Buffers for Biological Systems ©2010, Promega Corporation. Confidential and Proprietary. Not for Further Disclosure. What do buffers do? Buffers function to resist changes in hydrogen ion concentration Biologists often think of buffers as doing much more: •providing essential cofactors •providing critical salts •providing essential nutrients for cells and tissues However, you cannot overlook the basic function: resisting changes in hydrogen ion concentration. If you do, your reactions could be compromised. Confidential and Proprietary. Not for Further Disclosure. What makes a buffer system? A buffer consists of a weak acid and its conjugate base (or a weak base and its conjugate acid). Weak acids and bases do not dissociate completely in water, but instead exist in solution as an equilibrium of dissociated and undissociated species. For acetic acid, we would express this equilibrium like this: HAc (acetic acid) ↔ H+ + Ac– Hac can release H+ to neutralize OH– and form water. The conjugate base Ac– can react with H+ ions added to the system to form acetic acid. In this way pH is maintained as equilibrium of the three species is maintained. Confidential and Proprietary. Not for Further Disclosure. How buffers work All buffers have an optimal pH range over which they are able to moderate changes in hydrogen ion concentration. This range is a factor of the dissociation constant of the acid of the buffer (Ka) and is generally defined as the pKa (–logKa) value plus or minus one pH unit. pKa can be determined using the Henderson-Hasselbalch equation. So… a buffer system with a pKa of 3.4 would have an optimal pH range of 2.4–4.4 and would not be an effective buffer at 7.0. We present the derivation of the H-H equation in the P&A Guide Buffers chapter. Confidential and Proprietary. Not for Further Disclosure. Buffers with more than one dissociation constant Polyprotic acids are acids that can lose more than one hydrogen ion. Since more than one hydrogen ion can dissociate in solution, the acid has more than one pKa value. Multiple pKavalues are usually denoted as pKa1, pka2, etc. If the pKa values are close together, the optimal pH for the buffer will be a continuum determined by the range of pKas. Citric acid is a polyprotic acid that can lose three protons, and the pKas for each dissociation are close together, so a citric acid buffer will be effective over a continual pH range, from approximately 2–7.4. Citrate pKa1 3.13 Citrate pKa2 4.76 Citrate pKa3 6.40 Confidential and Proprietary. Not for Further Disclosure. What makes a “Good” buffer In 1966 Norman Good and colleagues developed criteria for buffers for biological systems. 1. A pKa between 6 and 8. 2. Solubility in water. 3. Exclusion by biological membranes. 4. Minimal salt effects. 5. Minimal effects on dissociation from changes in temperature and concentration. 6. Minimal interactions between buffer components and critical reaction components. 7. Chemical stability. 8. Light absorption outside of wavelengths used for assay readout. 9. Components should be easy to obtain and prepare. Confidential and Proprietary. Not for Further Disclosure. Optimal buffering at a neutral pH Most biochemical reactions have an optimal pH in the range of 6–8, so buffers for these reactions need to have pKas that support buffering at these pH values. Some buffers and their pKa values (in water at 25°C): Acetate 4.76 PIPES 6.76 MOPS 7.20 HEPES 7.48 Tris 8.06 Borate 9.23 Confidential and Proprietary. Not for Further Disclosure. from: Stoll and Blanchard. 1990. Meth. Enzymol. 182, 24–38. Solubility in Water Most biochemical reactions occur in aqueous conditions, so your buffering components should be soluble in water. If for some reason, you will be using a solvent other than water, make sure you understand how that solvent affects the dissociation of your buffer components. Confidential and Proprietary. Not for Further Disclosure. Minimal salt interactions If the system to be studied requires salts, appropriate ions can be added. However, using an ionic buffer can adversely affect the reaction if reaction studied is affected by salts. Confidential and Proprietary. Not for Further Disclosure. Minimal effects on dissociation from changes in concentration Changes in dissociation resulting from changes in concentration are usually small, and most buffers can be made as stock solutions that are diluted to working solutions. However, some buffers do show a significant change in pH upon dilution. For instance, the pH of Tris decreases approximately 0.1 pH unit per tenfold dilution, and the pH could change dramatically if you dilute a working solution and are at the limits of the optimal buffering range of the Tris. Confidential and Proprietary. Not for Further Disclosure. Minimal effects on dissociation from changes in T Changes in temperature can affect dissociation as well. Again Tris buffers are instructive (and problematic). For example, if you prepare a Tris buffer at pH 7.0 at 4.0°C and perform a reaction in that same buffer at 37°C, the pH will drop to 5.95. If you have a Tris buffer prepared at 20°C with a pKa of 8.3, it would be an effective buffer for many biochemical reactions (pH 7.3–9.3), but the same Tris buffer used at 4°C becomes a poor buffer at pH 7.3 because its pKa shifts to 8.8. So the take home message: Prepare the buffer at the temperature at which you intend to use it. BTW: Tris is NOT one of Good’s buffers. Confidential and Proprietary. Not for Further Disclosure. Minimal interactions between buffer components and critical reaction components If a complex forms between the buffer and a required cofactor, say a metal cation like zinc or magnesium, your reaction might be compromised. For example calcium precipitates as calcium phosphate in phosphate buffers. Not only would any Ca2+-requiring reactions be compromised, but the buffering capacity of the phosphate buffer also is affected. Having excessive amounts of a chelating agent in an enzymatically driven reaction could cause problems (e.g., a high concentration of EDTA in a PCR amplification). Citrate is a calcium chelator, so avoid citrate buffers in situations where calcium concentrations are critical. Watch buffer components that have reactive R groups. For instance Tris has a reactive amine group. Remember buffers are not inert! Confidential and Proprietary. Not for Further Disclosure. Chemical Stability The buffer should be stable and not break down under working conditions. It should not oxidize or be affected by the system in which it is being used. Try to avoid buffers that contain participants in reactions (e.g., metabolites). Confidential and Proprietary. Not for Further Disclosure. Preparing buffers Prepare buffers at the appropriate temperature and concentration. If you dilute a buffer or use it at a different temperature than the one at which it was prepared, measure the pH after dilution and equilibration to the new temperature. Adjust the pH of the buffer system correctly. Not all buffers are prepared the same way. Be sure you understand how your buffer system works and that you do not introduce any new ions into the system during pH measurement. See the P&A Guide for more information. Make sure you know how to use and care for the pH meter. Confidential and Proprietary. Not for Further Disclosure. Preparing buffers Some buffering components may have to be heated or put in alkaline or acidic conditions before they will dissolve. Some buffers cannot be autoclaved because they will degrade upon heating (so they will need to be filter sterilized). When working with acids and bases be sure to wear the appropriate protective clothing and eyewear. Do not try to neutralize strong acids with strong bases. If you are using a solvent other than water, be sure you know how that solvent affects the pKa of the buffer system. Confidential and Proprietary. Not for Further Disclosure. Using buffers Check all stored buffers before use. Swirl the bottle to check for any contaminants that may have settled to the bottom during storage. Remeasure pH if you are diluting a buffer before using it in your reaction. Keep detailed notes on buffer preparation so that you can replicate your experiments (or troubleshoot confusing data). Indicate grade of materials used, supplier, and lot no. if known. Indicate what acid or base was used to pH the buffer and its concentration. If additional components were added to the buffer indicate at what point pH was measured. Confidential and Proprietary. Not for Further Disclosure.