Basic Principles of HPLC
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Transcript of Basic Principles of HPLC
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Basic Principles of HPLC
Shahzad Bashir2009-ag-1231
Center for Agricultural biochemistry and biotechnology
UNIVERSITY OF AGRICULTURE
FAISLABAD
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Outline
• What is HPLC?
• Types of HPLC
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HP
igh
erformance
L iquid
Chromatography
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HP
igh
ressure
L iquid
Chromatography
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What is HPLC?
• The most widely used analytical separations technique
• Utilizes a liquid mobile phase to separate components of mixture
• uses high pressure to push solvent through the column
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Components
• Mobile phase reservoirs
• HPLC Pump(s)
• Sample injector (manual or auto)
• Column
• Detector
• Mobile phase waste container
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Mobile phases
• Water
• Methanol
• Acetonitrile
• Additives, salts, acids, bases
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HPLC Pump(s)
• A high pressure pump is used to generate a specific flow rate to mobile phase, typically millimeter per minute.
Sample injector
• An injector is able to introduce the sample into the continuously flowing mobile phase stream that carries the sample into HPLC column.
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Columns
• Length (5-15 cm)
• The column contains the chromatographic packing material needed to effect the separation.
• This packing material is called the stationary phase.
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Detectors
• UV
• Fluorescence
• Electrochemical
• Mass Spectrometric
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Chromatograms
Restek® ULTRA C-18 and CN Columns (250mm x 4.6mm, 5µ),
Mobile Phase: (1:1 Methanol:Water), 1.5 mL/min.
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Types of HPLC
• Reverse-phase (polar mobile phase/non-polar stationary phase/somewhat polar analytes)
• Normal Phase (non-polar mobile phase/polar stationary phase/non-polar analytes)
• Ion-Exchange (salts/ionic stationary phase)• Size-exclusion (aqueous/gel for large MW
solutes, >104)
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PopularitySensitivityAccurate quantitative determinationsuitability for separating nonvolatile and
thermally sensitive specieswidespread applicability in industry, to many
fields of science, and to the publicIdeally suited for proteomic analysis.
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Advantages to HPLC
Higher resolution and speed of analysis
HPLC columns can be reused without repacking or regeneration
Greater reproducibility of results.Easy to operation and data analysisAdaptability to large-scale
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Advantages of HPLC are result of 2 major advances
Stationary supports with very small particle sizes and large surface areas
Appliancation of high pressure to solvent flow
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THANKS