High Performance Liquid Chromatography

High performance
Liquid
Chromatography
PRESENTED BY MUHAMMAD TALHA SALIK
CHROMATOGRAPHY
 Laboratory techniques for the separation of mixture

 Chroma mean “color” and graphein mean “ to write/study
 The chromatographic system consists of two immiscible
phases:
 Stationary Phase
 Mobile Phase
Phases of Chromatography
 Stationary phase is a  Mobile phase is a

porous solid matrix. fluid (liquid, gas) and
It is a system on its movement is
which the materials effected by gravity,
to be separated are applied pressure or
selectively adsorbed. capillary.
Some chromatography terms
Analyte:
 Substance that is to be separated during chromatography
Immobilized phase:
 Stationary phase which is immobilized on the support particles or
on the inner wall of the column tubing
Some chromatography terms.
Mobile phase:
 Phase which moves in a definite direction. (liquid/gas/fluid).
 Consists of the sample being separated/ analyzed and the solvent
that moves the sample through the column.
Effluent:
 Mobile phase leaving the column.
• stationaryphase is the solid surface of the paper
•And the liquid phase is of the mobile phase.
Types of Chromatography
 Column chromatography
 Paper chromatography
 Thin layer chromatography
 High performance liquid chromatography
 Gas chromatography
Types of Chromatography
 Liquid chromatography
 ion-exchange chromatography,
 gel chromatography,
 affinity chromatography,
 partition chromatography
 adsorption chromatography
APPLICATIONS
 Play important role in pharmaceutical industry

 Use to prepare huge quantities of pure material
 Analyze the purified compound for trace contamination
 Use in quality analysis in food industry
Such as ; proteins , amino acids and vitamins.
 Also involve in biochemical and fuel industry and
biotechnology .
High - Performance Liquid
Chromatography (HPLC)
 It is a technique used to separate, identify and quantify

components in a mixture And purify on the basis of
polarity
Principle:
 Modern high-performance liquid chromatography (HPLC) uses
pressure for fast separations, controlled temperature, in-line
detectors, and gradient elution techniques
Phases
 Statinory phase :tube shaped column that is

filled and packed with solid adsorbent material
like silica molecules.
 Mobile Phase: it is a liquid solution .It is
initially 90% water and 10% organic solvent
making it hydrophilic but gradually it is changed
to hydrophobic solution of 10% water and 90%
organic solvent
Components
 Pump
 Column
 Sample injector
 Detector
 recorder
Pumps
 A pump forces the mobile phase through the column at a much

greater velocity than that accomplished by gravity flow columns
Columns
 The stationary phase is packed into long stainless steel columns
 The most common material used for column packing is silica gel
Sample Injectors
 A small syringe can be used to introduce the sample into the path
of the mobile phase that carries it into the column
 Many HPLC instruments have loop injectors that can be
programmed for automatic injection of samples
 Inject the liquid sample within range of 0.1- 100 ml of volume
under high pressure.
Detectors
 Modern HPLC detectors monitor the eluate as it leaves the

column and, ideally, produce an electronic signal proportional to
the concentration of each separated component.
 Spectrophotometers that detect absorbances of visible or UV light
are most commonly used as detectors
Recorders
 The recorder is used to record detector signal versus the time the
mobile phase passed through the instrument, starting from the
time of sample injection.
 The graph formed is called a chromatogram
 The retention time is used to identify compounds when compared
with standard retention times run under identical conditions.
 Peak area is proportional to concentration of the compounds that
produced the peaks.
HPLC Uses
 Identification , quantitation and analysis of hemoglobin

variants , drugs , toxic substances , amino acids , carbohydrate
and hormones.
 In DNA different analysis
 Urine analysis and antibodies analysis in blood
 Show the presence of tumors and their size and location
 Use in detection of diseases , hypertension, heart disease by
hormonal level
 Check blood sugar level for diabetes monitoring
Disadvantages of HPLC
 Cost
 Complexity
 Low sensitivity for some compounds
 Irreversibly adsorbed compounds not detected
References
 Clinical chemistry Principles , Techniques , correlations

 Internet search resourses

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High performance

 Laboratory techniques for the separation of mixture

 Stationary phase is a  Mobile phase is a

 Play important role in pharmaceutical industry

 It is a technique used to separate, identify and quantify

 Statinory phase :tube shaped column that is

 A pump forces the mobile phase through the column at a much

 Modern HPLC detectors monitor the eluate as it leaves the

 Identification , quantitation and analysis of hemoglobin

 Clinical chemistry Principles , Techniques , correlations

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