Polyprotic Acids

Polyprotic Acids

protic Acids Have More than One Acidic Group

Carbonic acid
H2CO3 + H2O
HCO3- + H2O

H3O+ + HCO3pKa1 = 6.4

H3O+ + CO32- pKa2 = 7.2

Phosphoric acid
H3PO4 + H2O
H2PO4- + H2O
HPO42- + H2O

H3O+ + H2PO4pKa1 = 2.12

H3O+ + HPO42- pKa2 = 7.21
H3O+ + PO43- pKa3 = 12.68

Tryptophan (W)

O
OH

N
H

pKa1 = 2.35

NH3
pKa2 = 9.33
+

Amino Acids
Small

Nucleophilic

Hydrophobic

Aromatic

X-ray Crystal Structure

of Collagenase Binding
to Collagen.

Amide

Acidic

Basic

Amino Acids

X-ray Crystal Structure

of Collagenase Binding
to Collagen.

General Polyprotic Acid

H2A HA H pKa1

Solution Composition

HA A2 H pKa2

Principle
Species
In other words, what chemical
species has the highest
concentration at the given
pH?

pKa1

pKa2

pH

H2A

HA-

A2-

Isoelectric Point

oelectric point is the pH at which a polyprotic acid has a net charge of

For a simple diprotic amino acid: pI = (pKa1 + pKa2)
For other higher polyprotic acids, the calculation is more complex.

(A) AmyL

(B) AmyLQS50.5

(C) AmyLQS55.6
Distribution of surface charge on:
(A) AmyL
(B) AmyLQS50.5 (pI 10.0)
(C) AmyLQS55.6 (pI 5.0).
Surface charge distributions were
calculated using the GRASS software;
negative charge is shown in red and
positive charge is shown in blue. The
conservation of negative charge in the
active-site cleft explains why the
enzymatic activity is conserved in
AmyLQS50.5, despite its high overall pI.

Active Site
Biochem. J. (2000) 350 (3139)

Isoelectric Point
Isoelectric Point of
Human Proteins

The near-neutral pI region has presumbably been depleted by

selective pressure -- cytoplasmic proteins are least soluble at their
isoelectric point.
Proteins may thus be evolutionarily trapped on one peak or the
other.

Isoelectric Focusing
A protein dissolved in buffer at its pI has no net
charge and thus no net electrophoretic mobility.
Positive Electrode

Isoelectric focusing employs a pH gradient

extending the length of an electrophoresis gel. A
protein stops migrating when it enters the zone in
which the surrounding pH equals its isoelectric
point, pI. At any other point in the gradient, the
protein acquires a charge (positive or negative)
which causes it to migrate toward its pI (green
2-D Gel Separation
and blue arrows).

MW
Proteins from Human
Retinal Pigment Epithelium
(RPE) Cells

pH

Negative Electrode

Proteomics
Unlike the genome, the proteome is
exquisitely
sensitive
to
cellular
conditions and will consist of proteins
having abundances dependent upon
stage
in
the
cell
cycle,
cell
differentiation,
response
to
environmental conditions (nutrients,
temperature, stress, etc.), or disease
state(s).

Jensen, P. K.; Pasa-Tolic, L.; Anderson, G. A.; Horner, J. A.; Lipton, M. S.;
Bruce, J. E.; Smith, R. D.; Anal. Chem. 1999; 71; 2076-2084.

LC-MS

Figure 1. (A) IE-MS electropherogram for a lysate from

E. coli. The positive ESI mass spectra in (B)-(D) are
from single scans under their respective peaks. The
insets of (B) and (C) show the high-resolution spectra for
a single charge state.

Polyprotic Calculations
In General Chemistry You Should Have Learned:
1) How to calculate the pH of a polyprotic acid solution H nA
Set up ICE table
2) How to calculate the pH of a diprotic salt solution NamHnA
pH ~ (pKa1 + pKa2) (will discuss more)
3) How to calculate the concentration of all species of a
diprotic acid
Set up two or more ICE tables
4) The relationships between Kas and Kbs for polyprotic
acids
Ka1 x Kb2 = Kw
(diprotic acid)
Ka2 x Kb1 = Kw
Ka1 x Kb3 = Kw

(triprotic acid)

Polyprotic Problems
Example 1:
Calculate the pH of 0.10 M tryptophan
hydrochloride.

pKa = 2.35
OH

NH3 Cl
+
pKa = 9.33

N
H

Example 2:
Calculate the composition of all species in a 0.10 M tryptophan
hydrochloride solution.
Example 3:
Calculate the pH of 0.10 M monosodium tryptophan
chloride.

O
O Na
N
H

NH3 Cl
+

Titrations of Polyprotic Acids

To calculate the pH at any step in the titration of a polyprotic
acid:
1) Use stoichiometry & amount of base added to compute stage of
titration
2) Focus on the principle species & its proton transfer equilibrium

Diprotic Acids
Carbonic Acid
H2CO3 + H2O
HCO3HCO3- + H2O

H3O+ +
H3O

+ CO

Stoichiometr
ic Points

23

Initial

Buffer
Regions

Triprotic Acids
Phosphoric
H3PO4 + H2O
Acid
H2PO4H2PO4- + H2O
HPO42HPO42- + H2O

H3O+ +
H3O+ +
H3O+ + PO43-

Sample Problem
Example:
100 mL of 0.100 M phosphoric acid, H3PO4 was titrated with the following
volumes of 0.100 M NaOH. Calculate the pH at each point.
pKa1 = 2.12 pKa2 = 7.21 pKa3 = 12.68
(a) 50 mL of NaOH added
(b) 100 mL of NaOH added
(c) 175 mL of NaOH added
(d) 350 mL of NaOH added

Answers:
(a) pH = 2.12
(b) pH = 4.67
(c) pH = 7.69
(d) pH = 12.05
100 mL NaOH
added

H3PO4

200 mL NaOH
added

H2PO4(a)

(b)

HPO42(c)

300 mL NaOH
added

PO43(d)