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Chromatography

Chromatography is used to separate components of complex mixtures for qualitative and quantitative identification.

Liquid Chromatography (HPLC) is used to identify and measure soluble substances in mixtures.

Gas Chromatography is used to identify and measure gasses and volatile compounds in mixtures.

Methods of Separation

Normal Phase: a mode of chromatography commonly using two types of packing materials, in which the solvent or mobile phase is less polar than the stationary phase or packing material of the column. Normal phase chromatography is generally used for the separation of organic molecules that are not soluble in water or aqueous solutions.

Non-Bonded Normal Phase (Adsorption): the oldest method of chromatography, in which separation is achieved by selecting absorption of the polar or polarizable function groups of the sample molecules directly onto the surface of a polar inorganic packing material such as silica (Si). Different molecules adsorb to varying degrees and are therefore separated.

Bonded Normal Phase: a mode of chromatography in which a polar organic phase is chemically bonded to the silica substrate. The bonded phase material contains polar functional groups such as amino (NH2) or cyano (CN) groups to which the sample molecules are attracted. The degree of retention on the column is a function of the polarity of the bonded phase, particle size, and pore diameter of the substrate particles.

Reverse Phase: a mode of chromatography in which the solvent or mobile phase is more polar than the stationary phase. Reverse phase chromatography is generally used for the separation of non-polar or weakly polar water-soluble molecules; however, more polar molecules can also be separated by using a more polar bonded phase or such special techniques as ion pairing. The solvents required in reverse phase chromatography often denature biological macromolecules.

Hydrophobic Interaction (HIC): a form of reverse phase chromatography in which separation is based on hydrophobisity; however, gradients of decreasing salt concentration are used to elute the column, resulting in separation under non-denaturing conditions.

Ion Exchange: a mode of chromatography in which molecules are separated according to their charges. The packing material, which may be silic- or resin-based, is covered with a bonded phase containing ionic sites. This gives a fast separation of electrolytes and such biological molecules as proteins, peptides, amino acids, and nucleic acids. Biological activity is generally retained since denaturing organic solvents are not used.

 

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