Buffers: What are they and how do they work?

Buffers are aqueous solutions that resist changes in pH by reacting with excess hydrogen ions to balance the concentration of H⁺ and OH^- in a solution. Chemically, buffers consist of a weak acid and its conjugate base or a weak base and its conjugate acid. When an acidic solution is added to a buffer, the weak acid reacts with the excess H⁺ ions to form more of the conjugate base, thus decreasing the concentration of H⁺ ions to regain a balance with the OH^- ion concentration.

Buffers work best when they have an acid dissociation constant (pK_a) value near the pH of your target solution. The relationship between pH, pK_a, and the ratio of acid and conjugate base concentrations can be described by the Henderson-Hasselbalch Equation [1]:

pH = pK_a + log₁₀([A-])/([HA])

where [A^-] is the concentration of the conjugate base and [HA] is the concentration of the weak acid.

It is also worth noting that the measured pH of a buffer is dependent on temperature. More specifically, the pK_a decreases with increasing temperature, therefore it is recommended that buffer solutions are designed with the temperature in which the reaction will occur in mind [2].

Why are buffers important?

Experiments performed in molecular biology labs require the successful completion of many chemical reactions, which in turn require a favorable pH range. Unfavorable pH ranges can catalyze unwanted reactions. For example, acidic conditions can induce DNA depurination, which is a common problem for both living cells and synthetically produced nucleic acids [3,4]. That is why we recommend storing IDT oligos in TE buffer (IDTE, pH 7.5 or 8.0), which maintains a constant, near-neutral pH.

What is the difference between a buffer and a reagent?

The term “reagent” is commonly referred to as any solution or consumable material that is used for scientific purposes (just like how “ingredients” are used when cooking!). This isn’t far off from the technical definition—a reagent is any substance that is intended to participate in a chemical reaction. Alcohols, such as ethanol and methanol, are examples of common laboratory reagents that can be used in a vast range of chemical processes, from fermentation to fueling engines. Solid substances can be reagents too. For example, when making a reaction mix for PCR, your primers, DNA polymerase, and dNTPs are all considered reagents. Thus, reagents encompass a broad category of materials with diverse chemical roles, while buffers are specifically capable of resisting pH changes and mediating the balance of hydrogen ions. To put it simply, all buffers are reagents but not all reagents are buffers.

Good’s buffers

Although there are many solutions with great buffering capacity, when someone refers to the solution as a “good” buffer." They might be referring to one of a specific set of 20 buffers, appropriately (and somewhat amusingly) named Good’s buffers after the researcher Dr. Norman Good. Dr. Good published the list based on a set of 10 criteria that he and his colleagues developed in the 1960s [5-7]. The criteria include characteristics that would be particularly useful in research environments, such as selecting for buffers that do not easily accumulate in or permeate cell membranes and would not interfere with spectrophotometric assays due to their light absorbance wavelengths. Tris buffer, commonly found in labs as TE or TBS, is on Good’s list. However, there is no need to limit yourself to Dr. Good’s selections—for example, phosphate buffers are very commonly used in biological research despite not being an official Good’s buffer. 

Is water a good buffer?

Unlike the solutions on Good’s list, water is not a good buffer. Water molecules break down to produce H⁺ and OH^- ions in an equal 1:1 ratio, so there are no “extra” ions available to react with additional H⁺ or OH^- ions when an acid or base is added to the water. However, water that is not entirely pure, such as tap water, may possess some limited buffering capacity due to the presence of dissolved minerals.

Deionized, reverse osmosis, and/or distilled water are even worse buffers than tap water because many of their dissolved solutes (including ions) have been removed. Pure water at 25°C is completely neutral with a 7.0 pH, but unlike buffer solutions, it is not able to resist fluctuations in pH upon the addition of acids or bases.

Wondering which buffer to use for rhAmpSeq™ experiments, annealing DNA oligos, or other experimental procedures? Check out our buffer FAQs or contact us today!