pH adjustment of mobile phase

[kvalliappan]

Hi,

I would like to know which of the following is the right approach in adjusting the pH of the mobile phase:

1. Adjust the desired pH in the aqueous phase and then add the
organic phase.

2. Mix aqueous and organic phase togethter and then adjust the pH .


Thanks for your time

[JA]

Resource

Example

i.e. The "best", most reproducible way, is to prepare the aqueous component of your mobile phase by weighing out the buffering constituents and dissolving them up in a known volume of water. Then you can add your organic component, if necessary.

Second case is to follow the practice of adjusting your aqueous mobile phase to a desired pH.

Lastly (the least common way, it seems) is to adjust the pH of the complete aqueous/organic mobile phase.

[Uwe Neue]

In nearly all cases that are relevant for the practitioner of HPLC, it is best to measure and adjust the pH in the completely aqueous componets of your mobile phase. The reason is simply the fact that the pH and the pK of you buffer change, as you add an organic solvent. However, in water, you know what the pK of your intended buffer is, and you can adjust your buffer easily to a suitable pH.

Example: you want to make a good phosphate buffer at neutral pH. Then you adjust the buffer in water to pH 7, because this is where the pK of phosphate is known to be in water.

[Bill Tindall]

I would agree with the previous responses with the following caution. When an organic solvent is added to an aqueous buffer the pH in the mixed solvent, compared to water, may go up, down and stay nearly the same, depending on the chage type of the buffer components and the basicity of the solvent added. The pKa of the compounds to be separated may in turn go up, down or stay nearly the same depending on their charge type. For example, if the analyte is an amine and the buffer is phosphate(worst case), the desired pH you might predict for the separation from aqueous pKa data will be in substantial error in a solvent rich in methanol or acetonitrile. Phosphate buffers get considerably more basic when mixed with methanol or acetonitrile while the pKa of amines is not greatly affected in these water/solvent mixtures. These changes will only be an issue if the separation is done at a mobile phase pH near the pKa of the analytes.

If the separation has been developed and all that is required is to rerpoduce the buffer day to day, then follow the approach suggest by JA. It is by far the quickest and most precise.

However, if you are reproducing a procedure from someone else then you need to know, or guess, whether they specified a pH in the water before mixing (likely if it is a US procedure), or after.

If you want to change buffer components, or you are concerned with details of pH sensitive chemistry that might occur in the mobile phase, for example decomposition, then measuring or matching pH in the mobile phase is essential.