Biochemical Techniques and Instrumentation

2.7.2.4.1. Stationery phase

Stationery phase materials are spherical particles of uniform size. They are made of chemically modified silica, alumina or styrene/divinylbenzene copolymers. They exist in three forms:

1. Microporous supports: Particle size of this support is 5-10 μm. They are most commonly used in HPLC.
2. Pellicular support: Particles are spherical, non-porous beads with a particle size of 5 μm. They are good for the separation of proteins and large biomolecules.
3. Bonded phases: Certain functional groups are chemically bonded onto the stationery phase such as silica. There are two types of bonded phase columns - normal and reverse phases.

1. Normal-phase partition chromatography

Normal phase has a highly polar stationary phase made of triethylene glycol or water. Mobile phase should be non-polar solvent such as hexane or i-propyl ether. As the compound polarity decreases or solvent polarity increases, the retention time will decrease.

2. Reversed-phase partition chromatography

Reverse phase is very common and has a non-polar stationary phase made of hydrocarbon. Mobile phase should be polar solvent such as water, ethanol, acetonitrile or tetrahydrofuran. As the compound polarity decreases or solvent polarity increases, the retention time will increase.