Isothermal Titration Calorimetry is often considered the gold standard for studying interactions between molecules, primarily because of its capability to measure thermodynamic and kinetic quantities with one experiment.
Isothermal Titration Calorimetry (ITC) is an experimental procedure used to calculate the heat of a reaction at different concentrations of the reactants. One reactant is added gradually to a solution containing the other reactants. The reaction occurs, and a device called a calorimeter monitors the heat released or absorbed due to this addition.
The heat of a reaction is defined as the amount of heat that must be added or deducted from the reaction system to maintain the reactants at a constant temperature. When the reaction is performed at a constant pressure, this heat represents a thermodynamic quantity called enthalpy of the reaction (ΔH).
This information can be used to determine the binding affinity of the reaction (the tendency of the reactants to react and form chemical bonds with each other), the binding stoichiometry (ratio of reactants binding with each other in the reaction) and molar enthalpy change (enthalpy of reaction per mole of the reactants reacting with each other).
These in turn can be used to calculate other thermodynamic quantities like the Gibbs Free Energy change (ΔG, a measure of the spontaneity of a certain reaction) and the entropy change (ΔS, the measure of randomness of a system).
The experimental setup consists of two titration cells – one is a control cell and contains only a buffer solution, while the other is an experimental cell containing the buffer and some (but not all) of the reactants. A buffer solution is a solution whose pH remains constant despite the addition of an acid, base or water.
Both of these cells are placed in an adiabatic jacket – this means that no heat transfer is possible between the reaction system and the surrounding environment. Thermopiles made of semiconductor material measure the temperature differences between the control cell and the experimental cell. A heat sink equalises the temperature difference caused by the reaction.
The thermopiles generate a potential difference proportional to the difference in temperature at their two junctions. In this case, this is the difference between the control and experimental cells.
The reactant not already present in the experimental cell is then titrated - that is, added slowly in very small amounts – to both the cells. Heat changes occur due to dilution, the addition of reactant, as well as the reaction itself. This causes a temperature difference, which is detected by the thermopiles and converted into a potential difference.
This difference is proportional to the power at which the heat sink works to transfer heat to or from the experimental cell. The power spike required to equalise the temperatures is recorded with time. This is called a thermogram. The area under each spike is added up and plotted against the molar ratio of the titrant (reactant added to the solution during titration). All the quantities mentioned at the beginning of the article can be determined using this graph.
This remarkable process is used for interactions between proteins and DNA, studying reactions of enzymes and analysing various drugs. Truly, it is an indispensable procedure in experimental chemistry as well as biology.