ISOTHERMAL TITRATION

CALORIMETRY

PRESENTED BY:
Ms. PRAJAKTA S.PAWAR.

GUIDED BY:
DR. S. V. GANDHI.

1st Sem, M.Pharm. 2010

Dept. of P’ceutical Chemistry
AISSMS College of Pharmacy,
Pune.

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CONTENTS:

1) Introduction
2) Thermometric Titrations
3) Isothermal Titration Calorimetry[ITC]
I. Instrument
II. Principle
III. Standard Operating Procedure
IV. Critical Parameters
V. Advantages
VI. Disadvantages
VII. Applications
4) References

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CALORIMETRY:

Calorimetry is the science of measuring the heat of chemical

reactions or physical changes.
Calorimetry is performed with a calorimeter.
The word calorimetry is derived from the Latin word calor,
meaning heat
The reaction system is considered as the environment.
Q (surr) =Q(cal)+Q(contents)

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CALORIMETER INSTRUMENT:

A calorimeter is a device used for calorimetry.

Differential scanning calorimeters, isothermal
microcalorimeters, titration calorimeters and accelerated rate
calorimeters are among the most common types.
A simple calorimeter just consists of a thermometer attached to
a metal container full of water suspended above a combustion
chamber
To find the enthalpy change per mole of a substance A in a
reaction between two substances A and B, the substances are
added to a calorimeter and the initial and final temperatures
(before the reaction started and after it has finished) are noted.
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Multiplying the temperature change by the mass and specific
heat capacities of the substances gives a value for the energy
given off or absorbed during the reaction
It does not account for the heat loss through the container or
the heat capacity of the thermometer and container itself. In
addition, the object placed inside the calorimeter show that the
objects transferred their heat to the calorimeter and into the
liquid, and the heat absorbed by the calorimeter and the liquid
is equal to the heat given off by the metals.

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CALORIMETER TYPES:

1. Adiabatic calorimeters

2. Reaction calorimeters
3. Bomb calorimeters
4. Calvet-type calorimeters

5. Constant-pressure calorimeter
6. Isothermal titration calorimeter
7. Differential scanning calorimeter
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CALORIMETER INSTRUMENT:
Reaction calorimeter
Use : Measuring the heat of reaction.

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THERMOMETRIC TITRATIONS:

Thermometric titrations are one utilises heats of rection to

obtain titration curves.
The curves are obtained by plotting changes in solution
temperature against volume of titrant.
Thermal titrations have been successfully applied to
neutralisation , complexation , redox & precipitation titrations.
The most important advantage of these titrations is that they
can be used in non-aqueous media because dielectric constant
of the solvent has no effect on these titrations.

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Thermometric titrations depends only on the heat of
reaction(∆H ) given by:
∆H = ∆G + T∆S
The success of thermometric titration depends upon the
amount of heat (∆H ) evolved or absorbed during the reaction.

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ISOTHERMAL TITRATION CALORIMETRY:

Isothermal Titration Calorimetry (ITC) is the gold standard for

measuring biomolecular interactions.  ITC simultaneously

determines all binding parameters (n, K, ∆H and ΔS) in a
single experiment – information that cannot be obtained from
any other method.
When substances bind, heat is either generated or absorbed. 

ITC is a thermodynamic technique that directly measures the

heat released or absorbed during a biomolecular binding event. 
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Measurement of this heat allows accurate determination of
binding constants (KB), reaction stoichiometry (n), enthalpy
(∆H) and entropy (ΔS), thereby providing a complete
thermodynamic profile of the molecular interaction in a single
experiment. 
Isothermal Titration Calorimetry (ITC) provides complete
thermodynamic characterization of the binding of a drug to its
therapeutic target, including enthalpy (∆H) and entropy (T∆S),
free energy (∆G) and binding affinity (KB).

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These thermodynamics parameters are related to each other by
the following
equations
∆G = ∆H –T∆S
∆G = -RT lnKB
For spontaneous reactions, ∆G is negative, and ∆G is directly
related to the binding affinity. The tighter the binding, the more
negative the ∆G.

