Mobile Phase Buffers in Liquid Chromatography: A Review of Essential Ideas

Buffered solutions that resist changes in pH are very important to various aspects of the practice of liquid chromatography (LC). In this installment, I discuss several essential principles related to when and why buffers are important, as well as practical factors such as commonly used buffering agents that are recommended for use with different types of detectors. This installment is intended as a prelude to subsequent installments that will explore specific troubleshooting scenarios that are impacted by effective use of buffered solutions.

In my conversations with people in all corners of the field, I find too often that there is information and understanding missing from users’ thinking about, and uses of, buffers. I think it is easy to imagine why this happens—for example, we see liquid chromatography (LC) users transplanted from other fields or disciplines, who may not have ever been exposed to the idea of buffer capacity. It can be really eye-opening, however, to field questions from users wondering why their peak shapes are bad, or their retention times are not repeatable, only to find that the problems originate with poor choices related to buffers. They have the latest LC instrument make and model. They have a shiny new mass spectrometer that gives them accurate mass and high resolution. But the major weakness of the method and analysis is the buffer. This is a situation we should avoid, of course. In response to my questions about choices related to buffers in situations like this, I also find too often responses along the lines of “That’s what the person before me did,” or “This method came from a different group, and we have to do it that way.” I certainly understand there are situations where one must implement a method that has been handed to them for a variety of possible reasons, and there is nothing I can do in this article to change that. However, I think there are many opportunities to prevent these kinds of poor choices from being made in the first place through education and formation of good habits. Upon inheriting a method, please ask, “Do these conditions make sense? Is there a better way? Has the community learned anything in the recent past that might influence what conditions we use here?” Let’s not continue propagating the old errors, myths, and bad habits of the past.

Much has been written about buffers in this “LC Troubleshooting” column, this magazine, the LCGC Blog more broadly, and in lots of other places in the past. This is an instance where I think it is fair to say that the problem is not so much a lack of information as it is digesting and sifting through the large volume of information out there to figure out what is relevant to your particular situation. This installment of “LC Troubleshooting” tries to help with that. I give an overview of the main aspects to think about, brief summaries of the prevailing wisdom in the field, and then point to examples of relevant, trustworthy sources for more information for those who want to dig deeper. The discussion is colored in a way that is most relevant to reversed-phase (RP) and hydrophilic-interaction (HILIC) separations. Of course, many of the aspects are relevant to other separation modes as well, such as ion-exchange and size-exclusion separations, but we don’t have space to address them in detail here. Perhaps we’ll follow up with those details in a different month and year.

What, and What For?

Before getting into any other details, we should establish what exactly we are referring to when we say “buffer,” and also establish why we need buffers at all. The textbook definition of a buffer is a solution that resists changes in pH upon the addition of acid or base to the solution. The extent to which a particular solution acts as a buffer depends on a handful of factors, including the nature of the buffering agent (which itself must be a Bronsted acid or base), its concentration, and the pH of the solution relative to the pK_a of the buffering agent. This buffering ability can be quantified using the concept of buffer capacity (see next section). First, though, let’s consider the chemistry that is responsible for this buffering against pH changes using the simple example of a solution of acetic acid and sodium acetate in water. If we add 10 millimoles of acetic acid and 10 millimoles of sodium acetate to enough water to make a liter of solution, we will find that at equilibrium the concentrations of protonated acetic acid (HAc for short) and deprotonated, negatively charged acetate ion (Ac^– for short) are roughly equal, and the pH of the solution will be approximately 4.8. We illustrate the idea that the quantities of these species are related by showing the following chemical reaction:

Now, if we consider what happens when we add some strong acid to the system, perhaps through the addition of some hydrochloric acid (HCl), which will introduce some H⁺ (because HCl is fully dissociated in water), Le Chatelier’s Principle tells us that some of the Ac^– will be consumed by reaction with H⁺ to produce HAc. In equation 1, the H⁺ we add from HCl is a product in the reaction, and adding a product species to a system at equilibrium will shift that equilibrium such that species on the reactant side of the equation (HAc in this case) is produced from species on the product side (Ac^– and H⁺ in this case). In this way, the H⁺ ions that are added to the solution from the addition of HCl are “soaked up” by the Ac^–, and the net change to the concentration of H⁺, which dictates the pH of the solution, is small. In this case, the solution acts as a good buffer, resisting a major change in pH, even through strong acid has been added to the solution.

