Increased baseline

[sebastian111]

I have some problems with the quantification of AB by FPLC.
I used the following program
- 0.5 ml/min.
- Equilibrate: 2 CV 2xPBS.
- Wash: 2 CV 2xPBS
5 CV 100 mM Citrat pH 5.0
- Elution: 5 SV 100 mM Citrat pH 2.8
- Reequilibrate: 15 SV 2xPBS
After the elution step the baseline decreases down to round about 5 mAU, before the elution step it is at 0 mAU. Therefore it is hard to quantitate if my sample peak is very low.
Do somebody know how i can eléminate this problem?

[anupama]

Try to inject your mobile phase & see that profile. I t can give you idea & you can play with molar ratio too.I saw this type of problem with citrate buffer not on FPLC.

[sebastian111]

A colleague already tried this before, and you also saw this problem after the elution citrat buffer was injected (a run without a sample), but this problem doesn´t occur if you only inject the wash citratbuffer. They only distinguish in the pH value!? But i need a pH of 2.8 to elute the antibodies...Some other proposals?