why my RPHPLC keeps making air bubbles... - (Oct/10/2007 )

Hi guys,
I feel helpless now....
I am purifying peptide that is around 40 amino acids. But the column keeps making bubbles now and then.

The first time bubbles come up I was using Proteo analytical column. And I was using gradient RPHPLC, mixing water and 100% Acetonitrile. Using gradient from 50% Acetonitrile to 95%. And I use flow rate of 0.53ml/min. Then someone told me that may be caused by mixing water with organic solvent at a low speed. Then I swich flow rate to 1ml/min , switch solvent A from water to 50% acetonitrile, and used 95% acetonitril to wash all the air bubbles out. Then air bubbles stopped.

This time, I am using the same column, but mixing water and 100%acetonitrile from 5% Acetonitrile to 95%. Using flow rate 1ml/min. Air bubbles start to come out again. Then I think I may want to mix water and low concentration Acetonitrile. So I swithed solvent B from 100% ACN to 50%. But air bubbles still are coming out.

The thing is I am purifying a hydrophilic short peptide, and I don't want it to come out with the TFE peak, so I have to start from low organic solvent concentration.

But where do these air bubbles come from?? I always degas all the solvent before using!

Could anyone give me any suggestions? The Proteo column is almost new, only used for like 25 times. And used to have high resolution. And I always check column to make sure no leaking.

Thank you very much


latina

Hello latina!
Try to disconnect column from your system and start flow at 1 ml min. So you can check your flow cell on the presence air bubbles here.
Can you configurate your system to allow upstream of eluent through the column. So it will give you a simple way to accelerate air bubbles removal. I advise you to work with upstream eluent flow direction. About the cause of air bubble formation - If your HPLC system not equipped with on line degasator so it will be better to improve your degasation process. BTW did you warm up AcCN to remove air bubbles.

like you did originally, make up your solvent a with the acetonitrile (95:5, water:acn). this may prevent the formation of bubbles (which are coming from the mixing of water with acn and are released from solution when the medium exits the column, due to pressure drop).

you can also attach a backpressure regulator onto the system (after the monitor) so that the bubbles won't form until after passing through your monitor.

In addition, you can try to degas your solvents really well and purge the pump.