But…so what? Why is it important for the mobile phase to have this buffering ability in LC? The short answer, and I think the aspect that probably gets the most attention, is that mobile phase pH can dramatically affect the separation selectivity for ionogenic compounds—that is, compounds that are prone to gain or lose protons as the pH of the solution is changed. In RP and HILIC separations, these gains and losses of protons are also accompanied by changes in the charge state of an analyte (for example, see equation 1 where acetic acid goes from zero charge to a -1 charge upon the loss of a proton), which in turn dramatically affects the water solubility of the compound and retention under RP and HILIC conditions (see the section below on selectivity effects for more information about this aspect). However, there are many other effects of buffers on LC separations and detectors, and some of these can actually be more important from a troubleshooting and method optimization point of view. A short list includes effects on peak shape, retention repeatability and reproducibility, and signal-to-noise (S/N) ratio and detection limits (particularly when using mass spectrometric detection).

The Concept of Buffer Capacity

The extent of the ability of the buffer to mitigate changes in pH because of the additions of acids or bases can be quantified using the concept of buffer capacity (β), as expressed in equation 2 (1). In this expression, [H⁺] and [OH^–] are the concentrations of hydrogen and hydroxide ions in solution, K_HA is the dissociation constant of the acidic form of the buffering agent (acetic acid, for example), and C_HA is the “formal” concentration of the buffering agent, which, in the case of the acetic acid/acetate buffering system, would be the sum of the actual concentrations of the protonated acetic acid and deprotonated acetate ion in solution. Figure 1 shows a plot of the buffer capacity versus pH for the acetic acid/acetate system. It turns out that βmaximizes when the solution pH is equal to the pK_aof the buffering agent. We see this in Figure 1 with the maximum in the curve around pH 4.8. The buffer capacity also increases at very low and high pH; in these regions the high concentrations of H⁺ and OH^- actually act as good buffers themselves.

FIGURE 1: Plot of the buffer capacity versus pH for an acetic acid/acetate buffering system. The buffer capacity maximizes when the pH is equal to the pKa of the acidic component of the buffering agent.

The buffer capacity concept is also very useful for illustrating the point that just because a solution contains a buffering agent does not mean that the solution is actually a good buffer at every pH. Indeed, Figure 1 shows that the acetic acid/acetate system has very little buffer capacity (that is, it is a terrible buffer) in the pH range of about 7 to 10. When I talk with people about their mobile phases, I frequently hear them describe a solution prepared by adding ammonium acetate to water, without further pH adjustment. The pH of this solution will be approximately 7, and Figure 1 shows that this solution actually has little ability to buffer. It is true that the ammonium ion can contribute to buffering, but the useful range of the ammonium/ammonia system only extends down to approximately pH 7.5. We should be careful to manage our expectations about what such a solution can and cannot offer to a LC method in terms of pH control of the mobile phase.

Effects of Buffers on Primary Separation Metrics

I think the two direct effects of buffers on primary separation metrics that receive the most attention are the effects of buffer pH on selectivity, and the effects of buffer type, pH, and concentration on peak shape.

