Chromatography Forum

Hello, I am new to your forum and a novice in LC.

Our lab has an UPLC system from Waters with a degasser in the system, does this completely remove the need to degas mobile phases before you run the instrument?

You might get some mixed responses here. Personally if the LC had a built in degasser, I never degassed my mobile phases.

Easy way to test is to run a test injection using mobile phase that was degassed by you and then again using mobile phase that wasn't degassed by you (relying on the instrument). If you notice a significant difference in the quality of the baseline, that will be your answer.

Personally if the LC had a built in degasser, I never degassed my mobile phases.

Same here.

Same here.

same here

Dear Bjorn

You would be very surprised when filtering "HPLC GRADE" solvents and later inspecting the filters the dirt you'll find in them. If you use common salts p.a. grade for the buffers the story is worse. I prefer to waste time filtering and no to be sorry later.

Best regards

Fernando

I prefer to waste time filtering and no to be sorry later.

I think everyone would agree with that, but the original question was about degassing, not filtering.

You might have problems if you allow the instrument to mix the solvents; especially at high flow rates. It might be a good exercise to briefly sonicate the solvents even if there is a degasser.

I sense the possibility of confusion here.

There are (at least) three ways to "degas" HPLC mobile phases before they go to the instrument.

Helium purging.

Filtration under vacuum.

Ultrasound.

There is a good chance that you will be filtering the aqueous phase if you have added anything to it. You might also filter the organic phase. In which case you get degassing for free.

Ultrasound is quick and easy, if you have an ultrasonic bath. If can heat up the liquid and lead to loss of volatile organics from pre-mixed phases.

Helium purging needs expensive gas and associated plumbing. A trickle purge with helium can protect mobile phase from the atmosphere while in use, but it will strip organics out of pre-mixed phases.

Peter

Hi Tom

You are very right but the only way to degass that I have in the lab is to filter w/membrane under vacuum.

Best regards

Fernando

I remember reading a publication a decade ago (or 2 or 3) where the authors showed that one can actually contribute more particles/impurities by doing in-house filtration, and advised against that for HPLC or better grades.

Dear gents,
I agree with Peter Apps (mostly).
We normally do vacuum/filtration for a new mobile phase.
If we later use the same mobile phase, we only sonicate the solution for 5-10 min.
Alfred

Hi Alfred

One thing we disagree about is 5- 10 min sonication. Although you still get bubbles after that time and it is easy to convince yourself that gas is still coming out of solution, what is really happening is that the liquid is boiling at sonic hotspots. You could leave it in there for an hour and it would still be doing it.

A surge of gas comes out of solution in the first 5 - 10 s, the bath and liquid start warming up after about 30 s, that is not good.

Peter

(1) Ultrasonic degassing: Several decades ago, a group of us shared a lab with an HPLC technician who very carefully sonicated her solvents in an ultrasonic waterbath to degas them. The noise was really unpleasant, and in a fit of frustration one day, we decided to test if it worked. We took a bottle of water, shook it thoroughly to get 100% saturation with atmospheric gases, and confirmed this with a hand-held oxygen saturation electrode. Reading: 100%. We then sonicated for, I think, half an hour, but I can't remember exactly. I also can't remember exactly the final reading, but it was either 98% or 99% saturation. Sonication in a water-bath does not remove any dissolved oxygen from aqueous solvent at all. It might remove nitrogen, but I find it hard to see why the two gases should behave so differently. It is probably completely ineffective for aqueous mixes. It might work with more powerful sonication than a typical lab cleaning-bath.

I definitely trust the online degasser. In fact when I look at people putting ammonium hydroxide through an online degasser, I do worry exactly what comes out! The original ammonium hydroxide stock-bottle solution has probably been losing ammonia steadily since it was bought. It's impossible to pipette accurately, and then we put a solvent that's basically a dissolved gas through an instrument designed to remove dissolved gasses, and we assume the concentration is what we thought we made up. Scary!

(2) Filtering: I gave up filtering organic solvents a very long time ago, on the grounds that I was adding more rubbish than I take out. I gave up filtering aqueous mixes that contain only liquid components (i.e. formic acid and acetic acid) when we switched to using an 18.2MOhm water purifier with a 0.2u filter on its outlet. I still filtered anything that contained solid components (ammonium formate etc.). More recently a Waters applications specialist advised us to stop filtering anything for our triple-quad instrument, saying he'd encountered more problems from people introducing spurious contaminants from filters than he'd encountered blocked systems. Being lazy, I have happily followed his advice, and haven't encountered any problems. I do feel very unprofessional about it!

Small sonication baths do not always work properly even if they make correct noises. Sonication will remove dissolved gasses the same way as boiling will.
What type of oxygen electrode did you use?

http://www.chromacademy.com/troubleshoo ... 101999.pdf
Have a look at figure 2.
Is this important?
http://www.chromatographyonline.com/mob ... =&pageID=2
"Although in-line degassers are effective in reducing the total gas burden of the mobile phase to acceptable levels, dissolved air still remains in the mobile phase. For some applications, this residual air can cause problems, as is noted below." ... "In some applications, such as sub-200-nm detection by UV, fluorescence, and reductive electrochemistry, the residual dissolved oxygen in vacuum-degassed mobile phases may be sufficient to compromise detection."

Personally, I usually rely on the inline-degasser with no additonal degassing.
As this thread has also concentrated on filtering, I usually don't filter any mobile phases, neither organic nor aqueous. I rely on the inline-filters, which are usually cleaned or replaced (only) once a year. With columns >3µm, I don't have particular problems with clogging this way. For anything <3µm, I usually use an additional on-column inline-filter or a precolumn.