Date post: | 12-Feb-2017 |
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HPLC is characterized by the use of high pressure to push amobile phase solution through a column of stationary phase
allowing separation of complex mixtures with high resolution.
INTRODUCTIONINTRODUCTION
Varian HPLC SystemVarian HPLC System
9010 SolventDelivery System
9050 VariableUV/Vis Detector
HPLC SolventReservoirs
HPLCColumn
RheodyneInjector
9060 Polychrom(Diode Array) Detector
ComputerWorkstation
Keep an eye onthese 4 screens!
PRINCIPLEThe principle of separation in normal phase mode and reverse phase mode is adsorption.
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.
Normal vs. Reversed Phase ChromatographyNormal vs. Reversed Phase Chromatography
Normal Phase Reversed Phase
Stationary phase Polar (silica gel) Non-polar (C18)
Mobile phase Non-polar(organic solvents)
Polar(aqueous/organic)
Sample movement Non-polar fastest Polar fastest
Separation based on Different polarities(functionality)
Differenthydrocarbon content
TLC TLC vs.vs. HPLC HPLC
TLC HPLC TLC HPLC Type of Analysis qualitative only qualitative &
quantitative Stationary Phase 2-dimensional
thin layer plate 3-dimensional
column Instrumentation minimal! much! with many
adjustable parameters Sample Application spotting
(capillary) injection
(Rheodyne injector) Mobile Phase Movement capillary action
(during development) high pressure
(solvent delivery) Visualization of Results UV lightbox “on-line” detection
(variable UV/Vis) Form of Results spots, Rf’s
(retention factors) peaks, Rt’s
(retention times)
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How can We Analyze the How can We Analyze the Sample?Sample?
Carbohydrates1. fructose2. Glucose3. Saccharose4. Palatinose5. Trehalulose6. isomaltose
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mAU
time
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SeparationsSeparationsSeparation in based upon differential migration between the stationary and mobile phases.
Stationary Phase - the phase which remains fixed in the column, e.g. C18, Silica
Mobile Phase - carries the sample through the stationary phase as it moves through the column.
Injector
Detector
Column
Solvents
Mixer
Pumps
High Performance Liquid ChromatographWaste
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SeparationsSeparationsInjector
Detector
Column
Solvents
Mixer
Pumps
Chromatogram
Start Injection
mAU
time
High Performance Liquid Chromatograph
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SeparationsSeparationsInjector
Detector
Column
Solvents
Mixer
Pumps
Chromatogram
Start Injection
mAU
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SeparationsSeparationsInjector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
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SeparationsSeparationsInjector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
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SeparationsSeparationsInjector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
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SeparationsSeparationsInjector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
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SeparationsSeparationsInjector
Detector
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Solvents
Pumps
Mixer
Chromatogram
Start Injection
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SeparationsSeparationsInjector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
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SeparationsSeparationsInjector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time
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SeparationsSeparationsInjector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
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SeparationsSeparationsInjector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time
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SeparationsSeparationsInjector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time
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SeparationsSeparationsInjector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time
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SeparationsSeparationsInjector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time
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SeparationsSeparationsInjector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time
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SeparationsSeparationsInjector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time
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The ChromatogramThe Chromatogram
Injection
to
tR
mAU
time
tR
to - elution time of unretained peak
tR- retention time - determines sample identity
Area or height is proportionalto the quantity of analyte.
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HPLC ApplicationsHPLC Applications
Chemical
Environmental
PharmaceuticalsConsumer Products
Clinical
polystyrenedyesphthalates
tetracyclinescorticosteroidsantidepressantsbarbiturates
amino acidsvitaminshomocysteine
Bioscienceproteinspeptidesnucleotides
lipidsantioxidantssugars
polyaromatic hydrocarbonsInorganic ionsherbicides
ADVANTAGES1. Separations fast and efficient (high resolution power)
2. Continuous monitoring of the column effluent
3. It can be applied to the separation and analysis of very complex mixtures
4. Accurate quantitative measurements.
5. Repetitive and reproducible analysis using the same column.
6. Adsorption, partition, ion exchange and exclusion column separations are excellently made.
Refrences
www.worldofteaching.com
www.worldofteaching.com
www.educationbhaskar.com