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Validation of HPLC
Transcript of Validation of HPLC
Cyprian Nwokoro & Swapnil Shinde
MSc. Drug Discovery & Development
University of Sunderland
What is HPLC...?
Principle of HPLC
Selection of solvent
Pump or Degasser :
The role of the pump is to force a liquid (called the mobile phase) through the liquid at a specific flow rate, expressed in milliliters per min (mL/min).
•Normal flow rates in HPLC are in the 1 to 2 mL/min range.
•Typical pumps can reach pressures in the range of 6000-9000 psi (400-to 600-bar).
•During the chromatographic experiment, a pump can deliver a constant mobile phase composition or an increasing mobile phase composition.
Column: (“Heart of the Chromatograph” )
The column’s stationary phase separates the sample components of interest using various physical and chemical parameters.
ColumnsTypes of columns in HPLC:
• Analytical [i.d. 1.0 -4.6-mm; lengths 15 –250 mm]
• Preparative [i.d. > 4.6 mm; lengths 50 –250 mm]
• Capillary [i.d. 0.1 -1.0 mm; various lengths]
• Nano [i.d. < 0.1 mm, or sometimes stated as < 100 μm]
Materials of construction for the tubing
• Stainless steel (the most popular)
• Glass (mostly for biomolecules)
• PEEK polymer (biocompatible and chemically inert to most solvents)
•The injector serves to introduce the liquid sample into the flow stream of the mobile phase.
•Typical sample volumes are 5-to 20-microliters (μL).
•The injector must also be able to withstand the high pressures of the liquid system.
•An auto sampler is the automatic version for when the user has many samples to analyze or when manual injection is not practical.
Refractive Index (RI) Detection
How HPLC works...?
Experiment and Results
100ml Acetonitrile and
400ml Dihydrogen Phosphate
0.01gm Chlorpheniramine in
100 ml above mobile phase