An isothermal titration calorimeter is composed of two identical cells made of a highly efficient thermally conducting and chemically inert 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. 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. In an endothermic reaction, the opposite occurs; the feedback circuit increases the power in order to maintain a constant temperature (isothermal operation).
Observations are plotted as the power needed to maintain the reference and the sample cell at an identical temperature against time. As a result, the experimental raw data consists of a series of spikes of heat flow (power), with every spike corresponding to one ligand injection. These heat flow spikes/pulses are integrated with respect to time, giving the total heat exchanged per injection. The pattern of these heat effects as a function of the molar ratio [ligand]/[macromolecule] can then be analyzed to give the thermodynamic parameters of the interaction under study.
To obtain an optimum result, each injection should be given enough time for a reaction equilibrium to reach. Degassing samples is often necessary in order to obtain good measurements as the presence of gas bubbles within the sample cell will lead to abnormal data plots in the recorded results. The entire experiment takes place under computer control.
Direct titration is performed most commonly with ITC to obtain the thermodynamic data, by binding two components of the reaction directly to each other. However, many of the chemical reactions and binding interactions may have higher binding affinity above what is desirable with the c-window. To troubleshoot the limitation of c-window and conditions for certain binding interactions, various different methods of titration can be performed. In some cases, simply doing a reverse titration of changing the samples between the injection syringe and sample cell can solve the issue, depending on the binding mechanism. For most of the high or low affinity bindings require chelation or competitive titration. This method is done by loading pre-bound complex solution in the sample cell, and chelating one of the components out with a reagent of higher observed binding affinity within the desirable c-window.