Buffers

Phosphate Buffers

The phosphates are among the most widely used buffers. These solutions have high buffering capacity and are very useful in the pH range 6.5 to 7.5. Because phosphate is a natural constituent of cells and biological fluids, its presence afford a more "natural" environment than many buffers. Sodium or potassium phosphate solutions of all concentrations are easy to prepare. The major disadvantages of phosphate solutions are
1) precipiation or binding of common biological cations (Ca2+ and Mg2+) and
2) inhibition of some biological processes, including some enzymes.

Tris Buffer

The use of the synthetic buffer Tris [tris(hydroxymethyl)aminomethane] is now probably greater than that of phosphate. It is useful in the pH range 7.5 to 8.5. Tris is available in a basic form as highly purified crystals, which makes buffer preparation especially convenient. To prepare solutions, the appropriate amount of Tris base is weighed and dissolved in water. For 1L of a 0.1M solution, 12.11g (0.1 mole) of Tris base is weighed and dissoved in 950 to 975 mL of distilled water. The pH is adjusted by addition of acid (concentrated hydrochloric if Tris-HCl is desired), with stirring, until the appropriate pH is attained. Water is added to a final volume of 1L and a final pH check is made. Although Tris is a primary amine, it causes minimal interference with biochemical processes and does not precipitate calcium ions. However, Tris has several disadvantages, including
1) pH dependence on concentration, since teh pH decreases 0.1 pH unit for each 10-fold dilution;
2) interference with some pH electrodes;
3) a large <pKa/'C compared to most other buffers. Most of these drawbacks can be minimized by adjusting the pH after dilution to the appropriate concentration, purchasing electrodes that are compatible with Tris, and preparing teh buffer at the temperature at which it will be used.

Carboxylic Acid Buffers
The most widely used buffers in this category are acetate, formate, citrate, and succinate. This group is useful in teh pH range 3-6, a region that offers few other buffer choices. All of these acids are natural metablites, so they may interfere with the biological processes under investigation. Also, citrate and succinate may interfere by binding metal ions (Fe3+, Zn2+, Mg2+, etc.) Formate buffers are especially useful because they are volatile and can be removed by evaporation under reduced pressure.

Borate Buffers
Buffers of boric acid are useful in the pH range 8.5-10. Borate has teh major disadvantage of complex formation with many metabolites, especially carbohydrates.

Amino Acid Buffers
The most commonly used amino acid buffers are glycine (pH 2-3, and 9.5-10.5), histidine (pH5.5-6.5), glycine amide (pH 7.8-8.8), and glycylglycine (pH 8-9). These provide a more "natural" environment to cellular components and extracts; however, they may interfere with some biological processes, as do the carboxylic acid and phosphate buffers.