Hello everybody /:o)

I'm trying to develop some methods that i do not have any specification for them. When i notice that i have to use a buffer, i try K phosphate or Na phosphate.. but what pH should I choose? I'm just testing, 2,5 than 5 than 8 than 10. Is there a rule of thumb for that?? I know that the pka of the substance can be very useful to determine it, but how? and where can i find this pka. Sometimes i look it up in Merck index and i do not find it...

thanks for help

Renata from Brazil

Renata,

What type of column you are using?
normal silica would leach out at >8
:(
let me check out pKa, should be readily available.

A

Hi...

I've written about all methodologies in general.... I commonly use C18....

Renata

Hi Renata
HPLC separation is best performed when analyte is either fully ionised or totally non-ionised. To obtain stable retention times the pH of mobile phase should be ±2 pH units from analyte’s pKa

Here's a good rule of thumb that I tend to apply:

For acidic analytes use an aqueous buffer at pH 2
For basic analytes use an aqueous buffer at pH 10
10-50mM buffer is usually adequate. 20mM is good to start. [0.01M = 10mM] K Phosphate buffer has a low UV absorbance but remember most inorganic buffers are more soluble in methanol than acetonitrile or THF.

As mentioned by 'A' at pH 7-8 silica can dissolve into mobile phase but this phenomenon is less likely with type B silica and endcapped columns.

Hi Renata,

Thank you phil for your clarification.Sorry for my ignorance.
Renata, can check this out (type in 'pKa,list' in MSN search)
http://www.chem.uic.edu/ghosh/links.htm

If the analyte you working on is nowhere to be found (books/websites) then too bad, I guess you can try determining it by measuring the pH of a known concentration of analyte.

A

thanks for everybody!!!!!!

Renata