Low pressure mixing

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

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I'm working on a new procedure that uses
MPA: 20mM acetate in water and MPB: 100% ACN.
The gradient goes from 5% B to 70%B.
I'm using a low pressure mixing system without the inline degasser.
Mobile phases are thoroughly degassed.
I'm getting lots of air bubbles leaving my GPV, causing pressure changes, and low pressure.
I recently replaced the GPV with a refurbished one and this is still happening. So, I believe this is due to mixing.
From other discussions on this topic, I read that high pressure could resolve the bubbles because they are only produced at atmospheric conditions.
SO, I'm trying to figure out a fix. Here's what I'm thinking..
1) premix mobile phases
2) fix/replace degasser (agilent 1100)- would this help? Does it change the pressure of the lines?
3) replace some other pump part (already have new AIV, outlet ball valve, and seals.)
4) take a vacation and let someone else deal with it.
5) Buy a binary pump.
6) Add back pressure?

Since this has happened for other methods too, I'm hoping to have a good solution. I'm going to pre-mix today, but I would love to hear your thoughts or anything I haven't thought of.
Thanks!
DeniseHeinz wrote:
I'm working on a new procedure that uses
MPA: 20mM acetate in water and MPB: 100% ACN.
The gradient goes from 5% B to 70%B.
I'm using a low pressure mixing system without the inline degasser.
Mobile phases are thoroughly degassed.
I'm getting lots of air bubbles leaving my GPV, causing pressure changes, and low pressure.
I recently replaced the GPV with a refurbished one and this is still happening. So, I believe this is due to mixing.
From other discussions on this topic, I read that high pressure could resolve the bubbles because they are only produced at atmospheric conditions.
SO, I'm trying to figure out a fix. Here's what I'm thinking..
1) premix mobile phases
2) fix/replace degasser (agilent 1100)- would this help? Does it change the pressure of the lines?
3) replace some other pump part (already have new AIV, outlet ball valve, and seals.)
4) take a vacation and let someone else deal with it.
5) Buy a binary pump.
6) Add back pressure?

Since this has happened for other methods too, I'm hoping to have a good solution. I'm going to pre-mix today, but I would love to hear your thoughts or anything I haven't thought of.
Thanks!


Always use organic in all your mobile phases! Otherwise you will cause column collapse. Thus;

MPA 95% 20 mM Acetate/5% ACN
MPB 5% 20 mM Acetate/ 95% ACN
Air is always dissolved in water. Its mixing with ACN will allow the dissolved gas to evolve out of the mixture. This may be occurring in your pump.
Why did you remove the degasser ? That is your problem. Nothing wrong with the GPV (bad diagnosis) and no need for a binary pump (which also benefits from degassing too). You need to continuously degas the mobile phase. This will prevent cavitation and increase baseline stability. You must reduce the amount of gas inside the liquids. The mobile phase can be degasser using an inline vacuum degasser (as long as it is chemically compatible with the mobile phase used) or continuous sparging with high purity helium gas.

Since your gradient only max's at 70% ACN (30% buffer), no issues with phase collapse exist.
[quote="Multidimensional"]Why did you remove the degasser ? That is your problem. Nothing wrong with the GPV (bad diagnosis) and no need for a binary pump (which also benefits from degassing too). You need to continuously degas the mobile phase. This will prevent cavitation and increase baseline stability. You must reduce the amount of gas inside the liquids. The mobile phase can be degasser using an inline vacuum degasser (as long as it is chemically compatible with the mobile phase used) or continuous sparging with high purity helium gas.

Since your gradient only max's at 70% ACN (30% buffer), no issues with phase collapse exist.[/quote]

We removed the degasser because it quit working. I'm working on repairing it today. My degasser is an inline vacuum degasser.
And, I did a little more homework and I see that even though we are thoroughly degassing the mobile phase after preperation, it's not enough. And, the reason I see bubbles with mixing is that the solubility of dissolved oxygen goes down in mixed solvents. For people wanting to learn more about dissolved gasses and mobile phase mixing, here's a quick link with a summary:
https://www.shimadzu.com/an/hplc/suppor ... 5/023.html

I've been working with HPLC for over 15 years, and I'm ashamed I didn't fully understand this concept!

My GVP did fail the acetone mixing test during my calibration (could be from the degassing issue); so, we had another reason to buy one besides the air bubble problem. Luckily I found one for $600!

Thanks for the help!
Inline vacuum degasser modules (and sonication) do not substantially remove oxygen from the solution (it reduces it a bit because it reduces the amount of air). Continuous helium sparging has and always will be the best degassing technique for ultra low level UV detector or Fluor detection where oxygen quenching are well know to occur. Helium displaces oxygen in the solution, but is not soluble in it so makes the perfect degassing "gas".

