Job plot: Difference between revisions

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{{Short description|Type of graph used in analytical chemistry}}

{{Context|date=October 2009}}

Within [[chemistry]], a '''Job plot''', otherwise known as the '''method of continuous variation''' or '''Job's method''', is a method used in [[analytical chemistry]] to determine the [[stoichiometry]] of a binding event. The method is named after Paul Job and is also used in [[instrumental analysis]] and advanced [[chemical equilibrium]] texts and research articles. Job first published his method in 1928, while studying the associations of ions in solution.<ref>{{Cite journal|last=Job|first=Paul|year=1928|title=Formation and Stability of Inorganic Complexes in Solution|journal=Annales de Chimie |series=10|volume=9|pages=113–203}}</ref> By plotting the [[Ultraviolet–visible spectroscopy|UV absorbance]] of a solution of {{chem2|Tl(NO3)/NH3}} against the [[mole fraction]] of {{chem2|Tl(NO3)}}, he produced a graph which provided information about the equilibrium complexes present in solution.

== Theory ==

A '''Job plot''' (also known as the '''method of continuous variation''' or '''Job's method'''; named after P. Job) 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.

[[File:Job Plot 1.jpg|thumb|A simple Job Plot showing how a physical property (P) changes upon changing the mole fraction of compound A (Χ_A).]]

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 totalsum of the [[molar concentration]]s of the two binding partners (e.g. a [[protein]] and [[ligand]] or a metal and a ligand) areis 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<ref>Huang, C.Y. Determination of Binding Stoichiometry by the Continuous Variation Method: The Job Plot. ''Methods in Enzymology'' (1982) '''87''', 509-525.</ref> if sufficiently high concentrations are used. This method is named after P. Job, who first introduced this methodology in 1928.<ref>Job, P. ''Annali di Chimica Applicata'' (1928) '''9''', 113-203</ref>

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.<ref name=Renny>{{cite journal | last1 = Renny | first1 = J. S. | last2 = Tomasevich | first2 = L. L. | last3 = Tallmadge | first3 = E. H. | last4 = Collum | first4 = D. B. | year = 2013 | title = Method of Continuous Variations: applications of job plots to the molecular associations in organometallic chemistry | journal = Angew Chem Int Ed Engl | volume = 46 | pages = 11998–2013 }}</ref>

There are several conditions that must be met in order for Job's method to be applicable:<ref name="MacCarthy">{{cite journal|last=MacCarthy|first=Patrick|coauthors=Zachary D. Hill|date=February 1986|title=Novel Approach to Job's Method|journal=Journal of Chemical Education|volume=63|issue=3|pages=162–167}}</ref>

#The system must conform to [[Beer's law]]

The maximum (or minimum) on the plot corresponds to the stoichiometry of the two species if sufficiently high concentrations are used.<ref>{{cite book | last1 = Huang | first1 = C.Y. | year = 1982 | title = Determination of Binding Stoichiometry by the Continuous Variation Method: The Job Plot | series = Methods in Enzymology | volume = 87 | pages = 509–525 }}</ref> 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.<ref name = "Renny" /> Further, after determining the equilibrium constant, we can determine what complexes (ratio of A and B) are present in solution.<ref name = "Stoichiometry">{{cite journal | last1 = Facchiano | first1 = A. | last2 = Ragone | first2 = R. | year = 2003 | title = Modification of Job's method for determining the stoichiometry of protein – protein complexes | journal = Analytical Biochemistry | volume = 313 | issue = 1 | pages = 170–172 | doi = 10.1016/s0003-2697(02)00562-6 | pmid = 12576074 }}</ref> 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]].<ref>{{cite journal | last1 = Hauser | first1 = A | year = 2004 | title = Ligand Field Theoretical Considerations | journal = Adv Polym Sci | volume = 233 | pages = 49–58 }}</ref> An early work of I. Ostromisslensky describes essentially this approach.<ref>{{Cite journal |last=Ostromisslensky |first=Iwan |date=January 1911 |title=Über eine neue, auf dem Massenwirkungsgesetz fußende Analysenmethode einiger binären Verbindungen |url=https://doi.org/10.1002/cber.19110440141 |journal=Berichte der Deutschen Chemischen Gesellschaft |volume=44 |issue=1 |pages=268–273 |doi=10.1002/cber.19110440141 |issn=0365-9496|url-access=subscription }}</ref>

#One complex must predominate under the conditions of the experiment

#The Total concentration of the two binding partners must be maintained constant

== Requirements ==

#pH and ionic strength must be maintained constant

There are several conditions that must be met in order for Job's method to be applicable.<ref name="MacCarthy">{{cite journal|last=MacCarthy|first=Patrick|author2=Zachary D. Hill|date=February 1986|title=Novel Approach to Job's Method|journal=Journal of Chemical Education|volume=63|issue=2|pages=162–167|doi=10.1021/ed063p162|bibcode=1986JChEd..63..162H}}</ref> 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|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]] has been advised against.<ref>{{Cite journal|last1=Brynn Hibbert|first1=D.|last2=Thordarson|first2=Pall|date=2016-10-25|title=The death of the Job plot, transparency, open science and online tools, uncertainty estimation methods and other developments in supramolecular chemistry data analysis|url=http://xlink.rsc.org/?DOI=C6CC03888C|journal=Chem. Commun.|language=en|volume=52|issue=87|pages=12792–12805|doi=10.1039/c6cc03888c|issn=1364-548X|pmid=27779264|doi-access=free}}</ref>

==References==