High-pressure liquid chromatography (HPLC) is a technique that uses a solid stationary phase and a liquid mobile phase (acetonitrile, methanol, phosphate buffer etc..) to separate components in a mixture. In the HPLC setup, the stationary phase is often housed in specialized columns, commonly referred to as HPLC columns, which are typically constructed from high-quality stainless steel. Liquid chromatography columns in HPLC are designed to withstand the high pressures required for efficient separation. Chemical interactions between the stationary phase and the chemical composition of the components in the mixture result in the components traveling at different speeds in the HPLC column and separating or eluting at different times from the column stationary phase.
The analytical column in HPLC serves as the heart of the chromatographic system. HPLC columns come in various dimensions and particle sizes, catering to different analytical needs and the nature of the compounds being separated.
Common LC Column Formats
Nano LC Columns
High powered sensitivity for exceedingly small samples (Column ID 50-75 μm)
Micro LC Columns
Increased sensitivity for small samples (Column ID 0.15-.05 mm)
UHPLC Columns
High speed separations of analytes (Column ID 1.0-2.1 mm)
Analytical LC Columns
General, all-purpose separations (Column ID 2.1-4.6 mm)
Semi-Prep and Preparative LC Columns
Large scale isolation and purifications (7.8-100 mm)
Separation Mode | Description: |
---|---|
Reversed Phase | Used to separate hydrophobic compounds. |
Normal Phase | Used to separate hydrophobic compounds and matrices that are retained too strongly by reversed phase. |
HILIC | Used to polar organic compounds that are poorly retained by reversed phase. |
Ion Exchange | Used to separate charged compounds. |
Ion Exclusion | Used to separate organic acids, carbohydrates, sugars, starches, and oligosaccharides. |
Chiral LC | Used to separate enantiomers. |
Size Exclusion Chromatography (SEC) | Used to separate biomolecules and polymers. |