Chromatography Forum

First of all, I´m not a bloody beginner (at least I thought so...) when it comes to chromatography but this issue just keeps falling on my feet again and again.
So, I´m having an old (maybe 12 years old) Beckman HPLC (System Gold binary pump 125 with Diode array detector 168). I´m using it for easy quality control purposes after protein purification. Usually I´m running a 2% gradient from 0 to 100 % B using an old Grace Vydac C18 column.
The Hplc system did not come with a degasser (Why?! I talked with a guy from Beckman and he told me that Beckman did not deemed it necessary...), so I hooked up a Shimadzu online degasser to remove dissolved oxygen from the solvents. I´m using Water / Acetonitrile mixtures with 0.1% TFA.
Now here is the problem. I keep getting bubbles from somewhere. They show up after a while ~at approx 50% B and then the whole chromatogram becomes erratic and useless.

What I have tried so far (and did not work too well):
1. Clean the degasser with some low percentage phosphoric acid
2. Tighten up connections
3. Avoid mixing of pure water with pure ACN - Instead I´m now mixing 20% ACN with 80% ACN (rest in both bottles is water)
4. Degass offline prior to the experiment on a vacuum line under stirring.
5. Replace the ACN with Ethanol

Well so far I have failed solving this problem. The Degasser works fine when used on an isocratic system (just PBS for Gelfiltration).
Does anyone have a suggestion for me what else to try? I don´t have to money for a new degasser...
Thanks in advance.

I guess the mixing chamber has a high volume so rest of oxygen can be undesolved. Easy and cheap is to connect a 30 to 50cm capillary to your detector outlet. Please check back pressure!!! This restriction should keep bubbles away. Good luck.

Hi,
I have connected a narrow capillary to the UV detector and it helped! No more issues with bubbles. Thank you so much for helping me!
Best regards

Why is that? could you explain?

Air (not sure if the rule applies to all gasses) is more soluble in aqueous solution under pressure. It also becomes less soluble when said aqueous solution is mixed with solvents like ACN. Between the pump and the end of the column, there is enough pressure to counteract the formation of bubbles that occurs when the aqueous phase is mixed with the orgranic solvent. However, after the mix exits the column and enters the flow cell, it is under very little pressure and the dissolved gasses can form bubbles. Kind of like when you open a bottle of a carbonated beverage.

When you place a thin capillary tube after the flow cell, it generates enough backpressure to keep the gasses in solution until the leave the flowcell. Hence, no more bubbles, at least where they matter.