Pulsed-Field Gel Electrophoresis involves electrophoresis in agarose, where two electric fi elds are applied alternately at different angles for defined time periods (e.g. 60 s)

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#Id: 2782

#Unit 13. Methods in Biology

The retention time tR for each analyte has two components

 i - dead time, tM. &

ii - adjusted retention time, t’R.

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#Id: 2783

#Unit 13. Methods in Biology

t’R is characteristic of the analyte and is the difference between the observed retention time and the dead time:

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#Id: 2784

#Unit 13. Methods in Biology

Retention factor (k) is simply the additional time that the analyte takes to elute from the column relative to an unretained or excluded analyte that does not interact with the stationary phase and which, by definition, has a k value of 0. Thus

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#Id: 2785

#Unit 13. Methods in Biology

The ratio VS/VM is referred to as the volumetric phase ratio, b. Hence:

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#Id: 2786

#Unit 13. Methods in Biology

Chromatography columns are considered to consist of a number of adjacent zones in each of which there is sufficient space for an analyte to completely equilibrate between the two phases, Each zone is called a theoretical plate (having no N).

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#Id: 2787

#Unit 13. Methods in Biology

The length of column containing one theoretical plate is referred to as the plate height, H, which has units of length normally in micrometers.