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Normal Phase Chromatography

Normal Phase Chromatography is the original type of liquid chromatography, as it was also used in the early years of chromatography. It can sometimes also be referred to as adsorption chromatography if the separation mechanism is based on adsorption due to the stationary phase selected. Normal phase chromatography is generally characterised by stationary phases with polar surface properties and a mobile phase with a non-polar character.

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Hibar RT 100x3.0mm Superspher 60 Å Si 4µm HPLC Column
1.51924.7142
Hibar RT 100x3.0mm LiChrospher 60 Å Si 5µm HPLC Column
1.51924.7109
Hibar RT 100x3.0mm LiChrospher 60 Å Si 12µm HPLC Column
1.51924.7106
Hibar RT 100x3.0mm LiChrospher 60 Å Si 10µm HPLC Column
1.51924.7104
Hibar RT 100x3.0mm LiChrospher 100 Å Si 10µm HPLC Column
1.51924.7101
Hibar RT 50x3.0mm Purospher 90 Å Si 5µm HPLC Column
1.51923.7180
Hibar RT 50x3.0mm Purospher STAR 120 Å Si 5µm HPLC Column
1.51923.7175
Hibar RT 50x3.0mm Purospher STAR 120 Å Si 3µm HPLC Column
1.51923.7174
Hibar RT 50x3.0mm Superspher 100 Å Si 4µm HPLC Column
1.51923.7143
Hibar RT 50x3.0mm Superspher 60 Å Si 4µm HPLC Column
1.51923.7142
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Technical Data

Basics

The stationary phases of normal phase chromatography

Unmodified silica gels or aluminium oxide particles are often used as stationary phases, as the adsorption effect is the main separation mechanism with these materials and can therefore be referred to as "true" adsorption chromatography.

Other materials that can be used in normal phase mode (i.e. with non-polar mobile phases) are specially modified silica gels (cyano, amino, diol, nitro), which will not be discussed in detail here. More detailed information can be found under the heading "Medium Polar Phase Chromatography".

Mobile phases of normal phase chromatography

Almost all common organic solvents and their mixtures can be used as mobile phases in normal phase chromatography, as long as they are not strong acids or bases. Aliphatic hydrocarbons, such as hexane or heptane, have the weakest elution power. As the polarity of the solvent increases, so does its elution power. Polar solvents used include acetic acid ethyl ester, acetone, THF or various aliphatic alcohols.

What is the separation in normal phase chromatography based on?

Due to the ability of the stationary phase to form hydrogen bonds, electrostatic and other intermolecular interactions with the analytes, retention in normal phase chromatography is based on the different degrees of adsorption of the analytes on the stationary phase. Polar analytes are therefore retained more strongly than non-polar analytes, as they are more able to enter into polar interactions with the stationary phase.

Schematic mode of operation of normal phase chromatography

How do I select the correct normal phase column?

Polar stationary phases are used in normal phase chromatography. The simplest phase is pure silica. The interaction is based on adsorption. However, the disadvantage of pure silica phases is that small amounts of water in the mobile phase can accumulate on the stationary phase and thus change the retention and selectivity. Therefore, the reproducible operation of pure silica columns is often difficult. In addition, a long equilibration period is often required to operate the columns from pure silica. Other possibilities are Medium polar phases such as cyano, amino or diol, which can also be used in normal phase chromatography.

 

The different phases for the normal phase initially differ in their polarity: Silica > Amino > Diol > Cyano

 

There are also special applications in which different phases are used:

 

  • Silica: separation of very non-polar compounds (carotenoids or lipids), separation of extremely polar compounds (are not retarded in RP), separation of isomers (achiral), use in the DC and in flash chromatography
  • Amino: fat-soluble vitamins, alternative selectivity to silica
  • Diol: steroids, alternative selectivity for hydrogen bonds and usually longer retention compared to silica
  • Cyano: steroids, different selectivity for double bonds, non-polar phase for normal phase chromatography

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