gets deliver to the column
in forward stroke.

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Mr. S. P. Shinde
3. Constant Pressure Pumps
o In these types of pumps, the mobile phase is driven through the
column with the use of pressure from the gas cylinder.
o A low-pressure gas source is needed to generate high liquid
pressures
o The valving arrangement allows the rapid refill of the solvent
chamber whose capacity is about 70mL
o This provides continuous phase flow rates

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There are two types of valves.
1. External loop valve injector
2. Internal loop valve injector

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Mr. S. P. Shinde
Requirements for an Ideal HPLC Column :
1. It should have uniform column packing.
2. It should have spherical particles.
3. The particle diameters should be ranging from 3 to 10um.
4. The porosity of the particles should be in the range 50-70%, extending
to 80% for the size-exclusion chromatography.
5. The column should withstand the pressure during operation.
6. It should give reproducible results.
7. It should be easy to handle.
8. The particles should not shrink or swell with the nature of the eluent.
9. It should have a uniform particles pore size distribution.
10. Particles should be available with a range of mean pore diameters of 60-
100 A0
11. Column packing should be chemically inert.
12. It should be easily available and cost effective.

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Mr. S. P. Shinde
Types of Columns :
(i) Guard Column
o Guard column is the column placed before
the analytical column.
o These columns also called as pre columns.
o They are used to protect the analytical
column from the impurities and other
contaminants from solvent.
o It also removes the components that bind
irreversibly to the stationary phase.
o The guard columns are compulsorily used
during the bioanalytical studies to protect
the analytical column from biological
matrix.

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Mr. S. P. Shinde
(ii) Analytical Column
o Analytical column is considered as the heart of an HPLC system.
o The reason is this is the part where the separation of the mixture takes place.
o The efficiency of the separation is purely depends on the column.

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Mr. S. P. Shinde
Types of Analytical Columns :
(a) Small bore Columns : Columns having the diameter less than about
2 mm. They are also known as 'microcolumns'. that they are operated
at much lower mobile phase flow rates
(b) 3X3 Columns Short : (3.3 x 4.6 mm) columns packed with 3μm
bonded silica stationary phases have sufficient efficiency for many
separations.
(c) Monolithic Columns : It has porous channels rather than being
packed with beads. Their construction is more similar to rod with
random channelling

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Mr. S. P. Shinde
Types of Packaging used in HPLC Column
a) Porous Packing :
o Porous microparticles are the most commonly used stationary phase
particles in modern HPLC.
o They consist of fully porous particles that can be either irregular or
spherical in shape.
o The diameters are ranging from 3 to 10 μm.
o The pores provide the surface with which the sample interacts.
o Particles with smaller pore size exhibit a larger surface area and
therefore have greater retention.
o Large particles like proteins require a large pore size.
o The particles are composed of silica, alumina, the synthetic resin
polystyrene-divinylbenzene, or an ion-exchange resin.
o Silica is most common packing used in LC.

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b) Pellicular Packings :
o It consist of a solid spherical bead of relatively large inner diameter
with a thin outer layer of stationary phase.
o Particles were spherical, nonporous, glass or polymer beads with
typical diameters of 30 to 40μm.
o They are much useful for the ion-exchange chromatography.
o The advantages include ease of packing, long term stability and low
cost.
c) Bonded Phases :
o The supports for majority of the bonded phase packings used in
partition HPLC are prepared from rigid silica, or silica-based
compositions.
o These solids are formed as uniform, porous, mechanically sturdy
particles commonly having diameters of 1.5-10μm.

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Mr. S. P. Shinde
Detectors is a device which detects the eluted components emerging from
column.
An ideal HPLC detectors should meet the following requirements.
1. It should respond to wide variety of components.
2. It should be compatible for the temperature and flow rate changes.
3. The sensitivity should be high.
4. It should have low dead volume.
5. It should give at least selective response and specific is preferable.
6. It should give reproducible results.
7. It should be compatible with liquid flow.

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Mr. S. P. Shinde
Types of Detectors
Bulk property
Detectors
Refractive
index Detectors
Solute property
Detectors
UV- visible
Absorption
Detectors
Fluorescence
Detectors
Amperometric
Detectors
Other Detectors
Infrared
Absorption
Detectors
Evaporative
Light Scattering
Detectors
(ELSD)
Mass
Spectrometric
Detectors

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Mr. S. P. Shinde
1. Bulk property Detectors
• Refractive Index Detectors
o Detection occurs when the light is bent due to samples eluting
from the columns, and this is read as a disparity between the two
channels.
o It is not much used for analytical applications because of low
sensitivity & specificity.
o When a solute is in the sample compartment, refractive index
changes will shift the light beam from the detector.

