Reverse-phase chromatography principles

Amersham Pharmacia Biotech (ed), Reversed Phase Chromatography Principles and Methods, APB, Uppsala, 1999. 

Basic Principles in Reversed Phase Chromatography, Amersham Biosciences Online Education Centre, 2002. 

Hearn MTW (1998), Fligh-resolution reversed-phase chromatography, In Janson JC, Ryden L (Eds), Protein Purification Principles, High-Resolution Methods, and Applications, 2nd Ed., Wiley-VCH, Weinheim, pp. 239-282. 

Reversed Phase Chromatography (RPC), Principles and Standard Conditions... 

Ionic samples may be separated by reversed-phase chromatography, provided that they contain only weak acids or only weak bases (in addition to neutral compounds) present in undissociated form, as determined by the chosen pH this is known as ion suppression . Ion-pair chromatography is an extension of this principle. An organic ionic substance is added to the mobile phase and forms an ion pair with a sample component of opposite charge. This is, in fact, a salt but its chromatographic behaviour is that of a nonionic organic molecule ... 

Figure 7.8. HPLC analysis of capsaicins, the principle components responsible for the heat in hot pepper, in two samples of hot sauces using reversed-phase chromatography with acidified mobile phase and UV detection. Reprinted with permission from reference 18.

Reversed-phase chromatography is the most common of all the modes as it can usually be used to separate most types of compounds in HPLC. It is based on the principle that like dissolves like . It uses a nonpolar column, e.g. C-18, and relatively polar mobile phases. 

In reversed-phase chromatography, a nonpolar stationary phase is used in conjunction with polar, largely aqueous mobile phases. Between 70 and 80% of all HPLC applications utilize this technique. Its popularity is based largely on its ease of use equilibration is fast, retention times are reproducible, and the basic principles of the retention mechanism can be understood easily. Most stationary phases are silica-based bonded phases, but polymeric phases, phases based on inorganic substrates other than silica, and graphitized carbon have found their place as well. 

Reversed-phase chromatography is the most widely used separation principle in HPLC. 

The principle behind reversed-phase chromatography is the partitioning of the sample components between the hydrophobic stationary phase and the hydrophilic mobile phase. This is the same principle as liquid-liquid extraction. Whether or not the sample components are more attracted to the stationary phase or mobile phase depends on the components relative solubility in hydrophobic and hydrophilic solvents. The relative amount of time the component spends in each phase determines the component s retention time. The hydrophobic components will have longer retention times. 

Tswett s initial column liquid chromatography method was developed, tested, and applied in two parallel modes, liquid-solid adsorption and liquid-liquid partition. Adsorption ehromatography, based on a purely physical principle of adsorption, eonsiderably outperformed its partition counterpart with mechanically coated stationary phases to become the most important liquid chromatographic method. This remains true today in thin-layer chromatography (TLC), for which silica gel is by far the major stationary phase. In column chromatography, however, reversed-phase liquid ehromatography using chemically bonded stationary phases is the most popular method. 

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