Chromatofocusing

A  technique  which  is  related  to  ion-exchange  chromatography,  but which  separates  on  a  different  principle,  is  Chromatofocusing.  In Chromatofocusing,  use  is  made  of  the  buffering  capacity  of  the  ion- exchange substituent groups, themselves.  The column is equilibrated with starting buffer, the sample applied, and immediately the finishing buffer is applied.  Displacement  of  the  starting  buffer  by  the  finishing  buffer generates  a  moving  pH  gradient  in  the  column.  Proteins  which  fall behind their pI on this gradient will no longer bind to the  column  and will be swept along faster than  the  pH gradient.  Proteins  which move  ahead of the pH gradient, by contrast, will bind  strongly  to  the  column  and will be immobilized until overtaken by the pH gradient.  The net  result  is that proteins will be eluted from the column at their respective pI values.

In  practice  it  is  found  that  simple  displacement  of one  buffer  with another in a conventional exchanger causes too sharp a change in  pH.  A shallower pH gradient,  more  suitable  for  Chromatofocusing  separations, can  be  generated  by  using  ampholytes as  the  eluting  buffer,  and  a substituent group, such as polyethyleneimine, which titrates  over a larger pH range.  Ampholytes are mixtures of randomly substituted poly  amino- poly carboxylic acids.  They  are  also used in isoelectric  focusing  and  in isotachophoresis.  This  requirement  for  ampholytes  makes  Chromatofocusing more expensive than normal ion-exchange chromatography.

References

-Dennison, C. (2002). A guide to protein isolation . School of Molecular mid Cellular Biosciences, University of Natal . Kluwer Academic Publishers new york, Boston, Dordrecht, London, Moscow .