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Mr. S. P. Shinde
2. Solute property Detectors
a. UV- visible Absorption Detectors

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UV-visible Detector
o UV visible detector is widely used as it detects large number of
compounds because most drugs have appropriate structural
characteristics for light absorption.
o These are useful for aromatic compounds and other type of
unsaturated systems.
o These are classified as fixed or variable wavelength detectors.
o Fixed wavelength detectors employ filter as a source to provide
appropriate wavelength.
o Most common fixed wavelength detectors are based on 254 nm.
o Variable wavelength detectors are employ a spectrophotometer to
provide dispersion of light and selection of any wavelength in UV
visible regions.
o Diffraction gratings are frequently used for wavelength dispersion.

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Photodiode Array (PDA)
o A photodiode array (PDA) is a linear array of discrete photodiodes on an
integrated circuit (IC) chip.
o Array detectors are especially useful for recording the full Uv- vis is a
absorption spectra of samples that are rapidly passing through a sample
flow cell, such as in an HPLC detector.

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b. Fluorescence Detectors:
o It is based on the fluorescent radiation emitted by some compounds.
o The excitation source passes through the flow cell to a photo detector
while a monochromator measures the emission wavelengths.
o More sensitive and specific.
o The disadvantage is that most compounds are not fluorescent in
nature.

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c. Amperometric Detector:
o Amperometric detectors works based on the reducing and
oxidizing property of the sample when a potential is applied.
o The diffusion current recorded is directly proportional to the
concentration of the compound recorded.
o ADVANTAGE: Highly sensitive detector.
o DISADVANTAGE: This detector is applicable only when the
functional groups present in the sample can be either oxidized or
reduced.

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Other Detectors
A. Infrared Absorption Detectors
o IR spectrophotometer and FTIR spectrophotometer have been used
for HPLC.
o IR detector cells are similar in construction to those used in the UV
instruments, except that the IR cuvettes are made up of Sodium
chloride or Calcium fluoride.
o Cell path lengths range from 0.2 to 1.0 mm and volumes from 1.5 to
10μL.
o A major limitation in the use of the IR detectors is the low
transparency of many useful solvents. Also, the use of aqueous
mobile phases is restricted.

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B. Evaporative Light Scattering Detector (ELSD)
In this, the column effluent is, converted into a fine mist by a flow of
nitrogen or air using a nebulizer. The fine-droplets are passed through a
heated tube where the mobile phase evaporates, leaving fine particles of the
analytes. This is then passes through a laser beam. The scattered radiation is
then detected at the right angles to the flow by a silicon photodiode.

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Mr. S. P. Shinde
C. Mass Spectrometric Detectors
Now a days the highly sophisticated mass spectrometric detectors are
widely used due to their sensitivity and reliability. When mass
spectrometer is used as a detector for LC (LC-MS), it can greatly aid in
identifying species as they elute from a chromatographic column. Various
interfaces are used to couple LC with MS.

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o Tubings are important for the HPLC instrument to achieve proper
separation.
o Selection of tubing is one of the major parts in achieving desired
separation.
o Ultra-small diameter stainless steel capillary tubes are generally
used as tubings for HPLC and UPLC techniques.
o The tubings should be free from impurities, such as oil, grease
and other foreign material.
o Such impurities may lead to cross-contamination and inaccurate
sample reading.
o For accurate results the factors such as inner and outer diameter of
tubing, uniformity of inner surface materials and alignment of
tubing with the bore of receiving port etc.
Tubings

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o Recorders are used to record the responses obtained from
detectors after amplification
o They record the baseline & all the peaks obtained with respect to
time (Rt)
o But the area of the individual peaks cannot be known.
o Integrators: improved version of recorders with data processing
capabilities.
o Rt, height and width of peaks, peak area, % area.
o Integrators provide more information on peaks than recorders

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o Quantitative analysis involves the determination of concentration
of the analyte present in the sample quantitatively.
o In HPLC, quantitative determination involves the measurement of
peak height or peak area.
o The sample is injected into the HPLC system and after separation;
peaks are obtained in the chromatogram.
o The area or height of the peak is plotted versus the concentration
of the substance.
o For peaks that are well resolved, both peak height and area are
proportional to the concentration.
Quantitation Techniques

