Chromatography Forum

Hi guys,

I am developing a new gradient for one of the applications. I am not going to explain method details as this is irrelevant to my question.
In accordance with manufacturers recommendations I am using volatile buffer for this application, namely Ammonium Bicarbonate buffer adjusted to pH=7.6 with formic acid (pH adjusted in aqua’s phase). The method seems to work fine apart from one peaks retention time decreasing (other peaks to shift as well but not considerably). The decrease is proportionate to the ''age'' of mobile phase. I have performed a number of test runs to support my hypothesis and have figured out that the drift is due to the change in pH. The drift is quite considerable, approx. 3 seconds per hour and taking into account that I have to run the method for a total of 24 hours I end up with one of the peaks eluting over a minute earlier compared to my first injection. I have mentioned that if the amount of mobile phase is higher then the rate of the drift is slower but still present.

Anyway, the question is: is there a way to stabilize the volatile buffer to prevent it from changing its pH?

Thanks in advance
Alek

Hi ExLet,

Perhaps, as ammonium bicarbonate is of middle pH, protecting the eluent with a blanket of nitrogen or helium (no sparging - just a careful layer of the gas over the eluent) or with a soda-lime tube as once was common in Ion Chromatography for protection of basic eluents could help?

It may not be a problem of evaporation, but of the eluent's pH changing due to ingress of atmospheric carbon dioxide.

Please see what others think...and Good Luck!

Thanks mattmullaney, I'll keep it as an option.

I fully agree with MattMullaney. I had a similar Problem some years ago. Question: do you have Aircondition in your lab?

Question: do you have Aircondition in your lab?

I wish but no...

What is going to be more effective, gas or soda lime glass tube/beads?

Dry Nitrogen! Try to get the bottle into a water bath with constant temperature. It will help.
Good luck

Guess it's my turn to agree with Gerhard! With the older Dionex pumps we kept the eluent bottles under gentle positive pressure with nitrogen AND used soda lime tubes. If only one method of carbon dioxide protection can be enacted, please choose the helium/nitrogen blanketing technique.

Thanks for you replies guys. I do have another question though. Is pH going to be more stable if I replace my current buffer say with Phosphate buffer, or all of the buffers are relatively unstable at pH 7.6 regardless of whether it is volatile or non volatile buffer?

If your buffer is Ammonium Bicarbonate the pka in 50% Methanol is 10,3 and pH range is 9,3 - 11,3, 5mM are recommended.
I would Change the buffer to 4-Methyl-Morpholine 10mM, here pka is around 8,4 in 50% Methanol, pH range is 7,4 - 9,4. That should work well and can be used with MS detection. Phosphate has 2 pka's one at 2,15 and the second at 7,2 (in 50% Methanol) and is recommended for pH range 6,2 - 8,2, but not useful with MS detection and at elevated temperatures you will shorten column lifetime.
I would go with the 4-Methyl-Morpholine!

If your buffer is Ammonium Bicarbonate the pka in 50% Methanol is 10,3 and pH range is 9,3 - 11,3, 5mM are recommended.
I would Change the buffer to 4-Methyl-Morpholine 10mM, here pka is around 8,4 in 50% Methanol, pH range is 7,4 - 9,4. That should work well and can be used with MS detection. Phosphate has 2 pka's one at 2,15 and the second at 7,2 (in 50% Methanol) and is recommended for pH range 6,2 - 8,2, but not useful with MS detection and at elevated temperatures you will shorten column lifetime.
I would go with the 4-Methyl-Morpholine!

Gerhard, the issue is that our lab is currently not equipped with nitrogen or helium, and my manager is trying to avoid the use of gas, even if I’ll have to sacrifice a column. Is 4-Methyl-Morpholine buffer in 50% methanol going to be stable enough to be used without nitrogen blanket?

I would say - YES. What is the Minimum and the Maximum temperature in your lab? you know that pH can/will Change according to temperature changes!
If you have a column Thermostate and a water bath you should work at slight elevated temperature to work at a constant temperature over the day. I would highly recommend to invest first in a small bottle of Helium. Or do you have a vacuum degasser?

I would say - YES. What is the Minimum and the Maximum temperature in your lab? you know that pH can/will Change according to temperature changes!
If you have a column Thermostate and a water bath you should work at slight elevated temperature to work at a constant temperature over the day. I would highly recommend to invest first in a small bottle of Helium. Or do you have a vacuum degasser?

Minimum temperature is 20oC (for now, during winter will get down to 15oC)
Maximum temperature is 28oC
Yes, I do know that pH is temperature dependant value, however it is not relevant in this case as I have monitored the temperature of mobile phases during runs
Column temperature for the method is 35oC and I warm up the mobile phase to an approx 30oC in the water bath during degassing. Mobile phase is degassed using sonication only without vacuum