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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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.

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High Performance Liquid ChromatographWaste

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SeparationsSeparationsInjector

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High Performance Liquid Chromatograph

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SeparationsSeparationsInjector

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The ChromatogramThe Chromatogram

Injection

to

tR

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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

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www.worldofteaching.com

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