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The different calibration methods are used in quantitative analysis
Quantitation
Techniques

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o A standard feature of data systems is to report percent peak area.
o This is obtained by adding the areas of all peaks in a chromatogram
and reporting each peak as a percentage of the total.
o This report format is convenient for screening a chemical reaction for
completion and approximate product purity as well as other
applications.
o A related report is area normalization, in which one peak is chosen as
a reference peak and all other peaks are reported as a percentage of
the reference peak.
o Area normalization is common for methods used to test drug stability
or assay impurities in drug products.
1. Area Normalisation Method

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Mr. S. P. Shinde
o The standard additions method is referred to as 'spiking' method.
o It is commonly used to determine the concentration of an analyte in a
complex matrix such as biological fluids, soil samples, etc.
o The matrix may contain other components that interfere with the
analyte signal causing inaccuracy in the determined concentration.
o The several aliquots of sample are prepared in separate volumetric
flasks of the same volume.
o The first volumetric flask is diluted with suitable diluents and
remaining flasks are spiked with the standard solution followed by
dilution.
2. Standard Addition Method

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1. Add same quantity of unknown sample to a series of flasks.
2. Add varying amounts of standard (made in solvent) to each
flask, ex. 0,5,10,15mL)
3. Fill each flask to line, mix and measure.

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Continue…
o The instrument response is then measured for all of the diluted
solutions.
o A curve is plotted by taking volume of standard added in the
X-axis and instrument response in the Y-axis.
o The concentration of analyte in the sample is calculated by
linear regression analysis.

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o Internal standard is a known amount of compound, different from the
analyte that is added to an unknown sample.
o In this method, an equal amount of an internal standard, a component
that is not present in the sample, is added to both the sample and
standard solutions.
o The internal standard selected should be chemically similar to, have
similar retention time and derivative similarly to the analyte.
o Additionally, it is important to ensure that the internal standard is stable
and does not interfere with any of the sample components.
o The internal standard should be added before any preparation of the
sample so that extraction efficiency can be evaluated.
o Quantification is achieved by using ratios of peak height or area of the
component to the internal standard.
3. Internal Standard Method
known
Conc.
known
Area
unknown
Area
unknown
in
Std.
Internal
of
Area
known
in
Std.
Internal
of
Area
unknown
Conc. 

=

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Mr. S. P. Shinde
o Standard is not mixed along with the sample to be separated.
o Separate solution of standard and sample are prepared.
o External standard should be 100% pure (AR grade) or it should be
of known purity.
o Both solution are injected separated in the column which result in
produces two separate chromatogram.
o The height of sample is compared with height of standard and their
concentration are determined.
4. External Standard Method

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System Suitability
o It is used to verify that the chromatographic system is suitable for the
intended analysis.
o That is to ensure that the complete testing system including
instruments, electronics, reagents, column & analyst is suitable for
intended application.
o System suitability test is an essential part of HPLC & GC methods

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1. Retention Time (tR)
o Retention Time (tR) is the duration taken by the sample components from
the point of injection into the column to reach the detector.
o When the solute has higher affinity to the stationary phase the retention
time is longer and when the solute has higher affinity to the mobile
phase the retention time is shorter.
o In reverse phase chromatography, the more lipophilic compounds are
retained longer whereas in normal phase chromatography their retention
will be shorter.
2. Retention Volume (VR)
o The retention volume is the volume of mobile phase required for elution
of a component.
o It may be calculated from the retention time and the particle or
macropore size, porosity, and specific surface flow rate in mL/min : area.
VR = tR x F

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3. Resolution (RS)
o The resolution is the separation of two components in a mixture.
4. Capacity Factor (k’)
o The capacity factor is the ratio of amounts of the solute at the stationary and
mobile phases within the analyte band inside the chromatographic column.
Where;
CS = the conc. of the solute at the stationary phase
Cm = The conc. of the solute at the mobile phase
Φ = The ratio of the volume of stationary (Vs) and mobile (Vm) phase all
within the chromatographic band.
Or
φ
Cm
Cs
k' =
Vm
Vs
φ =

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5. Relative Retention Time (RRT)
o The ratio between the retention time of any peak and the known
component is used instead of void time is referred to as the Relative
Retention Time (RRT).
6. Peak Capacity (Pc)
o Peak Capacity is defined as number of peaks that can be separated
within a retention window for a specific predetermine resolution.

