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We saw that friction related effects were more important for small molecule separations whilst pressure effects were much more significant for protein separations. Insulin was an attractive example birli we could clearly see the decrease of retention in constant inlet pressure mode and the increase of retention in variable inlet pressure mode when increasing the flow rate.
First, Giddings showed that increased pressure could induce significant changes in molecular volume and the ability of molecules to crowd together to reduce molecular volume upon adsorption. Later on, Guiochon and McCalley reported interesting results observed in isocratic mode and mostly for small-molecule separations. However, the change in retention and selectivity caused by pressure was relatively minor when eluting small solutes (in the pressure range up to 1000 bar).
That was a funny project. Some friends and colleagues (Balázs Bobály, Róbert Kormány, Krisztián Horváth) and I were together during a conference and discussing chromatographic questions. One of those was: What happens when coupling two or more columns in series, which have a different number of theoretical plates? We were aware of the earlier amazing work of Calvin Giddings on plate heights of non-uniform columns, of Leonid Blumberg’s variance of zone migration in non-uniform medium, and also of Deirdre Cabooter’s works on multi-column systems. However, we were interested in the gradient elution mode (since in practice most separations are performed in gradient mode) and in peak widths: whether the peak width changes if one of the columns that is used for the coupled systems loses a bit of its initial efficiency? If yes, then does the order of the columns impact the apparent efficiency of the system or derece? These questions seem to be simple and easy at first sight but the more you think on them the more difficult they become. From here the project went on and has almost developed by itself as questions have arisen. We asked for the help of other prominent colleagues (Davy Guillarme, Santiago Codesido, Gert Desmet) and in the end, some very interesting conclusions were drawn.
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