It might seem like a detail, but often success of failure of an experiment depends on the proper choice of buffer. It is not just the pH regulation that matters, but also the interactions of the buffer with other components in the solution. You might think that phosphate is the best choice for biochemical studies since phosphate is such an important part of physiological buffers. However, phosphate can have interactions with ions (e.g. calcium) and can lead to reduced colloidal stability. White phosphate may work well in a living cell that does not mean that it will work well in the chemical laboratory. Phosphate can have a 2- or even 3- charge, which leads to reduced stability of proteins and other macromolecules. For this reasons many organic buffers are used. These are desribed in the video. These are often organic sulfonates, which are used because they do not contribute to colloidal instability (i.e. aggregation and precipitation).
There are two main methods of buffer preparation. The first consists of mixing the acid and its conjugate base (e.g. acetic acid and sodium acetate). In this method one calculates the appropriate ratio of the two species so that one will satisfy the Hendersen-Hasselbach equation and achieve the target pH. The second method involves carrying out a titration using strong base to partially neutralize an acid. When the neutralization has treached the target pH one may stop and consider that the solution is buffered. Both of these methods must also take into account the issue of buffer strength. The concentrationa of the acid and its conjugate base must be large enough to maintain the target pH when excess acid or base is added. One may use the Hendersen-Hasselbach equation to determine what the appropriate buffer strength is, i.e. what concentrations of the acid and conjugate base are necessary. Similar comments apply to a base and its onjugate acid. These issues are discussed in the video on buffer preparation.
Phosphate buffer is still the most common buffer in the chemistry laboratory. All of the warnings that have been given in previous sections are cautionary notes, but they do not mean to suggestthta phosphate should be avoided. Rather you should understand the issues involved in the choice of buffer and be aware that things can wrong. That way you will be ready to try alternatives if there is a problem in your experimental protocol. Since phosphoric acid is a polyprotic acid we first discuss polyprotic acids. This is done also because a number of polyprotic acids are excellent bueffers. citrate is one that we will discuss, but there are several others that are in common use.
As the final segment in the topic of buffers we discuss the details of a phosphate buffer. One major point of the presentation will be to justify the use of pKa2 as a separate equilibrium that has no interference from pKa1 or pKa3.