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7. Peak Asymmetric Factor (Af) and Tailing Factor (T)
o The chromatographic peak is assumed to have a Gaussian shape under
ideal conditions.
o The front side deviation from the Gaussian peak is known as peak
fronting & rear side deviation is known as peak tailing.

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8. Selectivity Factor (α) or Separation Factor
o It describes the separation of two species on the column.
o The relative retention of a two peaks in a chromatogram is called
Selectivity.
o Chemically distinguish between sample components.

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Trouble Shooting

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There are five major of symptoms to help you quickly identify the source
of the problem(s)
Problem
Source
Pressure
Leaks
Chromatogram
Injector
Miscellaneous

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1 - Abnormal Pressure
Causes Solutions
Power off Turn on power
Fuse blown Replace fuse
Broken piston Replace piston
Air trapped in pump head Purging & Degas solvents
Insufficient mobile phase Replenish reservoir
Faulty check valve(s) Replace check valve(s)
Major leak Tighten or replace fittings
Problem-1: No pressure reading, no flow

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Mr. S. P. Shinde
Causes Solutions
Faulty meter Replace meter
Faulty pressure transducer Replace transducer
Causes Solutions
Injector blockage Clear blockage or replace injector
Column temperature too low Raise temperature
Blocked guard column Remove/replace guard column
Blocked in-line filter Remove/replace in-line filter
Problem-3: Steady, high pressure
Problem-2: No pressure reading

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Causes Solutions
Flow set too low Adjust flow rate
Leak in system Locate and correct
Improper column Use proper column
Column temperature too high Lower temperature
Problem-4: Steady, low pressure

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Mr. S. P. Shinde
2 - Leaks
Problem-1: Leaky Fittings
Causes Solutions
Loose fitting Tighten
Dirty fitting Disassemble and clean Replace
Problem-2: Leaks at Pump
Causes Solutions
Loose Purge valve Tighten valve
Pulse damper failure Repair or replace
Loose check valves
Tighten check valve (do not over
tighten)

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Mr. S. P. Shinde
Problem-3: Injector Leaks
Causes Solutions
Improper syringe-needle Use correct syringe diameter
Waste-line blockage Replace waste line
Problem-4: Column Leaks
Causes Solutions
Loose end-fitting Tighten end-fitting
Column packing in ferrule Disassemble, rinse ferrule, reassemble
Problem-5: Detector Leaks
Causes Solutions
Blocked waste line Rebuild or replace
Leaky fittings Tighten or replace

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3 - Chromatogram
Problem-1: Peak Tailing
Causes Solutions
Column Contamination Wash Column as per std protocols
Wrong mobile phase pH Adjust pH

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Problem-2: Peak Fronting
Causes Solutions
Column overload Amount of sample can be decreased
Formation of channels in the column Change the column

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Problem-3: Split Peak
Causes Solutions
Contamination on guard and
analytical column inlet.
Remove guard column replace if
necessary.

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Mr. S. P. Shinde
Problem-4: Baseline Drift
Causes Solutions
Impurities in column
Clean the contaminated column with a
strong solvent
High viscous mobile phase Lower viscosity solvent can be used
Poor column efficiency Use mobile phase of lower viscosity

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Mr. S. P. Shinde
Problem-5: Baseline Noise
Causes Solutions
Continuous noise : detector lamp
problem
Replace the UV lamp or clean the
detector cell.
Periodic : pump pulses Check pulse damper and check valves.
Random : accumulation of impurities
Before injection sample clean up
should and backflush column with a
strong solvents.

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Mr. S. P. Shinde
Problem-6: Broad Peaks
Causes Solutions
Large volume of injection
Reduce the volume of injection or
solvent
Poor column efficiency Lower viscous solvents can be used
Retention time to long
Gradient elution or strong mobile
phase can be used

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Mr. S. P. Shinde
Problem-7: Ghost Peaks
Causes Solutions
Column may be contaminated Column washing
Peaks of previous run may be eluted Column washing
Contaminated water might have been
used
Use only double distilled water for
HPLC

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Mr. S. P. Shinde
4 - Injector
Problem-1: Manual injector hard to turn
Causes Solutions
Damaged rotor seal Rebuild or replace valve
Rotor too tight Adjust rotor tension
Problem-2: Manual injector hard to load
Causes Solutions
Blocked loop Replace loop
Dirty syringe Clean or replace syringe
Blocked lines Clear or replace lines

