I am trying pH 9-10 separations, monitoring at 206 nm, and am getting very noisy baselines. I can't change the wavelength, so I have to change the MP to reduce the noise. The best combo I have found so far is Ammonium acetate pH'ed with amonium hydroxide. The organic is ACN, and I am using a gradient. I pH'ed the aqueous, and did not adjust the "apparent pH" after adding the organic.

I'm wondring if Ammonium chloride would be a better choice, or will I just have to put up with noise? Suggestions?

Dear Wanda;

It is surprising that you are able to use Acetate salts at such low wavelength. Acetate gives you strong UV background below 225nm. Obviously, the noise is related to tjhis problem.

If you are not able to change the wavelength, then change the salt in your mobile phase, or just use some dilute NaOH or NH4OH to give you some buffering capacity. Most of the real buffer systems such as Borate are likely to have strong Ub background at 206nm.

Good Luck;

Benjamin

While it's not widely known, hydroxide absorbs in the low ultraviolet. Working at high pH and low wavelengths are likely the cause of your problem. While I agree that acetate is probably not a good choice at this wavelength, neither is chloride as both absorb at low wavelengths. A UV transparent anion such as methanesulfonate would probably be a better choice but you'll need to make use of a "HPLC grade" of this anion as many sources of this anion contain sulfonation byproducts which will absorb in the ultraviolet. I would try to stick to the lowest possible pH.

Wanda,
You might want to try ammonium bicarbonate for the high pH separations. It generally has good purity which means fairly low backgrounds and has very good solubility in water and fairly high organic concentrations.

Regards,
Mark

Check the absorbance of your mobile phase with a spectrophotometer to see what the absorbance is at the wavelength you are using. You can get a noisy baseline when there is little lamp energy left as a result of absorbing most of the lamp energy by the mobile phase. If the absorbance in a 1 cm cell is near 2 you have lamp energy problem.

Phosphate, carbonate or borate would be better choices for buffer; I would use phosphate. However, upon adding methanol or acetonitrile to carbonate or phosphate they will become more basic. (amine and ammonia buffers get more acidic upon adding organic) That is to say that a pH9 phosphate buffer in water will become higher pH buffer as organic is added. Therefore, you will have to account for this change when preparing the buffer. One way to do this would be to measure the pH of the actual mobile phase you are now using (water plus organic) and match it with the phosphate or carbonate buffers measured after organic addition.

Dear Wanda,

as far as I know, any ammonium salt has a very strong absorbtion at low UV.
To check the absorbance of your mobile phase, condition your column with ACN/Water, start a monitor baseline (or balance your UV detector) and condition the column with your desired mobile phase. Then you'll see the absorbance of the mobile phase.
Next thing to consider is that the UV absorbtion is related to the concentration of your buffer. If you're able to use only very low concentration of buffer, maybe it is possible to use ammonium phosphat without a noisy baseline.
Good luck.

Ammonium ion does not absorb in the ultraviolet above 200 nm, the effect is simply related to the basicity of the cation promoting the presence of hydroxide anion which does adsorb in the ultraviolet.

We have worked with ammonia and ammonium bicarbonate at high pH with low UV detection. If I find the UV cut-off for the carbonate, I'll let you know.
Actually, I just found it: ammonium bicarbonate at pH 10 is suitable for down to 210 nM at the standard 10 mM concentration commonly used in MS

Thanks to everyone! Great suggestions and explanations. You've been a great help.

Hello everybody,

my comment about the strong absorbance of ammonium salts is wrong.
Sorry.