Purification subfractionation

In preparative HPLC, fraction collection is likely to be employed for two extreme applications (a) the purification of one or a few major components or (b) the isolation of trace components or impurities in the presence of main components. The first problem is generally solved by the millivolt level (threshold) collection mode with time or drop subfractionation of each peak. A... 

In Figure 3 is documented the resolution of a polyclonal antibody sanple exhibiting only a few closely spaced isoelectric bands. The antibodies were raised to the bacterial carbohydrates derived from Micrococcus lysodeiktikus in a rabbit from a colony outbred for simplicity of their clonotype patterns. After purification by affinity chromatography, the antibodies exhibited three major bands, with very close isoelectric points. To effectuate their separation, commercial Airpholine was subfractionated in the RIEF and a narrow cut, pH range 7.5 to 8.5, was used for the fractionation (8). This illustrates the resolution achievable in critical separations. 

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