Chromatography Forum

I have a system very sensitive to pH change so I wish I have a strong buffer(40mM). But my pH is 5.7. It seems I have three choice: actate, phosphate and citrate.

acetate (pKa 4.75) should be able to cover 5.7;( as my other thread mentioned the JT baker NH4OAc I have has a contamination, the peak for which is absent with phophate or citrate)

phosphate (pKa2 6.82) should be bad; but Synder HPLC book Appendix IV page735 prepare phosphate buffer pH low till 5.6 while acetate up till 5.6 only.

Does this mean acetate is good only up to 5.6 (not 5.75) while phosphate can be good as low as pH5.6 (lower than 5.82)?

Citrate should be OK but higher viscosity and UV.

What is your suggestion?

If you only look at pH and pKa than citrate is the buffer of choice. Both acetate and phosphate are less then optimal for pH=5.7

We use extensively citrate buffer at pH=6.0, 0.02mol/L and have no problems (wavelength=254nm). Flow rates usually range between 0.25 to 2.0mL/min.

Check with your HPLC manufacturer about compatibility issues with citrate. Citrate is more aggressive towards stainless steel than acetate or phosphate. I don't know if the viscosity of citrate buffers is significantly higher than that of other buffer systems.

And many references say KHPO4 pka2=7.2

Several problems and issues:

First of all, buffer capacity falls off exponentially as you get further away from the pKa. At 1 pH unit, the buffer is only 10% ionized (or un-ionized). At 1.5 pH units, it's about 3%, and at 2 pH units it's 1%.

Second, pH units are listed (and pH is usually measured) in 100% aqueous solvent. When you add organic solvent, the equilibrium shifts to favor the unionized form (for anionic buffers, that means the pKa shifts down).

Finally, the UV-cutoff problems with things like citrate (and, to a lesser extent, acetate) are really only important if you are detecting at fairly low wavelength. Also, I agree with rhaefe that you're unlikely to see any viscosity issues at 40 mM.

Bottom line: if your detection wavelength is above about 230-240 nm, citrate is probably the best choice. If low wavelength is a requirement, it's probably a coin toss between acetate and phosphate, with acetate having a slight edge.

Thanks, Tom

Maybe it is not critical but citrate gives less flat base line than the other two.

Maybe it is not critical but citrate gives less flat base line than the other two.

You're the only one who can decide whether or not it's acceptable

If a basic nitrogen doesn't mess with your chromatography, you could also try piperazine or MES. Carbonate is also an option, but volatility can be tricky.