It can be anything: the sample, the running conditions, or the adsorbent. If you are sure about the first two, I would try cleaning the adsorbent with NaOH

Hi Palan, are you using AKTA or FPLC for your runs, or are you using gravity?

If there is a problem with the resin clogging, then you should see increased backpressure in the pump systems and decreased flow if you are using gravity. Other signs of a fouled resin is that the column shrinks more than usual during a run and the peak is broader than usual.

 If this is the case, then follow Omar's suggestion of cleaning the column with NaOH.

If this is not the case, then something happened to the sample that you applied. Maybe somebody forgot to add proteinase inhibitors to the sample before storage?

There may be two things 1. Coloum clogging 2. Protein sample stoarge for  7 days at 4℃. Can you tell the proteins you are extracting from? 

I agree with the answers given by Mehta.. Your column resins may be clogged and as you have kept sample stored at refrigerated temperatures for a week which may have lead to the loss of your desirable derivatives. Run for freshly prepared sample might give you desired peaks.

Yes I agree with Girija and Naresh as you store your protein at 4℃ almost one week there is chances of degrading your desirable protein. 

If you use Bradford assay for determination of protein concentration it may show same for first and after few days because it reacts with amino acids and show the same concentration and this is may not be your desirable protein.   

Omar, thank you for your suggestions. Because we didn't clean the adsorbent, it could be the reason. But we always make NaOH flow through the lines after every run, including that case, I'm not sure it is a real reason.

Steingrimur, we are using AKTA. Same as Omar, clogging could be the reason. Or sample also can be the reason. I thought the sample is fine because after depth filtration, for sample preparation with Q FF, the concentration and purity(SE-HPLC) of outputs in both of runs were same.

But as you, Naresh, Girija, and Ramesh said, the sample can be changed. Actually, before depth filtration, the concentration and purity are getting lower and lower than before even if it has been stored 4℃.

I must change the sample to more fresh one and wash the instrument and resin with NaOH before use.

Thanks again everyone. :)