HPLC , pumps used in hplc , column used in hplc, detector used in hplc.

1. HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY
PRESENTED BY:
Khaja Anees Ahmed
M Pharm 1st semester
Dept. of Pharmaceutics
HKE’S
MTRIPS Gulbarga.
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2. CONTENTS
Introduction
Principle
Instrumentation
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3. INTRODUCTION
• HPLC stands for High performance liquid chromatography and it is also
known as high pressure liquid chromatography.
• It is extension of conventional liquid chromatography.
• HPLC is a analytical technique used to separate, identify & quantify the
mixture of compounds.
• It gives high performance due to the small particle size of stationary phase
(30µm).
• High pressure is applied for rapid separation of compound that is 1000-4000
psi.
• It is used in biochemistry and analytical chemistry to identify, quantity and
purify the individual components of a mixture.
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4. PRINCIPLE
• The principle of separation in normal phase mode and reverse phase mode is
adsorption.
• When a mixture of components are introduced into a HPLC column, they
travel according to their relative affinities towards the stationary phase.
• The component which has more affinity towards the adsorbent, travels slower.
• The component which has less affinity towards the stationary
phase travels faster.
• Since no two components have the same affinity towards the stationary phase
and the components are separated.
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5. INSTRUMENTATION
1. Mobile phase reservoir
2. Pumps
3. Mixing unit and solvent degassing
4. Injector
5. Column
6. Detectors
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8. MOBILE PHASE RESERVOIR :-
• In HPLC, the reservoir are usually made up of stainless steel or borosilicate
glass with the capacity of 500 ml to 1 L of solvent ( HPLC grade ).
• Beside, they are duly equipped with adequate mean for the removal of
dissolved gaseous product viz O2 and N2 that may produce undesired small
bubbles in the column as well as detector system engaged.
• Used to store the mobile phase.
• Two types of solvent reservoirs are
• i. Binary system
• ii. Quarternary system.
• HPLC grade solvents are used, these are highly pure.
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9. PUMPS
• The role of the pump is to force a liquid called the mobile phase through the
liquid chromatography at a specific flow rate, expressed in milliliter per min
(1ml/min).
• Normal flow rates in HPLC are in the 1-2ml/min range.
• Typical pump can reach pressure in the range 6000-9000psi.
• During the chromatographic experiment, a pump can deliver a constant mobile
phase composition (isocratic) or an increasing mobile phase
composition(gradient).
Types of pumps
• Reciprocating pump.
• Displacement pump.
• Pneumatic or constant pressure pump.
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10. RECIPROCATING PUMP :-
• The reciprocating pump it consist of a small chamber where the solvent is
moved in and by an eccentric system or gear.
• The forward stroke shut down the inlet check valve, whereas the outlet check
valve open the flood gate and allow the respective intended mobile phase to
pumped in to the Colum efficiency.
• In the particular instance, the mobile phase is
in direct contact with the piston.
• Reciprocating pump critically give rise to
pulsed flow.
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11. DISPLACEMENT PUMP :-
• It consists of large, syringe like chamber equipped with a plunger activated by a
screw driven mechanism powdered by a stepping motor.
• A positive-displacement pump makes a fluid move by trapping a fixed amount
and forcing (displacing) that trapped volume into the discharge pipe.
• Some positive-displacement pumps use an
expanding cavity on the suction side and a
decreasing cavity on the discharge side.
• Liquid flows into the pump as the cavity
on the suction side expands and the liquid flows out of the discharge as the cavity
collapses.
• The volume is constant through each cycle of operation.
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12. MIXING UNIT
• Mixing unit is used to mix solvents in different proportions and pass through the
column.
There are two types of mixing units.
1) Low pressure mixing chamber- which uses helium for degassing solvents.
2) High pressure mixing chamber- It does not require helium for degassing
solvents.
• Mixing of solvents is done either with a static mixer which is packed with beads
or a dynamic mixer which uses magnetic stirrer and operates under high
pressure.
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13. INJECTOR
• The injector serves to introduce the liquid sample into the flow of the mobile
phase.
• Typical sample volumes are 5-20 microliters.
• The injector must also be able to withstand the high pressure of the liquid
system.
• Several devices are available either for manual or auto injection of the sample.
Types of injector
1. Septum injector
2. Stop flow injector
3. Rheodyne injector
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14. 1. Septum injector:
• It is used for injecting the sample through a rubber septum.
• This is not common, since the septum has to withstand high pressure.
2. Stop flow injector:
• In which the flow of mobile phase is stopped for a while and the sample is
injected through a valve device.
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15. 3. Rheodyne injector:
• It is the most popular injector. This has a fixed volume loop like 20µL or 50µL
or more.
• Injector has 2 modes, i.e., load position when the sample is loaded in the loop
and inject mode when the sample is injected.
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16. Column Material
• Most widely used columns are made up of stainless steel which can withstand
high pressure.
• Column length: Varies from 5cm to 30cm
• Column diameter: Ranges from 2mm to 50mm
• Particle size: From 1µ to 20μ
• Particle Nature: Spherical, uniform
sized, porous materials are used.
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17. • Normal Phase Columns: These columns have a polar stationary
phase and are used for separating polar compounds. Common
stationary phases include silica gel bonded with polar functional
groups like amino (NH2) or diol groups.
• Ion Exchange Columns: These columns contain ion exchange resins
as the stationary phase and are used for separating charged compounds
based on their ionic properties.
• Affinity Columns: These columns contain a stationary phase with
specific affinity ligands that selectively bind to target molecules based
on their biological or chemical properties.
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18. DETECTORS
• Detectors used depends upon the property of the compounds to be
separated. Different detectors available are
a) UV detector
b) Refractive index detector
c) Flourimetric detector
d) Conductivity detector
e) Amperometric detector
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19. Refractive Index Detector
• Detector is used to measure changes in the refractive index of the mobile phase
as analyte molecules pass through the detector.
• This type of detector is particularly useful for compounds that lack
chromophores or fluorophores, making them invisible to UV or fluorescence
detectors.
• The RI detector is sensitive to changes in the concentration of solutes in the
mobile phase, allowing for the detection and quantification of analytes based
on their refractive index differences from the solvent.
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20. Conductivity Detector
• A conductivity detector in HPLC measures changes in the electrical
conductivity of the mobile phase as analyte molecules pass through the detector.
• This type of detector is particularly useful for ionizable compounds, such as
acids, bases, and salts.
• When ions are present in the mobile phase, they affect the conductivity of the
solution.
• The conductivity detector detects these changes and can quantify the
concentration of ions in the sample. This detector is especially valuable for
analyzing ionic compounds or mixtures containing ions.
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21. REFERENCES
• Instrumental methods of chemical analysis by B. K. Sharma, Published by
Satyendra Rastogi, Meerut, India.
• Instrumental methods of chemical analysis by Gurudeep.R. Chatawal, Sham.K.
Anand.
• Text book of Pharmaceutical Analysis, fourth edition by Dr. S Ravi Sankar.
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