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Mr. S. P. Shinde
5 - Miscellanous
Problem-1: Smell
Causes Solutions
Leak
Check system for loose fittings, change
pump seals if necessary
Problem-2: Warning lamps
Causes Solutions
Pressure limit exceeded Check for blockage
Other warning lamps See service manual

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Mr. S. P. Shinde
Applications of HPLC
1 – Pharmaceutical Industries
❑ It is used in the departments such as
• R & D
• Quality control
• F & D
❑ Assay of pharmaceutical drugs
❑ Multi components analysis
❑ Analysis of polyphenols, steroids, vitamins.
2 – Stability Studies
❑ Stability of the products
❑ Degradation studies such as
• Acid
• Base
• Oxidation
• Photo degradation

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Mr. S. P. Shinde
3 – Bioanalysis
❑ Used to determine the drug in biological matrices such as
• Blood
• Plasma
• Urine
• Serum
• Faeces
❑ It is useful in pharmacokinetics and bioequivalence studies
4 – Natural Product Analysis
❑ Standardization of herbal extract
❑ Standardize poly herbal formulations

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Mr. S. P. Shinde
5 – Food Analysis
❑ Analysis of honey sample
❑ Food formulation
❑ Dairy products
❑ Sugar analysis
❑ Analysis of nutrition value in nutraceuticals.
6 – Drug Interaction Studies
❑ Useful in drug-drug interaction studies
❑ Herb-drug interaction studies

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Mr. S. P. Shinde
7 – Preparative Analysis
❑ Used to isolate the separated component from mixture
❑ Isolation of phytoconstituents from herbal extract
8 – Forensic Sciences
❑ To identify and quantify the components in
• Blood
• Plasma
• Serum
• Urine
9 – Cosmetics
❑ Many cosmetic products can be analysed
❑ Both qualitative and quantitative analysis is carried out.

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Mr. S. P. Shinde
Ultra Performance Liquid
Chromatography (UPLC)

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Mr. S. P. Shinde
Ultra Performance Liquid Chromatography (UPLC)
Introduction
• Ultra Performance Liquid Chromatography was introduced in 2004.
• Advanced in instrumentation and column technology.
• Made to achieve increase in resolution, speed and sensitivity in liquid
chromatography.
• Innovation in particle technology and instrument design to meet and
overcome various challenges associated with the conventional LC.
• The technique lead to rapid analysis, increased sensitivity with
precise result and made successful analysis.

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Mr. S. P. Shinde
• Decreases run time and increases sensitivity.
• Provides the selectivity, sensitivity, and maintaining resolution
performance.
• UPLC’s fast resolving power quickly quantifies related and
unrelated compounds.
• Use of multi residue method.
• Reduces process cycle time.
• Less solvent consumption.
• It improves three areas- Speed, resolution, sensitivity.
➢ Speed - 1.5min
➢ Pressure - 15000psi
➢ Sensitivity- 3-5µl
advantages

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Mr. S. P. Shinde
• Due to increased pressure requires more maintenance and reduces the
life of the columns of this type.
• In addition, the phases of less than 2µm are generally non-regenerable
and thus have limited use.
disadvantages

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Mr. S. P. Shinde
The Principle involved in UPLC is based on van Deemter equation which
describes the relationship between flow rate of mobile phase and plate
height or column efficiency.
H = A + B/v + Cv
Where,
H= Height equivalent to Theoretical plate
A= Eddy diffusion
B= Longitudinal diffusion
C= Equilibrium mass transfer
v= Velocity (flow rate)
Van deemter equation that describes the relationship between linear
velocity (flow rate) and plate height (HETP or column efficiency)
principle

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Mr. S. P. Shinde
• It is more selective and sensitive technique than HPLC due to smaller
particle size.
• It has high resolution performance and faster resolving power.
• It ensures rapid analysis due to decreased run time typically 2-3 min with
reduced cost of operation.
• The analysis time was reduced up to nine times with UPLC column of
1.7μm particle size compared to the conventional HPLC column with 5μm
particle size.
• The very high pressures up to 100 MPa involved in UPLC system, has no
negative influence on analytical column or other components of
chromatographic system.
• UPLC increases sensitivity and decreases the consumption of solvent and
increases sample throughput.
• It provides real-time analysis in step with manufacturing processes.
Advantages over hplc

High Performance Liquid Chromatography (HPLC)

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