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High Performance (Pressure) Liquid Chromatography (HPLC)

The General Approach

Collect blood sample
Separate plasma (partial clean-up)
Extraction into organic solvent (polarity/pH)
Injection onto column
Separation on column
Quantitation with detector
Record and analyse results

HPLC Instrumentation

Solvent Reservoir
Pump
- High pressure - 1000 to 5000 psi
Injector
- Low pressure - stop flow
- High pressure value
Column
- Normal Phase - organic (water-free) mobile phase
  - Silica gel - non-aqueous
  - Adsorption
- Reverse phase (C8, C18) - aqueous mobile phase
  - Partitioning
- Ion-exchange - aqueous mobile phase
- Molecular sieve - aqueous mobile phase
  - Size
Detector
- Specific
  - Absorbance
  - Fluorescence
  - Electrochemical
- Non-specific
  - Refractive index
  - Radioactivity
  - Conductivity
Recorder

Click on Figure 3.5.1 to
download a demonstration HyperCard stack
written by Keith Brain at the Welsh School of Pharmacy
Please let Keith know if you are using this in your teaching.
Requires HyperCard or HyperCard Player from Apple

Fig 3.5.1 Diagram illustrating a Typical HPLC System
Redrawn from: Pieper and Rutledge, Laboratory Techniques for Pharmacists, Upjohn 1989, page 27, figure 6

Figure 3.5.2 Example Chromatogram

Figure 3.5.3 Example Standard Curve - Peak Height Ratio versus Concentration

References

This file was last modified: Thursday 30 Jan 2003 at 12:43 PM