Chromatography Forum

hi

i was just wondering whats the effect of sample pH on Ion exchange chromatography profile (lets say for example sample A in solution of pH 4 and sampleA ina solution of pH 5/6 ...its an cation exchange chromatography and mobile phase hase pH 5.0)

regards
rick

Depends on the nature of the sample:
- how many ionizable groups?
- what kind of ionizable groups?
- pKa of those ionizable groups?
And on the details about the cation exchanger
- strong or weak exchanger?
- if a strong exchanger, what is the capacity?
- if a weak exchanger, what is the pKa?

All the rest is common sense: what will be the effect of the pH change on ionization of the stationary phase and the analyte.

Sorry if my question was little ambiguous ….

I was just want to get some doubts out of my head

what will happen if I inject my sample in a sample buffer of pH 5, pH 4 and pH 6 ( in my case, the sample is a protein of pI 5.6 column is strong cation exchange Tosho SP-5PW)..i understand there will be a difference in ionization in different pH but will it have a significant effect on chromatography profile as we are subjecting all of sample to a mobile phase with pH 5.0 ??

thanks a lot

For small injection volumes, it should not make much difference, as the sample will mix with the mobile phase and be at or close to pH 5 when it hits the columnn (i'm assuming the ionic strength is constant).

For larger injection volumes (too large to mix completely with the mobile phase between injector and column), I would expect to see some on-column reconcentration with the pH 4 (at that pH, your protein would be strongly bound; i.e., for constant ionic strength, a pH 4 buffer would be a weak eluant) and possibly some peak broadening and/or shape problems with the pH 6 (at that pH, your protein will be approximately unretained; i.e., for a constant ionic strength, a pH 6 buffer would be a strong eluant for your protein).

All of the above arguments are hypothetical. Remember that your stationary phase can only interact with the *outside* of the protein. At the pI, your protein has a net charge of zero, but depending on the distribution of charged groups and the conformation of the protein, the surface may have either a net positive or negative charge.