Phosphate Buffers (Molecular Biology)

Phosphate buffers are commonly used in biological work. Phosphoric acid, 0=P( OH)3 has pKa values of about 2, 7, and 12. All three hydroxy groups are identical and, indeed, all four oxygen atoms are identical on the NMR time scale as a result of rapid hydronations and dehydronations. Thus, 2 is the p^ for the first H+ to dissociate, even though it is coming equally from each of three groups. The next p^ is raised to 7 because it is much harder for H+ to dissociate from the charged HsPOj ion than from the neutral H3P04 molecule and, similarly, for the third pK Hence, the Henderson-Hasselbalch equation for orthophosphate is

The large separation of the pKa values means that, for a buffer of concentrations of H3PO4 and PO4 are negligible, and the pa values of 2 and 12 become irrelevant.

An advantage of phosphate as a buffer is that salts such as NH2PO4 and Na2HPO4 are solid and both can be weighed out in the appropriate quantities; hence, the reproducible preparation of phosphate buffers need not involve adjustments of pH. Care must be taken, however, that the salt used, hydrate or anhydrous, does not lose or gain water on storage. Another advantage is that phosphate is transparent in the UV, down to wavelengths at which proteins may be estimated by their peptide bond absorbance, where carboxylate ions also absorb.

The main disadvantage is that with a doubly charged buffering species, the pKa of 7.2 is three times more sensitive to changes in ionic strength than a buffer with only singly and zero charged components. Another disadvantage is that phosphate can support algal or fungal growth as a nutrient. Phosphate will also sequester and precipitate many cations, especially Ca (see Calcium Signaling).

1. Diphosphate (pyrophosphate)

A variant of phosphate buffer is diphosphate (pyrophosphate). The acid, HO P(=0){0H) O P{=0){ OH) OH^ has two strongly acidic groups, with pX^ values near 2, as with orthophosphate. The -P(=0)( OH : O groups generated are electron-withdrawing, and so the next OH group has its p\K lowered from the 7.2 of orthophosphate to 6.2 (at an ionic strength of 0.1 M; with its more strongly charged ions, the dissociation constants of diphosphate are even more sensitive than orthophosphate to changes in ionic strength). The P(=0)( O ) -group, however, is slightly electron-donating, so that the final pKa is raised to 8.4. Hence, the Henderson-Hasselbalch equation for pyrophosphate in the neutral pH region is

Diphosphate can be hydrolyzed to orthophosphate, and the equilibrium strongly favors orthophosphate, but diphosphate is stable for long periods of time in neutral and alkaline conditions, even on heating.