Buffer Solutions

Characteristics of the pH

Not only in the laboratory but also in daily life we come across acidic and basic foods, beverages and cosmetics. Apart from largely neutral solutions such as the human saliva, pH values are divided into two areas:

• Acidosis: denotes a value below the pH value of 7.35. Examples: gastric acid (pH 1.2 to 3.0) & carbon dioxide (pH at about 4.68)
• Alkalosis: describes a value above pH 4.45. Examples: human blood (pH 7.35 to 7.45) & cosmetic soap (pH between 9 and 10)

Buffer solutions are substances that can avert a drastic change in the pH value after the addition of an acid or base. Suitable buffer solutions are either solutions composed of a weak acid and the alkali metal salt of this acid or mixtures of a weak base and the salt of this base.

Temperature influences the pH value

The pH value of a buffer solution can change depending on the substance and temperature. Therefore, it is advisable to set the pH value at the temperature at which you will be working later. In most animal cells, the physiological pH value at 37°C is between 7.0 and 7.5. Tris (see above) is a buffer particularly sensitive to temperature. Set to 7.5 at 37°C, its pH rises to approximately 8.5 in the test system at 0°C. At 0°C, cell extracts are often used in vitro (Scopes, 1994). Generally, good buffers are less sensitive to temperature, and carboxylic acid buffers (citrate, formate, and succinate) are even less sensitive.

In practical terms, this means the buffer should be brought to the appropriate temperature, and the pH electrode should be calibrated at that temperature. Many modern pH meters have an integrated function that allows users to set the pH value at room temperature for different working temperatures (e.g., +4°C or +37°C). However, this approach has limitations. The dpKa/dT value, or the change in pKa as a function of temperature, is not the same for all buffers. For instance, the pKa value of Tris increases by 0.028 units when the temperature rises by 1°C, whereas the pKa value of HEPES increases by only 0.014 units. Inaccuracies are unavoidable with this method because these values should be considered by the pH meter.

Titration

The pH value is typically adjusted using NaOH/KOH or HCl. Adding the acid or alkali slowly while stirring vigorously prevents high concentrations of H⁺ or OH^− ions from forming locally. Otherwise, the buffer substances may be altered chemically in such a way that they become inactive or have an inhibitory effect in their modified form. When both the protonated (acid) and non-protonated (base) forms of a buffer are available, the pH can be adjusted by mixing the two substances.

If monovalent cations are present or are to be investigated, the pH can be adjusted using tetramethyl or tetraethylammonium hydroxide. Acetate, sulfate, or glutamate can be used instead of HCl, though enzyme interference is more likely to occur.

Adjusting the Ionic Strength

As with adjusting the pH value, select an electrolyte to adjust the ionic strength of the buffer solution, if necessary, because the ionic strength increases depending on the electrolyte used. Salts of tetramethyl- or tetraethylammonium are suitable for this purpose because the large cations interact less effectively with the negative charges of the enzymes. Acetate, as a large anion, interacts poorly with alkali metals.

Buffer additives

Changes in the pH value are to be expected if other components are added to the buffer (e.g., EDTA, DTT, or Mg²⁺), which is why it should be measured again. In living cells, glutathione prevents the oxidation of proteins by various substances. When lysing cells, a reducing agent such as β-mercaptoethanol (5–20 mM) or DTT (1–5 mM) must usually be added. B-mercaptoethanol oxidizes within 24 hours of being added to the buffer. Therefore, it is advisable to include this substance in the buffer only during protein processing and use DTT for long-term protein storage.

To prevent bacterial or fungal growth, especially in buffers with a pH between 6.0 and 8.0, it is recommended to either perform sterile filtration (0.22 μm) or add 0.02% (3 mM) sodium azide. When added to concentrated stock solutions during dilution to the working concentration, sodium azide is diluted to such an extent that it generally no longer causes problems.

pH Meter Check

Nowadays, accurate pH meters with digital displays are widely available for adjusting the pH value of a buffer solution. The meter is calibrated using two pH standards that cover the buffer's range. If the accuracy of the measurement is in question, it can easily be remedied by standardizing the pH meter with a 50 mM phosphate buffer, which is then diluted tenfold. The pH value should now be 0.2 units higher.

Aqueous solutions containing nutrients for microbes are at risk of becoming contaminated with germs. This includes many buffer solutions. Various methods are used to preserve the solutions:

• Heat sterilization by autoclaving
• Sterile filtration through filters with pore sizes ≤0.22 µm
• Refrigerated storage
• Storage as a highly concentrated stock solution.

Preservatives, such as sodium azide, are often added to commercial products.

Autoclaving Buffer Solutions

Some buffer substances are stable enough to be autoclaved without any problems, provided that all the components and reaction products of the buffer solution are also stable. For instance, ammonium and pyridine buffers are unstable and cannot be autoclaved. Carbonate buffers also cannot be autoclaved because they release CO₂ in open systems at temperatures above 20°C. At temperatures above 100°C, the buffering effect is completely lost. Filtration is also the preferred method for sterilizing the following buffer substances: HEPES, HEEPS, imidazole, MOPS, taurine, and TEA.