Mobile Phase

In LC the mobile phase is often called the solvent, but since solid samples have to be made into a solution by dissolution in a sample solvent, the use of the term solvent for the mobile phase may cause confusion. However, in order to avoid solubility prob lems when the sample is injected on to the column, it is safest, whenever possible, to make the sample solution using the same liquid as is to be used as the mobile phase, in which case no confusion should arise.

The time taken by a sample to pass through the column (the total elution/retention time: tR) is a function of the mobile-phase velocity and the volume of mobile phase required to elute the component from the column, the total retention volume (VR), is given by:

where F is the volume flow rate of mobile phase measured at the column outlet at ambient temperature (Ta) and ambient pressure (Pa). Since column temperatures are usually close to ambient, temperature corrections on flow rate are rarely applied. Volume flow rates are measured from the time required to collect a given volume of mobile phase or with an electronic flow meter. Unlike gas chromato-graphy, liquid mobile phases can be considered as incompressible and the flow rate is uniform throughout the column.

Linear flow rates (u) are measured from the retention time, called the hold-up time, of an unretained substance (tM), i.e. one which moves at the same velocity as the mobile phase:

where y = viscosity, L = column length, u = average mobile phase velocity, AP = pressure drop across the column and tM = residence time for an unretained substance.

Introducing the particle diameter (dp), the flow resistance parameter ($) is given by:

where L is the column length.

However, whereas in gas chromatography it is reasonable to assume that a substance like nitrogen or helium will have negligible solubility in the stationary phases used, in LC finding a species which is soluble in the mobile phase and will not have at least some retention on the stationary phase - therefore giving too high a hold-up time - often proves difficult. Furthermore, with a porous stationary phase, excluded components (not uncommon with mobile phases with a high water content) do not access the total mobile-phase volume and may actually run ahead of the mobile phase, giving too low a hold-up time.

Porosity can be used to verify the hold-up time. The total porosity (s) of a column is the volume fraction occupied by the mobile phase:

where F = volume flow rate, dc = column diameter and L = column length. For a totally porous material s & 0.75 and & 0.4 for porous layer beads.

Thus, values of the hold up time obtained for a given component should give these values according to the type of packing material used. Values < 0.75 indicate the molecule is excluded from the pores of the stationary phase, and values > 0.75 indicate that retention on the stationary phase has occurred.

Choice of mobile phase is fundamental to the separation in LC. Mixtures of solvent, with up to four components are commonly used, so that as well as the chemical nature of the components, miscibility considerations are important. Physical properties, such as viscosity, volatility, refractive index and UV absorption can also limit the choice of mobile phases.

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