This document discusses HPLC (High Performance Liquid Chromatography). It begins by providing an introduction to Talsaniya Roman, the author, and their presentation on HPLC. It then covers various topics related to HPLC in more detail over several pages, including the definition of chromatography, different types of HPLC classification like normal phase vs reversed phase, different stationary and mobile phases, detectors commonly used in HPLC like UV, refractive index, fluorescence, and conductivity detectors, and key components of an HPLC system like the pump, injector, and column. The document provides information on HPLC in a technical presentation format.

1. Talsaniya Roman
Date 12-8-2019
Chemisty department of PG center sir p.p institute of science
Maharaj Krishnkumar sinhji bhavnagar university
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2. HPLC(liquidChromatography)
This presentation demonstrates the
Type ,Part,principal,instrumentation
of most use full analytic instrument
Hplc.
It is usefull for seperation of plant
pigment ,also for qualitative and
quantities analysis of vitamin ,drug
and pesticide. But most for study of
Proteins structure.
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3. DEFINATION OF CHROMATOGRAPHY
Chromatography is based on the principle where
molecules in mixture applied onto the surface or into the
solid, and fluid stationary phase (stable phase) is
separating from each other while moving with the aid of a
mobile phase.
The factors effective on this separation process include
molecular characteristics related to adsorption (liquid-
solid), partition (liquid-solid), and affinity or differences
among their molecular weights.
Because of these differences, some components of the
mixture stay longer in the stationary phase, and they
move slowly in the chromatography system, while others
pass rapidly into the mobile phase, and leave the system
faster.
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4. Three components thus form the basis of the chromatography
technique.
Stationary phase:This phase is always composed of a “solid” phase
or “a layer of a liquid adsorbed on the surface solid support”.
Mobile phase:This phase is always composed of “liquid” or a
“gaseous component.”
Separated molecules
The type of interaction between the stationary phase, mobile
phase, and substances contained in the mixture is the basic
component effective on the separation of molecules from each
other.
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6. HPLC
 first type of Classification
two modes are defined depending on the
relative polarity of the two phases: normal
and reversed-phase
chromatography.In normal phase
chromatography, the stationary bed is
strongly polar in nature (e.g., silica gel), and
the mobile phase is nonpolar (such as n-
hexane or tetrahydrofuran). Polar samples are
thus retained on the polar surface of the
column packing longer than less polar
materials.Reversed-phase chromatography is
the inverse of this.The stationary bed is
nonpolar (hydrophobic) in nature, while the
mobile phase is a polar liquid, such as
mixtures of water and methanol or
acetonitrile. Here the more nonpolar the
material is, the longer it will be retained.
Second Classification Base on principal
Aadsorption chromatography the stationary
phase is an adsorbent (like silica gel or any
other silica based packings) and the separation
is based on repeated adsorption-desorption
steps.
ion-exchange chromatography the
stationary bed has an ionically charged surface
of opposite charge to the sample ions.This
technique is used almost exclusively with ionic
or ionizable samples. The stronger the charge
on the sample, the stronger it will be attracted
to the ionic surface and thus, the longer it will
take to elute.The mobile phase is an aqueous
buffer, where both pH and ionic strength are
used to control elution time.
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7. Chiral andsizeexclusionchromatography
size exclusion chromatography the
column is filled with material having
precisely controlled pore sizes, and the
sample is simply screened or filtered
according to its solvated molecular size.
Larger molecules are rapidly washed
through the column; smaller molecules
penetrate inside the porous of the
packing particles and elute later. Mainly
for historical reasons, this technique is
also called gel filtration or gel
permeation chromatography although,
today, the stationary phase is not
restricted to a "gel".
Chiral hplc is use to separate of
sterioisomer by using penomena called
inclusion complexing
Concept: formation of a diastereomeric
complex in a chromatographic equilibrium
such that the nonchiral interactions are at
minimum strength and the differential chiral
interaction is at maximum strength.
Identifying those points of interaction
between the stationary phase and the
racemate guides you in the choice of CSPs
and the best conditions under which to
operate. Non-chiral interactions generally
anchor a molecule and, therefore, assist in
the formation of the diastereometric
complex. Both π-π interactions driven in
normal phase phase solvents and inclusion
complexation driven in reversed phase
modes are the first significant areas to
address the potential of an appropriate
chiral stationary phase
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8. HPLC
Affinity
chromatography is an important biophysical
technique that enables the separation,
identification, and purification of the
components of a mixture for qualitative and
