Preparative Chromatography - Sample Mass Overload > Page 18


It is also seen that up to 16 mg of benzene could be injected onto the column before the peak merged into that of naphthalene. In fact, by sacrificing a small amount of benzene significantly more benzene (probably at least 30 mg) could have been injected before the major portion of the peak was contaminated with naphthalene. This compromise, which involves sacrificing a small quantity of the selected component to obtain a larger load, is common in preparative chromatography, and arises from the tailing that again results from the formation of a non-linear adsorption isotherm. In samples where the two components are present in the mixture at similar levels and are less well resolved, as in the separation of a pair of enantiomers, this compromise must be taken with caution. As both peaks will be overloaded, and asymmetrical, the tail of one peak will merge with a high concentration at the sharp front of the following peak, and thus significant contamination of the second peak will occur. The results of the overload experiment are better examined quantitatively. Curves relating the retention distance of the front and back of each peak to the sample load are shown in figure 8.The retention distances of the front and back of each peak (measured at the points of injection, 0.6065 x peak height) are shown plotted against sample mass. The change in retention with mass of benzene injected is clearly demonstrated, the maximum effect being for the solute anthracene (the last eluted peak ) and the minimum for benzene itself. It is interesting to note that there is little change in the band width of the last eluted peak anthracene.


After J. Chromatogr., Ref. (3)


Figure 8. The Effect of Mass Overload of Benzene on the Retention of Benzene, Naphthalene and Anthracene