variability high pH mobile phase LC-MS

[Ruth]

Dear forum-members,

When using low pH mobile phases for LC-MS/MS analyses, I always use buffered water (set at a pH where the buffer is active) and organic solvent containing some acid to reduce their influence on pH.
I experienced that if I removed just one component, the variability of the area of peaks was very high (coefficient of variantion larger than 15%).

I am interested in using high pH mobile phases now, can you suggest me which to use?
In literature I find water with ammonium bicarbonate set at pH 9-10 and an organic solvent. Should I add some additives to the organic solvent too? The first results using water 10 mM ammonium bicarbonate pH 9 and methanol suggest variation...

Thanks for your suggestions!

[Uwe Neue]

Ammoniumbicarbonate has a better buffer capacity at pH 10.

What is the largest concentration of your organic solvent? If you do not need to go to 100% organic, you can use ammoniumbicarbonate in both the A and B solvents.

[Ruth]

The gradient I would use has a maximum of 90%B. Can I add another 10 mM ammoniumbicarbonate to the methanol then? Should I also set the pH of the organic eluent at 10 (like the water)?

Thanks!

Ruth

[Uwe Neue]

Ruth,
here is what you do, and you will also immediately learn if there is precipitation problem.

You prepare a 100 mM solution of the pH 10 ammonium bicarbonate buffer. You dilute this 10:1 with water to create your aqueous mobile phase. You take the same solution, and dilute it 10:1 with acetonitrile. If you get precipitation, this won't work, and you need to work with a more dilute buffer in mobile phase B. You can now test the other extreme: take the now 10 mM aqueous mobile phase and dilute it 10:1 with acetonitrile. Now, I do not expect any precipitation at all, but your final buffer is 10x more dilute than the buffer in mobile phase A.

You can then play with anything in between.

I have some data that indicate that the first approach works without precipitation, but I do not have all the details that I would like to have to confirm this. This is why I recommend the experiment.

Never confuse yourself by trying to measure the pH in the presence of an organic solvent, unless you have become an expert in the subject.

[Christian Eckhoff]

I have two questions for Uwe re. use of ammonium bicarbonate:

1. Can you use ammonium carbonate instead? It seems when you use bicarbonate and adjust to pH 10 with ammonium you are just scrubbing of the proton in the bicarbonate first so why not go straight to the carbonate from the beginning?
2. Can you recommend a stability period for a pH 10 ammonium (bi)carbonate buffer? One day, one week, maybe even one month?

Thank you!

[Uwe Neue]

Ammonium carbonate does not exist. It contains a large amount of a carbamate, or whatever the thing is called. Look it up in the Merck index.

Ammonium bicarbonate solutions adjusted with ammonia to Ph 10 are stable, provided reasonable precautions, such as closed plastic containers.

[Christian Eckhoff]

The Merck Index refers to Hartshorn. I confirmed on PubChem that Hartshorn is a mixture of ammonium bicarbonate and ammonium carbamate. Elimination of one molecule of water from the bicarbonate produces the carbamate.
Diammoniumcarbonate does exist. In PubChem it is also called Hartshorn salt. I have a jar in the lab, MW 96.08. It comes in big chunks and has a strong smell of ammonia. I reviewed the MSDS from Baker. It is stable until exposed to air when it decomposes to ammonia and ammonium bicarbonate.

[Ruth]

Dear,

I've been working on some other stuff, but I recently tried Uwe Neues suggestion:

I prepared 100 mM ammonium bicarbonate and diluted it 1:10 with water and 1:10 with methanol. No preciptation was seen and variability was less than when using pure methanol as B.

I did everything at pH 9.0 and this works fine (freshly made everyday)! I could switch to pH 10, but since pH 9 works fine and I guess the lower pH is better for the column, I think I will continue using this mobile phase!

[grzesiek]

why not consider ammonium acetate, it works perfectly for most of applications

[Ruth]

@ grzesiek: I tried a mobile phase containing ammonium acetate at pH 4. The peak area was signifiantly lower than when using high pH mobile phase...

[grzesiek]

use ammonium acetate at high pH, ammonia buffer has pK something about 9