Lecture Notes 3A Dr. M.

Eze
3502 2005-06

Slide 3A-01

Titration Curves Reveal pKa of Weak Acids:

Consider titration of:

0.1M acetic acid (HAc) solution with 0.1M NaOH at 25oC.

Two reversible equilibria

H2O W H+ + OH - Eqn 3.1

HAc W H+ + Ac- Eqn 3.2

and their equilibrium constants

Kw = [H +][OH -] = 1x10-14 M2 (see Eqn. 1.4 b)

Ka = {[H+][Ac-]/[HAc]} = 1.74 x 10-5 M Eqn 3.3

Slide 3A-02

Before addition of NaOH, some of the protons are available, as predicted by Eqns 3.2 &
3.3

Add NaOH: H+ is consumed

– upsets Eqn 3.1
- Eqn 3.2 is driven to the right, forming H + + Ac-

At ½ titration, pH = pKa (see Henderson-Hasselbalch eqn)

Within the range (pKa+1) to (pKa-1), [or (pKa+0.5) to (pKa-0.5)]

pH changes little with NaOH added

= BUFFERING REGION

Slide 3A-03

Further titration leads to end point, around pH 7

All weak acids display the same shape of titration curve

They differ in the position of curve on pH axis:

-Dependent on
-the pKa
-acidity of the proton

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Thus, stronger acid will reach ½ titration at lower pH (i.e., titration curve lower on the pH axis)

than a weaker acid.

Note the predominant ionic forms at the various points (I.e., pH) during titration.

Slide 3A-04

pH

NH4+
pKa = 9.25

NH4+

CH3 COOH
pKa =4.76

CH3COOH

0 0.25 0.5 0.75 1.0

Moles base added per mole acid

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 Slide 3A-05

BUFFER:
1. solution in water of

Weak acid + its conjugate base

OR
Weak base + its conjugate acid

2. Resists changes in pH when small amounts of acid (H +) or base (OH -) are added.

3. E.g., Acetic acid + Acetate ion

Slide 3A-06

+ -
Kw = [H ][OH ]
- H2O
[OH ]

Acetate
Acetic acid HAc Ac- -
CH3COO H CH3COO

+ - H+
Ka = [H ][Ac ]/[HAc]
(From: Nelson & Cox 2005 “Lehninger Biochemistry Principles of Biochemistry“)

Slide 3A-07

Mix equal concns of acetic acid and acetate ion

(as at midpoint of titration curve, where pH = pKa)

Add small amount of H +; Ac - is protonated to HAc

This keeps Kw constant.

Because H + is consumed, pH remains constant.

Add a little OH -; HAc is deprotonated to Ac -

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This keeps Kw constant.
Because OH - is consumed, pH remains constant.

Only the ratio [Ac -]/[HAc] changes during buffering

A buffer at pH = pKa will buffer added acid or base to same extent.

Slide 3A-08

Note Very Well: both pH and pKa are log values.

[PA]/[PD] %[PA]
pH Retative to pKa [Ac-]/[HAc] %[Ac -]

pH = pKa + 4 10000/1 99.99

pH = pKa + 3 1000/1 99.90
pH = pKa + 2 100/1 99.01
pH = pKa + 1 10/1 90.91
pH = pKa 1/1 50.00
pH = pKa - 1 1/10 9.09
pH = pKa - 2 1/100 0.99
pH = pKa - 3 1/1000 0.10
pH = pKa - 4 1/10000 0.01

Familiarity with this concept, and its applications will be of help when calculating net charges on
molecules at different pH values.

Slide 3A-09

Exercise:

Calculate the charge on an acetic acid molecule at pH 4.6 [Assume pKa =4.76]

Solution:

This means, the amount of the acetate present

pH = pKa + log{[Ac -]/[HAc]}

4.6 = 4.76 + log{[Ac -]/[HAc]}
10-0.16 = [Ac-]/[HAc]
0.6918/1 = [Ac -]/[HAc]

Fraction deprotonated (i.e., existing as CH3COO-)

= 0.6918/1+0.6918 = 0.4089
Charge on the molecule = (0.4089 x –1) = - 0.4089

Slide 3A-10

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 Buffering Systems:
Useful at pH = pKa+0.5 (not more than pKa+1) range
I.e., only 1 pH unit on either side of the pKa
= Buffering Region.

