Influencing Factor of Reversed-Phase Chromatography

In reversed-phase chromatography, the stationary phase is a non-polar bonded phase, such as octadecylsilane (C18, ODS), octyl (C8) bonded silica; mobile phase water as the base solvent, adding a certain amount of water mixed with the dissolved polar regulator
A weakly polar bonded phase, a bonded phase such as an ether group and a dihydroxy group, is used for reversed phase or normal phase chromatography.

Influencing factor

1. Molecular structure of solute (polarity)

  • The weaker the polarity, the stronger the hydrophobicity, and the larger k, the larger the tR.
  • The more the carbon number of the homolog, the weaker the polarity, and the larger the k;
  • The introduction of a polar substituent reduces the hydrophobicity and the k value becomes smaller.

2. stationary phase

  • The hydrophobicity of the bonded alkyl group increases as the carbon chain is extended, and the k of the solute also increases.
  • The greater the concentration of the alkyl group bonded to the surface of the silica gel, the larger the k of the solute.

3. Mobile phase

  • The stronger the polarity, the weaker the elution ability, and the larger the k of the solute;
  • Solvent type: water is a weak solvent, and alcohol is a strong solvent;
  • Solvent ratio: the proportion of water increases, causing k to increase;
  • Addition of salt: increase the k of the neutral solute.