Why is pH important for HPLC buffers?

Critical points :

1️⃣ In chromatography, the principle "like dissolves like" applies, meaning nonpolar analytes interact well with nonpolar stationary phases, leading to higher distribution constant (kd) values and improved separations.

2️⃣ Neutral or ion-suppressed analytes, being more nonpolar than ionized ones, exhibit enhanced retention on nonpolar reversed-phase (RP) stationary phases.

3️⃣ The pH of the mobile phase significantly affects the ionization state of ionizable analytes, necessitating buffer usage to maintain the analyte in the desired state.

4️⃣ At a pH equal to its pKa, an analyte exists in both ionized and neutral states, resulting in multiple kd values and poor chromatography with broad, tailing peaks.

5️⃣ Acidic analytes in acidic environments (pH at least two units below their pKa) retain their protons, leading to ion suppression and improved retention on RP-HPLC.

6️⃣ In more basic environments, acidic analytes dissociate into their conjugate base, becoming ionized and resulting in reduced retention on RP-HPLC.

7️⃣ Basic analytes in basic environments (pH at least two units above their pKa) remain in their ion-suppressed, neutral form, improving retention on RP-HPLC.

8️⃣ In more acidic environments, basic analytes become protonated and ionized, leading to reduced retention and earlier elution on RP-HPLC.

9️⃣ The pH also affects the column stationary phase, with very low pH causing stripping of the bonded stationary phase, high pH damaging the silica support, and mid pH ranges causing ionization of silanol groups, increasing tailing observed with analytes containing basic functional groups.

𝗔𝗱𝗱𝗶𝘁𝗶𝗼𝗻𝗮𝗹 𝗱𝗲𝘁𝗮𝗶𝗹𝘀

To understand why pH is important for successful chromatography of certain analytes, we must first understand the fundamental concepts of how chromatography works.

Chromatography is based upon the distribution, or partition, of analytes between the mobile phase and the stationary phase: The equilibrium of analytes between two different phases, referred to as kd, is represented by the equation:

This takes into account the concentration of the analyte bound to the stationary phase, and the concentration of the same analyte travelling in the mobile phase. It is similar to the idea of liquidliquid extraction, where an analyte will partition between an aqueous and organic layer to the same degree, provided all other variables, such as temperature, agitation time and pH, are constant.

The difference with liquid chromatography is that one layer is moving; for reverse phase (RP) HPLC the organic layer is equivalent to the column stationary phase (C18 for example), and the more polar mobile phase is part of a dynamic system. The continuous flow of mobile phase in HPLC means that when analytes bound to the stationary phase enter the mobile phase, they are transported along the column.

The movement of the mobile phase in a definite direction, driven by the pumps, therefore means that an equilibrium is never reached: There is instead a continuous dynamic with some proportion of analytes interacting with the stationary phase and the remaining analytes of the same type moving through the column in the mobile phase.

This movement of analytes away from those in the stationary phase disrupts the kd equilibrium, causing the analytes in the stationary phase to reenter the mobile phase, which again disrupts the equilibrium so some analytes then reenter the stationary phase,This is how analytes travel

through the column.

The level of interaction of analytes with the stationary and mobile phases occurs to varying degrees, depending on the analyte, which is how separation of different sample components is achieved.

This interaction of analytes with the stationary phase, and the same analytes’ preference towards the mobile phase, determines how quickly the analytes moves through the column. If there are strong interactions with the stationary phase, and not much affinity for the mobile phase, the analyte will have a high kd value, and will take longer to move through the column: It will be immobile, interacting with the stationary phase, not progressing through the column. If there are only weak interactions and the analyte prefers to be in the mobile phase, then movement through the column will be much faster this gives a low kd value.

The final point to consider before we look at the effect of pH on HPLC separations is the idea that a nonpolar analyte will prefer to interact with a nonpolar stationary phase than a more polar mobile phase. Remember that for chromatography “like dissolves like” nonpolar analytes will stay on a nonpolar stationary phase until the mobile phase is similar enough, in terms of in polarity (or nonpolarity), for the analytes to interact with it.

