Gas bubbles at 40min

[acetone]

Hi,
My system seeing the gas bubbles at 40min of 60 min gradient elution.
Flow rate: 0.5 ml/min
A: NaOAc buffer Ph6 B; MeOH
Shimadzu 10AVP system
Degass by He, always see bubbles at 40min no matter how long the mobile phases degassed.
Please supply some hints.
Regards,

Acetone

[tom jupille]

First of all, are you sure they really *are* bubbles (as opposed to junk eluting from the column)?

Assuming they are bubbles:

Make sure that you degas continuously (not just "one shot"), because once you stop the He, your solvents begin to re-equilibrate with the atmosphere.

Keep the runs of teflon tubing between the reservoirs and the pump(s) as short as possible. Teflon is somewhat permeable to air (that's why I like the in-line membrane degassers that sit immediately upstream of the pump!).

[ZeroAir]

Yes, buy a degasser to sit on top the pump. Problem solved.

[Peter Skelton]

It's also a good idea to mix your A solvent to the starting mixture of your gradient, and re-adjusting the gradient programme to keep the slope the same.