What makes buffers effective

Using the Henderson-Hasselbalch equation gives:. What is the effect of adding of 0. The strong acid, HCl would dissociate completely in water:.

The new concentration would be:. The equilibrium would then be re-established:. There is a small change in pH, from 4. This same amount of HCl added to water shows a much bigger effect. For example, we could make a solution that is 0. We can use the Henderson-Hasselbalch equation to calculate the pH of this buffering solution.

Since we are calculating the pH, we have to treat this as an acid dissociation reaction. These two examples should point out an important consideration in making buffers. The pH of a buffer is close to the p K a of the acid used in the buffer.

If we want to buffer an acidic solution, use an acid with a small acidic p K a. To buffer a basic solution, use an acid with a large basic p K a. The acetate buffer would be effective of the pH range from about 3. Outside of these ranges, the solution can no longer resist changes in pH by added strong acids or bases. The reactions we have to work with are:.

Most biochemical experiments have an optimal pH in the range of 6—8. The optimal buffering range for a buffer is the dissociation constant for the weak acid component of the buffer pK a plus or minus pH unit.

Solubility in water. Biological reactions, for the most part, occur in aqueous environments, and the buffer should be water-soluble for this reason. Exclusion by biological membranes. However, if this is an important criterion for your particular experiment, it is helpful to remember that zwitterionic buffers positive and negative charges on different atoms within the molecule do not pass through biological membranes. Minimal salt effects. In other words, the buffer components should not interact or affect ions involved in the biochemical reactions being explored.

Minimal effects on dissociation from changes in temperature and concentration. Usually there is some change in dissociation with a change in concentration. If this change is small, stock solutions can usually be diluted without changing the pH of the buffer.

However, with some buffers changes, in concentration produce more dramatic changes in pK a , and stock solutions cannot be diluted without significantly affecting pH. For instance, the pH of Tris decreases approximately 0. Temperature changes can be a problem too, and again, Tris provides a cautionary example of a commonly used buffer because it exhibits a large shift in dissociation with a change in temperature. For example, if you prepare a Tris buffer at pH 7.

So the take home message: Make the buffer at the temperature you plan to use it. If your experiment will involve a temperature shift, select a buffer with a range that can accommodate any shift in dissociation as a result of the change in temperature.

Well defined or nonexistent interactions with mineral cations. If the buffer and cations in your system react, your buffer becomes less effective because it cannot handle additional hydrogen ions. If a complex forms between the buffer and a required cofactor, say a metal cation like zinc or magnesium, your reaction also might be compromised. For instance, having excessive amounts of a chelating agent in an enzymatically driven reaction could cause problems like too high a concentration of EDTA in a PCR amplification, for instance.

Tris buffers again give us problems, because Tris contains a reactive amine group. If you are trying to make Tris buffer that is RNase free, the amine group on the Tris molecule will react with diethylpyrocarbonate, the chemical typically used to pretreat aqueous solutions that will be use for RNA work. Take home message: Buffers are not inert. Be careful which ones you chose. 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. Light absorption. The buffer should not absorb UV light at wavelengths that may be used for readouts in photometric experiments. His work set the standard for the many benefits of of buffers in biological research. No matter what buffer you choose , you need to consider effects of temperature and environment on the buffer and ensure that the buffer you choose will be compatible with your system.

For additional help, access the Student Resource Center. Promega products are used by life scientists who are asking fundamental questions about biological processes and by scientists who are applying scientific knowledge to diagnose and treat diseases, discover new therapeutics, and use genetics and DNA testing for human identification.

Originally, founded in in Madison, Wisconsin, USA, Promega has branches in 16 countries and more than 50 global distributors serving countries. Good information about buffers! About the dissociation constant of the weak acid in the system, I also found additional good information about this I hope this will also catch your interest : dissociationconstant. Between PBS and tris buffered saline, which is a better buffer? I want to try TBS. Hi, Since you intend to inject your protein of interest into a lab animal as part of your experiments you should defer to the animal use guidelines for your institution and seek the advice of your veterinarian on this matter.

Anything injected into an animal, including the buffer, should be approved by IACUC and requires that you consider aspects like toxicity, pH, possible presence of endotoxin, and injection route. However, because you will need to follow the rules and regulations approved by your institution, then your best course of action is to seek guidance directly from your vivarium veterinarian.

Most biological reactions take place at a pH between 6 and 8, so ideal buffers have pKa values in this range to provide maximum buffering capacity there. Likewise, select a buffer with a pKa slightly higher if you expect your experiment to raise your working pH.

Buffers should be very soluble in water, and minimally so in nonpolar solvents fats, oils, and organic solvents. This prevents the buffer from accumulating in cell membranes, vesicles and other nonpolar compartments in biological systems. A buffer should not readily pass through cell membranes—this also reduces the disproportionate accumulation of buffer in subcellular structures.

Highly ionic buffers can cause problems or complications in biological systems. Avoid citrate and phosphate buffers in reactions that are calcium-dependent Tris also chelates calcium. Buffer concentration, temperature, and ionic strength the measure of ion concentration should have minimal impact on the dissociation of the buffer. If the buffers form complexes with cationic ligands, the complexes should be soluble. Ideally, at least some of the buffering compounds will not form complexes.

The buffers should be chemically stable, resisting enzymatic and non-enzymatic degradation. Buffers should not absorb visible or ultraviolet light at wavelengths between nm and nm. This prevents interference with spectrophotometric assays.

Applichem Biological Buffers.