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Our hypothesis was that, in the case of large biomolecules, the changes of molecular conformation caused by the pressure or high flow rates (thermal effects) will play a much more important role in changing retention and selectivity. Especially when considering their “bind and elute” type retention behavior. In our work, this huge conformational change was experimentally verified both by fluorescence emission measurements and by the analysis of native and reduced forms of various proteins which possess the same molecular weight but a different conformation.
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One of the main limitations in liquid chromatography today is still the significant extra-column volume contribution of commercial chromatographic systems to band broadening. I believe that current instruments have reached their limits and therefore one might expect some completely new integrated system designs (for example, a slot for the column, on-column injection and on-column detection) alongside developments of column technology.
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