Effect of pH on reversed phase HPLC

The pH of your mobile phase can have dramatic effect on both retention time and chromatographic peak shape as it affects the ionization state of the analytes, and so the chemistry of the interactions occurring within the column. It is therefore very important if you are working with analytes that may be affected by pH changes that you maintain the mobile phase pH, once it has been measured by a calibrated pH meter. Mobile phase modifiers such as trifluoroacetic acid (TFA) or triethylamine (TEA) are common, but they won’t maintain pH for this you need buffers, or a solution that will resist a change in pH when small volumes of acid or alkali are added to it, or when it is diluted with water. A buffer solution contains a mixture of a weak acid and its conjugate base (or a weak base and its conjugate acid). When considering the effect of pH on ionizable analytes it is useful to remember the acid dissociation equilibrium.

If this is considered in terms of an analyte with a weak carboxylic acid functional group:

From the above equations it is clear that a weak acid has two possible states; either the neutral form (AH/RCOOH), or if dissociated it will become ionized (A/RCOO). If the acidic moiety is ionized, or charged, then it becomes more polar and will have poor retention on a typical RPHPLC stationary phase.

The equilibrium between these two ionization states can be pushed in either direction, primarily by manipulating the pH. As the pH is reduced (made more acidic), the additional H+ ions (protons) present in solution will push the equilibrium of the weak acid to the left, towards its neutral, ionsuppressed form: There are enough protons in solution that the RCOOH will keep its H+ and not become ionized. If the pH was increased, the equilibrium would move to the right and the acid would dissociate to provide H+

and restore the balance.

Similarly the same principle applies if you have an analyte with a basic functional group:

This weak base also has two possible states; the neutral form (RNH2), or the ionized (RNH3+). If the basic moiety becomes ionized, in this case through becoming protonated, it becomes polar and so will have poor retention on a typical RPHPLC stationary phase. To summarise if you have an acidic compound in your sample and you fix the pH low enough that it remains in the neutral form, it would display a longer retention time on a RP nonpolar column than if you fix the pH too high (basic). If the pH was too high, the acidic moiety would dissociate, becoming ionized and more polar. Like dissolves like, so

the ionized form would be retained less on a nonpolar stationary phase. As you increase the pH of the mobile phase (decrease the number of protons, H+), the retention of acidic compounds will decrease (equilibrium will shift so they will lose a proton and become ionized). At the same time, the retention of bases will increase as they deprotonate (give away extra H+) and so become neutral. As pH decreases bases gain a proton – removing H+ from the acidic environment.

Effect of pKa on reversed phase HPLC

We need to take this one step further to fully understand the importance of setting a mobile phase pH, and ensuring it remains at that pH.

Any compound containing acidic and/or basic functionality has a specific ‘ionization constant’, or Ka, which indicates the degree that the acidic or basic moieties will ionize in an aqueous solution. If an analyte is readily ionizable, it will have a large influence on the concentration of H+ in the solution. The greater the ionization, the ‘stronger’ the acid or base.

The pKa is the logarithmic acid dissociation constant and is the commonly used form of this ionization constant. If an acidic analyte has a high pKa, it is less likely to ionize or dissociate and so is a weak acid (typically with pKa values between 2 and 12 in water). If a basic analyte has a high pKa however, this analyte is more likely to ionize and so is a strong base. It is important to note that it is not possible to discern if an analyte is basic or acidic by its pKa; very weak acids will have high pKa values, it is the functional groups that determine the acidity/basicity of a compound.

The reason the pKa of your analytes is so important in HPLC is because when the pH is set close to the pKa, the analytes will be present in solution in both neutral and ionized forms. Those in the neutral, nonpolar, form will be well retained by the nonpolar RP stationary phase, but those in the ionized state will have less retention, a different kd. Therefore, all analytes of the same type will not be travelling through the column in the same manner resulting in very poor peak shapes and irreproducible chromatography.

A good rule of thumb is to fix the pH for an acidic compound at least two pH units below the pKa, and for a basic compound it should be set at two pH units above the pKa. This ensures that the pH is far enough away from the pKa that any small changes in mobile phase pH will not have a dramatic effect on analyte retention and peak shapes; as can be seen using the diagram below for acetic acid. The ideal pH for a mobile phase sits within the plateau region for the neutral form of the analyte. Think about the tolerance given for the pH of your mobile phases, e.g. pH 3.5 ± 0.1%. If an SOP states a much tighter tolerance than this, it is likely to be because the pH is very close to the pKa of an analyte in the sample, and any minor deviations from the set point will have a negative effect on the chromatography and reproducibility of the analysis.

