SPECTROPHOTOMETRIC

DETERMINATION
OF THE STOICHIOMETRY OF A
COMPLEX

Lay, Henry
Sison, Kris Jester Franco IV
Group 7

Objectives
Determine

the formula
of a colored complex by
continuous

variation

method
mole ratio method
slope ratio method

TERMINOLOGIES

Spectroscopy-

general term used to describe

techniques based on the measurement of
absorption, emmission, or luminescence of ER

Spectrometry-

Measurement of the absorption

of ultraviolet, visible, or infrared radiation

Spectrophotometry-

spectrometry of the
absorption of UV, visible, or infrared radiation

TERMINOLOGIES

Continuous Variations Method- method which

keeps the overall volume and moles of reactants are
constant but the mole ratio varies systematically

Mole-ratio Method- method where the analytical

concentration of one reactant is constant while the
other is varied

Slope-ratio Method- assumes a reaction is driven

to completion by excess reactant, Beers law is
obeyed, and only the complex absorbs the
wavelength used in the experiment

SPECTROPHOTOMETRY

Based on the absorption of light by an analytical

species.

Measures via spectrophotometer the amount of

light absorbed by the sample

The measurement can be related to relevant

properties such as concentration

IMPORTANCE OF
SPECTROPHOTOMETRY
Applied

in chemical and clinical labs more than

any other method
Identification and determination of many
inorganic, organic, and biochemical species
Easy and convenient: relatively fast method to
get quantitative measurements
Selective: analyte usually unique to a wavelength
High sensitivity and high accuracy: because of
modern instruments

LIGHT

Waves

Photons

In

uv-vis spectrophotometry, the wavelengths used range

from about 250-750nm, the visible range itself is about 400700nm.

Colored

solutions are very good candidates for

spectrophotometric analysis.

Spectrophotometer

When light is absorbed by sample, the radiant

power [energy per unit area] of the beam of light
is decreased.
From the source, light passes through a
monochromator to select a single wavelength.
The monochromatic light with power Po strikes a
sample of a specific, and the power emerging
from the other side is P.
P<= Po

PERCENT TRANSMITTANCE
AND ABSORBANCE

Transmittance fraction of the original light that

passes through the sample
T=P/Po
%T=100T
A quantity directly measured by the
spectrophotometer as P and Po is directly
mesured by the electro nic detector system

PERCENT TRANSMITTANCE
AND ABSORBANCE

Absorbance (A) = log (Po/P) = -log T

When no light is absorbed, P = Po, and A = 0

A.k.a optical density
Directly proportional to concentration

Beers Law : A = bc

Beer Lambert Law

Absorbance depends on six factors:

Identity of absorbing substance

Concentration
Pathlength b
wavelength
Solvent identity
Temperature

For

solutions which are dilute enough, the above

factors combine together to yield a linear
relationship between absorbance, concentration,
and pathlength, known as the Beer-Lambert Law:

Beer Lambert Law

Accounts for the diverse strengths of absorbance

of chemical species likewise the variable absorbing
strength for different wavelengths of light
molar absorptivity () is unique for a given
solution species and a particular wavelength of
light
The greater the molar absorptivity, the higher the
absorbance.
States that A is directly proportional to the
concentration of the absorbing species.

Beer Lambert Law

Is limited to dilute solutions (<0.01M) of most

substances.
In highly concentrated solutions, solutes
influence one another due to proximity, and
their properties somewhat change.
Non-absorbing solutes can also interact with
absorbing ones and alter their absorptivity
If absorbing molecule participates in
concentration dependent equilibrium,
absorptivity changes with concentration

Beer Lambert Law

Is limited to dilute solutions (<0.01M) of most

substances.
In highly concentrated solutions, solutes
influence one another due to proximity, and
their properties somewhat change.
Non-absorbing solutes can also interact with
absorbing ones and alter their absorptivity
If absorbing molecule participates in
concentration dependent equilibrium,
absorptivity changes with concentration

Methodology
Preparation of Reagents

Methodology
Continuous Variation Method
6 50mL volumetric flasks labeled A-F

Iron solution -- > acetate buffer(5mL) -->

hydroxylamine(1mL) --> 0.0007M
phenanthroline

Dilute to mark and mix thoroughly

Sit for 10 minutes, measure absorbance

at 508 nm w/ distilled water as reference

Methodology
Continuous Variation Method
flask

Fe(mL)

