LabProcedure_174506540.ppt

GENERAL LABORATORY PROCEDURES A.PH, AND BUFFERS Measurement of pH A pH measurement is usually taken by immersing a glass combination electrode into a solution and reading the pH directly from a meter. At one time, pH measurements required two electrodes, a pH-dependent glass electrode sensitive to H+ ions and a pH-independent calomel reference electrode. Selection of a Biochemical Buffer Phosphate Buffers The phosphates are among the most widely used buffers. These solutions have high buffering capacity and very useful in the pH range 6.5 to 7.5. Because phosphate is natural constituent of cells and biological fluids, its presence affords a more “natural” environment than many buffers. Sodium or potassium phosphate solutions of all concentrations are easy to prepare using the Henderson-Hasselbalch equation. Selection of a Biochemical Buffer Phosphate Buffers The major disadvantages of phosphate solutions are (1)precipitation or binding of common biological cations (Ca 2+ and Mg 2+), (2) inhibition of some biological processes, including some enzymes, (3) limited useful pH range. Selection of a Biochemical Buffer Zwitterionic Buffers (Good’s Buffers) N.E. Good and his colleagues recognized the need for a set of buffers specifically designed for biochemical studies (Good and Izawa, 1972). Good’s buffers are widely used, but their main disadvantage is high cost. Selection of a Biochemical Buffer Several Synthetic Zwitterionic Buffers Selection of a Biochemical Buffer Selection of a Biochemical Buffer Zwitterionic Buffers (Good’s Buffers) Tris: The use of the synthetic zwitterionic buffer Tris [tris(hydroxymethyl)aminomethane] is now probably greater that of phosphate. It is useful in the pH range 7.5 to 8.5. Selection of a Biochemical Buffer Zwitterionic Buffers (Good’s Buffers) Tris: To prepare solutions, the appropriate amount of Tris base is weighed and dissolved in water. For 1 L of 0.1 mol/L solution, 12.11g of Tris base is weighed and dissolv