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ITC INSTRUMENT:

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ITC INSTRUMENT ADVANTAGES:

1. Small instrument – requires less than 3 ft of bench space.

2. Completely computerised operation.
3. Must be located in room with constant temperature.
4. No user – serviceable parts except syringe.

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ITC INSTRUMENT:

Diagram of ITC cells and syringe

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Two coin shaped cells in adiabetic chamber kept at exactly the
same temperature.
Sample can be heated fast, but cooling is slow : sample should
be colder than the experimental temperature when loaded.
Reference cell contains water or buffer nothing is added.
Sample cell contains macromolecule in solution.
Solution is stirred with the paddel-shaped tip of the needle to
ensure rapid mixing of components.

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SCHEMATIC OF AN ITC INSTRUMENT:

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An isothermal titration calorimeter is composed of two

identical cells made of a highly efficient thermal conducting
material such as Hastelloy alloy or gold, surrounded by an
adiabatic jacket.
Sensitive thermopile/ thermocouple circuits are used to detect

temperature differences between the reference cell (filled with
buffer or water) and the sample cell containing the
macromolecule.
Prior to addition of ligand, a constant power (<1 mW) is

applied to the reference cell. This directs a feedback circuit,
activating a heater located on the sample cell.
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During the experiment, ligand is titrated into the sample cell in
precisely known aliquots, causing heat to be either taken up or
evolved (depending on the nature of the reaction)
Measurements consist of the time-dependent input of power
required to maintain equal temperatures between the sample
and reference cells.
In an exothermic reaction, the temperature in the sample cell
increases upon addition of ligand. This causes the feedback
power to the sample cell to be decreased (remember: a
reference power is applied to the reference cell) in order to
maintain an equal temperature between the two cells.

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In an endothermic reaction, the opposite occurs; the feedback
circuit increases the power in order to maintain a constant
temperature (isothermic /isothermal operation).
Observations are plotted as the power in μcal/sec needed to
maintain the reference and the sample cell at an identical
temperature. This power is given as a function of time in
seconds.
As a result, the raw data for an experiment consists of a series
of spikes of heat flow (power), with every spike corresponding
to a ligand injection.
These heat flow spikes/pulses are integrated with respect to
time, giving the total heat effect per injection.

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The pattern of these heat effects as a function of the molar ratio
[ligand]/[macromolecule] can then be analysed to give the
thermodynamic parameters of the interaction under study.
The entire experiment takes place under computer control.

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PRINCIPLE:

The ITC instrument is a heat-flux calorimeter operating

according to the dynamic power compensation principle, i.e., it
measures the amount of power (μcal/sec) required to maintain
a constant temperature difference (close to zero) between the
sample and the reference cell.
 Initially, the feedback system continuously applies a small
power to the sample cell, which determines the baseline level.
Each injection of the syringe solution (usually termed as
ligand) triggers the binding reaction and, depending on the
binding affinity and the concentration of reactants
(macromolecule, M, and ligand, L) in the cell, a certain amount
of macromolecule /ligand (ML) complex is formed.

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The formation of complex is accompanied by the release
(exothermic reaction) or the absorption (endothermic reaction)
of heat that causes a difference in temperature between the two
cells
Then, the feedback system either lowers or raises the thermal
power applied to compensate such temperature unbalance.
After each injection, the system reaches equilibrium and the
temperature balance is restored.

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Therefore, the recorded signal shows a typical deflection
pattern in the form of a peak. Integrating the area under the
peak, assuming the baseline as reference, provides the amount
of heat associated with the injection.
As the reactant in the cell becomes saturated, the heat signal
diminishes until only the background heat, due to an unspecific
phenomena (e.g., ligand dilution, liquid friction), is observed.

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PROCEDURE OF ITC:
This procedure contain detail description of setting up and
using the ITC instrument. The time required for performing a
complete ITC experiment (setting up the instrument, running
the experiment, and analyzing the results) is ∼2 hr. this
procedure detailed and specific explanations are provided. it
can serve as a guide for other models.

