# ITC (Isothermal Titration Calorimetry)

Mechanics

Oct 27, 2013 (4 years and 8 months ago)

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A single experiment sufficient to obtain all of
the thermodynamic components

Stoichiometry

of the interaction (n)

Association constant (K
a
) & Dissociation constant
(
K
d
)

Enthalpy (
Δ
H
b
)

Free energy (
Δ
G
b
)

Entropy (
Δ
S
b
)

Heat capacity of binding (
Δ
C
p
)

A
system
is defined as the matter within a
defined
region of space (i.e.,
reactants,
products
, solvent)

The
surroundings
is the matter
in the rest of
the
universe

The
total
kinetic energy
due to the motion
of
molecules
(translational, rotational,
vibrational
)
+
the total
potential energy
associated with
the
vibrational

and electric
energy of atoms within molecules or crystals
.

Δ
U
= W + Q

Δ
U
= W + Q

Q
P

=
Δ
U

W

Q
P

=
Δ
U

P(V
2
-
V
1
)

Q
P

=
Δ
U

P(
Δ
V)

Q
P

=
Δ
H

The enthalpy
is the heat absorbed or emitted
by a system at constant pressure

Exothermic reaction

Δ
H
is negative

Emits heat

Endothermic reaction

Δ
H
is positive

Absorbs heat

Reaction continues

Temperature change
occurs

Power supply is given
to maintain a constant
temperature
difference between the
reaction cell and the
reference cell

Power supply is
measured

Exothermic reaction:

Emit heat

Negative peak on ITV

Endothermic reaction:

Absorb heat

Positive peak on ITC

Quantitative technique that can directly
measure:

the binding affinity (K
a
)

enthalpy changes (ΔH)

binding
stoichiometry

(n) of the interaction between
two or more molecules in solution

Gibbs energy changes (ΔG), and entropy
changes (ΔS), can be determined using the
relationship:

ΔG =
-
RTlnK
a

= ΔH
-
TΔS (where R is the gas constant
and T is the absolute temperature).

As promised…

The Energy is conserved

The total energy of a system and its
surroundings is constant

In any physical or chemical change, the total
amount of energy in the universe remains
constant, although the form of the energy
may change.

Δ
U
= W + Q

Δ
E
represents the change in the energy

Q the heat absorbed by the system

W the work done on the system

Q
P

=
Δ
U

W

Q
P

=
Δ
U

P(V
2
-
V
1
)

Q
P

=
Δ
U

P(
Δ
V)

Q
P

=
Δ
H

The enthalpy
is the heat absorbed or emitted by a
system at constant pressure

The total entropy of a system and its
surroundings always increases for a
spontaneous process

Δ
S
total

=
Δ
S
system

+
Δ
S
surroundings

Δ
S
surroundings

=
-

Δ
H
system
/T

Δ
S
total

=
Δ
S
system

-

Δ
H
system
/T

-
T

Δ
S
total

=
Δ
H
system

-

T

Δ
S
system

Δ
G =

Δ
H
system

-

T

Δ
S
system

ΔG<0 spontaneous change

ΔG=0 equilibrium

For a reaction to be spontaneous, the entropy
of the universe,
ΔS
total
, must increase:

Δ
S
system

>

Δ
H
system
/T or

Δ
G =
Δ
H
system

T

Δ
S
system

< 0

The
free energy must be negative for a
reaction to be spontaneous!

Reactant

Product

Association constant:

Δ
G =

RT
lnK
eq

K
eq

= 10

Δ
G/1.36

at

state,

at

which

ΔG=
0

Immobilization or labeling
필요

없다
.

K
d
,
Δ
H can be measured

Can be applied to different
reaction temperature and
pH

Enormous amounts of
binding partner

Only medium affinity

Limitation for membrane
proteins

High price