Job plot

A Job plot (also known as the Method of Continuous Variation or Job's Method) is used to determine the stoichiometry of a binding event. This method is widely used in analytical chemistry, instrumental analysis, and advanced chemical equilibrium texts and research articles.

In solutions where two species are present (i.e. species A and species B), one species (A) may bind to the other species (B). In some cases, more than one A will bind with a single B. One way to determine the amount of A binding to B is by using a Job plot.

In this method, the total molar concentration of the two binding partners (e.g. a protein and ligand or a metal and a ligand) are held constant, but their mole fractions are varied. An observable that is proportional to complex formation (such as absorption signal or enzymatic activity) is plotted against the mole fractions of these two components. The maximum (or minimum) on the plot corresponds to the stoichiometry of the two species if sufficiently high concentrations are used. This method is named after P. Job, who first introduced this methodology in 1928.

There are several conditions that must be met in order for Job's method to be applicable:

1. The system must conform to Beer's law
2. One complex must predominate under the conditions of the experiment
3. The Total concentration of the two binding partners must be maintained constant
4. pH and ionic strength must be maintained constant