quantitative analysis.
It is a separation technique in which a mobile
phase carrying a mixture is caused to move in
contact with a selectively absorbent
stationary phase.
Affinity chromatography is a type of
liquid chromatography for the separation,
purification or specific analysis of sample
components.
It utilizes the reversible biological interaction
or molecular recognition called affinity which
refers to the attracting forced exerted in
different degrees between atoms which
cause them to remain in combination
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9. TypebaseonElution
ISOCRATIC SEPARATION
The separation of the mixture
compounds is done by using one mobile
phase and constant throughout the
elution, it is called as isocratic elution.
It consists of single, two or more
solvents of similar polarity to elute the
sample through the column.
If the separation of the sample mixture is
observed in the bands on the column,
the development process may be
stopped at that moment and separated
constituents are extracted by using
suitable solvents.
 GRADIENT SEPARATION
 The separation of the mixture of
components is done by using two or more
mobile phases and the flow of solvent is
stepwise manner depending on ratios are
sent into the column.
 This separation technique is started with a
low concentration of the organic
components of the mobile phase.
 Upon increasing the concentration of the
organic components of the mobile phase,
the competition for adsorption was
increased.
 The commonly used organic solvents
include methanol, acetonitrile, THF, etc.
 The accelerated compounds from the
column decrease the retention time of slow
eluting components.
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11. HPLC
 Solvent reservoir
The pure solvent or a mixture of two or
more solvent having appropriate eluting
power are placed for sample components.
The selection is mainly based on the
polarity of the stationary phase, nature of
sample components, and upon the
chromatographic method.
For isocratic elution, one or two or more
solvents with the fixed composition are
used.
For gradient elution, the composition of
the solvents used is changed continuously.
Glass or stationary steel reservoirs are used
in modern HPLC for storing the mobile
phase.
Degassers and filtration systems are often
provided with reservoirs.
 Degasser
Bubble formation on mixing of
solvents can lead to a number of
problems in HPLC analysis which
can be prevented by degassing of
mobile phase.
 Commonly used degassing practices
for HPLC mobile phase are:
 Helium purging
 Vacuum degassing
 Sonication
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12. In HPLC, the mobile phase is passed through the stationary column
under high pressure, means of pumps.The HPLC pumps should
possess the following features.
 They should generate high pressure up to 6000 psi.
 They should provide a flow rate from 0.1 to 10ml/min, flow
reproducibility up to 0.5%.
 They are easy to dismantle and repair.
 There are two type of pump as below
DISPLACEMENT PUMP
They maintained by a constant flow rate of
the mobile phase throughout the column.
The constant flow in the column is maintained
by a plunger in the syringe-like chamber.
The plunger is driven by a motor.
RECIPROCATING PUMP
It consists of a small chamber in which the
mobile phase is pumped by the forward and
backward movement of a piston which runs
an electric motor
The flow of the mobile phase in the chamber
is regulated by two valves.
Piston directly pushes the mobile phase with
each other or it pushes the diaphragm which
lies between it and the mobile phase.
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13. DISPLACEMENT PUMP
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15. Column
The Guard column is placed between
the sample injector port and the
analytical column filled with the same
packing material.
It is made up of stainless steel that
helps with high pressure of about
5000 psi and the length of the column
is usually between 5-20 cm.
Small internal diameter 2-8 mm of
precolumn gives good results and
helps in increasing their working
efficiency.
Generally large particle size columns
are placed with 20-30% of silicon
phase and the pressure drop across
the column is negligible when
compared to the analytical column.
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16. Analyticcolumn
Analytic column
Column length : 5cm to 30cm
Particle size : 1 micro to 20 micro
Column diameter : 2mm to 50mm
Particle nature :spherical ,uniform size ,porous material are used
Column Particle Size Pore Size
Specific surface
area
Carbon content End capped Usgage pH
C18
3/5/8/10/15/20/50/75
μm
100Ǻ 330m²/g 16% Yes 2-8
C18H 5/8/10μm 100Ǻ 330m²/g 20% Yes 2-11
C8 3/5/8/10μm 100Ǻ 330m²/g 12% Yes 2-8
C18H 3/5/8/10μm 100Ǻ 330m²/g 9% Yes 2-11
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17. SampleInjector
 The sample is injected with an injection valve,
where autosampler with a microprocessor is
present in the HPLC technique.
 The collected sample is sent into the column head
and the packing of column material remains
stable.
 They are mostly three types of sample injectors
used in HPLC, they are as follows.
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20. DetectorUsedInHPLC
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 There is versatile detector use but most use detector
For HPLC is listed below .
1. UV visible detector
2. Reflective index detector
3. Fluorescence detector
4. Conductivity detector
5. Mass Spectometero
Fluorescence detectors

