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HPLC – High-Performance Liquid Chromatography
This technique is a form of column chromatography, it pumps a mixture in a solvent at high pressure through a column with chromatographic material.
The HPLC can separate and identify different compounds present in any sample that can be dissolved in a liquid.
HPLC is a very sensitive, efficient, and accurate technique.
Types of Chromatography (different column resin compositions):
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Normal Phase: The column is filled with polar silica particles and the buffer is non-polar. The polar particles in the injected sample will stick to the silica more and have a longer retention time.
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Reverse Phase: The column is filled with hydrophobic particles and the running buffer used in the system is polar. Hydrophobic particles will stick to the resin more.
Required Material:
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HPLC autosampler vials
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Centrifugal filters
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Eppendorf vials
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HPLC machine
In a HPLC there are the following variables:
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Flow rate
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Pressure
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Solvent buffers
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Column type
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Detection parameters
Protocol:
Mobile phase preparation:
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To 1.5 litres of deionized water add 400ml of acetonitrile/ methanol.
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To this solution add 2.4ml of glacial acetic acid.
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Using purified deionised water, dilute the solution to make a total of 2 litres.
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Add 40% sodium hydroxide and read pH - pH should be 4.2.
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Filter the mobile phase under a vacuum to remove the solids that could clog the chromatographic column.
Sample injection and data collection:
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Prepare your sample and slowly inject 100µl of the sample into the system.
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Load the stationary phase to the column.
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Start the HPLC and wait for the components to develop.
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On completion the data is collected and saved, and the syringe can be removed.
Results and calculations:
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Calculate the concentration of all the separated components.
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The triangular method determines the peak areas for all standards and samples.
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Calculate the time taken for each component to reach the peak.
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Draw calibration curves of peak area vs. concentration for all components.
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Calculate the least-squares fit for calibration curves.