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The greater the separation factor value is over 1.0, the better the separation, until about 2.0 beyond which an HPLC method is probably not needed for separation. Resolution equations relate the three factors such that high efficiency and separation factors improve the resolution of component peaks in an HPLC separation.
The separation is based on the differential partitioning between the mobile and the stationary phases. Subtle differences in a compound's partition coefficient result in differential retention on the stationary phase and thus affect the separation. [1] Chromatography may be preparative or analytical.
Liquid chromatography (LC) is a method that in some ways is more powerful than GC, but can be coupled to mass spectrometry just as easily. In LC, the concerns involving sample preparation can be minimal. In LC, both the stationary and mobile phase can affect the separation, whereas in GC only the stationary phase should be influential.
In liquid chromatography, the mobile phase velocity is taken as the exit velocity, that is, the ratio of the flow rate in ml/second to the cross-sectional area of the ‘column-exit flow path.’ For a packed column, the cross-sectional area of the column exit flow path is usually taken as 0.6 times the cross-sectional area of the column.
Reversed-phase liquid chromatography (RP-LC) is a mode of liquid chromatography in which non-polar stationary phase and polar mobile phases are used for the separation of organic compounds. [1] [2] [3] The vast majority of separations and analyses using high-performance liquid chromatography (HPLC) in recent years are done using the reversed ...
The 3 factors are not completely independent, but they are very close, and can be treated as such. So what does this mean? It means that to increase resolution of two peaks on a chromatogram , one of the three terms of the equation need to be modified.
The modification of silica gel stationary phase led to many creative ways of modifying stationary phases in order to influence the separation characteristics. The most notable modification was the chemical bonding of alkane functional groups to silica gel to produce reversed-phase media. [11]
A theoretical plate in many separation processes is a hypothetical zone or stage in which two phases, such as the liquid and vapor phases of a substance, establish an equilibrium with each other. Such equilibrium stages may also be referred to as an equilibrium stage , ideal stage , or a theoretical tray .