A buffer solution, consisting of "weak acid and its conjugate base" or "weak base and its conjugate acid," can slow down the pH change when adding a certain amount of other substances. One of the most commonly used buffer solutions in biological experiments is PBS, which is composed of Na2HPO4 and KH2PO4 buffer pairs and has a strong buffering capacity within the pH range of 5.8-8.0.
In order to ensure that the buffer solution has enough buffering capacity, when preparing the buffer solution, the following should be done:
To achieve a concentration ratio of the conjugate acid-base pair close to 1, an appropriate buffer pair should be selected based on the pH range that needs to be maintained, and the pKa of the weak acid in the buffer pair should be equal to or close to the required pH.
For example, if a buffer solution with a pH of 7.0 is needed in a biological culture medium, H2PO4- and HPO42- can be selected as suitable buffer pairs as H2PO4- has a pKa2 of 7.21. If a buffer solution with a pH of 9.0 is required, NH3·H2O-NH4Cl buffer pair (pKa(NH4+) = 9.25) can be selected.
It can be seen that the pKa of the weak acid is the main basis for selecting a buffer solution. The table lists several commonly used buffer solutions.
The acid that is commonly used as a buffer solution consists of a weak acid and its conjugate acid salt, which has buffering effect. The common buffer systems are:
A weak acid and its salt (such as HAc—NaAc)
A weak base and its salt (NH3·H2O—NH4Cl)
The aqueous solution composed of the acid salt of a multi-weak acid and its corresponding secondary salt (such as NaH2PO4—Na2HPO4).
Buffered electrolyte salts, such as the Tris (tris(hydroxymethyl)aminomethane) system, carbonate, citric acid, phosphoric acid, acetic acid, and barbituric acid, are typically employed as buffer solutions in the realm of biological products.
When choosing a buffer system in biochemical experiments or research work, it should be carefully selected because sometimes the factor that affects the experimental results is not the pH value of the buffer solution, but some kind of ion in the buffer solution. Buffering agents such as borate, citrate, phosphate, and Tris can all cause unwanted chemical reactions.
Borate: Borate forms complex salts with many compounds, such as sucrose.
Citrate: Citrate ions are prone to bind with calcium, so it cannot be used when there is calcium ion present.
Phosphate: It is an enzyme inhibitor or even a metabolite in some experiments, and heavy metals can easily precipitate from the solution in the form of phosphate. And its buffering capacity is very low above pH 7.5.
The role of a buffer solution, including the tromethamine tris buffer, is to adjust the pH of the solution within a limited range, so that the acidity of the sample solution meets the range specified by the analysis method. For example, phenol can react with 4-aminodiphenylamine to form orange-red azodye in the presence of an alkaline medium and iron cyanide, and a pH of 10.0 ± 0.2 is most suitable to prevent interference from aromatic amine. Therefore, a buffer solution is added to the sample solution to adjust and control the pH of the sample solution to 10.0 ± 0.2.
In practical work, some analysts, due to their lack of understanding of the mechanism of buffer solution, use strong acid or strong base solutions such as hydrochloric acid or sodium hydroxide to adjust the pH of the buffer solution to the required value when the prepared and used buffer solution does not match the specified value, thinking that this can quickly bring the buffer solution to the desired pH value. However, the result is just the opposite—although the pH value of the solution is adjusted correctly, its buffer system is destroyed, and it loses its buffering effect.
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