BUFFERS
LABM 201; Clinical Chemistry I
Fall Semester
Rose Daher, PhD
Friday, January 26, 2024
LECTURE OUTLINE
1. ACIDS AND BASES
2. ACID BASE EQUILIBRIUM
3. BUFFERS
4. POLYPROTIC ACIDS
5. EXERCISES
6. USEFUL SITE:
HTTP://www.bio.cmu.edu/courses/03231/biochemF03.htm
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Rose Daher, PhD
Clinical Chemistry I
1. ACIDS AND BASES

Acid: can donate a proton

Base: can accept a proton

Example:
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Rose Daher, PhD
Clinical Chemistry I
1. ACIDS AND BASES


The compound M-O-H is

an acid if it looses its proton

a base if it releases an OH- group
Whether a compound is an acid or a base depends on the relative strength of the M-O bond versus
the O-H bond


The strength of the acid increases with the electronegativity of M

↑ electronegativity  more electrons are drawn from the OH group

This electron density stabilizes the M-O bond
Group I elements (Li, Na, K) all have electronegativities of one or less.


Thus, the M-O bond is more ionic in nature and LiOH, NaOH, KOH are very basic
When M is a non-metal (e.g. C, S, N, Cl) the compound is usually acidic

The more electron-withdrawing M is, the stronger the acid
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Clinical Chemistry I
1. ACIDS AND BASES




Ethanol is very weak acid
Acetic acid somewhat stronger
Trichloroacetic acid is very strong.
A quantitative measure of the strength of an acid is given by the acid dissociation
constant, Ka
Increased Acidity
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Clinical Chemistry I
2. ACID BASE EQUILIBRIUM
HA

H+
+
A-
Henderson-Hasselbalch Equation
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Clinical Chemistry I
2. ACID BASE EQUILIBRIUM

pKa is dissociation constant for weak acids and depends on degree of dissociation,
pH and temperature.
Acid
HCl
Oxalic
Acetic
Ammoinium (NH4+)
Methanol
Benzene
8
pKa
-7
1.23
4.76
9.25
16
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Type
Very strong
Strong
Weak
Very weak
Extremely weak
Not really an acid
Clinical Chemistry I
3. BUFFERS




A buffer is something that resists change
A pH buffer is a substance that permits solutions to resist large changes in pH upon
the addition of small amounts of H+ or OH- ions
Buffers play an important role in cellular processes because they maintain the pH at
an optimal level for biological processes
Strong acids are poor buffers
 Buffers must be Weak Acids or Weak Bases


An acidic buffer contains a weak acid and a salt of the weak acid (conjugate base)
A basic buffer contains a weak base and a salt of the weak base (conjugate acid)
HA

weak acid
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H+
+
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Aconjugate base
Clinical Chemistry I
3. BUFFERS

The two species (conjugate acid and conjugate base) resist large changes in pH by
partially absorbing additions of H+ or OH- ions to the system

The amount of change depends on the strength of the buffer and the [A-]/[HA] ratio

The buffering capacity of a weak acid is best at pH values near (within 1 pH unit
of) its pKa

The buffering capacity decreases as the dissociation becomes more complete
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Clinical Chemistry I
Titration Curve of 100 ml of Amino Acid solution
4. POLYPROTIC ACIDS

Many compounds can release more than one proton in the pH range of 0-12
 Examples are phosphate (pKa = 2.14, 7.20, 12.38), carbonate, and dicarboxylic acid


If the pKa s are separated by 2 or more pH units (i.e. phosphoric acid) each
ionization can be treated separately
Depending on the structure of the polyprotic acid the pKa for each ionization can
vary widely
Acid
Phosphoric
Oxalic
Succinic
Carbonic
12
pK1
2.14
1.23
4.21
6.37
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pK2
7.20
4.19
5.63
10.20
Clinical Chemistry I
5. EXERCISES
1.
Calculate the pH of an acetate buffer composed of 0.2M sodium acetate and 0.05M acetic acid:
HAc
↔ H+ + Ac- , pKHAc = 4.76
2.
Prepare an acetate buffer whose concentration is 0.2M and has a pH of 5.0. pKHAc = 4.76;
MWHAc = 60 g/mol; MWNaAc = 82 g/mol; buffer volume = 1 liter.
3.
An enzyme-catalyzed reaction was carried out in 0.2M “Tris” buffer [Tris (hydroxymethyl)
aminomethane] pH = 7.8. As a result of the reaction, 0.03 mol/L of H+ was produced.
Tris+ →
a.
b.
c.
d.
e.
Tris0 + H+ ; pKa = 8.1
What was the ratio of Tris+ (Conjugate acid) / Tris0 (Conjugate base) at the start of the reaction?
What are the concentrations of Tris+ and Tris0 at the start of the reaction?
Show the reaction by which the buffer maintained a near constant pH?
What are the concentrations of Tris+ and Tris0 at the end of the reaction?
What was the pH at the end of the reaction?
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Clinical Chemistry I
5. EXERCISES
4. Describe the preparation of 2 liters of 0.4M phosphate buffer pH = 6.9, starting from :
a. 1.2M K2HPO4 solution and 2M H2SO4 solution
H3PO4 → H+ + H2PO4-,
Ka1 = 7.11 x 10-3
H2PO4- → H+ + HPO42-, Ka2 = 6.34 x 10-8
5. An enzyme catalyzed reaction was carried out in a solution buffered with 0.25M
phosphate, pH = 7.2. As a result of the reaction, 0.05 mol/L of acid was formed.
a. What was the pH at the end of the reaction?
b. What would the pH be if no buffer were present?
c. Write the chemical equation showing how the phosphate buffer resisted a large change
in pH.
6. Calculate the volume of 0.2M sodium acetate and 0.2M acetic acid solutions required to
prepare 100 ml, 0.2M acetate buffer, pH = 4.70 . Acetate buffer pKa = 4.76.
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Clinical Chemistry I
5. EXERCISES
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Clinical Chemistry I