How to Make 100 ml of 100 mM Tris-HCl and 100 ml of 100 mM Tris Base and Analyze pH Changes from 0 to 50 Degrees Celsius

To prepare 100 ml of 100 mM Tris-HCl and 100 ml of 100 mM Tris base, you will follow these steps:

Materials Needed:

• Tris base
• Hydrochloric acid (HCl)
• Distilled water
• pH meter or pH paper
• Beakers and graduated cylinders
• Thermometer

Preparation of 100 mM Tris-HCl:

1. Weigh out 12.1 g of Tris base (Tris[hydroxymethyl]aminomethane).
2. Dissolve the Tris base in about 80 ml of distilled water.
3. Carefully add hydrochloric acid (HCl) dropwise while constantly stirring the solution until you reach a pH of 7.4. You may need to calculate the amount of HCl needed to achieve this pH.
4. Once you reach the desired pH, transfer the solution to a 100 ml volumetric flask and make up the volume to 100 ml with distilled water.

Preparation of 100 mM Tris Base:

1. Again, weigh out 12.1 g of Tris base.
2. Dissolve in about 80 ml of distilled water.
3. No pH adjustment is needed since this is Tris base. Just make up the volume to 100 ml after dissolving.

Combining the Solutions:

Now, you have both 100 mM Tris-HCl and 100 mM Tris base. When you mix equal volumes of both solutions (50 ml of each), you will get a final volume of 100 ml of a mixed buffer solution. The resulting pH will depend on the ratio of the acid and base components in the solution, and it is usually around 7.4.

Monitoring pH Changes:

To observe the changes in pH as the solution is heated from 0 to 50 degrees Celsius, follow these steps:

1. Prepare a water bath or heating device that allows you to gradually increase the temperature.
2. Use the pH meter to measure the initial pH at 0 degrees Celsius.
3. Heat the solution slowly, and take pH measurements at regular intervals (e.g., every 5 degrees up to 50 degrees Celsius).
4. Record the pH values and analyze how the pH changes with temperature. Generally, the pH of Tris buffers decreases as temperature increases.

This experiment will help you understand the buffering capacity of Tris in response to temperature changes, which is important for various biochemical applications.