Within chemistry, a Job plot, otherwise known as the method of continuous variation or Job's method, is a method used in

analytical chemistry Analytical chemistry studies and uses instruments and methods to separate, identify, and quantify matter. In practice, separation, identification or quantification may constitute the entire analysis or be combined with another method. Separati ...

to determine the stoichiometry of a binding event. The method is named after Paul Job and is also used in

instrumental analysis Instrumental analysis is a field of analytical chemistry that investigates analytes using scientific instruments. Spectroscopy Spectroscopy measures the interaction of the molecules with electromagnetic radiation. Spectroscopy consists of many ...

and advanced

chemical equilibrium In a chemical reaction, chemical equilibrium is the state in which both the Reagent, reactants and Product (chemistry), products are present in concentrations which have no further tendency to change with time, so that there is no observable chan ...

texts and research articles. Job first published his method in 1928, while studying the associations of ions in solution. By plotting the UV absorbance of a solution of against the

mole fraction In chemistry, the mole fraction or molar fraction (''xi'' or ) is defined as unit of the amount of a constituent (expressed in moles), ''ni'', divided by the total amount of all constituents in a mixture (also expressed in moles), ''n''tot. This ex ...

of , he produced a graph which provided information about the equilibrium complexes present in solution.

Theory

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 sum of the

molar concentration Molar concentration (also called molarity, amount concentration or substance concentration) is a measure of the concentration of a chemical species, in particular of a solute in a solution, in terms of amount of substance per unit volume of sol ...

s of the two binding partners (e.g. a

protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, res ...

and

ligand In coordination chemistry, a ligand is an ion or molecule ( functional group) that binds to a central metal atom to form a coordination complex. The bonding with the metal generally involves formal donation of one or more of the ligand's elec ...

or a metal and a ligand) is held constant, but their

mole fractions In chemistry, the mole fraction or molar fraction (''xi'' or ) is defined as unit of the amount of a constituent (expressed in moles), ''ni'', divided by the total amount of all constituents in a mixture (also expressed in moles), ''n''tot. This e ...

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. χ_A is the mole fraction of compound A and P is the physical property being measured to understand complex formation. This property is most oftentimes UV absorbance. The maximum (or minimum) on the plot corresponds to the stoichiometry of the two species if sufficiently high concentrations are used. The plot also provides insight to understand the equilibrium constant (K_eq) of complex formation. A greater curvature leads to a more evenly distributed equilibrium, while a more triangle-shaped plot signifies a large K_eq. Further, after determining the equilibrium constant, we can determine what complexes (ratio of A and B) are present in solution. In addition, the peak of the Job Plot corresponds to the mole fraction of ligands bound to a molecule, which is important for studying

ligand field theory Ligand field theory (LFT) describes the bonding, orbital arrangement, and other characteristics of coordination complexes. It represents an application of molecular orbital theory to transition metal complexes. A transition metal ion has nine valen ...

. An early work of I. Ostromisslensky describes essentially this approach.

Requirements

There are several conditions that must be met in order for Job's method to be applicable. Firstly, the property being studied must vary in direct proportion to the concentration of the species. In the case of UV-visible spectroscopy, for example, this means that the system must conform to the Beer-Lambert law. In addition, the total concentration of the two binding partners, the pH and ionic strength of the solution must all be maintained at fixed values throughout the experiment. Finally, there must be only one complex in solution which predominates over all others under the conditions of the experiment. This requirement means that only systems with high association constants, or systems in which only one stoichiometry can form, are suitable for analysis by Job plot. As such, the use of the Job plot in

supramolecular chemistry Supramolecular chemistry refers to the branch of chemistry concerning chemical systems composed of a discrete number of molecules. The strength of the forces responsible for spatial organization of the system range from weak intermolecular forces ...

has been advised against.

References

{{Analytical chemistry Scientific techniques