Chromatography Forum

I estabolised the HPLC method by using the phosphate buffe (pH=5.5, adjust by phosphoric acid)
Now I want to transfer the method to the LC/MS.

I want to know what is the buffer I can chose and how to prepare it.

Thanks

Ammonium acetate (pka of acetate is around 4.7) will work for your buffer. I would use about 10 mM of ammonium acetate and buffer it at pH 5.5 with ammonia or even better I would calculate the amounts needed from each buffer to achieve the desired pH and stick with it.

Thanks!
I will try it. Could you please tell how to preparae it?

I'll do but not earlier than this weekend. I have a program in my old computer (called phoebus) that can do that very well but I need to put the computer together (I haven't retired yet that old computer because the licenses are linked with that computer). Of course you can calculate the same values but I'm too lazy to do that.

As promised, here you are:

For the preparation of 1L of ammonium acetate at pH 5.5 with an ionic strength of 10 mmol/L, prepare 1M solutions of acetic acid and ammonia and then use 11.6 mL of the 1M acetic acid solution and 10 mL of the 1M ammonia solution. Add the balance in water and you will have a solution of the above specifications and according to the software you'll end up with a solution with 3.2 mmol/L*pH of buffer capacity and 1.04 mS/cm of conductivity.

If your stock solutions are well prepared and fresh, these are the pH etc values that you will end up.

Dear Kostas,

Could you tell me where I can find that program virtually to prepare buffers with desired pH value and ionic strength? Or could you otherwise send it to my mail if it isn't too large?

Thanks in advance,
Regards

Hi Zokinato,

I thought that the software is not sold anymore, but I search a little bit after you asked and found that it is still sold (for way too much in my opinion). You can however download and use it for 30 days for free.

Here are a couple of sites of interest:

http://www.analis.be/products/product.asp?prod=624 (go to documentation for the free trial)
http://www.analis.be/tempFiles/61427983 ... hoebus.pdf

There are actually other website that you can do similar things for free but it is not as user friendly and/or do not give you as much information. A good software to have, especially if you are doing capillary electrophoresis.

Thanks Kostas for the web site.
It is pretty interesting and useful software.

Regards

Just one proviso: If you choose the software approach, please don't assume that the pH you'll get will be the pH you want. When you're starting with a highly volatile thing such as an ammonia solution, which is in any case only supplied +/- 1-2% by the manufacturer, it's very hard to be sure exactly what concentration you will be adding.

This probably won't matter; if you keep your recipe constant, the retention times should be constant. In any case, a good method should be set up robustly, such that small changes in pH don't cause big changes in retention time.

However, it would be great to measure the pH from time to time. Personally, I get very upset by published methods which give a recipe and a pH, but the pH cannot be reproduced by following the recipe.