21. I. CriteriaForGoodDetector
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It should have either specific or general response to compounds in
a mixture.Sensitivity towards solute over mobile phase.
It should have a response to solute and not the mobile phase.
It should have a low cell volume for memory effects minimization.
It should respond linearly to solute concentration
it sould not contribute to zone spereading
it sould be not be affected by temperature variation and flow rate.

22. UV Detector
Thistypeofdetectorisonlyusedfor compoundcontaining
chromophore.Thedetectormeasurestheconcentrationsormassflows
oftheseparatedanalytes,andconvertsthemintoelectronicsignals.
Theavailabilityofreliableandsensitivedetectorsismostlyresponsible
forthesuccessofHPLCasapervasiveanalyticaltechniqueinscientific
discoveryandqualitycontrolapplications.Constructionshowbelow.
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23. RefractiveIndexdetector
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The detection principle involves measuring of the change in refractive index of the
column effluent passing through the flow-cell. The greater the RI difference between
sample and mobile phase, the larger the imbalance will become.Thus, the sensitivity will
be higher for the higher difference in RI between sample and mobile phase
The optical schematic of the deflection detector is shown in below.This detector based on
the deflection principle of refractometry, where the deflection of a light beam is changed
when the composition in the sample flow-cell changes in relation to the reference side (as
eluting sample moves through the system).When no sample is present in the cell, the light
passing through both sides is focused on the photodetector (usually photoresistor).As
sample elutes through one side, the changing angle of refraction moves the beam.This
results in a change in the photon current falling on the detector which unbalances it.The
extent of unbalance (which can be related to the sample concentration) is recorded on a
strip chart recorder.

24. Fluorescencedetectors
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Figure below shows the optical schematic of a typical
fluorescence detector for liquid chromatography.The
detectors available on the market differ in the
method in which the wavelengths are controlled. Less
expensive instruments utilize filters; medium priced
units offer monochromator control of at least
emission wavelength, and full capability research-
grade instruments provide monochromator control of
both excitation and emission wavelengths
Fluorescence detectors are probably the most sensitive among the existing
modern HPLC detectors. It is possible to detect even a presence of a single analyte
molecule in the flow cell. Typically, fluorescence sensitivity is 10 -1000 times higher
than that of the UV detector for strong UV absorbing materials. Fluorescence
detectors are very specific and selective among the others optical detectors.This is
normally used as an advantage in the measurement of specific fluorescent species
in samples.

25. Conductivitydetector
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Solutions containing ionic components will conduct
electricity. Conductivity detector measures electronic
resistance and measured value is directly proportional to
the concentration of ions present in the solution.Thus it is
generally used for ion chromatography.

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