It is important to point out that it may not be as simple to follow the two pH units rule when working with bases that have pKa values above 8. If the rule is followed, the pH of the mobile phase would need to be at pH >10, which is outside of the working pH range of most silica columns. Similarly, if an acidic analyte has a pKa of 3, a mobile phase of pH 1 may well be too low for the column. These two extremes of pH cause different effects, but both are equally as damaging to standard silicabased columns. At high pH, the silica support inside the column will be damaged due to dissolution of the siloxane bridges in the silica backbone the silica will start to fall apart. At low pH, the bonds holding the stationary phase onto the silica support will be cleaved, resulting in loss of chromatographic performance.

There are three main solutions to this issue. In all of these examples it is crucial that the pH is fixed to maintain the ionizable functional groups in one state. The important factor is that whatever the pH is, it is far away from pKa!

✴️In the case of weak bases, the first option relies on exploiting any additional hydrophobic interactions between the analyte and the RP stationary phase to maintain sufficient retention. If the basic moiety is part of a large nonpolar molecule, it may be possible to analyze it at a low pH, in the ionized state: The hydrophobic interactions should overcome enough of the polarity introduced by the protonation of the base to ensure adequate retention.

The added benefit of analyzing bases at low pH, is the lone silanol groups (SiOH) on the silica support will be ionsupressed. At a pH above their pKa (~pH 4), lone silanol groups act like an acid and will dissociate to give ionized SiO that interacts strongly with basic moieties and results in bad peak tailing. At low pH, the SiOH

remains neutral. If the additional hydrophobic interactions do not provide sufficient retention for the analyte, one alternative would be to use a polarembedded, or polar endcapped, column. These columns have polar groups within, or endcapped onto, the silica backbone of the column, and so allow very highly aqueous (100%)

mobile phases to be used without the risk of phase collapse (selfassociation). The highly polar, ionized bases are more likely to be retained by the increased polarity provided by these more polar RP stationary phases.

✴️The second option for working at extremes of pH requires the use of specialized columns, such as Agilent’s ZORBAXExtend or ZORBAXStableBond, that incorporate additional chemistries into the stationary phase to protect the silica and/or the bond retaining the stationary phase (for more information www.crawfordscientific.com/Zorbax_HPLC_Columns.htm). It is important to note that while these columns can be used at extremes of pH, it is never advisable to store columns at these extremes

✴️The final option is particularly relevant to analysis of samples containing mixes of weak acids and bases where at least one functional group will be ionized at whatever pH is chosen, or analysis of strong acids/bases, or analytes that have multiple functional groups. In cases such as these, the pH of the mobile phase is used to fix the relevant analytes in the ionized state, so that they will interact (ionpair) with a modifier added to the mobile phase that carries the opposite charge. This interaction leads to an overall neutral molecule that will then be successfully retained by a RP HPLC column. If you use trifluoroacetic acid (TFA) in your mobile.

phases, it lowers the pH to ionsuppress any acids, while also ionpairing with any ionised bases.

Other common ionpair reagents are quaternary ammonium compounds (e.g. tetrabutylammonium phosphate) to ionpair with acids and supress weak bases, and alkylsulphonic acids to ionpair with bases and suppress weak acids. Triethylamine (TEA) is another commonly used modifier, often referred to as a ‘sacrificial base’ that will ionpair with ionized silanol groups on the stationary phase, reducing peak tailing effects.

A disadvantage of using ionpair reagents is that you are chemically modifying your stationary phase and so should always have ionpair dedicated columns. This ensures that analyses not requiring ionpairing are not affected by this change in column chemistry.

Also very high concentrations of ionpair reagent can result in any neutral analytes present in the sample having limited access to stationary phase, resulting in decreased retention. Some ionpair regent also have significant UV activity, and so may absorb light at the analytical wavelengths being used with UV detection (for example the UV cutoff for TFA is 210 nm).

𝙎𝙪𝙢𝙢𝙖𝙧𝙮

✅ When working with ionizale analytes, maintaining the pH of the mobile phase is crucial to control ionization levels.

✅ For acidic analytes, set the pH at least two units below the pKa; for basic analytes, set it at least two units above the pKa.

✅ If adjusting the pH isn't feasible with the current column, consider alternative column chemistries stable at extreme pH levels or offering increased retention for polar compounds.

✅ Ion-pairing analytes can enhance retention, as can using column stationary phases with specific functional group chemistries that ion-pair with analytes.