HPLC (known as high performance liquid chromatography, high
pressure liquid chromatography) is used to separate the phases of
a solid or liquid analyte, regardless of its stability and
volatility. HPLC equipment is popular for its regarded ease of set
up, use, and configuration potential. The principle of HPLC testing
focuses on what occurs when the analyte (compound being analyzed)
and a test solvent are pushed under pressure through a column
within the HPLC equipment. The solvent strength and the particular
flow rate within HPLC equipment affect retention times.
Primary Applications of HPLC
- Identification of compounds
- Characterization of molecular bonds within compounds
- Purity testing
- Molecular quantification
Principles of HPLC Testing Methods
Normal-phase (also known as liquid-solid) HPLC measures the
absorption of the analyte on a polar surface. Reversed-phase (RPC)
HPLC elutes the analyte based on the relative properties of its
mobile phase and the bonded stationary phase. Ion exchange (IEC)
HPLC separation occurs by means of ion exchange between the bonded
ion group and counter ions. Size exclusion (SEC) HPLC invokes
separation based on the size of the molecule being tested and the
sieve material used within the HPLC column.
Pharmaceutical Application of HPLC Testing
Every tested compound exhibits a specific retention time
upon exiting the column. This retention time, graphically
represented as a retention "peak," is unique to each compound and
can be used to identify a molecule. HPLC is useful in
compound identification and pharmaceutical purification testing,
as it is estimated that over 60% of existing molecules have been
uniquely identified by HPLC testing.
Purchasing and Configuring HPLC Equipment
HPLC systems may be integrated/all-in-one or consist of an HPLC instrument and compatible components. It may be necessary to configure an older model HPLC system with very specific HPLC detectors. Vendors offer a range of compatible detectors such as UV-Visible, conductivity, and refractive index detectors. Newer systems such as ultra-high performance liquid chromatographers (UHPLC) are built with high-efficiency considerations.
Follow Us
Keep up with our latest articles, news and events. Plus, get special offers and
more delivered to your inbox.