Effect of pH on Selectivity

Although there are several potential effects of mobile phase pH on selectivity, which can come from changes in the ionization state of the analyte, the stationary phase, or both, changes in the ionization of the analyte are those we typically point to first. A simple view of this effect is shown in Figure 2, where I have illustrated two RP separations of a simple mixture of neutral (N), acidic (A), and basic (B) compounds. We see that at pH 2 the neutral is separated, but the acid and base are coeluted. If the acidic compound has a carboxylic acid functional group, it will be protonated and neutral at pH 2. The basic compound will be protonated and positively charged. When we move to pH 7, however, we see the retention time of the acid decreases dramatically, because it has a pK_a between 2 and 7, loses a proton upon the move to pH 7, and becomes negatively charged and much more water soluble. The base, however, remains unchanged in the move from pH 2 to 7, because its pK_a is well above 7, and the ionization is unaffected by the move from pH 2 to 7. This simple illustration emphasizes the importance of considering mobile phase pH as a variable during method development. Readers interested in learning more about this aspect are referred to reference (2).

FIGURE 2: Simple illustration of the effect of mobile phase pH on selectivity of an RP separation when the sample contains ionogenic compounds.

Effects on Peak Shape

There are also several different mechanisms through which a buffer can affect peak shape in RP and HILIC separations. These include effects on the chemistry of interaction partners influencing retention (for example, whether or a silanol group on the surface of a silica particle is charged or not), and shielding of injected analytes from charged molecules adsorbed to the stationary phase surface, which can affect “loadability” (that is, the amount of analyte mass that can be injected before poor peak shapes are observed). There is extensive literature on these topics. For representative articles that discuss these aspects, readers are referred to references (3) and (4).

Practical Factors

In addition to the conceptual aspects of what a buffer is, its effective pH range, and its primary effects on actual separations, there are many “practical factors” to consider when choosing a buffering agent and how to use it.

Buffers and Bugs

Microbes will grow readily, and sometimes rapidly, in solutions that are buffered at or near physiological conditions (for example, dilute phosphate buffers around pH 7 are microbe-friendly). This means that, if you are trying to buffer mobile phases under these conditions, you will need to do something to prevent microbial growth, otherwise microbes can cause serious damage to LC columns and other instrument components. The most common approaches to prevent microbial growth (aside from working far from pH 7, which is not always possible) are to use additives that will kill the bugs or prevent their growth in the first place, but not affect the chromatography (for example, sodium azide), or add some (10% or so is a good starting point) organic solvent such as methanol or acetonitrile to the buffered solution. Of course, this is not always an option. Another approach is to maintain sterile conditions for the buffer solution at the time of preparation (for example, through filtering) and throughout its use (for example by preventing infiltration of microbes from laboratory air). Readers interested in learning more about these details are referred to references (5) and (6).

Solubility

Different buffering agents vary tremendously in terms of their solubilities in water. This becomes more interesting when we introduce organic solvents into the picture, as is the case with mobile phases used for RP and HILIC separations. For example, my research group has found that ammonium acetate is quite soluble in even neat methanol, whereas ammonium formate is not nearly as soluble. One place where these solubilities are very important, and I think this might be surprising to many chromatographers, is when we mix two or more solvents in LC pumps. For example, it is common to use something like 50 mM sodium phosphate in water in one pump channel, and neat acetonitrile (ACN) in another pump channel. Sodium phosphate is not soluble in all proportions of mixtures of ACN and water, thus we have to be extremely careful that we don’t instruct the pump to produce an ACN/buffer mixture that will cause the phosphate to precipitate inside the pump. Doing so can rapidly cause damage to the pump because of the abrasive nature of the precipitates. The most extensive, systematic study I am aware of on the solubility of buffering agents commonly used in LC in organic solvent/water mixtures was published by Schellinger and Carr nearly two decades ago (7), and I highly recommend incorporating the findings of this work into your knowledge base for LC work.

