Normal-Phase vs Reversed-Phase HPLC

One of the most important choices in chromatography method development is deciding between Normal-Phase (NP) and Reversed-Phase (RP) HPLC. The following comparison outlines the main differences:

Normal-Phase HPLC

The stationary phase is more polar than the mobile phase.

Common column types include Silica, Amino (NH2), Diol, and Cyano (CN).

The mobile phase usually consists of non-polar solvents such as hexane or ethyl acetate.

Polar compounds are retained for a longer time.

Reversed-Phase HPLC

The stationary phase is less polar than the mobile phase.

Typical columns include ODS (C18), C8, C4, Phenyl, and Cyano (CN).

The mobile phase often contains polar solvents such as water or acetonitrile.

Non-polar compounds are retained for a longer time.

The retention order of analytes is essentially reversed when switching between NP and RP systems—this principle lies at the core of chromatographic separation.

Choosing the appropriate phase depends on factors such as analyte polarity, sample solubility, and the desired separation outcome.

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