EXPERIMENT NO.

1
SPECTROPHOTOMETRIC DETERMINATION OF THE
STOICHIOMETRY OF A COMPLEX
Gautane, J.C., Rabanal. G. A., Sepagan, J.

ABSTRACT

Absorption spectroscopy is one of the most efficient methods for determining the
chemical composition of complex ions in solution and their formation constants. The only
requirement is that either one or both of the reactants or the product must be able to
participate in a rival equilibrium without producing an absorbing species.

The three most popular techniques used to study complex ions are the slope-ratio
method, the mole-ratio method, and the method of continuous variations, often known as
Job's method. The limiting reagent controls the metal-ligand complex concentration in any
solution, with stoichiometric mixing of the metal and ligand resulting in the highest
concentration.

If the complexation reaction is observed at a wavelength where the metal-ligand

complex is the only one that absorbs, an absorbance vs. mole fraction graph will show
two linear branches, one when the ligand is the limiting reagent and the other when the
metal is the limiting reagent. During the process, two reactants are combined and
evaluated spectrophotometrically. The final absorbance of each mixture is computed at a
certain wavelength and plotted against the mole fraction. The curves produced by the plot
are known as Job's plots. The intersection of these two branches, which turned out to be
1:3, represents the stoichiometric mixing of the metal and ligand.

Keywords: Spectrophotometric determinations, absorbance, absorption spectroscopy,

Job’s Method, continuous variations.
1. INTRODUCTION

There are a number of ways to determine the composition of complex ions

in solution and their formation constants such as; the mole-ratio method, slope-
ration method and the method of continuous variations or Job’s method. This
experiment used Job’s method in order to determine the formula of the complex, it
utilizes the application of spectrophotometric techniques due to the formation of a
highly-colored complex. Spectrophotometry is used to quantify the light absorbed
by the molecules in order to identify the concentration of the species present in the
solution.

In this method, two reactants with varying rations are combined and
examined spectrophotometrically. The resulting absorbance of each mixture is
determined at a given wavelength and is plotted against the mole fraction. The
curve produced in the plot is called Job’s plots (Olson & Bühlmann, 2011). The
ratios obtained from the plot correspond to the mole fractions of the components
in the complex solution thus, one can deduce the formula of the complex.

This experiment aims to determine the formula of the colored complex using
the UV-Vis spectrophotometer.

2. EXPERIMENTAL
2.1 Materials and Reagents
The reagents used 0.7 M hydroxylamine hydrochloride solution, an
acetic acid and sodium acetate buffer solution with a pH of 4.0 and a molar
concentration of 0.10 M, 0.0007 M of ferrous ammonium sulfate, and
0.0007 M of 1,10-phenanthroline.
The instruments used are 5 mL and 10 mL serological pipettes with
aspirators, an analytical balance, six (6) 100-mL volumetric flasks, 1000 mL
beaker, and a UV-Vis spectrophotometer (Perkin Elmer).
 2.2 Continuous Variation Method
The six volumetric flasks were labeled A through F, and the following
solutions were pipetted in the following order: Fe solution, 10-mL buffer, 2
mL hydroxylamine hydrochloride, and 0.0007 M phenanthroline solution.
Table 1 shows the amount of iron solution and 1,10-phenanthroline solution
transferred to each flask.
Table 1. Amount of iron and phenanthroline solutions transferred.
Flask A B C D E F
mL of iron 0.00 4.00 8.00 12.00 16.00 20.00
solution
mL of 20.00 16.00 12.00 8.00 4.00 0.00
phenanthroline

After the corresponding amount of solutions were transferred, each

flask was diluted to mark with distilled water. Then, the solutions were mixed
thoroughly. After 10 minutes, the absorbance of each solution was
measured at 508 nm using distilled water as the reference. Lastly, the mole
ratio of the components was calculated, and the absorbance plot was
constructed.

3. RESULTS AND DISCUSSION

Coordination compounds are atoms, ions, or molecules that include one or

more metal centers bound to ligands (electron-donor atoms, ions, or molecules)
(Constable & Housecroft, 2013). These complexes may have a charge or be
neutral. The complex is stabilized by nearby counter-ions when it is charged. A
metal ion is located in the middle of a complex ion, which is surrounded by
various other molecules or ions.
 The amount of light reflected or absorbed by these types of chemicals can
be measured or calculated using spectrophotometry (Hammond, 2014). The
spectrophotometer may also be used to calculate the concentration of these
chemical compounds. The most used oxidation-reduction indicator in chemistry
is ferroin, which is a tris-complex of 1,10-phenanthroline (phen) and Fe2+ ions
(Mudasir et al., 2015).

