Buffer for cation exchange chromatography - acetic acid or sodium acetate? - (Feb/26/2008 )
Hi all,
again some basic questions in field of HPLC/FPLC protein purification - particularly in cation exchange chromatography.
1. In the handbook of "Ion Exchange Chromatography & Chromatofocusing" (GE Healthcare) is given as a buffer for pH range of 4.3-5.3 the acetic acid.
However in some exmples (in that Handbook) they show using 20mM sodium acetate and in other examples using 20mM acetic acid (in elution buffer with LiCl or NaCl).
So, what is the practical difference (in resolution, in binding, in elution etc.) in the use of sodium acetate or directly acetic acid?
2. I suppose, that using a buffer with pH of 5 is not any problem for protein with pI approx. 8. Is it right? Or is there any danger of, for example, too strong binding the protein to column (Mono S) in such a acidic buffer (and cosequently on the quality of separation from other proteins)?
Thank you in advance for your answers and suggestions.
vic
again some basic questions in field of HPLC/FPLC protein purification - particularly in cation exchange chromatography.
1. In the handbook of "Ion Exchange Chromatography & Chromatofocusing" (GE Healthcare) is given as a buffer for pH range of 4.3-5.3 the acetic acid.
However in some exmples (in that Handbook) they show using 20mM sodium acetate and in other examples using 20mM acetic acid (in elution buffer with LiCl or NaCl).
So, what is the practical difference (in resolution, in binding, in elution etc.) in the use of sodium acetate or directly acetic acid?
2. I suppose, that using a buffer with pH of 5 is not any problem for protein with pI approx. 8. Is it right? Or is there any danger of, for example, too strong binding the protein to column (Mono S) in such a acidic buffer (and cosequently on the quality of separation from other proteins)?
Thank you in advance for your answers and suggestions.
vic
You want to use the salt, pH'ed with the acid to have the correct operating pH. Remember that acetic acid is weak, so its pH varies much more with concentration.
pH 5 should be fine with your protein. Separation will occur with the salt gradient. Remember, the pH you use is only to ensure that all available groups are protonated, so don't foregt to pH the sample before you load it onto the column!