powerpoint presentation on high performance liquid chromatography which include its definition, classification, principles of seperation, instrumentation and application.

1. High
Performance
Liquid
Chromatography 1

2. HPLC
Prepared By-
Miss. Chitralekha
Sonawane
Guided By-
Dr. Sonali Mahaparale
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3. Contents
Introduction
Advantages and Disadvantages
Classification of HPLC
Instrumentation
Factors influencing
Applications
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4. Introduction
Chromatography is a technique in analytical
chemistry used to separate, identify, and
quantify each component in a mixture.
HPLC is really the automation of traditional
liquid chromatography under conditions
which provide for enhanced separations
during shorter periods of time, utilizing very
small particles, small column diameters, and
very high fluid pressures.
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5. Advantages Disadvantages
• Needs a small sample
with a high accuracy
and precise.
• Two interacting phases
• Room temperature
Analysis
 Accurate results.
 Quick method for analysis
of sample.
 Need a skill to run
the instruments
 Solvents consuming
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6. Classification of HPLC
1) Based on modes of
chromatography
2) Based on principle of
separation
3) Based on elution technique
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7. Types of Modes
 Normal phase mode
Stationary phase is polar, Ex. Silica, alumina
Mobile phase is non-polar, Ex. Hexane ;
dichloromethane; isopropanol; methanol
 Reveres phase mode
Stationary phase is non-polar, Ex. Silica gel with
C18/C8
Mobile phase is polar Ex. water ; methanol;
acetonitrile; tetrahydrofuran (THF)
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8. Principle of seperation
 Adsorption chromatography
 Ion exchange chromatography
 Ion pair Chromatography
 Affinity chromatography
 Molecular Exclusion Chromatography
 Partition Chromatography
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9. Adsorption Chromatography
It utilizes a mobile liquid or gaseous phase that
is adsorbed onto the surface of a stationary solid
phase. The equilibrations between the mobile
and stationary phase accounts for the separation
of different solutes.
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10. Ion Exchange Chromatography
Resin (the stationary solid phase) is used to
covalently attach anions or cations onto it.
Solute ions of the opposite charge in the mobile
liquid phase are attracted to the resin by
electrostatic forces.
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11. Ion Pair Chromatography
In this chromatography a reverse phase column
is temporally converted into ion exchange
column by using ion pairing agents like pentane,
hexane, sulphonic acid etc.
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12. Affinity Chromatography
It utilizes the specific interaction between one kind of solute
molecule and a second molecule that is immobilized on a
stationary phase. For example, the immobilized molecule
may be an antibody to some specific protein.
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13. Molecular Exclusion Chromatography
The liquid or gaseous phase passes through a
porous gel which separates the molecules
according to its size.
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14. Partition Chromatography
This form of chromatography is based on a thin
film formed on the surface of a solid support by
a liquid stationary phase. Solute equilibrates
between the mobile phase and the stationary
liquid.
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15. Elution Technique
 Isocratic elution
Employs a single solvent or solvent mixture of
constant composition.
 Gradient elution
Here two or more solvent systems that differ
significantly in polarity are employed. After
elution is begun; the ratio of the solvents is
varied in a programmed way, sometimes
continuously and sometimes in a series of steps.
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16. Instrumentation
1. Solvent Reservoir
2. Pumps
3. Sample Injection System
4. Columns
5. Detector
6. Waste
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17. 1
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18. Solvent Reservoir
 Glass reservoirs contain 500ml or more solvent.
 Degassers
 Vaccum pumping system
 A distillation system
 A device for heating and stirring
 A system for sparging
• Solvent treatment system
 Isocratic
 Gradient
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19. Pumps
General Requirements;
 Deliver high volumes (flow rates) of solvent (1to
10 mL/min)
 Deliver precise and accurate flow (<0.5%
variation)
 Deliver high pressure (to 6000 psi)
 Inert toward solvents, pulse free flow, be reliable
Types;
1) Reciprocating pucmp
2) Displacement pump
3) Pneumatic pump 1
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20. Reciprocating pump
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21. Displacement Pump
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22. Pneumatic Pump
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23. Sample Injection System
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LOAD INJECT

24. Columns
Analytical Columns
 Length from 10 to 30 cm.
 Inside diameter is 4 to 10 mm.
 Particle size of packings is 5 or 10 m.
 Columns of this type contain 40,000 to 60,000 plates/meter.
Types of columns packaging
 Porouse- Silica Gel , alumina, Ion exchange resine.
 Particular- Glass or polymer(Polymethacrylate, Polyvinyl
alcohol)
Guard Columns
 The composition is similar to that of the analytical column.
 Particle size is usually larger.
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25. Detectors
 Bulk Property Detectors
Refractive Index Detector
Light Scattering Detectors
 Solute Property Detectors
UV-VIS absorption Detector
Fluorescence Detector
Mass Spectroscopic Detector
Electrochemical Detector
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26. Refractive Index Detector Electrochemical Detector
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27. Light Scattering Detector Fluorescence Detector
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28. UV-VIS Absorption Detector Mass Spectroscopic Detector
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29. Chromatogram
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30. Factors Influencing
 Internal diameter of column
 Pump pressure
 Sample size
 The polarity sample, solvent and column
 Temperature
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31. Applications
 Pharmaceuticals industry- Stability, Quantity of drug
and testing of biological fluids.
 Analysis of natural contamination – Phenol and
mercury from sea water.
 Forensic test – Steroid present in blood urine and
sweat.
 Clinical test- Monitoring of hepatic chirosis patient
 Food and essence manufacture- Sweetener analysis
in the fruit juice.
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32. References
 https://www.shodex.com/en/kouza/b.html
 http://hplc.chem.shu.edu/NEW/HPLC_Book/Detect
ors/det_ri.html
 https://en.wikipedia.org/wiki/High-
performance_liquid_chromatography
 https://www.rpi.edu/dept/chem-eng/Biotech-
Environ/IONEX/be_types.htm
 http://lab-training.com/landing/free-hplc-training-programme-
9/
 Skoog, Holler, Crounch “INSTRUMENTAL ANALYSIS”, India
edition, Cengage learning, page no. 893-931.
 P. D. Sethi, “HPLC QUANTITATIVE ANALYSIS AND
PHARMACEUTICAL FORMULATION, CBS Publication, page
No.- 1-50.
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