Figure 1. Molecular Geometry of Ferroin

It is stable and soluble in aqueous solution with a pH range of 2–9 for this
vividly red octahedral complex ion. It has a molar absorption coefficient of 1.11
x 104 mol/cm1 at 510 nm and no pH-related peak shift. The oxidized form of
ferroin, [Fe(phen)3]+, has a low molar absorption coefficient at 600 nm of 830
mol-1 cm-1.

To keep the pH of the solution at 4 and prevent the production of Fe(OH)2

and Fe(OH)3 (F Au-YEUNG et al., n.d.), which are iron precipitates that occur
at higher pH levels, acetate buffer was also added. In addition, On the other
hand, the addition of the hydroxylamine hydrochloride solution caused the
reduction of Fe3+ to Fe2+ this is to avoid the deterioration of the ion by
spontaneous oxidation (Didukh et al., 2017).

The Job's approach, also known as the method of continuous variations, is

used to calculate the stoichiometry of a metal-ligand complex. The total moles
of metal and ligand, ntotal, in each solution that is prepared using this approach,
are identical (Renny et al., 2013). A graph of absorbance vs. the mole fraction
of ligand will exhibit two linear branches, one when the ligand is the limiting
reagent and the other when the metal is the limiting reagent if the complexation
reaction is observed at a wavelength where the metal-ligand complex is the
only one that absorbs (Powell, n.d.). A stoichiometric mixing of the metal and
the ligand is represented by the intersection of these two branches.

Table 2. Data gathered from the Spectrophotometer

Flask Metal Ligand XL Absorbance
A 0.0 20.0 1 0.0061
B 4.0 16.0 0.8 0.1622
C 8.0 12.0 0.6 0.1617
D 12.0 8.0 0.4 0.1193
E 16.0 4.0 0.2 0.0637
F 20.0 0 0.0 0.0094

As listed above, proportional variation of the volume of reagents utilized was

observed in the sampling, the fractions are also used to create a uniform X
variable which is plotted below. The ratio signifies the proportion of the ligand
to the amount of metal in the solution (see Eqn. 1).

𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝐿𝑖𝑔𝑎𝑛𝑑
𝑋𝐿 = (𝐸𝑞𝑛. 1)
𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝐿𝑖𝑔𝑎𝑛𝑑 + 𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑚𝑒𝑡𝑎𝑙

From the data above, it can be inferred that the equivalence between metal
and ligand occurred quickly, as the highest point is within the 0.8 threshold.
Also, from Table 2 the maximum absorbance observed was 0.1622 from Flask
B, and the minimum absorbance was 0.0061 from Flask A. From Flask B to the
rest of the other samples the absorbance gradually decreased as expected
from the varying proportions of Metal-Ligand solutions.
 Absorbance Plot of Metal-Ligand Chemical
0.18
0.16
0.14
Absorbance (a.u.) 0.12
0.1
0.08
0.06
0.04
0.02
0
1 0.8 0.6 0.4 0.2 0
XL

Figure 1. Plot for XL vs. Absorbance for Metal-Ligand solution

The empirical formulation of a coordination complex is ascertained using

the continuous variations approach (Job's method). Linear regression was
used since the graph can be separated into two line-equations (y=mx+b), in
adjacent line-equations usually the peak or intersection point is where the two
equations limit is maxima and minima. In the graph presented, the maximum
absorbance was used to be the last element of the linear regression of the first
line from 1.0 to 0.8 and the 1st element of the second line from 0.8 to 0.0. The
line equations are correspondingly:

Table 3. Data from line approximation

Linear Regression Line Equation Element Approximation
Elements
1.0-0.8 y= -0.7805x+0.7866 M 0.1795055991
0.8-0.0 y= 0.2018x+0.02254 L 0.7778275476

Since it is the ligand in the XL the corresponding x-value is the ligand with
approximately 0.7778275476 and the y-value is the metal with 0.1795055991.
This leads us to have about 1:3 of proportion or M1L3, it may also be considered
 to be 1:4 if we are to round up the value. The values could have better values
but were affected by systematic errors that were indetectable. The analyte's
reaction is reduced or increased by the matrix effect. Due to the sample's
inclusion of elements other than the analyte. The validity of conclusions drawn
from data gathered with that instrument may have been destroyed by
instrument drift. These modifications or variations are frequently predictable
and typically gradual.

4. CONCLUSION
Absorption spectroscopy is one of the most effective tools for finding the
formulae and formation constants of complex ions in solution. The only need
is that either the reactant or the product absorbs light, or that either the reactant
or the product can be forced to participate in a competitive equilibrium that does
not create an absorbing species. To determine the stoichiometry of the iron-
phenanthroline complex, Job’s method was utilized, and the absorbance was
plotted as a function of the mole-ratio of the metal and ligand. It was determined
that the ratio between the metal and the ligand was 1:3, thus following the
empirical formula ML3, the formula of the metal-ligand complex was
Fe(phen)32+.
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