210
publisher's indignation, I discovered that they were all
pirated copies. The forgery was complete, and since the
binding is an extremely high quality West German
product, they probably had to work extra hard at it. I
cannot restrain a certain admiration; but we all look
forward to the time, long overdue, when these countries
sign the international copyright agreement.
Meanwhile, in anticipation of a third edition, we are
keeping a weather eye on progress in all fields of
biochemistry, and we should be grateful to continue
receiving criticisms and suggestions from teachers and
research workers. Even without the prospect of a third
edition, it would be difficult to break the habit of
collecting and classifying new material, an exercise involv-
ing the library, the international conference, and the
shameless exploitation of colleagues. Many of the latter
generously provided material and/or gave their expert
opinions on written entries. On behalf of myself, Dr
Eagleson and the publishers, I should like to take this
opportunity of thanking them all.
T A Scott
A Simple and Universal Method for Making up
Buffer Solutions
CLIVE DENNISON
Department of Biochemistry
University of Natal
PO Box 375, Pietermaritzburg 3200
South Africa
Introduction
In the making up of buffers, there is a simple and
universally applicable approach which I am surprised is
not more widely used. The method involves weighing out
an amount of the appropriate weak acid or base, sufficient
to give the required final molarity, dissolving this in an
amount of distilled H20, close to the intended final
volume, and titrating to the desired pH with a fairly
concentrated solution of an appropriate strong acid or
base, before diluting to the final volume. Ingredients in
addition to the buffer salts should be added before
titration. Perhaps the rationale of the approach is best
explained by reference to some examples.
Example 1
The requirement is for a 0.1 M Na-phosphate buffer, pH
7.6. In the Henderson-Hasselbalch equation, pH = pKa +
log ([salt]/[acid]), the salt is Na2HPO4 and the acid is
NaHzPO4. A buffer is most effective at its pKa, which is
the point where [salt] = [acid]. From the equation it is
clear that if the [salt[ > [acid], the pH will be greater than
the pKa, and if [salt] < [acid], the pH will be less than the
pKa. Therefore if we were to make up a solution of the
acid, NaH2PO4, its pH will be less than the pKa, and
therefore will also be less than the pH at which the
solution will function as a buffer. To make a buffer from
BIOCHEMICAL EDUCATION 16(4) 1988
this solution, it will be necessary to titrate it with a base, to
a pH closer to the pKa. N a O H is a suitable base because it
maintains sodium as the cation:
NaH2PO4 + NaOH--+ Na2HPO4 + H20.
Once the solution has been titrated to the correct pH, it
may be diluted (at least over a small range, so that
deviation from ideal behaviour is small) to the volume
which will give the desired molarity. The Henderson-
Hasselbalch equation states that it is the ratio of salt to
acid, rather than their absolute concentrations, which
determine the pH. Note that (i) in this reaction the only
by-product is water, (ii) the molarity of the buffer is
determined by the mass of the acid, NaH2PO4, which is
weighed out, and the final volume to which the solution is
made up (for this example 15.60 g of the dihydrate would
be required per litre of final solution), (iii) the concen-
tration of the N a O H is of no moment, and so any arbitrary
concentration can be used (it should, of course, be
concentrated enough to effect the required pH change in
the available volume), and (iv) the reaction implies that
only a simple calculation of molarity and a single weighing
is required: only one solution needs to be made up and all
of the material weighed out is used in the buffer, ie there
is no waste.
It should be pointed out that it is not correct to weigh
out the 'salt', NazHPO4, in the first instance, as this gives
an unwanted by-product. If a solution of the salt is made
up, its pH will be above the pKa and it will require
titration with an acid to lower the pH. If HC1 is used, the
reaction will be:
Na2HPO4 + HC1--+ NaH2PO4 + NaC1,
yielding NaC1, of an indeterminate concentration, which
is not wanted in the buffer.
Sometimes, for example in ion exchange ionic-strength
gradient elution, it is required to have a gradient of, say,
[NaC1] superimposed on the buffer. Two buffers are then
required, for the two chambers of the gradient generator:
the starting buffer (ie the equilibration buffer, without
added NaC1, or with the starting concentration of NaC1)
and the finishing buffer, which is the same as the starting
buffer but which additionally contains the finishing con-
centration of NaC1. In making up the finishing buffer,
common ion effects (due to the sodium ion) must be taken
into account.
Example 2
The requirement is for an ionic-strength gradient finishing
buffer, 0.1 M Na-phosphate buffer, pH 7.6, containing
1.0 M NaCl. In this case the NaC1 is weighed out and
made up together with the NaHEPO4; common ion effects
are accounted for in the titration and complex calculations
are thus avoided. For 1 litre of buffer, NaH2PO4.2H20
(15.60 g) and NaC1 (58.44 g) are dissolved in about
950 ml of distilled H20, titrated to pH 7.6 with a fairly

concentrated N a O H solution (but of arbitrary concen-
tration) and made up to 1 litre.
