Preparative Chromatography - Sample Mass Overload > Page 19


The different effects of mass overload are now clearly revealed. Firstly, it must be emphasized that the curves in figure 8 represent the movement of the peak extremes and are in no way related to the peak shape. It is seen that the retention of the rear of the major peak, benzene, hardly changes with sample mass as this represents low concentrations of benzene and thus is eluted in the normal manner. However, the retention of the peak front is reduced progressively as the sample mass is increased. This results from both the formation of a non-linear adsorption isotherm and the increased elution strength of the mobile phase in contact with the benzene. In fact, as a result of solute–solute interaction the benzene is, in effect, partially eluting itself.

The effect of the mass overload of benzene on the other solutes is also clearly demonstrated. The presence of the high concentration of benzene in the mobile phase increases the elution rate of both the naphthalene and the anthracene. Its also seen, however, that the effect of the high concentration of the benzene on the closer eluting peak naphthalene is to produce band dispersion, whereas the anthracene band does not suffer significant dispersion and the retention of both the front and the rear of the anthracene peak appear to be linearly reduced with sample mass. The chromatograms shown in figure 7 also show that the anthracene peak maintains its symmetry throughout all sample sizes. The impact of the high concentration of benzene on the elution of the other solutes only occurs while the solutes are still in contact with one another at the beginning of the column. Once the separation has started to develop, and the three solutes are no longer in contact, then the naphthalene and anthracene will be eluted in the normal manner. This also explains the significant band dispersion of the naphthalene peak, as, being closer to the benzene peak, experiences the effect of the high concentration of benzene for a longer period and thus, is also effected by the change in character of the stationary phase due to the high concentration of benzene. The increased dispersion due to mass overload on closely eluting peaks is of particular importance in the separation of enantiomers and may become the major load limiting factor. It follows, that the phase system that provides the highest separation ratio should be used, irrespective of any increased (and apparently unacceptable) retention time, as the increase in retention time can often be compensated by the use of multi-sample development (which will be discussed later).