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Introduction to Buffer Solutions for HPLC Mobile Phase Selection

Introduction to Buffer Solutions for HPLC Mobile Phase Selection

 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.

Improving your HPLC Separations

Improving your HPLC Separations

Improvements to your existing HPLC method can be overwhelming at first. Whether it’s aiming to improve selectivity, improve reproducibility, or improving the cost-efficiency of your separations, a good place to start is to try alternative stationary phases/columns.

Develosil HPLC columns use high-quality silica gel that is manufactured in-house that can offer improvements to your HPLC method. We are in constant development to improve our silica gel manufacturing to improve your column performance. In the past couple years, Develosil has released a new column line: the HSR Series. The HSR series adopts a silica gel that has a large surface area to obtain high retention capacity. With the addition of organic solvent, you can achieve improvements in your selectivity.

Overall, the manufacturing from the Si gel to the final packed HPLC column offers you great benefits. For example, Develosil performs strict quality checks along the way to give you the reproducibility you need for your HPLC method. Also, since there is less middleman in the process, this gives you the cost savings that you need!

Develosil is dedicated to improving your HPLC separations. Contact us today to get started.