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Determine the appropriate concentrations of reactants:
1.Typically, the concentrations of reactants are in the micromolar range; if
the heat associated with the reaction is significant, however, the
concentrations can be lowered.
An important requirement for an ITC experiment is having appropriate
concentrations of interacting molecules such that the heat associated
with a given binding reaction is within the calorimetric determination
range.
 Sensitivity of the instrument varies depending on brand and model.
The Microcal VP-ITC instrument used in all the experiments described
in this unit has a limiting sensitivity of 0.1 μcal.
Therefore, to accurately determine the change of heat involved in the
interaction between molecules, each injection should have a minimum
of 1 μcal of associated heat
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To obtain a complete binding isotherm within the specified
number of injections, the ligand solution in the syringe should
be more concentrated than the macromolecule in the cell,so
that at the end of the experiment, the molar ratio of ligand to
macromolecule insidethe cell is 2 to 3 (for 1:1 stoichiometry).
Considering the volume of the sample cell (1.4ml), the typical
injection volume (10 μl), and the typical number of injections
(∼30), it is advisable to use a concentration of ligand in the
syringe 10 to 20 times higher than the solution in the cell.
This will guarantee that the reaction reaches the neutralization
point after 7 to 13 injections.

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Prepare samples:
Choose experimental conditions, taking into account the
stability and the solubility of the reactants and the biological
considerations of the system under study.
Prepare reactant solutions for the cell and the syringe under
identical conditions and with the same composition.
For the reactant solutions, buffer type, buffer concentration,
pH, ionic strength, and cosolvents must be the same.
In an ITC assay, consistency between the exact composition of
the buffer in the cell and the syringe is of crucial importance to
prevent dominance of unspecific heat effects.
The composition, concentration, pH, and ionic strength of the
buffer all affect the thermodynamic parameters, and the quality
of the experiment depends on maintaining a perfect match of
the buffer in the cell and syringe samples.
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One effective way of achieving this goal is to dialyze the
macromolecule against the desired buffer and then using the
filtered dialysis buffer to prepare the ligand solution.
Certain additives such as DMSO, which increases the
solubility of hydrophobic ligands, have an enormous effect on
the ITC signal, therefore, utmost care should be taken to keep
the concentration of DMSO in the cell and syringe nearly the
same.
2. Prepare the reactant solutions in the appropriate
volumes.Although the apparent volume of the sample cell is
1.4 ml, the volume of solution required for proper filling of the
cell is 2.2 ml. The volume required for the syringe is 0.5 ml.
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3.Degas all solutions (reactants and buffer solution used for
rinsing the cell) for 10 to 20min (stirring and temperature
control are optional) to avoid formation of bubbles in the
sample cell during the experiment
4.To prevent long equilibration delays, lower the calorimeter
thermostat setting slightly below the running temperature
(with a difference of 0.5◦ to 2◦C).Load solutions
5.Place degassed, distilled water or buffer solution in the
reference cell using a 2.5-ml, long-needle syringe.
6.Rinse the sample cell several times with buffer solution, then
remove all liquid.

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7. Fill the cell with macromolecule solution, taking care to
prevent the appearance of bubbles in the cell.The most
common configuration is one in which a macromolecule is
placed in the reaction cell and a low-molecular-weight ligand is
placed in the injecting syringe. For convenience,the injected
reactant located in the syringe is referred to as “ligand” and the
one in the reaction cell is referred to as “macromolecule,”
throughout this unit. The reader should be aware that other
configurations are possible

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8.Fill the calorimeter syringe with ligand solution according to
the maufacturer’s instructions. Rinse off excess ligand solution
on the surface of the needle with water and then carefully blot
the surface of the needle with a paper towel. A special plastic
syringe is used to fill the calorimeter injecting syringe. A
purge-refill cycle may be performed to ensure the absence of
air bubbles inside the syringe.

