Peptide not binding efficiently in IEX

[vvsgrat]

Hai,
I am facing problem in protein purification. Mine is a peptide of Molecular weight ~3 kDa and is cationic in nature (and most peptide bacteriocins are cationic). I use S-Ceramic Hyper D strong cationic column (Pall Scientific) with 10mM citrate buffet pH 4.0. Protein concentration 50mg/ml (loading capacity of the column is 75mg/ml) But my protein is getting bound partly only. Major is getting unbound (nearly 80-90%). I also tried with pH of 5.0 acetate buffer but is the same case. I also diluted the sample much with staring buffer (2X also) to bring the ionic strength of sample nearly to the buffer but it does not solve. How to go about for getting maximum binding.
In case of elution (Gradient 0-1M) it elute as a single peak with 0.1 to 1M (Broad peak). I even altered the flow rate but still its like same. Please help me for purification using IEX.

[leekaming]

Peptide usually purified using reverse phase column e.g. C18 column.
Depends on the nature of your peptide, there may be some exposed hydrophobic side chain, leading to soluble aggregate in aqueous buffer, thus cannot bind IEX efficiently.
So i suggest using reverse phase column.

[vvsgrat]

Peptide usually purified using reverse phase column e.g. C18 column.
Depends on the nature of your peptide, there may be some exposed hydrophobic side chain, leading to soluble aggregate in aqueous buffer, thus cannot bind IEX efficiently.
So i suggest using reverse phase column.

But it also boes not bind with C18 column (Waters). My sample is eluted out unbound. I did it recently. However, in past experiment the peptides bound with C18 column. now it is not getting bound. I dissolved the sample in buffer and eluted the sample with acetonitrile water having 0.1% TFA. Why this happens.

[leekaming]

I suspect that your peptide form big soluble aggregate and the aggregate cannot get into the pore of the resin. Try purify it with cation exchange column in urea buffer pH 4.
If soluble aggregate really the culprit, urea should be able to break the aggregate and the peptide can bind more efficiently.