As a chromatographer not many of us are aware of how critical pH is in your method development, sometimes it is even considered as an inconsequential factor, which could have a negligible effect on our analysis.

However, in truth pH can make or break your analysis, choosing the correct or more importantly optimized pH in a method can be a single most significant step when developing a method.

It doesn’t matter whether you are running a reverse phase, a HILIC or an ion-exchange mode pH of your sample and mobile phase always will be critical to accomplish the best possible results in your analysis.

Now we should go back in our memories and recall the simplest definition of pH that we have studied in school, which to our annoyance can be several. However, here as we are elucidating the significance of pH, will keep it very simple. Concept of pH was given in 1909 by a Danish Chemist S.P.L. Sorenson.

pH

• The very basic definition can be - pH is a measure of acidity or alkalinity of water soluble substances.

Some more complex ones are:

• The measure of hydrogen ion (H⁺) concentration of a liquid, relative to that of a given standard solution.
• Mathematically, the pH is the negative log of the hydrogen ion concentration [H⁺], expressed in morality.

pH = - Log[H⁺]

Now, the question arises, what is “p” in pH ?

Whenever, you see a “p” in front of a value like pH, pKa, pKb it means you are dealing with the negative Log of the value following the “p”.

The exact meaning of the “p” is disputed but according to the Carlsberg Foundation where Sorensen worked in 1909, pH stands for “Power of Hydrogen”. Thus, “p” can also stands for the Power of the value following “p”.

Thus, pH is defined by the negative Log of the Hydrogen Ion Activity in a solution.

Pure Water has a low conductivity and is only slightly ionized, however when water does dissociate it forms:

H₂O = H⁺ and OH^-

Where, H⁺ is the activity of Hydrogen Ions and OH^- is the activity of Hydroxyl ions.

The conc. of H⁺ and OH^- ions are equal at 25 degree Celsius 1 x 10^-7 ions/Liter.

pH scale is derived from the Water’s characteristic of auto-dissociation.

The above equation sets the pH scale to 0-14, which gives a convenient way to express 14 orders of magnitude of [H⁺] ions.

Degree of Ionization or Degree of Dissociation

To understand the true significance of pH in mobile phase, first we need to understand the Degree of Ionization or Degree of Dissociation of acidic or basic compounds. The pH of a compound can indicate whether you are dealing with an Acid or a Base, but it offers limited information indicating the true strength of the acid or the base, and that’s where comes the concept of degree of ionization or dissociation.

pKa = [- Log Ka]

pKb = [- Log Kb]

Ka = Acid dissociation constant or [H⁺] ion Conc.

Kb = Base dissociation constant or [OH^-] ion Conc. Ka for most weak acids = 10^-2 to 10^-14 and thus, pKa for most weak acids is between 2 to 14.

The stronger the acid is, it’s pKa value will be smallest and the stronger the base the larger will be its pKb value. Going forward we will be mostly using the term “pKa” to explain the concept.

All analytes that can be analyzed by reverse phase chromatography can be at least slightly ionized thus, they can be either “Weak Acid” or “Weak Base”. Therefore, when working with such polar compounds (weak acids or bases), you always should know the pKa or pKb values of the compounds, because that will help you decide the correct pH for the mobile phase.

In reverse phase chromatography to achieve the goal of resolution of analytes and a good peak shape, hydrophobic and some ionic interaction plays the major role in selectivity between the stationary phase and the analytes.

To achieve this goal, the primary objective of a chemist should be to get the desired analyte in a single state, deprotonated or un-ionized state.

Ionization state of analyte directly affects the degree of interaction of analyte with the stationary phase.

• When a molecule is ionized it is more polar and therefore it is more likely to participate in hydrogen bonding. In a reverse phase with aqueous mobile phase the analyte will spend less time in hydrophobic interactions with stationary phase and more time forming hydrogen bonds with the aqueous part of the mobile phase as compared to the neutral molecule, result will be less retention of polar molecules.

• When there are significant concentrations of both ionized and neutral forms of an analyte, the ionized form will have less hydrophobic interaction while neutral form will have more hydrophobic interaction, the result will be broad and asymmetrical peaks.
• The few free silanol groups or metal contaminants present in the silica may participate in ionic interactions with ionized state of the analytes, which may lead to poor peak shape or irreproducibility.

Now, it is possible to predict the ionization state of the analyte relative to the mobile phase’s pH value by knowing the analyte’s pKa value, in other terms we can control the ionization state of the analyte by simply adjusting the mobile phase pH.

Therefore, For Acids:

For Bases:

The values in the tables above can provide a guideline in deciding the pH of the mobile phase depending on the pKa value of the analyte. For maximum analyte neutralization the mobile phase pH must be at least 2 units above or below the pKa value.

As LC chromatography deals with partially or fully polar compounds, the two of the major challenges that presents in LC chromatography is retention of acidic compounds and peak shape of basic compounds, fortunately both of these can be overcome by just choosing and controlling the right optimized pH for your sample and mobile phase. Below picture aptly represents the benefits of choosing the right pH of mobile phase in relation to retention and peak shape of acidic and basic compounds.

Thus, when you will be next developing or optimizing a method consider pH as one of the most important factor and you will have positive circumstances for accomplishing your desired retention, resolution and peak shape even for your most challenging analytes.