One of the early steps for method development is the selection of a mobile phase that will work best to separate your analytes. When choosing a mobile phase, selection of an appropriate buffer is one of the key factors.


What is a buffer solution?

A buffer solution allows minimum changes in pH when a strong acid or strong base is added to the system. A buffer solution is an aqueous solution with either a weak acid and its conjugate base or a weak base and its conjugate acid. The equilibrium between the weak acid/base and its corresponding conjugate base/acid allows for small shifts of pH when the equilibrium is disrupted (adding acid or base!)

HA ⇌ H++ A

Example: Let’s take acetic acid. Acetic acid (CH3COOH) is a weak acid and the acetate ion (CH3COO), is its conjugate base. pKa=4.76. Ka=1.8 x 10-5. If we take 0.36 mol of acetate ion and 1 mol of acetic acid, we can calculate the pH with the Henderson-Hasselbalch solution


pH=pKa+log⁡([Base]/[Acid] )

Most buffer solutions should be prepared, so the concentration of the weak acid/base and its conjugate base/acid is close to concentration. This allows for the buffer solution have the capacity to handle small shifts of acidic and basic pH changes.

Why is buffer selection important in mobile phase considerations?

Ionizable analytes are affected by pH of the mobile phase. At certain pHs, each ionizable analyte may be in form of an ion or their unionized counterpart. If the pH isn’t stable, the retention time will vary!

How do I choose my buffer solution?

It’s best to choose a buffer solution that has a buffer range of the desired pH. The desired pH should be determined by looking at the each of analyte properties.
Also, when choosing a buffer solution, we need to see if the buffer is volatile if you are using mass spectrometry detection.

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