It is worth mentioning that the effect of operating pressure and the heat developed by friction birey also be modeled for protein separations, however this requires more experiments and more sophisticated models.
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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We hope that this information will make the life of practicing chromatographers easier! In the common practice of analytical R&D laboratories located in most biopharmaceutical companies, when developing a new method, several method variables are typically screened over a wide range. This is time consuming and above all unnecessary in many cases.
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