Ever since electronic vacuum degassers came onto the market in the 1980's, they have also brought with them a number of problems. Problems we never had with helium sparging can be seen with systems using vacuum degassers (such as baseline problems). Besides having to regularly clean and flush the vacuum chambers daily (and knowing what their volume is), many are NOT chemically compatible with many solvents and additives used in liquid chromatography (THF is deadly to the G1379-series). They also require regular service (~ every 5 0r 6 years) as the parts inside break down, often become contaminated and just plain wear out over time. Be sure and have the system professionally serviced as just replacing a part (i.e. vac pump) without replacing the internal vacuum tubing or verifying that the vac chambers are not contaminated or leaking will often result in the replaced part breaking again.
Agreed. The degasser is the most failed module in the Agilent stack. The Japanese made ones fail the most (serial number starts with JP vs DE (germany))
Over 50% of the time, the red light degasser can be fixed by trimming the vacuum tubing lines or replacing the line (very inexpensive)

I assume your pressure ripple is poor, I have found that when bubbles struggle to go into the skinny line of the MCGV (GPV) indicates a failed part of one of these 3:
1. outlet ball valve
2. AIV cartridge (basically a check valve)
3. AIV (active inlet valve)

All cheaper than a mcgv. consider a temporary swap from another system to evaluate
Hmmm - we had about 6 Agilent degassers from 1050 models on, and didn't have much trouble with them.

As to the pump, not only did we stock brand new outlet ball valve, AIV cartridge (both 1050 and newer), and AIV (active inlet valve), I also stocked other spare parts, needed to have high up-time. This included stuff like autosampler rotors, the coiled autosampler loop (had welds at the end go bad over time), purge valve, etc. I was pretty adept at troubleshooting and isolating the problem definitively, so this was valuable to us.
[quote="Multidimensional"]Inline vacuum degasser modules (and sonication) do not substantially remove oxygen from the solution (it reduces it a bit because it reduces the amount of air). Continuous helium sparging has and always will be the best degassing technique for ultra low level UV detector or Fluor detection where oxygen quenching are well know to occur. Helium displaces oxygen in the solution, but is not soluble in it so makes the perfect degassing "gas".

Ever since electronic vacuum degassers came onto the market in the 1980's, they have also brought with them a number of problems. Problems we never had with helium sparging can be seen with systems using vacuum degassers (such as baseline problems). Besides having to regularly clean and flush the vacuum chambers daily (and knowing what their volume is), many are NOT chemically compatible with many solvents and additives used in liquid chromatography (THF is deadly to the G1379-series). They also require regular service (~ every 5 0r 6 years) as the parts inside break down, often become contaminated and just plain wear out over time. Be sure and have the system professionally serviced as just replacing a part (i.e. vac pump) without replacing the internal vacuum tubing or verifying that the vac chambers are not contaminated or leaking will often result in the replaced part breaking again.[/quote]

So, I opened up the degasser, all my vacuum lines have cracks at the connections. I'm replacing the lines and checking voltages today. Do you have a helium sparging setups that works well with the agilents?
Replacing those lines will help a lot with your degasser function, but I am also a fan of premixing your A/B phases so that you can go from 0-100%B over the course of the gradient. This will provide less outgassing as well as a smaller pressure ripple overall.

Good luck-
Thanks,
DR
Image
Helium sparging is not brand specific. Any properly set up helium sparging system will work with any HPLC system. That said, the old HP 1050 stackable helium sparging module was very convenient to use on the 1100/1200-series stacks and Agilent even continued to sell them many years after they retired the 1050-series (esp for GPC applications). You can sometimes find them on the auction sites for $200 or less, with the bottle heads, but they are getting harder to find in good shape as the years go by. In any case, when using helium sparging, remember to use CONTINUOUS helium sparging (not on/off). Use a high pressure tank to feed a secondary, low pressure regulator (usually set at ~ 3 psi) to feed properly positioned stainless steel frits at the bottom of the bottles, away from the solvent pickups).
Some related articles on HPLC degassing may be of interest to you?

"INLINE HPLC DEGASSING MODULES"; https://hplctips.blogspot.com/2011/02/i ... dules.html

"An Often Ignored HPLC & LC/MS Contamination Source. Did you check your Vacuum Degasser?"; https://hplctips.blogspot.com/2015/08/a ... -lcms.html

"HPLC Solution Degassing, Sparging With the Wrong Gas (Gas Choice Matters)"; https://hplctips.blogspot.com/2013/05/h ... g-gas.html
hmmm, we had vacuum degassers on 1100 modules for years with very few problems, and now have vacuum degassers on Shimadzu Nexera systems, again with very few problems. We use UV and fluorescence detection (and MS, ELSD and CAD) without significant problems, but of course things can be method-specific, and depend on the level of sensitivity required.

Don't write off vacuum degassing. It does work, and it's a lot cheaper and more resource friendly than helium sparging (remember, helium is a non-renewable resource; the world will never see that helium again, and helium is still pretty useful for MRI instruments and NMR; helium is derived naturally from alpha emissions by radioisotopes in rock layers where it cannot escape; it's a rare resource; chucking it out the window should only be done as a last resort; Wikipedia defends helium usage by reporting a discovery in E. Africa - but it remains non-renewable, and it's high-risk to have all your supply derived from a single source, in a poorly-developed part of the world).

Of course you need to maintain your vacuum degasser or it will fail; its little vacuum pump will wear out as surely as the seals in the HPLC pump will wear, or the deuterium lamp in the PDA will lose intensity. We tend to forget about degassers and expect them just to sit in the background doing their stuff, but they aren't just a box with a light; they're active modules that need care.
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