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 from: Stoll and Blanchard. 1990. Meth.
Enzymol. 182, 24–38.
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 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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 Buffers
AP Chemistry

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 Common Ion Effect
Adding a common ion to an equilibrium system will
shift the equilibrium forward or reverse.
For a weak acid equilibrium this can affect the pH of the
solution.
For example: Adding NaF to a solution of HF.
HF H+ + F -

Adding F- ions will shift the equilibrium to reactants and

produce more HF and reduce [H+] ions in the process.
This makes the pH increase.
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 Buffers

Buffers are solutions in which the pH

remains relatively constant, even when
small amounts of acid or base are added
•Contain a weak acid (HA) and its
salt(NaA); H2CO3 & NaHCO3
•Or a weak base (B) and its (BHCl);
(NH3 and NH4Cl)
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 Buffers
A buffer system is better able to resist
changes in pH than pure water
Since it is a pair of chemicals:
•one chemical neutralizes any acid
added, while the other chemical would
neutralize any additional base
•AND, they produce each other in the
process!!!

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 How a Buffer Works
Consider the following buffer system
HCO3- + H+ H2CO3
If you add more H+ this buffer system it will react with the conjugate base
HCO3- to produce more H2CO3.
If you add base, OH-, it will grab an H+ from H2CO3 to produce more
HCO3- ion as follows
H2CO3 + OH- H2O + HCO3-

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 Buffer Capacity

The buffer capacity is the amount of acid

or base that can be added before a
significant change in pH
This depends on the amounts of HA and A-
present in the buffer
Most efficient buffer is when
[A-] =1
[HA]
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 Henderson-Hasselbach Equation
Derived from the equilibrium expression of a weak acid and the pH
equation.

[A-]
pH = pKa + log[HA]
This equation allows you to determine the pH of a buffer solution
pKa = -log Ka

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 The Common Buffer Problems
1. Compute pH of a buffer given the actual concentrations of the
conjugate acid and conjugate base.
The pH is easily calculated with:

[A-]
pH = pKa + log
[HA]

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Example: Determine the pH in which 1.00 mole of H2CO3 (Ka = 4.2 x 10-
7
) and 1.00 mole NaHCO3 dissolved in enough water to form 1.00 Liters
of solution.

[A-]
pH = pKa + log
[HA]
pH = -log (4.2 x 10-7) + log (1.00M)/(1.00M)

pH = pKa = 6.4
** pKa of a weak acid can help determine pH of buffer
you will make if it is mixed in a 1:1 mole ratio!
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 2. Make a buffer problem
How many moles of NaHCO3 should be added to 1 liter of
0.100M H2CO3 (Ka = 4.2 x 10-7 ) to prepare a buffer with a
pH of 7.00?
[HCO3-]
pH = pKa + log
[H2CO3]
7.00 = -log(4.2 x 10-7) + log [HCO3-]
(0.100)
0.60 = log[HCO3-]
0.001
[HCO3-] = 0.40 moles
100.6 = [HCO3-] should be added!
0.100
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 3. The pH shift problem:
If you add 2.00 mL of 0.100 M HCl to 100 mL of a buffer consisting of
0.100 M HA and 0.200 M NaA, what will be the change in pH?
Ka of HA is 1.5x10-5. pKa = 4.82.
The initial pH is:

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 If we add H+ to the equilibrium system it will react with the
strong conjugate base ‘A-, tor form more HA.
H+ + A-  HA
Set up an ICE chart to determine how the mole values will
change using stoichiometry!

mmoles HA
mmoles H+ mmoles A- Volume, mL
Initial 0.200 20.0 10.0 100+2 mL
Final 0 19.8 10.2 102 mL
Molarities 19.8/102 10.2/102

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