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Clean the ITC:

1) Wash the injection syringe with ≥150 ml water. Frequently, and

especially for low solubility reactants, wash the syringe with 50 ml
methanol (or water-organic cosolvent) and 300 ml water. Dry
syringe for ≥10 min.
2) Wash the plunger tip of the syringe injector with water and dry it.
3) Wash the sample cell with water (ten times or more) using the
long-needle syringe.
4) For periodic deep cleansing or when using a different
macromolecule or in cases where the sample precipitated or
aggregated in the sample cell, the sample cell should be washed with
a cleaning solution (compatible with the material the cells are made
of) according to the manufacturer’s instructions.
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Time Considerations:

The time required for a complete ITC experimentcan be

divided into different steps.
Preparing sample solutions requires 30 min.
Running the ITC experiment takes 2 to3 hr.
Cleaning the ITC instrument requires 20 min. analyzing the
results takes 30 min.

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CRITICAL PARAMETERS:
For a titration experiment to be successful and well designed,
the following critical points should be considered.
1) ITC measures the global heat effect associated with the
binding of two molecules. This includes the actual heat of
binding (binding enthalpy) and all other heat effects originated
from non-specific events (dilution of reactants, friction of
injected liquid, etc.). Therefore, it is important to ensure that
these contributions are minimized or considered in the
analysis.
2) Regarding the possibility of aggregation or association of the
reactants, the reactant with the lower solubility should be
placed in the calorimetric cell.

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3) As explained above, the appropriate range of concentrations and
the proper ratio of ligand/macromolecule concentrations needs
to be employed to guarantee completion of titration, reaching
the saturation point in a reasonable number of injections (<30).
4) A perfect match between the buffers has to be achieved to avoid
spurious heat effects due to protonation of different species and
mixing of different components that could be larger than the
actual heat effect associated with the binding reaction
5) From a practical point of view, it should always be emphasized
that a clean cell and a perfectly straight syringe are decisive to
avoid spurious results and to give excellent baselines with high
signal-to-noise ratio.

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ITC PROCESS:

ITC – BEFORE TITRATION

For ITC experiments, two binding
partners (e.g., protein A and protein B,
in the same buffer and at known
concentrations) are placed in the
injection syringe and the ITC cell. The
ITC cell is kept at a tiny temperature
difference compared to a reference cell
filled with buffer.

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ITC – AFTER TITRATION:

When protein B is injected, the two proteins

interact. If this interaction is exothermic, the ITC

uses less energy to heat the cell; if the interaction

is endothermic, the ITC uses more energy to heat

the cell. Precise measurement of the energy

required to maintain temperature of the cell

during the course of several injections leads to

the calculation of free energy, enthalpy, entropy,

Kd, and stoichiometry of binding from one single

experiment. Furthermore the reaction can be

measured without immobilization or labelling of

the binding partners.

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STEPS OF PROCESS ARE AS FOLLOWS:

1. Titration begins - The ligand, at this stage, is present in the

syringe and the protein macromolecule in the cell.
2. Returns to baseline- The signal returns to the baseline before
the next injection.
3. Titration begins- First injection- As the first injection made,all
the injected ligand is bound to protein.Here the protein –ligand
complex is shown.
4. Second injection- As the second injection is made, again all
injected ligand becomes bound to protein.

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7.Second return to baseline- Signal again returns to the baseline
before next injection.
8. Injections continue- As the injections continue, the protien
becomes saturated with ligand , so less binding occurs, and
only the heat of dilution is observed.
9. End of titration - When protein is saturated with ligand, no
more binding occurs and only heat of dilution isbserved

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ITC – WORKING:

ITC systems use a cell feedback network (CFB) to

differentially measure and compensate for heat produced or
absorbed between the sample and reference cell.
Twin coin-shaped cells are mounted in a cylindrical adiabatic
environment, and connect to the outside through narrow access
tubes .
A thermoelectric device measures the temperature difference
between the two cells and a second device measures the
temperature difference between the cells and the jacket.
As chemical reactions occur in the sample cell, heat is
generated or absorbed.
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The temperature difference between the sample and reference
cells (ΔT1) is kept at a constant value (i.e. baseline) by the
addition or removal of heat to the sample cell, as appropriate,
using the CFB system.
The integral of the power required to maintain ΔT1 = constant
over time is a measure of total heat resulting from the process
being studied.