Care for Your Column

When choosing a buffering agent, and especially the pH range of interest, we must keep the well-being of our columns in mind. The conventional advice for columns containing silica-based particles that have been modified by covalently bonding ligands to the silica surface is to work in the pH range of about 2 to 8. At pH levels below 2, the siloxane bonds tethering the ligands to the silica surface can be hydrolyzed, and at pH levels above 8, the silica itself will dissolve. Over the past two decades, there has been a flurry of research and development among manufacturers working to beat this limitation of conventional silica-based phases, and now it is easy to find products that can be used safely below pH 2 and above 8. However, each of these products is a little different, and users should pay close attention to the manufacturer’s advice provided with documentation inside the column box (or on their websites). Of course, there are many other exceptions as well. For example, materials based on organic polymers (for example, polystyrene-based phases) or other metal oxides (for example, alumina, titania, and zirconia) generally do not have the same pH limitations as silica, but have other practical limitations. Readers interested in learning more about these aspects are referred to references (8–10).

The Tug-of-War with Detectors

One of the cruel twists of nature that appears in the practice of LC is that the buffers that are optimal for the chromatography itself are often not optimal for the detector that is coupled to the outlet of the column. For example, phosphoric acid and phosphate salts are great buffering agents that are transparent to ultraviolet (UV) detection at least down to 210 nm. However, these same phosphate-containing buffers are effectively forbidden when using mass spectrometric (MS) detection because the phosphate is not at all volatile. On the other hand, MS-friendly buffering agents, such as dilute formic acid and ammonium salts of organic bases such as acetate and trifluoroacetate, absorb UV light pretty strongly around 210 nm, which leads to a number of complications, including baseline drift and noise. Readers interested in a more detailed discussion of these issues are referred to the excellent discussion in reference (11).

Baselines and Purity

Adding anything to a LC mobile phase, whether used for buffering the pH or some other purpose, increases the risk of introducing impurities that can later show up in the detector in the form of drifting baselines, new peaks in chromatograms that having nothing to do with injected samples, or even serious interferences with detection in general. One of the most spectacular examples of this from my own research group was a case where a new bottle of formic acid contained an impurity that suppressed ionization of proteins we were working with to the point that we thought something was seriously wrong with our mass spectrometer. In fact, simply switching back to a previous source of formic made the problem go away entirely. When choosing buffering agents and preparing the buffers, one should be careful to consider the availability of high purity reagents, and the ways that low level impurities might impact that the LC assay at hand. Readers interested in learning more about this aspect are referred to reference (12).

Making the Buffer in the Laboratory

When we go to actually make a buffer in the laboratory, there are multiple approaches we can use. The major distinction I see has to do with deciding whether to use a pH meter-guided approach or a gravimetric approach. Consider the preparation of a sodium phosphate buffer at pH 6. In the pH meter-guided approach, we might start by adding phosphoric acid to water, and then add sodium hydroxide until a pH meter tells us we have arrived at pH 6. This approach is very common in biochemistry laboratories. In the gravimetric approach, we would first calculate the masses of the mono- and dibasic sodium hydrogenphosphate salts needed to reach our target pH of 6. Then, we go to the laboratory, weigh out the salts, and add them to water. No pH meter is needed, other than to verify that a gross error was not made along the way. We find that the gravimetric approach is much more repeatable than the pH meter-guided approach and tends to be much faster as well. Readers interested in learning more about this aspect are referred to references (13) and (14).

Filter, or Not?

When preparing a buffered mobile phase, one must eventually decide whether or not to filter the solution prior to use with an LC instrument. On the surface, the answer seems simple—the solution should of course be filtered to remove any particulates or debris that might have been added to the solvent from the buffering agent. However, the filtration process itself can actually introduce more problems than it solves, if not executed properly. For example, filter materials can leach impurities into the solvent, and a filtration apparatus and environment that are not clean can also be a source of unwanted impurities in the filtered solution. In my laboratory, we use the guideline that we do not filter solutions after adding buffering agents that are liquids themselves (for example, formic acid, phosphoric acid, or ammonia), but we do filter after adding solid reagents such as sodium phosphate and similar salts, and we find this works pretty well on average. Readers interested in learning more about these details are referred to reference (6).

