No thanks! I would like to know more about CHROMacademy

 Over 3000 E-Learning topics / 5000 Articles & Applications
 
Why is pH important for HPLC buffers?

Key Points

  • In chromatography, “like dissolves like” i.e. non-polar analytes interact well with non-polar stationary phases and vice versa

  • Increased and improved interactions with the stationary phase leads to higher distribution constant (kd) values and generally improved separations

  • Neutral or ion-suppressed analytes are more non-polar than ionized analytes and so will have improved retention on non-polar reversed phase (RP) type stationary phases

  • The mobile phase pH can have a dramatic effect on the ionization state of ionizable analytes and so this must be fixed, by buffers, to maintain the analyte in the desired state

  • At a pH equal to its pKa, an analyte will be in both ionized and neutral states, resulting in multiple kd values for the same analyte and poor chromatography
    (broad, tailing peaks)

  • If an acidic analyte is present in an acidic environment (or at a pH at least two 2 units below its pKa) there will be sufficient protons (H+) in solution that the acidic analyte will retain its protons and so will be ion-suppressed - improving retention on RP-HPLC

  • If an acidic analyte is present in a more basic environment, with insufficient protons (H+) in solution, the acidic analyte will dissociate into its conjugate base: The acidic analyte will become ionized, resulting in reduced retention, earlier elution, on RP-HPLC

  • If a basic analyte is present in an basic environment (or at a pH at least two units above its pKa) there will be too few protons (H+) in solution for the basic analyte to become protonated, and so it will remain in its ion-suppressed, neutral, form - improving retention on RP-HPLC

  • If a basic analyte is present in a more acidic environment, where there are sufficient protons (H+) in solution, the basic analyte will become protonated. In this situation the basic analyte will be ionized, resulting in reduced retention, earlier elution, on RP-HPLC

  • The column stationary phase is also affected by pH. At very low pH (<2) the bonded stationary phase will be stripped from the silica support. At high pH (>8) the silica itself will be damaged by dissolution. At mid pH ranges (>5), any lone silanol groups (Si-O-H) present will act like an acidic moiety and will become ionized - increasing the tailing observed with analytes containing basic functional groups.

Further Information

The Distribution Constant

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:

The Distribution Constant

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 liquid-liquid 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 re-enter the mobile phase, which again disrupts the equilibrium - so some analytes then re-enter 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 non-polar analyte will prefer to interact with a non-polar stationary phase than a more polar mobile phase. Remember that for chromatography “like dissolves like” - non-polar analytes will stay on a non-polar stationary phase until the mobile phase is similar enough, in terms of in polarity (or non-polarity), 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:

acid dissociation equilibrium

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

acid dissociation equilibrium

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 RP-HPLC 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, ion-suppressed 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:

Effect of pH on reversed phase HPLC

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 RP-HPLC 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 non-polar 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 non-polar 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 pH on reversed phase HPLC


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, non-polar, form will be well retained by the non-polar 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.

Ionization states of Acetic acid

Ionization states of Acetic acid (pKa 4.5) across the full pH range. Image courtesy of chemicalize.org by ChemAxon.


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 silica-based 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.

Example of ionization states of a weak base

Example of ionization states of a weak base (ammonia, pKa 8.9) across the full pH range. Image courtesy of chemicalize.org by ChemAxon.


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 non-polar 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 (Si-O-H) on the silica support will be ion-supressed. At a pH above their pKa (~pH 4), lone silanol groups act like an acid and will dissociate to give ionized Si-O- that interacts strongly with basic moieties and results in bad peak tailing. At low pH, the Si-O-H remains neutral. If the additional hydrophobic interactions do not provide sufficient retention for the analyte, one alternative would be to use a polar-embedded, 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 (self-association). 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 ZORBAX-Extend or ZORBAX-StableBond, 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 (ion-pair) 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 ion-suppress any acids, while also ion-pairing with any ionised bases.

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

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

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

An alternative to ion-pairing is possible using modified column chemistry that has counter-ions built in as part of the stationary phase, such as the PrimeSep columns from SiELC. These columns are manufactured with a variety of functional groups that can be activated by changing the mobile phase pH, without the need for any additional chemical modifiers (for more information - www.crawfordscientific.com/Sielc_Primesep.html).


In Summary

  • When working with ionizable analytes it is crucial to fix the pH of the mobile phase to control the level of ionzation

  • With ACIDIC analytes, the pH should be set at least two pH unit BELOW the pKa

  • With BASIC analytes, the pH should be set at least two pH unit ABOVE the pKa

  • If this is not possible with the column being used, consider using alternative column chemistries that are stable at extremes of pH, or those providing increased retention for more polar compounds

  • Alternatively it may be possible to ion-pair analytes to improve retention, or use column stationary phases with specific functional group chemistries that will ion-pair with ionized analytes
loading data
loading data
loading data
loading data
loading data


Amy Claydon
 

This article was written by Amy Claydon.

After graduating with a Bioveterinary Science degree, Amy completed a Ph.D. in the field of proteomics; using nanoflow-HPLC-MS to explore biological protein turnover. Amy then completed two short post-doctoral positions, becoming familiar with various aspects of quantitative HPLC-MS as well as GC-MS analysis of rodent pheromone samples. She then moved to work within a research department at the Food and Environment Research Agency using various proteomics related techniques to assess food for its authenticity and potential adulteration.

Amy joined Crawford Scientific at the start of 2014 as a Training and Technical consultant, delivering courses on all aspects of chromatography and mass spectrometry; from fundamental concepts, through to troubleshooting and method development.

“Through spending almost 10 years within the same academic laboratory I was able to get a lot of hands-on experience will all sorts of HPLC systems and mass spectrometers; including Orbitraps, Triple-Quadrupoles and Ion Mobility systems. I was involved with upkeep and maintenance of the various platforms I used, as well as training other users, all helping to ensure everything remained in good working order as much as possible. Despite this, problems arise - and so I have had my fair share of issues to troubleshoot and resolve too!

group  subsCHROMacademy can deliver to corporate clients on a multi-user subscription basis.
Served up from secure servers to the corporate intranet or individual desktops.

  • Microsite - your own learning site powered by CHROMacademy
  • Your Landing Pages -with your logo and branding
  • Customized Assessments - Based on content agreed upon Certificate of Completion
  • Certification Programs - Offer your learners a goal to strive towards
  • LMS : Connect - Our Learning Management System is S.C.O.R.M. compliant and will connect to your system
  • Engagement Package - E-newsletter stimulation program derived from your content and ours
  • Full archive of Essential Guide webcasts & tutorials
  • 1000’s of eLearning topics - HPLC / GC / Sample Prep / Mass Spec
  • Ask the Expert - our experts will answer your chromatography questions within 24 hrs.
  • Assessments - test your knowledge
  • Application notes & LCGC articles
  • Troubleshooting and virtual lab tools

Request a quote

 

 
 Home | About UsContact Us | SubscribeTerms and Conditions | Advertise | Privacy Policy 

loading data

loading data

loading data

 

loading data


loading data