Which detector are u using? Use  followings buffer preparation and check once the current profile.

Anolyte (200 mM Phosphoric Acid)

Catholyte (300 mM Sodium Hydroxide)

Chemical Mobilizer (350 mM Acetic Acid)

Cathodic Stabilizer (500 mM Arginine)

Anodic Stabilizer (200 mM Iminodiacetic Acid)

Urea Solution (4.3 M Urea)

3 M Urea- cIEF gel

Hi Rekha!

Thank you for your  answer, The detector is PDA and i have used that buffers at the same concentrations but the problem persist :(

HI ! Have you tried to replace your capillary ? It looks like the coating of your neutral capillary is completely destroyed.

Be careful during the preparation of cIEF GEL. Can you please tell me the preparation of cIEF GEL. If problem persist then change your capillary. I am agree with Aurelia.

 Thank you so much both of you, the cIEF gel is from Beckman (PA800 plus) but i will try to replace the capillary

Hi, I know is bit late, but on topic: recently we got info from one of Beckman Coulter technicians that you shouldn't be using PDA since it destroys coating of neutral capillary. Instead you should install new capillary and use only UV detector.

Ironically, we bought PDA cause it was suggested with the best sensitivity.

I hope you managed to solve your problems even with using PDA :)

Hi! Thanks for the information, I didn´t know that and will consider that for the experiments!

This is kind of late but the noise in the baseline may be due to the ampholytes used. Pharmalyte 3-10 has given me a lower baseline noise than other ampholytes. Make sure the ampholyte solution is clear (strong yellow color indicates degradation).