Other Important Aspects

One aspect that is very important when considering buffers for RP and HILIC separations is that the addition of organic solvents to water can have a dramatic effect on the apparent pH of the resulting solution compared to the pH prior to the addition of solvent. This is a result of the effect of the organic solvent on the acid dissociation constants of components in solution, whether they are buffering agents of the mobile phase, or analytes that we inject into the mobile phase for analysis. Readers interested in learning more about this aspect are referred to references (13) and (15).

Summary

In this installment, I have reviewed the essential considerations for effective use of buffers in RP and HILIC separations. Although we often point first to the effects of mobile phase pH and buffers on separation selectivity and peak shape, there are actually a number of other practical factors that are also strongly influenced by buffer choice. The LC community has learned a lot over the past few decades about effective use of buffers, and users are encouraged to implement these learnings at the method development stage. When problems arise with existing methods, this knowledge base also serves as a tremendous resource for fixing buffer-related problems.

References

(1) Tindall, G. W. Mobile-Phase Buffers, Part II — Buffer Selection and Capacity. LCGC North Am. 2002, 20 (12), 1114–1118.

(2) Dolan, J. W. Back to Basics: The Role of pH in Retention and Selectivity. LCGC North Am. 2017, 35 (1), 22–28.

(3) Stoll, D., R. Essentials of LC Troubleshooting, III – Those Peaks Don’t Look Right. LCGC North Am.2022, 40 (6), 244–247.

(4) McCalley, D. V.; Stoll, D. R. But My Peaks Are Not Gaussian! Part III: Physicochemical Causes of Peak Tailing. LCGC North Am. 2021, 39 (11), 526–531,539.

(5) Dolan, J. W. Mobile Phase Problems. LCGC Magazine 1997, 5 (10), 874–878.

(6) Stoll, D. R. Filters and Filtration in Liquid Chromatography - What to Do. LCGC North Am. 2017, 35 (2), 98–103.

(7) Schellinger, A. P.; Carr, P. W. Solubility of Buffers in Aqueous–Organic Eluents for Reversed-Phase Liquid Chromatography. LCGC North Am. 2004, 22 (6), 544–548.

(8) Dolan, J. W. Column Care. LCGC North Am. 2008, 26 (8), 692–695.

(9) Dolan, J. W. Column Care and Feeding. LCGC Magazine1993, 11 (1), 22–24.

(10) Dolan, J. W. Detective Work, Part IV: Chemical Problems with the Column: Chemical Attack. LCGC North Am. 2016, 34 (2), 106–113.

(11) Snyder, L.; Glajch, J. L.; Kirkland, J. J. 7.2 Acidic and Basic Samples. In Practical HPLC Method Development; Wiley, 1997.

(12) Dolan, J. W. Buffers and Baselines. LCGC Magazine2001, 19 (6), 590–594.

(13) Tindall, G. W. Mobile-Phase Buffers, Part III — Preparation of Buffers. LCGC North Am. 2003, 21 (1), 28–32.

(14) Stoll, D. R.; Makey, D. Mobile Phase Buffers in LC: Effect of Buffer Preparation Method on Retention Repeatability. LCGC North Am. 2019, 37 (7), 444–449.

(15) Rosés, M. Determination of the pH of Binary Mobile Phases for Reversed-Phase Liquid Chromatography. J. Chromatogr. A 2004, 1037 (1–2), 283–298. DOI: 10.1016/j.chroma.2003.12.063

About the Author

Dwight R. Stoll is the editor of “LC Troubleshooting.” Stoll is a professor and the co-chair of chemistry at Gustavus Adolphus College in St. Peter, Minnesota. His primary research focus is on the development of 2D-LC for both targeted and untargeted analyses. He has authored or coauthored more than 75 peer-reviewed publications and four book chapters in separation science and more than 100 conference presentations. He is also a member of LCGC’s editorial advisory board.