Here we demonstrate three examples of separation of basic polar and non-polar molecules, that in a typical reverse phase chromatography settings, are either difficult to retain or produce poor peak shape and efficiency. BIST™ conditions can, instead, produce high efficiency, retention and symmetrical peak shape.
BIST™ can also separate structural isomers of multiple-charged compounds, such as sulfated cyclodextrin,
with a relatively simple gradient method on a short column. Previous methods for analysis of similar compounds
can require much longer columns, higher concentration buffers, and do not provide complete separation
of the constituent isomers.
The power of BIST™ lies in its ability to do these kinds of separations on short columns with simple mobile phases and low concentration buffers. Polymers with charged units, like polylysine, are difficult to separate using ion-exchange (IE) chromatography due to very strong and often irreversible interactions with the oppositely-charged stationary phase of the column. Usually, an extremely high concentration of the buffer, needs to be used to generate an ion-exchange process. This high buffer concentration isn’t usually desirable because of the higher viscosity of the MP and the likely formation of salt in the components of the HPLC system. With BIST™, polymers, like these, can be retained and analyzed with a small concentration of the buffer in the mM range using simple gradient of water.
Other large multi-charged molecules such as polyethyleneimine (PEI) can be very difficult to separate and
retain on standard columns. PEI is a complex mixture of molecules of different sizes and geometries. A simple BIST™
method with a step gradient allows for the retention of a single peak for each PEI fraction.
Colistin is another multi-charged molecule with structural isomers that BIST™ can retain. All that’s needed is the typical high-MeCN
concentration MP and a doubly-charged H2SO4 buffer. With this MP, the two isomers can be separated different only in one methyl group.
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