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NP/RP Chromatography
NP = normal phase chromatography (NP mode)
RP = reversed phase chromatography (RP mode)In the early days of chromatography silica gel was used as stationary phase. It was cheap to produce, relatively inert and has a high surface area. Silica gel was produced from sodium silicate by hydrolysis and desiccation. The stationary phase was composed of silica gel particles of 10 - 30 µm.
In the early 70s, a way to produce speric silca gel was discovered. Because of the round form of the speric silica, the columns could be packed tighter because the particles fit much better into each other. This allowed a more regular flow of the mobile phase. The composition of the particles could also be controlled better. Particles of 5 µm and 3 µm could be produced. This increased the efficiency of the columns but also back pressure.
Also in the early 70s bonded phases appeared, which means lipophilic groups are attached to the silica gel surface to give reverse phase silica gel which elutes polar components first.
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The difference between Normal Phase- and Reversed Phase Chromatography
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| Normal Phase Chromatography (NP) uses a polar stationary phase and a non-polar mobile phase. The polar analyte, which differs in polarity from the stationary phase, associates with the stationary phase and is retained. With increased polarity of the mobile phase, elution time increases. Non-polar molecules elute more rapidly whilst polar molecules are retained.
With the development of Reversed Phase Columns, Normal Phase Chromatography became less popular because of the lack of reproducibility. Recently, however, it has become useful again with the devolopment of HILIC bonded phases.
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| HALO columns have an RP phase. Because of the Fused Core Technology which fuses a porous layer to a solid silica core, the HALO phase allows a fast seperation as in UPLC yet under "normal" operating pressure below 400 bar and with regular HPLC systems. |
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| In Reversed Phase Chromatography (RP) the stationary phase is non-polar. For the production of a non-polar stationary phase, silane which has been substituted with long-chain hydrocarbons, is brought to react with silica gel. The polar surface of the silicagel is covered with a non-polar layer of alcanes. The polarity of the phase is reversed.
In contrast to NP, the retention time is longer for molecules which are non-polar in nature, allowing polar molecules to elute more rapidly. As mobile phase, polare compounds such as water and acetonitrile are commonly used.
For an iscratic seperation the composition of the mobile phase is not changed during seperation.
For a gradient seperation the composition of the moible phase is changed during seperation. This is the most commen technique used these days.
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