I would like to try a method with pH 12 but I have never worked with so basic mobile phases. Which buffer could I use, phosphate (pKa=12.3) or there are other buffers, too? And how can I set the pH to 12? What concentration of NaOH should I use?

Could anybody help me?

I WORK WITH PHOSPHATE 25mM,AT Ph 7, AND BUFFER IS DIFFICULT TO FILTER BECAUSE IT IS NEAR ITS SOLUBILITY LIMIT.
AFTER HAVE READ YOUR QUESTION, I TRIED, AND YOU CAN GO TO Ph 12 WITH KOH 1M. I DONīT KNOW HOW LONG ITīS GOING TO BE USEFUL, BECAUSE PHOSPHATES USE TO PRECIPITATE ON HIS OWN(WITHOUT MIXIN IT WITH ANY ORGANIC SOLVENT).
SORRY FOR MY ENGLISH, ITīS MORE THAN TEN YEARS SINCE I DONīT WRITE A WORD.
YOU KNOW YOU MUST BE CAREFUL WITH YOUR COLUMN.NOT EVERYONE RESIST THAT Ph.

Hello,
Phosphat buffer is common, but not the best choice for a new method development. Phosphat is non-volatile and you cannot use it for LCMS. And with elevated temperatures RP column lifetime is reduced. Try Pyrrolidine! pKa is 11.3 and pH range is pH10,3 to pH12,3! Pyrrolidine is volatile and so evan for LCMS you can use it. I would recommend a 10mM solution.
If you use a so called pH stable RP column, no matter if it is silica or something similar based, please be aware, that column lifetime could be very short. For such kind of applications still polymer based RP resins should be your first choice, and they are better in resolution, reproducibility and performance than most people might think. They are also available as Non-porous resin with 2,5ĩm particle size for short runtime.

Hope this helps. Good luck.
Regards
Gerhard Kratz
Tosoh Biosep GmbH

I have used Waters XTerra C18 column at pH 12 using TRIS buffer for several hundred injections without noticeable change in performance.

I agree with jclark I have used both Waters Xterra and Zorbax Extend columns at pH 11.5 for >400 injections and both columns have no noticable change in performance. Both of these columns have significantly higher plate counts than similar polymer columns.
Regarding buffers, you could also try piperidine, pKa 11.12. However before bothering with a "real" buffer, you might not need much buffering capacity and could just use a dilute strong base to adjust pH.

If you are using a MS detector then of course you need a volatile buffer, if you are using a uv detector then you need to consider the uv properties of your buffer. Before the newer base stable columns came out we tried to use organic buffers at high pH to extend column life, I don't think I would worry about it so much anymore. Good Luck.

Thanks everybody for the advices. One more question: which buffer could we use for LCMS at pH7? Besides phosphates, acetates and oxalates I don't really know what kind of other buffers exist, what is their pKa and which one can be used for LCMS (the organic ones(?)).

waters does not recoment the use of phosfate-buffers at high pH for xterra-columns.

Acetate would be your best bet for pH 7 buffer. You are kind of limited, because the buffer has to be volatile. Even unbuffered phosphoric acid can be bad for your system if it is used to often. Formic acid works well. Adjust the pH with ammonium buffers and ammonium hydroxide. You don't want to use oxalate.

Phosphate is tough on any column at elevated pH, regardless if it is Waters, Zorbax, Keystone, whoever. If you have to use it, minimize both the temperature and phosphate concentration.

For pH 12 work, try pyrrolidine. It's MS friendly as well. Ammonium bicarbonate (not carbonate) is a great high pH buffer, although its pKa of 10.3 levels it off for use at around 11 or so.

Is acetate volatile to use it with MS? And how could it be used for pH7 if its pKa is 4.8. Where could I read about the existent buffers and their use?

Acetate itself does not have any buffer capacity at pH 7. Use ammonium bicarbonate, and adjust the pH to 7 with acetic or formic acid for a real buffer at pH 7.

Uwe,

Does it work with MS?

Ammonium Bicarbonate buffers are safe to use with LC/MS.

To Dr. Neue:

If the pKa of ammonium bicarbonate is 10.3, how do we get a true buffer by adjusting pH of ammonium bicarbonate solution to 7 using acetic acid? Theory is that buffer range is pKa +/- 1. Please explain as I am also very eager to have a true buhffer at pH 7 for MS.

Ammonium carbonate is a buffer at pH 7.

The ammonium ion is simply a counter ion and is not a factor. At pH 7, the carbonate ion is essentially not present so the HCO3/CO3 pair with pK of 10.3 is not a factor either. The H2CO3/HCO3 pair is in a buffer region and carbonic acid has a pK of 6.3 so doing as Uwe suggested will produce a pH 7 buffer.

Hope that helps,
Marc

What buffers are recommended for LC-MS for pH 7.3 to 9.0? Are ammonia, triethanolamine and tris good candidates? Ammonia probably has too high a pKa for this purpose.

Thanks a lot in advance.

Marc explained very nicely the issue of a volatile MS compatible buffer at pH 7. In essence, it is a carbonic acid buffer. The problem that one has is the unavailability of carbonic acid. If you think about it, all what you need to do is to add some acid to the ammonium carbonate. Of course, there are limits to this, but at the concentrations that one is working in LC/MS, this is not a problem. In essence, think of it as the Alka-Seltzer of HPLC (of course, we try to avoid the bubbles)...

What is TRIS buffer?

TRIS is Tris(hydroxymethyl)aminomethane. An organic buffer that's pKa (at 25°C) is 8.06 and useful pH range is between 7 and 9. Used in CEC as it causes less joule heating than it's inorganic counterparts.

How do we prepare the tris buffer? What is the salt and the basic?