A buffer solution is a chemical system designed to resist significant changes in pH when a small amount of acid or base is added. Buffers are typically mixtures of a weak acid and its conjugate base, or a weak base and its conjugate acid. Tris(hydroxymethyl)aminomethane, commonly referred to as Tris, is a widely employed biological buffer effective in the near-neutral to slightly alkaline pH range. Tris-HCl buffer is used extensively in molecular biology and biochemistry for applications like DNA/RNA electrophoresis, protein purification, and enzyme assays, where maintaining a stable pH is required for molecular stability and reaction success.
Fundamental Principles of Tris Buffer
Tris functions as a buffer because it is a weak base that accepts hydrogen ions from the solution. Adding hydrochloric acid (HCl) to Tris base creates the Tris-HCl buffer system, consisting of the weak base (Tris) and its conjugate acid (Tris-H+). This system provides an effective buffering range typically between pH 7.0 and 9.0, aligning with the physiological pH of many living systems.
A buffer is most effective when its pH is close to the chemical’s pKa value, which for Tris is approximately 8.06 at 25°C. Tris is highly sensitive to temperature changes; the pKa decreases by about 0.031 pH units for every 1°C increase. This means the solution becomes more acidic as it warms. Because of this temperature dependence, the final pH must be measured at the temperature at which the buffer will be used.
Calculating and Preparing the Stock Solution
Preparation begins by calculating the mass of Tris base needed for the desired molarity and volume of the concentrated stock solution. Tris base has a molecular weight of 121.14 grams per mole ($\text{g/mol}$). For example, preparing 1 liter (L) of a 1 molar (M) Tris stock solution requires weighing 121.14 grams of Tris base powder.
The measured Tris base is added to a clean flask containing distilled or deionized water. The solid is dissolved in approximately 80% of the total required volume. This volume reservation accounts for the increase in volume that occurs during the subsequent addition of acid for pH adjustment. The mixture must be stirred continuously until the Tris base is completely dissolved, resulting in a highly alkaline solution.
Step-by-Step pH Adjustment and Finalization
Converting the basic Tris solution into the Tris-HCl buffer requires the slow, controlled addition of a strong acid, typically concentrated hydrochloric acid ($\text{HCl}$). This titration converts the Tris base into its conjugate acid (Tris-H+), establishing the buffering system. The process must be monitored with a properly calibrated pH meter, as the pH changes rapidly with each addition of concentrated acid.
The most important procedural detail is ensuring that the pH measurement is taken at the solution’s final intended usage temperature, which is often 25°C. Because the pH of Tris buffer is highly sensitive to temperature, adjusting the pH at the wrong temperature will result in an inaccurate, off-target pH. Concentrated $\text{HCl}$ is added dropwise, with constant stirring, until the pH meter displays the target pH value. Once the desired pH is reached, the final step is to bring the solution’s volume up to the mark using distilled water.
Quality Control and Storage
After the Tris-HCl buffer is prepared and the final volume is adjusted, quality control steps ensure its reliability for laboratory experiments. The final pH should be double-checked with the calibrated meter to confirm it is exactly at the target value for the specified temperature. Errors, such as measuring pH at the wrong temperature or overshooting the target, compromise the buffer’s performance.
For long-term stability and to prevent microbial growth, the prepared buffer is often filtered through a $0.22\text{ }\mu\text{m}$ sterile filter. The solution should be transferred to a clean, sealed container and stored at $4^\circ\text{C}$ to extend its shelf life. Each container must be clearly labeled with the buffer’s name (Tris-HCl), its concentration (e.g., 1 M), the final pH (e.g., pH 8.0), and the date of preparation.