How to choose a buffer?
When selecting a buffer system, its dissociation constant pKa value should be within one pH unit below the set pH, because when the pKa of the buffer changes more than one pH unit, its buffering capacity will be significantly reduced;
The buffer system should not affect the activity of the protein or the stability of the DNA, and avoid the interference of other ions;
In addition, the effect of temperature must be considered: temperature not only affects the buffer, but also affects the cells. Most animal cells have a pH of 7.0~7.5 under physiological conditions of 37°C, and can reach 8.0 when the temperature drops to 0°C. .
1. The advantages and disadvantages of phosphate buffer:
It is a widely used buffer. Because of its secondary dissociation, it has a wide range of buffered pH values. It can be configured with acidic, alkaline and neutral buffers of various pH values.
advantage:
Easy to mix into various concentrations
Wide applicable pH range
pH is less affected by temperature
Little change in pH after dilution
shortcoming:
It is easy to associate with common calcium ions, magnesium ions and heavy metal ions to form precipitation.
2. Advantages and disadvantages of Tris buffer:
Tris buffer is a buffer that is widely used in biochemical research. It is a weak base and the effective pH is usually in the "neutral" range.
advantage:
Strong alkalinity, you can use only this kind of buffer to configure pH buffers ranging from acid to alkali;
It has little interference to the biochemical process, and does not precipitate with calcium, magnesium ions and heavy metal ions;
It is easy to absorb CO2 in the air, so the prepared buffer should be tightly sealed;
This buffer has a certain interference effect on some pH electrodes, so use an electrode compatible with Tris solution.
shortcoming:
pH is greatly affected by concentration, diluted 10 times, pH change is greater than 0.1
Great temperature effect, such as pH=8.4 at 4℃, pH=7.4 at 37℃
3. The advantages and disadvantages of HEPES buffer:
HEPES is a non-ionic amphoteric buffer with good buffering capacity in the pH range of 7.2 to 7.4. It is commonly used in biochemical diagnostic kits, DNA/RNA extraction kits and PCR diagnostic kits.
advantage:
It can maintain a relatively constant pH value during open culture or cell observation, and has no toxic effect on cells.
shortcoming:
It may be toxic to some cells at high concentrations and is slightly more expensive than other buffers.
4. The advantages and disadvantages of PIPES buffer:
PIPES is a member of the piperazine buffer family and belongs to the same family as HEPES. It is a zwitterionic Good's buffer, effective in the pH range of 6.1 to 7.5. Since the pH range is slightly different from HEPES, the concentration of PIPES is only half of the concentration of HEPES.
advantage:
The buffer will not complex with metals, so it can be used in solutions containing metal ions;
It can be used for chromatographic analysis, electron microscope fixation of plant cells, and to replace glyphosate buffer.
shortcoming:
When the pH of the solution rises above 6.5, the free acid of PIPES is easily dissolved, but the sodium salt of PIPES is easily dissolved;
If you are using a buffer in the free acid form, use sodium hydroxide to convert it to sodium salt to improve solubility at lower pH;
Like HEPES, PIPES is not suitable for experiments involving redox reactions because it causes the formation of free radicals.