From your description above, solvent B is much lower in concentration than solvent A. Did you really mean this or does solvent B really contain one molar sodium chloride?

Regardless of your answer to the question above, I'm not clear on what your question is. Are you asking is it possible that you can have one peak using gel filtration and yet see multiple peaks when re-injecting this peak into an anion exchange column? If so, the answer is: yes. Gel filtration is a relatively low resolution technique that is not capable of resolving proteins that differ slightly in molecular weight whereas anion exchange can, at least in theory, resolve analytes of very similar molecular weight that differ in net charge under chromatographic conditions.

@CHRIS..solvent B has 1M Nacl. thanks for your answers. in other word i can say my protein has different confirmations under the chromatographic conditions. Molecular weight of all the the peaks obtained after anion exchange are same (confirmed by MS). 

What Chris is trying to say is that these proteins are likely to be different proteins all together. You say that you get only 1 peak from the SEC, that means everything in your solution is eluting at the same approximate size. However this does not mean they are all the same protein. When you carry out the Ionic exchange step the several peaks are most likely to be different proteins. Is you protein of interest tagged in any way or do you have antibodies specific to that protein, if so I would carry out a western blot on e peaks from the anion exchange and the one from SEC and see where your protein is.

i dont have antibody..my protein forms amyloid aggregates under specific conditions. i collected all three peaks from cation exchange chromatography and set up aggregation reaction. All these were forming amyloid aggregates. But, yes u r correct to confirm it i need to do western blotting or any activity assay on these fractions.

Interesting, You may well be able to deduce that yes there are differences in the conformation of your protein, this would indicate the change in exposed residues and then therefore the pI of the folded protein. With the formation of amyloid aggregates this can be affect by many factors, so you may be experiencing 'molecular aging' : http://www.ncbi.nlm.nih.gov/pubmed/23630590.

@ Simon

thanks...this would be nice to study for my protein

Hi,

The first advice I would have given was to first confirm that your protein of interest was indeed in the peak from the SEC column and each of the peaks from the anion exchange column. You could use a combination of activity/binding assays, western blots and mass spectroscopy.  It appears from your later responses that you were able to use an enzyme assay and mass spec to confirm your peak.  Additionally, analyze a sample of each of the peaks to determine if your sample is aggregating post-SEC.  Native PAGE is a popular technique, however, light scattering techniques such as DLS or MALS may be a better choice since you are working with amyloid proteins.  MALS will let you assess your samples in much more detail.

In regard to your original question, I agree with Simon that you may be observing multiple peaks as a result of differences in surface charge density.  Surface charge density could fluctuate because of conformational flexibility, non-specific interactions or other reasons.  Optimize your buffer conditions to include sufficient reducing agents, chelating agents, additional ions and, some glycerol or sucrose to minimize nonspecific interactions and maintain protein stability.  

Multiple peaks may indicate non-optimal ion-exchange conditions.  Your buffer pH may be affecting the elution profile.  Buffers for anion exchange are typically at least 1 pH unit above the protein’s isoelectric point (pI).  Your buffer at pH 6.5 may be too close to the pI of your protein for a strong interaction with the column resin.  Also, the pH of your buffer is close to the pKa of the imidizole ring of histidine (about 6.0), which can affect column binding and elution if there is a mixed population of protonated surface histidines.  There is a very interesting paper about the effect of histidine protonation on cation-exchange chromatography (see below).  The authors found that surface histidines on the mAB fragments being purified created diverse elution profiles that included peak shouldering and multiple peaks.  The profiles varied significantly depending on the buffer pH and column media used.  You may want to experiment with your ion-exchange step, extending the range of buffer pH’s and testing a variety resins (strong and weak anion and cation exchangers).  It is likely that optimizing your ion exchange chromatography will clarify if and why you are observing multiple populations.

Good luck!

@ELIZABETH.. Thanks for valuable explanation. I haven't put much effort on the standardization of conditions for ion exchange chromatography, as i believe my protein is pure form. But yes, i would surly try it.

Next, You also mention regarding different confirmations of the proteins due to exposure of different charged residues. And its is very true, but just for curiosity, can u share one or two basic literature regarding this.

Here are a few examples from the literature.  

Stability of tumor necrosis factor-α during ion exchange chromatography

Effects of urea induced protein conformational changes on ion exchange

chromatographic behavior

@Elizabeth..thanks you.great advise. I missed to mention this point earlier, my protein has stretch of N terminal, 10 histidine amino acids and may be due to irregular protonation of these histidines i am getting multiple peaks.. i mentioned three peaks earlier..but with these other 2 to 3 more small peaks were also present which can be seen at 214 nm. as per suggestions i am trying with Q FF sepharose. Earlier i was using SP FF.. May be i m wrong;  i don't know, but its not good to go with cation exchanger (SP FF)  if your protein has N terminal His tag.  

Hi ,

I strongly advocate removing the terminal histidine tag with a protease before SEC or IEX .  That is a very large tag, and it can end up disrupting protein folding/structure and downstream experiments.  I am particularly concerned because you are working with an amyloid protein.  Of course, the multiple peaks can be explained if the histidine tag is affecting protein folding or isn't fully protonated.  Just for your education, Q sepharose is a strong anion exchanger, and SP sepharose is a strong cation exchanger.  In your original post, I think you had said you were doing anion exchange but you were actually doing cation exchange if you were using SP sepharose.  I am writing an article for BiteSize Bio about ion exchange chromatography, and will send you a link once it's published.

@Elizabeth

i am looking forward about that article. Protein gets precipitate if i remove tag that was a main issue. hence people working on this protein generally do not remove tag. thanks for correcting me.