What is the limit Percentage of Buffer in LC-MS/MS

[mass]

Hi everybody,

I have a doubt regarding pesentage of Buffer(volatile) limit while doing impurity profiling combination with a solvent.

It varies instrument to instrument? if the buffer persentage is more what will happen eventhough we will keep the desolvation temperature 450°C.

Let me know regarding this in particularly ratio of Buffer and Solvent.

[JNF]

Hi Mass,


I recommend using (volatile) buffer concentrations up to 100 mM. Try to use the least possible. What can happen is that the excess ions from the buffer will "compete" for charge thus lowering your sensitivity.

Ciao
JNF

[mass]

Thanks JNF , for your reply

I am asking about the ratio of buffer, for a particular impurity i have to run 95% of Buffer(10mM amm. Acetate) and 5% of acetonitrile for 5min. then the gradient programme starts.

so my question is can i use 95%buffer ratio in mobile phase or not? If not what is the maximium ratio of buffer we can use in the LC-MS/MS system.
Is there is any creteria for this issue.

[MG]

OK, so you have 10mM * (0.95) = 9.5 mM of ammonium acetate spraying into your instrument during that time. I see no problem with that concentration.

You will have a loss of sensitivity if you exceed the 10-20 mM range of volatile buffer. But you won't harm your instrument, if that is what you are worried about.

[james little]

I routinely use 100% 10 mmolar ammonium formate adjusted to pH of 3.5 (600 ul of formic/liter) with addition of 3% organic to slow microbial growth. Then I go from 100% acqueous to 100% organic (usually either 100% methanol, 100% acetonitrile, or 50/50 mix).

On our quattro micro, no problem with formate buffer up to 50 mmolar, where the response starts to fall some. Also use just ammonium formate, or just formic (0.1% by volume to 1% in solvent A).

For basic drugs, the formate buffer between 10-40 mmolar adjusted to pH 3.5 gave the best response for most drugs.

At high % organic, the response can fall. Thus I normally add about 8-10% of the flow post-column of 25 mmolar ammonium acetate in pure methanol. Seems to help some of the components that elute at 100% organic. Also works when doing size exclusion with organic solvents.

[Uwe Neue]

James, I am surprised that your response drops at high organic. There are publications by Naidong Weng that say that you get HIGHER response in high organic, and we did a few limited experiments that confirmed that.

On the other hand, maybe it is also a question how you set up your MS. I am not an expert on this, and I appreciate advice.

[mass]

By the advises of MG and james, it shows that 100%buffer in mobile phase also works for MS and it doesnt form any salts at the ionsource, Is it right? or any opinions are there??

If 100% buffer is used then is there any problem in disolvation?

[MG]

I have never seen ammonium acetate or ammonium formate form solid deposits in an ion source, even with amounts as high as 100 mM.

Whether or not you have these buffers in your mobile phase, you will not generally have good sensitivity when your mobile phase is 100% aqueous. If your LC separation requires you to run at 100% aqueous for a portion or all of your run, you may be able to improve things by optimizing your source temperature and gas flows for that mobile phase.

[Victor]

Although this wasn't originally part of the question, you must be careful that the LC part of your analysis is not upset by a 100% aqueous buffer. For many phases, this can result in "phase collapse" probably better descibed as de-wetting of the stationary phase. In this case you may see under certain circumstances a loss in retention (or no retention) of your solutes. So this is in addition to any considerations about the MS side of things.

[mass]

Thanks to all, for giving valuble suggestions

Actually my gradient programme will starts with 95% of buffer and 5% of Acetonitrile for 5 min, then with in 5 min Sovent percent will rise to 40%.

I made the injection and got good results.

[james little]

In response to Uwe Neue about high organic response, it depends on the compound. We do some very hydrophobic plastisizers, cyclic oligomers of polymers, and surfactant like molecules and found the ammonium acetate in methanol enhanced their sensitivity.

Also, in some cases there were problems with uncertainty of whether we were seeing M+Na or M+H, the addition of ammonium acetate converted everything to M+NH4 and gave better response for these particular compounds. Another good way to determine the molecular ion is to increase the cone voltage or DP to very high voltages. The molecules will fragment, but the M+Na ions will be enhanced. We did a lot of breakdown curves and found in general M+H and M+NH4 optimize at lower cone voltages, but M+Na at higher.

The nice thing about the ammonium acetate in methanol is that it is very compatible with a wide variety of samples at high organic solvents even with methylene chloride. When we used water with ammonium acetate, it caused some analytes to precipitate. We haven’t systematically evaluated for a wide variety of compounds.

[Uwe Neue]

Ah, I see...

We typically keep the ammonium formate concentration constant. i.e. we run the gradient with three lines: water, organic, buffer, and the buffer is delivered at a constant rate.

OK, so high organic per se is nothing bad for the response, as long as you keep some source of ions around. We agree with each other.