Chromatography Forum

Hi all ,

I really don't know how to prepare buffer in mobile phase ( hplc ) .For example how can i prepare this :

1- mobile phase of 57%methanol and 43% 10mM ammonium carbonate buffer ,pH 9.6

2- whyi use buffer in mobile phase and not in solvent if sample ?

can you please supply me with some book useful in this

please help

thanks

Prepare 500 mL 10 mM carbonate buffer, pH 9.6 and then mix 570 mL methanol with 430 mL carbonate buffer (the one you just prepared in step 1).

Regarding the reason for not dissolving the sample material in the mobile phase, there are several potential answers. Some people do dissolve the sample in mobile phase consequently other don’t. I prefer my sample solution to be of a weaker eluting strength than the mobile phase. Sometimes it’s just more convenient to dissolve the sample in water and so on.

Best Regards

"then mix 570 mL methanol with 430 mL carbonate buffer (the one you just prepared in step 1). " - or use the buffer as your mobile phase A and methanol as mobile phase B and make your method to deliver A:B as 43:57 in the software you use

"2- whyi use buffer in mobile phase and not in solvent if sample ? " - cos the method says so

"ok i repered 500 ml carbonate but with what i adjust the PH with which base ?" - and what salt did you use? what salt did method description recommend?

but with what i adjust the PH with which base

OK, here is some more detail: The prescribed buffer is Ammonium Carbonate, so you adjust pH with ammonia.

the 10 mM will be diluted ??? am i right

You’re right in concluding that the buffer will be diluted and thus end its days as 10 mM. In fact it’ll become 4.3 mM buffer. But this is also the desired concentration – obviously. I think the instruction is quite clear. Don’t you think?

Best Regards

P.S. Maybe this topic should have been posted somewhere else?

that's absolutely helpfull i am really thank u all