Biochemistry Lab BIO3601

Lab 3
pH and Buffer
Calculations

Armando D. Solis, PhD

Biological Sciences Department
Acids and Bases
Acid: A chemical that donates a proton (H+) to solution.
(HA  H+ + A-).
Example: hydrochloric acid HCl  H+ + Cl-

Base: A chemical that donates an OH- to solution (or can

accept a H+).
Example: sodium hydroxide NaOH  Na+ + OH-
Acids and Bases

Acid – A molecule that donates a proton (H+)

Base – A molecule that accepts a proton.

Their reactions are subject to Keq:

Equilibrium constant
A+B C+D
Keq = [C]eq [D]eq
[A]eq [B]eq
Strong Acids and Bases

HCl, H2SO4, HNO3 are strong acids

NaOH, KOH are strong bases

Strong acids and bases undergo complete dissociation in

aqueous solution. Thus, pH can be determined directly from
concentration of acid or base.
Strong Acids

HA

H+

A-
Strong Acid
Weak Acids and Bases
Strong Acids completely dissociate.

Weak acids undergo incomplete dissociation.

Weak acids and bases do not fully dissociate; in fact, the

dissociation of most weak acids is relatively small, and only a
fraction of the acid is in the dissociated form.
Weak Acids
Partial dissociation of HA

HA

H+

A-
Acetic Acid Equilibrium

Ka is the direct measure of the acid’s strength,

which is the ability to donate a proton
pKa
The pKa is a measure of the strength of a weak acid. Although
all weak acids are weak, some are stronger than others. Since
Ka is the direct measure of the acid’s strength (ability to donate
a proton), the pKa follows the inverse trend:

larger Ka = stronger acid, but larger pKa = weaker acid

Titration Curve

pH Buffering Region

OH- added (equivalents)

 Key Questions
• What happens to the concentrations of the
acid and its conjugate base when pH is at pKa?
• What happens when pH is greater than pKa?
• What happens when pH is less than pKa?
Lactic acid has a pKa of 3.86.
• Which form (lactic acid or lactate) predominates
at pH = 2.2?
• How about at pH 7?
• How about at pH 3.86?
• Notice the charges on the acid/conjugate base
Weak bases and pKa
Remember the definition of pKa, as a measure of the strength
of an acid, and numerically the pKa= -log (Ka)

What does it mean for a weak base to have a pKa?

ammonia:

Treat the conjugate acid (ammonium ion) as the species that

will give up the proton, essentially reversing the reaction.
Thus, the pKa of this reverse reaction is a measure of the
strength of the conjugate acid.
Weak bases and pKa

ammonia
ammonium ion

Looking at this reaction, the reverse is:

NH4+  H+ + NH3 with pKa = 9.3
• Which form (ammonia or ammonium ion) predominates at
pH 11.2?
• How about at pH 6.5?
• How about at pH 9.3?
• Notice the charges on base/conjugate acid
Titration Curve Example
Titration of Acetic Acid with NaOH

pH
All data
shown and
Axes connected
labeled with a curve
with units,
when
appropriate NaOH (ml)
http://www.dartmouth.edu/~chemlab/chem3-5/acid1/full_text/chemistry.html
Buffer System
Henderson-Hasselbalch Equation

This equation can be derived directly from the equation of Ka of a weak

acid, by taking the log of both sides and then solving for pH.

When is the H-H equation useful? When you’d like to know the ratio or
individual quantities of the acid and its conjugate base at any pH given
the pKa of the acid.
A Note on Ratios and Fractions
The ratio between two components A and B expresses the
relative amounts of the two components. For example, if A =
1 and B = 5 then the ratio of A:B is 1/5. That is for every A,
there are 5 of B.

The fraction of A reflects what portion of the total of A and B

is composed of A. For example, if A = 1 and B = 5 then the
fraction of A = A / (A + B) = 1/( 5 + 1) = 1/6.

Thus, the ratio of A/B is 1/5, but the fraction of A is

1/6. Also, the fraction of B is 5/6.

Notice the difference in the denominator!

Using the H-H Equation

Lactic acid has a pKa of 3.86.

• What is the ratio of lactate/lactic acid at pH 7?
• What is the fraction of lactate at pH 7?
• At what pH does the concentration of lactate
equal lactic acid?
• At what pH will 99% of lactic acid dissociate?
Buffer Selection
Polyprotic acids

Carbonic acid, pKa = 6.1, 10.2

Titration of Polyprotic Acids
pH Meter