Electrospray Spectra of Co-Eluting Components

A potential problem encountered in these determinations is the ion suppression encountered in the presence of polar/ionic interfering materials which compete with the analyte(s) for ionization (see Section 4.7.2 earlier). Many of these analyses therefore involve some degree of off-line purification and/or an appropriate form of chromatography. Since it is not unusual to encounter closely related compounds that are not easily separated, it is also not unusual to employ both of these approaches, as in the following example. This illustrates the use of HPLC as a method of purification and demonstrates that highly efficient separations are not always required for valuable analytical information to be obtained. 

Part of the characterization of a protein encountered in the attempted development of a vaccine to the hepatitis E virus involved the determination of its molecular weight [7], 

The protein was purified by a dialysis procedure, denatured and analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Western blotting indicated that the protein of interest consisted of two components, one of which increased in concentration as the purification proceeded. The authors initially suggested that this could be due to the presence of a number of species produced by modification of the amino acid side-chains, for example, by glyco-sylation, or by modification of the C- or N- terminus. 

The purified protein was subjected to reversed-phase HPLC analysis by using a 150 x 1 mm Cis column with gradient elution from 0.1% aqueous trifluo-roacetic acid (TFA) to 0.1% TFA in acetonitrile, over a period of 55 min, at a flow 

The function of the post-column solvent addition was not discussed but is presumably to overcome, in the early stages of the gradient elution, the need to spray a largely aqueous mobile phase. 

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