Buffers for Biological Systems
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What do buffers do?
Buffers function to resist changes in hydrogen ion concentration
Biologists often think of buffers as doing much more:
•providing essential cofactors
•providing critical salts
•providing essential nutrients for cells and tissues
However, you cannot overlook the basic function: resisting changes in
hydrogen ion concentration. If you do, your reactions could be
compromised.
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What makes a buffer system?
A buffer consists of a weak acid and its conjugate base (or a weak base
and its conjugate acid).
Weak acids and bases do not dissociate completely in water, but
instead exist in solution as an equilibrium of dissociated and
undissociated species.
For acetic acid, we would express this equilibrium like this:
HAc (acetic acid) ↔ H+ + Ac–
Hac can release H+ to neutralize OH– and form water. The conjugate
base Ac– can react with H+ ions added to the system to form acetic
acid. In this way pH is maintained as equilibrium of the three species is
maintained.
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How buffers work
All buffers have an optimal pH range over which they are able to
moderate changes in hydrogen ion concentration.
This range is a factor of the dissociation constant of the acid of the
buffer (Ka) and is generally defined as the pKa (–logKa) value plus or
minus one pH unit.
pKa can be determined using the Henderson-Hasselbalch equation.
So…
a buffer system with a pKa of 3.4 would have an optimal pH range of
2.4–4.4 and would not be an effective buffer at 7.0.
We present the derivation of the H-H equation in the P&A Guide Buffers chapter.
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Buffers with more than one dissociation constant
Polyprotic acids are acids that can lose more than one hydrogen ion.
Since more than one hydrogen ion can dissociate in solution, the acid
has more than one pKa value. Multiple pKavalues are usually denoted
as pKa1, pka2, etc. If the pKa values are close together, the optimal pH
for the buffer will be a continuum determined by the range of pKas.
Citric acid is a polyprotic acid that can lose three protons, and the pKas
for each dissociation are close together, so a citric acid buffer will be
effective over a continual pH range, from approximately 2–7.4.
Citrate pKa1
3.13
Citrate pKa2
4.76
Citrate pKa3
6.40
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What makes a “Good” buffer
In 1966 Norman Good and colleagues developed criteria for buffers for biological
systems.
1.
A pKa between 6 and 8.
2.
Solubility in water.
3.
Exclusion by biological membranes.
4.
Minimal salt effects.
5.
Minimal effects on dissociation from changes in temperature and concentration.
6.
Minimal interactions between buffer components and critical reaction
components.
7.
Chemical stability.
8.
Light absorption outside of wavelengths used for assay readout.
9.
Components should be easy to obtain and prepare.
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Optimal buffering at a neutral pH
Most biochemical reactions have an optimal pH in the range of 6–8, so
buffers for these reactions need to have pKas that support buffering at
these pH values.
Some buffers and their pKa values (in water at 25°C):
Acetate
4.76
PIPES
6.76
MOPS
7.20
HEPES
7.48
Tris
8.06
Borate
9.23
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from: Stoll and Blanchard. 1990. Meth.
Enzymol. 182, 24–38.
Solubility in Water
Most biochemical reactions occur in aqueous conditions, so your
buffering components should be soluble in water.
If for some reason, you will be using a solvent other than water, make
sure you understand how that solvent affects the dissociation of your
buffer components.
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Minimal salt interactions
If the system to be studied requires salts, appropriate ions can be
added. However, using an ionic buffer can adversely affect the reaction
if reaction studied is affected by salts.
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Minimal effects on dissociation from changes in
concentration
Changes in dissociation resulting from changes in concentration are
usually small, and most buffers can be made as stock solutions that are
diluted to working solutions. However, some buffers do show a
significant change in pH upon dilution.
For instance, the pH of Tris decreases approximately 0.1 pH unit per
tenfold dilution, and the pH could change dramatically if you dilute a
working solution and are at the limits of the optimal buffering range of
the Tris.
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Minimal effects on dissociation from changes in T
Changes in temperature can affect dissociation as well. Again Tris
buffers are instructive (and problematic).
For example, if you prepare a Tris buffer at pH 7.0 at 4.0°C and perform
a reaction in that same buffer at 37°C, the pH will drop to 5.95.
If you have a Tris buffer prepared at 20°C with a pKa of 8.3, it would be
an effective buffer for many biochemical reactions (pH 7.3–9.3), but
the same Tris buffer used at 4°C becomes a poor buffer at pH 7.3
because its pKa shifts to 8.8.
So the take home message: Prepare the buffer at the temperature at
which you intend to use it.
BTW: Tris is NOT one of Good’s buffers.
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Minimal interactions between buffer components
and critical reaction components
If a complex forms between the buffer and a required cofactor, say a
metal cation like zinc or magnesium, your reaction might be
compromised. For example calcium precipitates as calcium phosphate
in phosphate buffers. Not only would any Ca2+-requiring reactions be
compromised, but the buffering capacity of the phosphate buffer also
is affected.
Having excessive amounts of a chelating agent in an enzymatically
driven reaction could cause problems (e.g., a high concentration of
EDTA in a PCR amplification). Citrate is a calcium chelator, so avoid
citrate buffers in situations where calcium concentrations are critical.
Watch buffer components that have reactive R groups. For instance
Tris has a reactive amine group. Remember buffers are not inert!
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Chemical Stability
The buffer should be stable and not break down under working
conditions. It should not oxidize or be affected by the system in which
it is being used. Try to avoid buffers that contain participants in
reactions (e.g., metabolites).
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Preparing buffers
Prepare buffers at the appropriate temperature and concentration. If
you dilute a buffer or use it at a different temperature than the one at
which it was prepared, measure the pH after dilution and equilibration
to the new temperature.
Adjust the pH of the buffer system correctly. Not all buffers are
prepared the same way. Be sure you understand how your buffer
system works and that you do not introduce any new ions into the
system during pH measurement. See the P&A Guide for more
information.
Make sure you know how to use and care for the pH meter.
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Preparing buffers
Some buffering components may have to be heated or put in alkaline
or acidic conditions before they will dissolve.
Some buffers cannot be autoclaved because they will degrade upon
heating (so they will need to be filter sterilized).
When working with acids and bases be sure to wear the appropriate
protective clothing and eyewear. Do not try to neutralize strong acids
with strong bases.
If you are using a solvent other than water, be sure you know how that
solvent affects the pKa of the buffer system.
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Using buffers
Check all stored buffers before use. Swirl the bottle to check for any
contaminants that may have settled to the bottom during storage.
Remeasure pH if you are diluting a buffer before using it in your
reaction.
Keep detailed notes on buffer preparation so that you can replicate
your experiments (or troubleshoot confusing data). Indicate grade of
materials used, supplier, and lot no. if known. Indicate what acid or
base was used to pH the buffer and its concentration. If additional
components were added to the buffer indicate at what point pH was
measured.
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