Buffer Preparation. Learning Objectives:

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1 Proteomics Buffer Preparation Buffer Preparation Maintaining the optimum ph during the biological sample processing is to maintain the proper functional and structural aspects of the sample. It is important to understand the theory behind the buffering action. Learning Objectives: After interacting with this learning object, the learner will be able to: Define buffer preparation and the mechanism involved. Operate to optimize the ph of the buffer. Assess the troubleshooting steps involved in the experiments. Note: The current IDD exists in two modes- interactive and automatic. Students taking lab course should select interactive (set as default), while the automatic mode may be selected for general users.

2 Buffer Preparation Mechanism of Buffer Action The mechanism of buffer action can be understood by considering an acidic buffer made of a weak acid like Acetic acid and its sodium salt Sodium acetate. Under normal conditions, weak acid exists as shown in the equation with the equation largely to the left. Sodium acetate however, exists as acetate ions and Na+ ions. When a small amount of strong acid like HCl is added to the buffer, the acetate ions from the dissociation of acetic acid and also sodium acetate combine with the H+ ions to form Acetic acid. Thus, the buffer is able to mop up the H+ ions thereby maintaining the ph of the system.

3 Buffer Preparation Mechanism of Buffer Action Again, by considering an acidic buffer made of a weak acid like Acetic acid and its sodium salt Sodium acetate, the mechanism of buffer action can be understood. Under normal conditions, weak acid exists as shown in the equation with the equation largely to the left. Sodium acetate however, exists as acetate ions and Na+ ions. When a small amount of strong alkali like NaOH is added to the buffer, the H+ ions from the dissociation of acetic acid combine with the OH- ions of NaOH to form Water. Thus, the buffer is able to mop up the OH- ions thereby maintaining the ph of the system.

4 Buffer Preparation Mechanism of Buffer Action The ka value can be found by the formula 4. The Ka for acetate buffer is already known. Substitute the values like concentration of sodium acetate, acetic acid, ka to find the ph. The calculation shown here would help better understand the method for buffer preparation and calculation of ph of a given solution.

5 Buffer Preparation ph meter Standardization The ph meter should be calibrated regularly to avoid any error in reading the actual ph of the solutions. For this, standard buffer solutions may be used depending on the ph range of the solution that are commonly used in the laboratory. Wipe the bulb of the ph meter with water and clean tissue paper to remove any salts which may interfere with the calibration of the ph meter. Now press the calibrate button on the ph meter and place the bulb in standard buffer solution of ph 4. Set the ph meter at 4 by pressing enter. Take the bulb out of the buffer solution and again wash it with water. Wipe the bulb with a dry tissue to remove any water on the bulb. Repeat the calibration step with standard solution of ph 10. The ph meter is now calibrated and ready to use.

6 Buffer Preparation ph Instrument Mechanism Working The Glass electrode in a ph meter consists of internal reference electrode (silver/silver chloride electrode) and a glass bulb at the end which is sensitive to the hydrogen ion concentration. Both sides of the bulb get protonated when the bulb is dipped in the solution whose ph is to be measured. The meter measures the sum of all the potentials in the system, including the potential across the glass, the internal electrode and the reference electrode. The equation used to relate the changing membrane potential to the concentration of hydrogen ions, or ph of the test solution, is derived from the Nernst equation which is shown below.

7 Buffer Preparation Buffer Preparation Prepare TBST by weighing NaCl and Tris base buffer of required amounts. Add Water upto 3/4th the total volume and dissolve the solutes. The addition of Tween 20 should be avoided till the ph of TBS is set to 7.6 using HCl since it is always better not to expose the bulb of the ph meter to detergents. After addition of sufficient volume of water to dissolve the solutes, place the beaker on a magnetic stirrer. Once the dissolution is complete, the ph of the buffer solution can be adjusted. Wash the ph electrode with distilled water thoroughly and wipe it with dry tissue paper. Now dip the electrode in the beaker containing TBS such that the bulb is totally immersed in the buffer. Adjust the ph to 7.6 using HCl. Care should be taken while adding HCl after the ph has reached 8 since the ph can quickly reach 7.6 and can go even lower. Just in case, the ph goes lower than 7.6, the ph can be increased using NaOH though its use should be avoided by taking care initially while adding HCl. Once the ph is set to 7.6, wash the electrode with distilled water and wipe it with a dry tissue paper. Place the electrode back into the KCl solution to protect it from getting dried thereby preventing any damage. Make up the volume of TBS from 750ml to 1000ml using water in a measuring cylinder. Add 1ml of Tween-20 to the TBS solution and mix the solution thoroughly. TBS-T of ph 7.6 is now ready for use.

8 Buffer Preparation Buffer Preparation

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