BioChemistry & Metabolism

Experiment 1

Introduction

The preparation of buffer solutions is a common task in the lab, especially in biological sciences. A buffer is a solution that resists a change in pH, because it contains species in solution able to react with any added acid or base, according to the principles of equilibrium. Thus, Buffer is a solution‌ ‌which‌ ‌tends‌ ‌to‌ ‌maintain‌ ‌a‌ ‌constant‌ ‌pH‌ ‌when‌ ‌excess‌ ‌acid‌ ‌or‌ ‌base‌ ‌is‌ ‌added.

Acidic buffer solution: An acidic buffer solution is simply one which has a pH less than 7. Acidic buffer solutions are commonly made from a weak acid and one of its salts - often a sodium salt.

Alkaline buffer solution: An alkaline buffer solution has a pH greater than 7. Alkaline buffer solutions are commonly made from a weak base and one of its salts. A frequently used example is a mixture of ammonia solution and ammonium chloride solution.

Although a buffer will resist a change in pH, eventually enough acid or base can be added to destroy it. The amount of acid or base needed to change the pH of a buffer is known as the "buffering capacity."

A simple phosphate buffer is used ubiquitously in biological experiments, as it can be adapted to a variety of pH levels, including isotonic. This wide range is due to phosphoric acid having 3 dissociation constants, (known in chemistry as a triprotic acid) allowing for formulation of buffers near each of the pH levels of 2.15, 6.86, or 12.32. Phosphate buffer is highly water soluble and has a high buffering capacity, but will inhibit enzymatic activity and precipitates in ethanol. The buffer is one of the most popular currently used, and is commonly employed in molecular and cell biology, chemistry, and material science, among many others.

Materials required

pH meter, K₂HP0₄.7H₂0, KH₂P0_4.H₂0, Distilled water, 1 N NaOH, 1 N HCL

Procedure

Preparation of 0.1 M potassium phosphate buffer at 25°C

* Phosphates sequester divalent cations such as Ca2+ and Mg2+.

1. Prepare

(a) 0.5L of 1M K2HPO4 at 174.18g mol-1 = 87.09g

(b) 0.5L of 1M KH2PO4 at 136.09g mol-1 = 68.045g

2. Mix the above mentioned two solutions to set desired pH and desired volume.

3. Make up the final volume with Distilled water.

4. Adjust pH of the solution to final desired pH using 1M NaoH and 1M HCl solutions.

Reference Material

1. Preparation of Buffers

2. Buffers and pH meter

Questions

1. How does a Buffer Control the pH of a Solution?

2. What Are the Criteria to Consider When Selecting a Buffer?

3. What are the essential components of a buffer?