I´m trying to separate peptides on HPLC using a 2.1mm x 25cm Vydac -18 column. The buffer system is A: 0.06%TFA/water and B: 0.052%TFA/80% acetonitrile. Flow rate 0.1 ml/min. The gradient I´m using is :
0-60min; 2-37.5% B
60-90min; 37.5-75%B
90-105 min; 75-98%B
This gradient has worked, but since I ran out of buffer and had to make new I´ve got a problem with bubbles forming during the gradient. I have degassed my solutions by vacuum + ultrasonic bath and helium sparging for 10 min, but bubbles i still forming after about 60 min of the gradient.
What can I do about it??

I would think flow rate is too slow for the 2.1 mm column. PLease try 0.25 ml/min.

Good luck

If you ran out of buffer by physically pumping CH. A dry, then you could have trapped air bubbles that are being dislodged from your system as the surface tension of your mobile phase decreases during your gradient. If you feel this could be the cause then you could try purging your system with IPA. IPA has a low surface tension and very good wetting properties and will sweep any air pockets from your system.

If you think it is outgassing then you could try adding a little back pressure to keep the gasses in solution. Add a backpressure regulator or a length of cappilary tubing downstream of the detector. Be careful to observe the backpressure limitations of your detector.

Try to use a online vakuum-degassing unit, if it´s possible.

We have had excellent results using vacuum degasing units, and I strongly recommend them.

Previously, we got good results using a few minutes of vigorous helium sparging to degas, followed by continuous helium purging of the mobile phase containers to exclude air. If you are using pure buffer and pure organic solvent as as your mobile phase components, then I strongly recommend this approach.

However if you are using mixtures of buffers and solvents, then you could find that the continuous purge will slowly carry off the solvent, changing your retention times. If this is your situation, ask again and I'll provide pointers.

Put a backpressure regulator

Helium sparging after vacuum degassing is effective at removing dissolved gas from mobile phase. You may be running into a problem that drove me to distraction with a unit in one of our satelite labs. I would prepare fresh MP and leave the lab with everything operating perfectly only to hear that the pump kicked out with air bubbles after I left. I finally figured out what was happening. The volume of helium being used to sparge the MP during use was not sufficient to maintain a head of helium in the bottle. Air sitting on the surface was dissolving in the liquid. I overcame the problem by maintaining a helium head pressure over the MP. A small low pressure regulator was put in line between the helium source and the eluent bottles. Releasing the pressure permited a huge volume of helium to degas the eluents. Tightening the caps kept the system under a slight positive pressure. The eluents were methanol, acetonitrile and water which were used for gradiant elution on the HPLC for many different analyses of various analytes. The bubble problem has never returned. Keep head pressure low and install back flow safety device to prevent eluent from flowing back to the regulator.

I see that problem before
the solution was on line vaccum degasser