Professional Documents
Culture Documents
Why Is PH Important For HPLC Buffers
Why Is PH Important For HPLC Buffers
Why Is PH Important For HPLC Buffers
Welcome mostafa mohamed
Enter your search value here Search
Over 3000 ELearning topics / 5000 Articles & Applications
Home HPLC GC Sample Prep MS Infrared Basic Lab Skills Bio Chrom Ask the Expert Forum Your Account Report Logout
Why is pH important for HPLC buffers?
Key Points
In chromatography, “like dissolves like” i.e. nonpolar analytes interact well with nonpolar 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 ionsuppressed analytes are more nonpolar than ionized analytes and so will have improved retention on nonpolar 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
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 ionsuppressed improving retention on RPHPLC
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 RPHPLC
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 ionsuppressed, neutral, form improving retention on RPHPLC
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 RPHPLC
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 (SiOH) 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:
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:
http://www.chromacademy.com/chromatography‐pH‐importance‐HPLC‐buffers.html 1/4
2/13/2018 Why is pH important for HPLC buffers?
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:
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.
http://www.chromacademy.com/chromatography‐pH‐importance‐HPLC‐buffers.html 2/4
2/13/2018 Why is pH important for HPLC buffers?
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 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.
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 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
http://www.chromacademy.com/chromatography‐pH‐importance‐HPLC‐buffers.html 3/4
2/13/2018 Why is pH important for HPLC buffers?
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 cut
off for TFA is 210 nm).
An alternative to ionpairing is possible using modified column chemistry that has counterions 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 ionpair analytes to improve retention, or use column stationary phases with specific functional group chemistries that will ionpair
with ionized analytes
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
HPLCMS to explore biological protein turnover. Amy then completed two short postdoctoral positions, becoming familiar
with various aspects of quantitative HPLCMS as well as GCMS 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 handson experience will
all sorts of HPLC systems and mass spectrometers; including Orbitraps, TripleQuadrupoles 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
Amy Claydon 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!
CHROMacademy can deliver to corporate clients on a multiuser 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 Enewsletter 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
Home | About Us | Contact Us | Subscribe | Terms and Conditions | Advertise | Privacy Policy
http://www.chromacademy.com/chromatography‐pH‐importance‐HPLC‐buffers.html 4/4