Making buffers solutions

Choosing the right buffer solution for proteins and their purification is a very important and delicate task.

On this page you will find both theoretical and practical considerations, as well as instructions on how to make buffer solutions in the Laboratory.

In the context of chronomatographic protein purification buffer solutions are the mobile phase.

Selection of the right buffer system for given protein and application is not easy. It requires a detailed knowledge of the system under investigation to choose pH and buffer system. In general you would like to choose the pH were the protein of interest is most stable, but how do you know that before you have purified the protein? You do not, unless you have some prior information on the protein or the protein family from the literature.

Choosing pH and buffers for ion-exchange experiments is introduced on the IEX-concept page.

Also the down-stream process can be important for the choice of buffer systems. For example, in protein mass spectrometry you will need a volatile buffer system (page 138 but page 140 in the PDF).
Norman Good and colleagues have defined the most important properties for biological buffers (buffers used in life science experiments). The most important properties of Good’s Buffers in chromatography are:

Stable dissociation. There should be a minimum influence of buffer concentration, temperature, and ionic composition of the medium on the dissociation of the buffer.

The buffer should not promote microbial growth. The buffers should be chemically stable, resisting enzymatic degradation. That means that possible microbial contamination cannot utilise the buffer as nutrient and can therefore not propagate. Buffers such as phosphate, acetate, ammonia all promote the growth of microbial contamination. 

No ultraviolet or visible absorption. Buffers should not absorb visible or ultraviolet light at wavelengths longer than 230 nm so as not to interfere with commonly used spectrophotometric monitoring.

Take a look at an easy overview of Good’s buffers. 

Further details on the buffers can be found on the supplier website: Sigma-Aldrich

When making and reporting buffers, it is important that the description is consistent. For example, 20 mM Tris pH 8 + 0.2 M NH4SO4 makes no sense. What is missing is the information on what form of the buffer is used, what chemical has been used to adjust the pH, and is ammonium sulfate added before or after pH is adjusted. The following way of writing makes it clear without any ambiguity. 20 mM Trisbase/HCl, 0.2 M NH4SO4, pH 8.0. This means Trisbase is used and ammonium sulfate is added before pH is adjusted with hydrochloric acid.

If you have over titrated (meaning the pH has gone below or above the value you are aiming at) can you then just add appropriate acid or base to correct pH? No! Why not? What type of error is introduced?  

Making buffer solutions in the lab
When have decided on the buffer systems, salts and other additives and the volumes to make you are ready to go to the lab. Or are you? Have you made the risk assessment, if not please follow this link.

You will find the chemicals in lab 151C and the pH-meter and the ultrapure water in lab 141. 

Buffer solutions have to filtered before use. Why? Usually at least a 0.45 µm filter has to be used. On this course we use 0.22 µm filters. The filter system is available in the lab.