Example 3
The requirement is for a 0.1 M Tris-HCl buffer, p H 8.0.
The first requirement is to identify the buffer components,
ie the salt and its parent acid or base. In this case Tris is
the base and Tris-HCl is the salt. The relevant reaction,
therefore, is:
ll2 OH CH2OH
i
HOH2C--C--NH2 + HC[ ~,
I
H O H 2 C - - C - - N H 3 Ct
I
CH20H
I
CH20H
For 1 litre of buffer, Tris base (12.11 g) is weighed out,
dissolved in about 950 ml distilled HaO, titrated to pH 8.0
with HC1 of arbitrary concentration, and made up to
1 litre.
Comments
This conceptual approach can be applied to almost any
buffer; for an acetate buffer, acetic acid could be weighed
out and titrated with NaOH. Some people seem to have
inhibitions about weighing out liquids. If this is bother-
some, the mass can always be converted to a volume from
the density.
A brief scan of some elementary textbooks revealed
that few of these presented the above practical approach
to making buffers. Boyer 1 presents this approach for Tris
buffers but does not explicitly state that the same
conceptual approach can be used with nearly all buffers.
The common impression conveyed to students - - perhaps
unwittingly - - by Bohinski, 2 Lehninger, 3 and SegeP is
that it is necessary to calculate the amounts of both
components of the buffer. Plummer 5 and Stenesh 6 re-
inforce this impression by giving tables of the volumes
required of both components of a known molarity.
However, to be fair, Stenesh 6 does, in a sentence, state
that it is possible to make buffers as suggested above.
None of these authors suggests a way of overcoming
common ion effects, a common enough requirement in
buffers for ion-exchange, for example, which may require
high salt concentrations.
Many people are aware of the approach to making
buffers outlined above and yet in laboratories in different
parts of the world I have seen others searching for data
tables or doing complex calculations before making up
two solutions, titrating them (hopefully the right way
around) and then discarding the excess of the one
solution. My objective in drawing attention to the above
method is to eliminate this fruitless effort and waste of
reagents and to make a plea for authors of textbooks to
emphasize the simple, practical, approach to making
buffer solutions.
In buffers intended for electrophoresis, it might be
necessary to prepare a buffer of defined ionic strength.
The ionic strength, I, of a solution is defined by
BIOCHEMICAL EDUCATION 16(4) 1988
211
I = 1/2~.,CiZi 2, where Ci is the concentration of each type
of ion (in mol dm -3) and Zi is its charge. It follows from
this that for a buffer comprising a weak monobasic acid
and its alkali metal salt, the ionic strength is equal to the
concentration of the salt (protons and hydroxyl ions can
be ignored between pH 4 and pH 10). Similarly for a
weak base (Tris, glycine) and its salt with an acid (HCI)
the ionic strength is again equal to the salt concentration,
ie
I = [salt] (1)
The molarity, M, of the buffer is defined as:
M = [salt] + [base] (2)
and therefore,
[base] = M - [salt] (3)
Substituting eqn 3 into the Henderson-Hasselbalch
equation gives,
pH = pKa - log [salt] + log (M - [salt]). (4)
Or, substituting eqn
pH = pK a - log I + log (M - I) (5)
Using similar arguments, it may be shown that in the case
of a buffer made from a weak acid, the relationship is:
pH = pKa + log I - log (M - I) (6)
The variables in eqns 5 and 6 are pH, ionic strength and
molarity and if any two of these are defined, the third is
fixed and can be calculated. For example, if the pH and
the ionic strength are first defined, the molarity of the
buffer required can be calculated. Once the molarity and
the pH are known, the buffer can be made up following
the titration procedure outlined above and the ionic
strength will 'automatically' be correct. For a more
detailed discussion of buffers of defined ionic strength, the
reader is referred to Perrin and Dempsey. 7
References
tBoyer, R F (1986) 'Modern Experimental Biochemistry', Addison-
Wesley Publishing Co, Reading, MA
2Bohinski, R C (1983) 'Modern Concepts in Biochemistry', fourth
edition, Allyn and Bacon, Boston, MA
3Lehninger, A L (1982) 'Principles of Biochemistry', Worth Publishers,
New York
4Segel, I H (1976) 'Biochemical Calculations', Second edition, John
Wiley and Sons, New York
5Plummer, D T (1978) 'An Introduction to Practical Biochemistry',
Second edition, McGraw-Hill, London
6Stenesh, J (1984) 'Experimental Biochemistry', Allyn and Bacon,
Boston, MA
7perrin, D D and Dempsey, B (1974) 'Buffers for pH and Metal Ion
Control', Chapman and Hall, London