Chromatography Forum

Hello fellow Chromatographers,

I am confronted with problems regarding Air bubbles. Currently, I am using the Agilent 1100 system.
Degasing is done with the "Merck Solvent Degasser L-7612" used for LaChrom system, since we don't have the inline degaser from Agilent.
I use a NH2-column for HILIC mode with the aim of analysing glycoprofiles of glycoproteins.
The solvents I use are: 100% Acetonitrile and Ammonium formiate 250mM (pH 4.4) and Isopropanol for purging.

So, the story: The old binary pump leaked and was substituted for a quarternary pump. Using the binary pump, the pressure was constant. Now with the quarternary pump, the pressure fluctuates strongly. The air bubbles appear after the the 4-channel mixing chamber.

History:
binary pump + degasing with supersonic = ok
quarternary pump + degasing with supersonic = very bad
quarternary pump + solvent degaser = better, but still random air bubbles

A few observations:
Purging with isopropanol to waste or pumping it through the column results in constant pressure.
Purging with Acetonitrile to waste or through column = constant pressure.
Acetonitrile+Ammonium formate = gets worse after a while.
(Gradient: ACN from 75% down to 50% in 25minutes). Especially, at the time when ammonium formiate has a higher percentage, the pressure fluctuates very strong.
I assume that is the reason why my dextran standard had different retention times with 3 runs at the same day.

Does anyone know what kind of problem I am dealing with this ?
Broken chamber or problems with solvent B ? I can also upload the chromatograms if anyone is interested.

Thanks, best Regards
Moe

Hello

I think that your problem is linked to:
-solvents and degassing
-differences between binary and quat pump

Binary pump is mixing solvents on high pressure side - mixer is after pump heads. So mixing is much better.

Quaternary pump is mixing solvents before pump head so mixing is not as good as for binary pump.

And main thing: air has different solubility in separate solvents (acetonitrile and buffer) and different in solvent mixture. So when you mix solvents air solubility is lower and that is why you see air bubbles.

I'd recommend:

1.Use degasser with bigger vacuum chamber
2.Do not use 100% buffer - instead use as mobile phase B 95:5 buffer:ACN

Regards

Tomasz Kubowicz

Thanks Mr Kubowicz,

I will try the approach with the premixing of the solvents. Unfortunately, we don't have a bigger vaccum chamber.

I attached some chromatographs with the mentioned problems.http://docdro.id/t6OsKUM

Thanks,
Moe

Hello

What I would do is pump diagnostic:
-run leak test to see if there is problem with valves

I think you can have problem with Active Inlet Valve (or cartridge inside) or outlet ball valve.

Regards

Tomasz Kubowicz

I did a check with a measuring cyclinder at the end of the tube. The flow was the same amount as specified in the HPLC software. So, the amount of the flow stays the same...even though the pressure changes so much.

Thanks
Moe

Hello

You need to run "leak test" for pump. The fact that flowrate is ok doesn't tell you that valves are working fine.

Regards

Tomas zKubowicz

Thanks Kubowicz!
I used 60:40 Buffer:ACN as solvent B and the fluctuations are gone. When I was mixing solvent and buffer together and using the ultrasonic bath, a great amount of gas was released, propbably responsible for the pressure fluctuations in the HPLC system.
The first 3 runs without the standard after this, the pressure started constant at 150bar and increased evenly, no steps or fluctuations.
After injecting the standard, the next 3 runs had small differences (about 10 bar) in the pressure, resulting in shifted peaks getting worse at the end of the run (first overlapping, then the shift gets bigger and bigger).
I will do the leak test after this weekend. Thanks again for your help, Mr Kubowicz!

Here are the chromatograms of the run with the ladder afterwards. http://docdro.id/9nlt7JE
The order of the pressure grap is pink, red and yellow.

Best Regards
Moe