Assessment of protein purity by chromatography and multiwavelength detection

Commercial protein preparations were analyzed for purity on a size-exclusion column coupled to a photodiode array detector. Two methods that are commonly supplied by the manufacturers of these detectors were used to establish the purity of the eluting peaks. These methods are based on (i) a visual inspection of the superposition of normalized spectra taken upslope, at the apex, and downslope and (ii) the variation of the ratio of the absorbance measured at two different wavelengths. The results obtained were compared with those of a newly developed method, based on a comparison of at least eight peak spectra by means of their correlation coefficients. It was found that the first two methods were not reliable in the case of protein peaks containing a spectrally similar impurity, while with the third method it was possible to detect the presence of only 2% (w/w) of a spectrally closely resembling protein contaminant (r = 0.9997), eluting with a chromatographic resolution of 0.37 sigma. Analysis of the samples with polyacrylamide gel electrophoresis indicated that the new method is at least equally suited to assess protein purity, provided a minimal resolution is achieved by the chromatographic system.