Iīm currently working on a separation process for a protein. The protein is very basic with high pI-values (two forms of protein - short and full because of a specific cleavage site). For both, short and full sequence, the isoelectric point is above 10.
I tried to separate with CEX column using a salt gradient with variation in gradient length and flow rate but it didnīt work.
I am not allowed to use SEC for the separation because, later on I have to upscale my process.
I also tried some pH-gradients but the chemicals I used absorbed at a wavelength of 214 nm (sequence of my protein is without an aromatic AA so it does not absorb at 280 nm).
If you have any ideas, inspirations,... please help me