Different acid-base pairs are needed for buffering different pH values. (Considering their
acid pKa values and buffering regions) E.g.,
pKa
Acetate CH3COOH W CH3COO + H - + 4.76
Phosphate H3PO4 W H2PO4- + H + 2.15
H2PO4 W HPO4 + H
- 2- + 6.82
HPO4 W PO4
2- 3- +H + 12.38
Borate H3BO3 W H2BO3 + H - + 2.14

Tris (Tris-hydroxymethyl)aminomethane
HOCH 2
+ -
HOCH 2 C NH3 Cl H + + Cl + Tris base pKa 8.1

HOCH 2 Tris acid

Slide 3A-11

Glycine (R = H): (& Other Amino Acids)

COOH - -
COO COO
+
H3 N CH
H+ + H 3+N CH H+ + H 2N CH
R
pKa 2.34 R pKa 9.60 R

Slide 3A-12

Titration Curves of Polyprotic Acids:

Treat :

1. Phosphoric Acd

2. Amino acid (e.g., glycine)

Students:

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3. Amino acid (e.g., Aspartic acid)

Slide 3A-13

Buffer Preparation: Here are 3 ways out of the many options possible

1. Mix weak acid and conjugate base in correct proportions to obtain the required
[PA]/[PD] ratio.

E.g., H 2PO4- + HPO42-

2. Mix weak acid and strong base: Strong base converts some weak acid to conjugate
base to establish the ratio

NaOH + H 2PO4- → HPO42- + H 2O + Na+

Use an amount of H2 PO4 - ) correctly calculated, such that the unreacted amount equals exactly the
amount to achieve the desired [PA]/[PD] ratio for your pH.

Slide 3A-14

3. Mix Weak base and strong acid:

Strong acid converts weak base to conjugate acidto establish the ration

Tris base + HCl → Tris.HCl

Buffer Capacity:

= Ratio of incremental amount of base added

(in equivalents)
To the corresponding pH change
i.e., (∆ equivalents of base added)/(∆ pH caused)

= # equivalents of base per unit pH change.

Slide 3A-15

So: Higher Buffer concn = Higher buffer capacity.

Since, pH = pKa + log{[PA]/[PD]}

pH is same if

[PA]/[PD] = ½, 2/4, 5/10, 0.1/0.2, 0.01/0.02

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But Buffer capacity is not same

Buffer capacity is highest at [PA]/[PD] = 5/10;

Decreasing as 5/10 > 2/4 > ½ > 0.1/0.2 > 0.01/0.02

Slide 3A-16

Cells and Tissues are Buffered by Weak Acids and Weak bases:

1. Proteins contain amino acids with functional groups which are weak acids or weak
bases.

Protein
Protein
CH 2 Imidazole
NH CH 2 side group
NH of histidine
+ H+ = weak acid
+ pKa pKa = 6.0
N 6.0
H N

2. Nucleotides e.g., ATP

3. Other metabolites

Slide 3A-17

Phosphate and Bicarbonate = Physiological Buffers:

In cytoplasm (all cells):

H2PO4- W HPO42- + H + pKa = 6.82

Buffer region 6.32 – 7.32

So, good for intracellular fluid (pH 6.9-7.4) of mammals.

Slide 3A-18

Blood Plasma by Bicarbonate System:

There are 3 equilibria

(i) H 2CO3 W HCO3- + H +

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Eqm Constant = K 1 = [H+][HCO3-]/[H2CO3]

Buffers like regular weak acid

(ii) Dissolved CO2

CO2(d) + H 2O W H2CO3

Eqm Constant = K 2 = [H2CO3]/[CO2(d) ][H 2O

Slide 3A-19

(iii) CO2 gas: controls [CO2(d) ]

CO2(g) W CO2(d)

Eqm Constant = K 3 = [CO2(d) ]/ [CO2(g) ]

All these three affect the pH of blood by controlling K 1.

[CO2(g) ] affects [CO2(d) ], which affects [H 2CO3]