Chromatography Forum

Ijust started using HPLCs, and a problem which i had been unable to solve is the formation of bubbles when methanol and water (in my case it was methanol and 1.25% acetic acid aq.) meet each other. No matter what i do (degass, reprepare, etc.) the bubbles are still there. What should i do?

If you degass your solvents AFTER the mix, you should have no problem!

I find that stirring the methanol/water mixture for 3-4 hours or overnight helps get rid of the bubbles. If bubbles persist beyond that, use an-line bubble trap or degasser.

Folive

Another method to minimize bubbles in the detector is to add a back-pressure device or a 0.5-1 meter piece of 0.010 inch tubing to the exit of the detector. The bubbles are caused by the pressure drop at the end of the column. Adding the narrow tubing to the detector exit usually produces enough back-pressure to keep the bubbles in solution. If you are collecting samples, the back-pressure device is not a good idea but the narrow tubing is okay as long as you account for the volume delay from the flow cell to the fraction collector.

As you said that you started using HPLC, I would add in the very good suggestion of skunked_once that you should be careful not to exceed 20 bar in post-detector back pressure as you might blow your UV cell. Make sure that the tubing you are using is not pluged or something...

I think that most modern UV cells can withstand a back pressure of 50 bar but I wouldn't go that far...

If you are using a low-pressure mixing gradient pump, the type that has a proportioning valve before the high-pressure pump, you must use some form of continuous degassing. One is an online vacuum degasser; modern designs are very good. Two is continuous bubbling with helium; effective but can be wasteful of heilum. Three is an initial bubbling with helium followed by static helium pressure; requires special bottle caps, but very effective.

The solubility of air is less in the mixture than in the individual solvents. Therefore a degassing of the individual solvents is not sufficient.

If you use a high-pressure mixing system, than the only place where air bubbles could occur is in the detctor. As mentioned before, a bit of restriction behind the detector will be OK. 5 cm of a small i.d. tubing is sufficient.

If you are using a low-pressure mixing system, then you need to use a pre-column degassing system, if you are dealing with variable mixtures of both solvents.

If you are running a single mixture isocratically, then the simplest way to degas is to put the solvent bottle into a ultrasonic bath and apply vacuum for about one minute. Lots of bubbels will pop up in the first moment, but then it will decline very rapidly. There is no benefit to do this for longer than maybe a minute. It you do it for a really long time, the methanol will evaporate and the solvent composition will not be what you had prepared.

thankyou very much... finally i can get of them. =)