Preparation of 1 M Tris.Cl, pH 7.2

Overview

  • Tris is also known as THAM, Tris, Tris base, Trizma, Tris(hydroxymethyl)aminomethane
  • Tris has a pKa of 8.07 at 25°C.
  • It has an effective pH range between 7.0 and 9.0. This buffer range correspond to physiological pH range.
  • Silver-containing single-junction pH electrodes (e.g., silver chloride electrode) are incompatible with Tris (Ag-tris precipitation clogs the junction).
  • Tris has been shown to inhibit activity of many enzymes.

Requirement

  • Reagents
    • Tris base (C4H11NO3, Molecular Weight: 121.14)
    • Conc HCl
    • pH paper (optional)
    • Deionized / Milli-Q water
  • Equipment and disposable
    • Measuring cylinder
    • Conical flask / Beaker
    • Magnetic stirrer
    • pH meter

Composition: 1 M Tris.Cl

Objective:

Preparation of 1000 ml of 1 M Tris.Cl, pH 7.2

Prior to start: Calibrate the pH meter with pH standard buffers

Preparation

Step 1: To prepare, 1000 ml of 1 M Tris.Cl solution, weigh out 121.14 gram Tris base (molecular weight = 121.14) and transfer to a 2 liter beaker/conical flask. Dissolve tris base in 800 ml deionized/Milli-Q water using magnetic stirrer.

Tips:

  • One can use manual shaking using a glass pipette. Magnetic stirrer makes the dissolving process easy and convenient.

Precautions:

  • Do not dissolve in 1000 ml of deionized/Milli-Q water. In most cases, especially when the solute amount is large, the volume of the solution increases when solute is dissolved in solvent. It is essential to dissolve the solute in a small volume of solvent (e.g., 80% of the required volume). After the solute is completely dissolved and pH is adjusted (if required), make up the correct volume with the solvent.

Step 2: Adjust the pH 7.2 with concentrated HCl.

Notes: The pH of 1 M Tris solution at 25°C will be between 10.0 – 11.5

Tips:

  • Approximately 45 ml HCl is required to adjust the pH.
  • One can set the desired pH of the solution at this point with the addition of HCl (approximately 40-45 ml conc HCl is required for setting 1M Tris solution pH 7 – 9)

Precautions:

  • Use appropriate measures to protect yourself while handling the concentrated HCl.

Step 3: Adjust the volume to 1000 ml with deionized/Milli-Q water.

Step 4: Autoclave the solution for 20 minutes at 15 lb/sq.in. on liquid cycle.

Storage:

Solution can be stored at room temperature for several weeks.

Applications:

The solution is used as a buffer for several biological applications. Some of the most common applications are

  • Preparation of TAE
  • Preparation of TBE
  • Preparation of TE
To prepare Tris-Cl, pH 7.2 of various volume (100 ml, 250 ml, 500 ml and 1,000 ml) and molarity (100 mM, 250 mM, 500 mM and 1M), follow the table.
Conc. / Volume 100 ml 250 ml 500 ml 1,000 ml
10 mM 0.12 g 0.3 g 0.6 g 1.21 g
100 mM 1.21 g 3.03 g 6.06 g 12.1 g
500 mM 6.05 g 15.13 g 30.25 g 60.5 g
1 M 12.1 g 30.25 g 60.5 g

121g