Chromatography Forum

Hi everyone, I have been testing new method for amino acid separation. To achieve required resolution, I used the following method:
Buffer: Ammonium dihydrogen phosphate 0.05M adjusted pH 5.5
Mobile phase A: Buffer
Mobile phase B: Acetonitrile
Here is my gradient program.

Flow rate is 1.5ml/min
After I use this method, the line containing Acetonitrile is ALWAYS damaged, it can't close tightly. So even when I don't use that line, solvent still can move in that line ( not much, but still enough to affect my results).
Could anyone tell me the factor causing those problems and how to avoid them. I greatly appreciate your answers!

It could be you are getting buffer precipitation in the mixing valve as you move from 100% ACN. Buffer solutions can be tempramental when mixed with ACN.

Back in the "olden days", I was taught not to start with 100% solvent in each reservoir but with 90:10 or 95:5 premixes which highlighted such buffer issues and reduced outgassing on mixing.

What Kind of HPLC System do you use. It seams that the tubing is not ACN resistant.
Please check the tubing and if necessary replace with a new Teflon tubing!
Good luck

It could be you are getting buffer precipitation in the mixing valve as you move from 100% ACN. Buffer solutions can be tempramental when mixed with ACN.

Back in the "olden days", I was taught not to start with 100% solvent in each reservoir but with 90:10 or 95:5 premixes which highlighted such buffer issues and reduced outgassing on mixing.

Yesss! I note one thing in your suggestion. Actually, in other gradient modes, I often mix buffer with sovent instead of using 100% sovent as this sittuation. This could be the problem. So, I think I will keep my Mobile phase A with 100% buffer and Mobile phase B with the ratio of Buffer:Acetonitrile = 5:95 or 10:90. Do you think it could solve my problem? ( but I also hope the Retention time of the later peaks aren't long). Thank you so much!

Floating check valve? ACN causes that over time, particularly w/ ruby/sapphire versions. If a thorough cleaning or replacement of the ACN side's check valve cartridge solves teh problem, consider switching to ceramic inserts.

Floating check valve? ACN causes that over time, particularly w/ ruby/sapphire versions. If a thorough cleaning or replacement of the ACN side's check valve cartridge solves teh problem, consider switching to ceramic inserts.

Thank DR, but look like it isn't the reason. In my work, I often use gradient program including buffer and Acetonitrile. But problem only occurs in the above sittuation.
Even if I replace a new check valve, after running that gradient, the problem will happen.

What kind of instrument do you use?
Maybe a faulty GPV?

Ace

Sorry for coming back late! But the problem is solved when I lower the concentration of buffer. I aslo use both buffer and Acetonitrile in my two mobile phase. Thank a lot to DR, your advise is useful for me.
By the way, I am doing another project with Isoratic mode. My mobile phase includes:
TEA solution(TEA 3% adjusted to pH 6.0 with Phosphoric acid): THF:Methanol = 62:30:8. The Flow rate is 1.0ml/min
For the fisrt running, everything was ok. But the next day, the baselinbe is drift. I spend a whole 6 hours for equilibration but useless.
My THF is stabilized with BHT and it is new so it couldn't be the problem. But the UV detector is at 215 nm, it is too close its UV Cut-off, I thought that was the problem because when I changed wavelength to 230nm, there was no drift.
But I must keep the Wavelength at 215nm ( my boss requires it!). Could anyone help me overcome this matter? It really annoys me. Thanks in advance.

If you cannot Change the wavelength and/or your Boss...... try to Keep the bottles with your solvents and buffers at a constant temperature, best to adjust to the same temperature as the column is opereated. Do you use a vacuum degasser?

If you cannot Change the wavelength and/or your Boss...... try to Keep the bottles with your solvents and buffers at a constant temperature, best to adjust to the same temperature as the column is opereated. Do you use a vacuum degasser?

I think the temperature is constant for both column and buffer. Also, there is a in-line vacuum degasser on my HPLC device. If only I could change wavelength

Did you try to change your THF?
Maybe use one without BHT?

Ace

Did you try to change your THF?
Maybe use one without BHT?

Ace

If I use THF without BHT, It will harm my devices, won't it? I would like to add some information. I have mixed and degased my Mobile phase very well. When I use that MP, it took me 1 hour to get stable baseline. But, the elution and peak shape was good after that. But it lasted only 4-5 hours then noise and drift appeared, from this monent on, elution and peak shape was poor, some small peaks was covered by the drift baseline. I wonder why my MP only stable in a short time??

For your isocratic method please use a magnetic stirrer to constantly mix your MP. I would recommend to use fresh distilled THF or THF HPLC grade. How pure are your chemicals? TEA and Phosphoric acid? TEA in most cases gives Long equilibration times.

Did you try to change your THF?
Maybe use one without BHT?

Ace

If I use THF without BHT, It will harm my devices, won't it? I would like to add some information. I have mixed and degased my Mobile phase very well. When I use that MP, it took me 1 hour to get stable baseline. But, the elution and peak shape was good after that. But it lasted only 4-5 hours then noise and drift appeared, from this monent on, elution and peak shape was poor, some small peaks was covered by the drift baseline. I wonder why my MP only stable in a short time??

A low wavelength means you will have to use preservative free THF. Periodically distilling it from a round bottom with some salt (I forget which one -ferrous sulfate?) in it will yield peroxide free THF. The trick(s) to keeping it that way are to helium sparge your mobile phase (vigorously for a minute or two when first put on your system, then just a trickle), nitrogen blanket any THF bottle you've used less than all of, and flush your HPLC with something else as soon as your run is complete. You might also consider replacing your solvent uptake plumbing with something more THF resistant than what is typically shipped with new LCs, depending on your THF concentration and length of sample sets.

PS - don't forget to do your periodic peroxide testing if you store unstabilized THF.

I think the problem is the low solubility of the buffer salt in ACN. Salts generally have a higher solubility in MeOH. Maybe you can substitute solvents. A buffer salt that is reasonably soluble in ACN is ammonium acetate.'

See here: http://www.sigmaaldrich.com/etc/mediali ... ochure.pdf