BUFFER FOSFATOS

When selecting a buffer, you should be aware of certain limitations to its use. Citric acid and
phosphate buffers readily form insoluble complexes with divalent cations, while phosphate can
also act as a substrate, activator or inhibitor of certain enzymes. Both of these buffers contain
biologically significant quantities of cations, e.g. Na + or K+. TRIS (Table 7.3) is often toxic to
biological systems: owing to its high lipid solubility it can penetrate membranes, uncoupling
electron transport reactions in whole cells and isolated organelIes. In addition, it is markedly
affected by temperature, with a l0-fold increase in H+ concentration from 4°C to 37°C. A number
of zwitterionic molecules (possessing both positive and negative groups) have been introduced
to overcome some of the disadvantages of the more traditional buffers. These newer compounds
are often referred to as 'Good buffers', to acknowledge the early work of Dr N.B. Good and eo-
workers: HEPES is one of the most useful zwitterionic buffers, with a pKa of 7.5 at 25°C.

These zwitterionic substances are usually added to water as the free acid: the solution must
then be adjusted to the correct pH with a strong alkali, usually NaOH or KOH. Alternatively, they
may be used as their sodium or potassium salts, adjusted to the correct pH with a strong acid,
e.g. HCI. Consequently, you may need to consider what effects such changes in ion
concentration may have in a solution where zwitterions are used as buffers.

Fig. 7.4 shows a number of traditional and zwitterionic buffers and their effective pH ranges.
When selecting one of these buffers, aim for a pKa which is in the direction of the expected pH
change (Tables 7.2, 7.3). For example, HEPES buffer would be a better choice of buffer than
PIPES for use at pH 7.2 for experimental systems where a pH increase is anticipated, while
PIPES would be a better choice for where acidification is expected.

Table 7.3 pKa values at 25°C of some acids and bases (upper section) and some large organic
zwitterions (lower section) commonly used in buffer solutions. For polyprotic acids, where more
than one proton my dissociate, the pKa values are given for each ionization step. Only the trivial
acronyms of the larger molecules are provided: their full names can be obtained from the
catalogues of most chemical suppliers.

*Note that this compound is hygroscopic and should be stored in a desiccator.

Preparation of buffer solutions
Having selected an appropriate buffer, you will need to make up your solution to give the
desired pH. You will need to consider two factors:

1. The ratio of acid and conjugate base required to give the correct pH.
2. The amount of buffering required; buffer capacity depends upon the absolute quantities
of acid and base, as well as their relative proportions.

In most instances, buffer solutions are prepared to contain between 10mmol L −1 and 200 mmol
L−1 of the conjugate pair. While it is possible to calculate the quantities required from first
principles using the Hendersori- Hasselbalch equation, there are several sources which tabulate
the amount of substance required to give a particular volume of solution with a specific pH value
for a wide range of traditional buffers (e.g. Perrin and Dempsey, 1974). For traditional buffers, it
is customary to mix stock solutions of acidic and basic components in the correct proportions to
give the required pH (Table 7.4). For zwitterionic acids, the usual procedure is to add the
compound to water, and then bring the solution to the required pH by adding a specific amount
of strong alkali or acid (obtained from tables). Alternatively, the required pH can be obtained by
dropwise addition of alkali or acid, using a meter to check the pH, until the correct value is
reached. When preparing solutions of zwitterionic buffers, the acid may be relatively insoluble.
Do not wait for it to dissolve fully before adding alkali to change the pH - the addition of alkali
will help bring the acid into solution (but make sure it has all dissolved before the desired pH is
reached).

Finally, when preparing a buffer solution based on tabulated information, always confirm the pH
with a pH meter before use.

Table 7.4 Preparation of sodium phosphate buffer solutions for use at 25°C. Prepare separate
stock solutions of (a) disodium hydrogen phosphate and (b) sodium dihydrogen phosphate, both
at 0.2 mol L−1. Buffer solutions (at 0.1 mol L−1) are then prepared at the required pH by
mixing together the volume of each stock solution shown in the table, and then diluting to a final
volume of 100mL using distilled or deionized water.