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GRAPH:

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INTERPRETATION:

Eighteen injections of ligand solution are added to protein

solution in the ITC cell. The area underneath each injection
peak (top panel) is equal to the total heat released for that
injection.
When this integrated heat is plotted against the molar ratio of
ligand added to macromolecule in the cell, a complete binding
isotherm for the interaction is obtained (bottom panel).
The one site model was used to fit the data. The solid red line
is the calculated curve using the best-fit parameters. The best
values for stoichiometry, binding constant, and enthalpy are
shown in the box.

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PURPOSE OF SELECING ITC:
Beyond binding affinities:  True affinity data via heat measurement offers a unique

insight into the biology and recognition processes, unobtainable with more limited
binding assays and techniques such as surface plasmon resonance (SPR).
Directly measure sub-millimolar to nanomolar binding constants : Measure

nanomolar to picomolar binding constants (109 to 1012 M-1) using the competitive
binding technique.
Application versatility:  Investigate any biomolecular interaction. True in-solution

technique:  No labeling or immobilization required.  No molecular weight limitations
or buffer restrictions.  Easily handles colored or turbid solutions and particulate
suspensions.
 

45
Easy to use: Unattended operation after sample loading.  All
functions are operated through software to minimize operator
involvement and facilitate fast and accurate analyses without
the need for expertise in thermodynamics.
ITC can determine:
Binding affinity -  Kd in range of millimolar to nanomolar
Number of binding sites
Can detect multiple and different binding sites
Enthalpy (ΔH) and entropy (ΔS) of binding
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APPLICATIONS:

1. Characterization of molecular interactions of small molecules,

proteins, antibodies, nucleic acids, lipids and other
biomolecules.
2. Lead optimization.
3. Enzyme kinetics.
4. Assessment of the effect of molecular structure changes on
binding mechanisms.
5. Assessment of biological activity.
6. Interactions between any two molecules can be studied with
ITC, including: Protein-small molecule
Protein-protein
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 Target-drug
Enzyme-inhibitor
Antibody-antigen
Protein-DNA
Protein-lipid
Small molecule-small molecule
7 Cell metabolism
8 Drug receptor interactions
9 Polymerizations, surfactants, metal chelation, etc.

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ITC ADVANTAGES:

1. It works in solution
True in-solution technique:  No labeling or immobilization
required.  No molecular weight limitations or buffer restrictions. 
Easily handles colored or turbid solutions and particulate
suspensions.
 
2. It dtermines the thermodynamics of the binding reaction.

3. It is an atomatic titration – setup and leave.

 
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 4. It is Convenient method for determining binding constants

5. ITC Can provide additional information about the nature of

the binding interface.

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DISADVATAGES:
1. It requires ΔH > ± 3-5 kcal/mol for precise determinations.
2. It requires macromolecule concentrations in the range 1µM –
1mM.
3. It consumes “large” amount of material.
4. It is insensitive to very tight or very weak binding.
5. It is slow process – 2 - 3 hours per run.

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REFERENCES:

1. Roger.E.Schrimer,Modern methods of Pharmaceutical

Analysis,Second Edition,Volume-1,1996.CRC Press,p.361
2. Skoog,Holler,Nieman,Principles of Instrumental
analysis,Published by Thomson brooks\ cole ,Fifth
Edition,2005 p.805
3. Willard,Merritt,Dean,Settle,Instrumental analysis,CBS
Publishers &Distributors,Seventh Edition,1986 p.777

4. B.K.Sharma,Instrumental Methods Of Chemical

Analysis,Page no.250
5. Robert.D.Braun, Introduction to instrumental Analysis,Page
no. 942.

52
6.http://www.google.com/itc/theory and instrumentation.accessed
on oct.14
7. http://www.yahoo.com/itc/theory and instrumentation.accessed
on oct.14
8. http://www.microcal.com/itc/animation slide.accessed
onoct.15
9. http://www.T A Instruments.com/ itc /instrument
figure.accessed on oct.17

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