Chromatography Forum

Up to how much strength of potassium dihydrogen phosphate(pH5.5), i can go in HPLC and please tell me how should i wash the column after every batch run...

That depends on the identity and concentration of the organic solvent (which will affect the solubility of phosphate). For use with UV detection, levels of 10 - 50 mM are common; 100 mM is not unheard of. These are values in the aqueous portion of the mobile phase (i.e., before you add organic). It is a good idea to check for buffer precipitation problems "off-line" in a test tube before trying it on the HPLC system.

Hopefully, you are also aware that pH 5.5 is well outside the buffering range of phosphate.

As for washing the column take a look at this article on column care.

http://chromatographyonline.findanalyti ... oryId=9678

That depends on the identity and concentration of the organic solvent (which will affect the solubility of phosphate). For use with UV detection, levels of 10 - 50 mM are common; 100 mM is not unheard of. These are values in the aqueous portion of the mobile phase (i.e., before you add organic). It is a good idea to check for buffer precipitation problems "off-line" in a test tube before trying it on the HPLC system.

Hopefully, you are also aware that pH 5.5 is well outside the buffering range of phosphate.

Thankyou. as far as buffer range of phosphate buffer is concerned i am aware of that, but i had tried different pH soluyions but the best response and separation is coming on that pH only. could you please explain how it can affect my analysis...

As for washing the column take a look at this article on column care.

http://chromatographyonline.findanalyti ... oryId=9678

Thankyou for that link that was really informative

i had tried different pH soluyions but the best response and separation is coming on that pH only. could you please explain how it can affect my analysis...

If your results are sensitive to pH, then I would be very concerned about phosphate at pH 5.5 .

In order to control pH, a buffer must be able to both donate and accept protons. That ability is maximum at the pKa of the buffer (the pH at which it is effectively 50% ionized). pH is a logarithmic function; I won't go into the math, but +/- 1 pH unit from the pKa and the molecule is either 90% or 10% ionized; +/- 2 pH units from the pKa and the molecule is either 99% or 1% ionized - which means that it can either donate or accept protons, but not both. The working range of a buffer is generally given as +/- 1 pH unit. Phosphate has pKa values of about 2 and 7. A better choice for pH 5.5 would be acetate (pKa 4.7).

If your separation is not very sensitive to pH, then it may not matter.

i had tried different pH soluyions but the best response and separation is coming on that pH only. could you please explain how it can affect my analysis...

If your results are sensitive to pH, then I would be very concerned about phosphate at pH 5.5 .

In order to control pH, a buffer must be able to both donate and accept protons. That ability is maximum at the pKa of the buffer (the pH at which it is effectively 50% ionized). pH is a logarithmic function; I won't go into the math, but +/- 1 pH unit from the pKa and the molecule is either 90% or 10% ionized; +/- 2 pH units from the pKa and the molecule is either 99% or 1% ionized - which means that it can either donate or accept protons, but not both. The working range of a buffer is generally given as +/- 1 pH unit. Phosphate has pKa values of about 2 and 7. A better choice for pH 5.5 would be acetate (pKa 4.7).

If your separation is not very sensitive to pH, then it may not matter.

Thank you, i will switch over to ammonium acetate as i had tried it....and also phosphate buffer may be the cause of fluctuation occurring in the analysis...