Tris Buffer
Introduction
Tris (hydroxymethyl) aminomethane is also known as THAM, with pKa 8.06. Tris (M.W: 121.14) is a white crystalline powder having molecular formula C4H11NO3. It forms a clear solution in water. Tris is used as a buffering system in most of the biological experiments.
Composition
| Reagent | 0.5M Tris buffer (1L) | 1M Tris buffer (1L) |
|---|---|---|
| Tris base | 60.57g | 121.14g |
| H2O | make upto 1000 ml | make upto 1000 ml |
Table 1. Composition of Tris buffer.
pH
The pH of the Tris buffer is adjusted based on the requirement. Predominantly, the pH range for Tris buffer is 7.2 to 9.0
When you dissolve Tris base, the pH is more alkaline. To bring down the pH, you may need higher volumes of concentrated HCl. In case of adjusting pH towards the alkaline range, use 1N NaOH.
Note:
Tris is one of the most temperature susceptible buffer systems. When you are setting the desired pH, you need to consider the deviation caused by temperature. For increment in each degree Celsius of temperature, pH drops by 0.028 pH units and vice versa. An example better explains the scenario, so let’s consider one.
You have made Tris buffer with pH to 7.4 at 25oC. For an experiment, you are using the same buffer at 37 oC. Since you have adjusted buffer pH to be 7.4 at 25 oC, at 37 oC, it would be 7.064
(12oC X 0.028 pH units = 0.336pH unit). So, if you want to use a buffer at 37oC, you have to prepare the buffer with pH 7.736 at room temperature, and it would be 7.4 at 37oC.
Preparation
- For 0.5 M (250ml)
- Take 200ml of distilled water in 500ml beaker and place it on a magnetic stirrer.
- Now add exactly weighed Tris base to the water and allow it to dissolve completely (clear solution)
- Now test the pH with pH meter by dipping an electrode into the solution.
- Adjust the pH to the desired value.
- Make up the volume to 250 ml.
Storage
Tris buffer can be stored at room temperature or at 4°C. Tris buffer is susceptible to bacterial attack. For better and long term storage, it is advisable to store at 4°C after Filter sterilization (recommended) or autoclave.