10

Phen(m
L)

10

Methodology
Mole ratio Method
6 50mL volumetric flasks labeled A-F

2mL Fe -->5mL acetate buffer --> 1mL

hydroxylamine --> 0.0007 M phenanthroline

Dilute to mark, mix thoroughly

After 10 minutes measure absorbance at

508 nm

Methodology
Mole ratio Method
Flask

Phen
(mL)

12

15

Methodology
Slope ratio Method
5 50mL volumetric flasks labeled A-E

5mL Fe > 5mL acteate buffer > 1mL

hydroxylamine > 0.0007 M phenanthroline

Dilute to mark and mix thoroughly

Stand for 5 minutes then read absorbance at 508

nm
flasks

Phen
(mL)

Methodology

Methodology

0.7

M hydroxylamine
hydrochloride (NH2)OH. HCl
4.8643g in 100mL

HOAc/NaOAc

buffer at pH=4

glacial acetic acid and NaOAc

Continuous Variation Method

Requires

that the total concentration of the

solution [ligand concentration + metal
concentration] is held constant for all samples
while ratio is varied
Assumes that only a single complex is present in
a solution

Results
Flask

Continuous

Volume of solution
(mL)
Iron (II)
Phenanthrol
ine

variations method

Mole Fraction
Iron (II)

Phenanthrol
ine

Absorban
ce

10

1.0

0.012

0.2

0.8

0.402

0.4

0.6

0.304

0.6

0.4

0.159

0.8

0.2

0.100

10

1.0

0.016

Results

Continuous

variations method

Continuous Variations Method

0.45
0.4
0.35
0.3
0.25
0.2
0.15
0.1
Absorbance
0.05
0

Mole Fraction of Iron (II)

Results

Continuous variations method

Xfe2+ = 0.25 = 1

Xc12h8n2
To

0.75

get the intersection for

phenanthroline, we subtract the iron
value from 1, so 1-0.25=0.75.
The ratio of iron(II) to phenanthroline
then becomes 0.25:0.75, which is 1:3

Results
Flask

Continuous variations method

A: iron(II) = 0mL/10mL = 0

phenanthroline = 10mL/10mL = 1.0

Flask

B: iron(II) = 2mL/10mL = 0.2

phenanthroline = 8mL/10mL = 0.8

Flask

C: iron(II) = 4mL/10mL = 0.4

phenanthroline = 6mL/10mL = 0.6

Flask D: iron(II) = 6mL/10mL = 0.6

phenanthroline = 4mL/10mL = 0.4

Flask E: iron(II) = 8mL/10mL = 0.8

phenanthroline = 2mL/10mL = 0.2

Flask F: iron(II) = 10mL/10mL = 1.0

phenanthroline = 0mL/10mL = 0

Continuous variations method

Flask

B:
Mol Fe(II)=
(0.0007M Fe)(2mL x 1L/1000mL)= 1.4x10-6
Mol phen=
(0.0007 M phen)(8mL x 1L/100mL)= 5.6 x10-6
mole fraction for Fe=

molFe
1.4 x10

0.2
6
6
totalmol (5.6 x10 ) (1.4 x10 )

Results : Mole Ratio

Method
simplest

of the spectrophotometric
techniques that have been used for the
study of complexformation equilibria

series of solutions are prepared which

contain equal formal concentrations of a
metal ion but different formal
concentrations of the complexing agent.

Results : Mole Ratio

Method

Results
Flask

Mole-ratio

Number of moles

Method

Phenanthroli
ne

Iron (II)

moles of
phenanthroline
moles of Iron (II)

Absorban
ce

7x10-7

1.4x10-6

0.5

0.047

2.1x10-6

1.4x10-6

1.5

0.152

3.5x10-6

1.4x10-6

2.5

0.244

5.6x10-6

1.4x10-6

4.0

0.257

8.4x10-6

1.4x10-6

6.0

0.236

1.05x10-5

1.4x10-6

7.5

0.253

Results

Mole-ratio Method

Mole-Ratio Method
0.3
0.25
0.2
0.15
Absorbance
0.1
0.05
0
0.5

1.5

2.5

moles C12H8C2/moles Iron (II)

7.5

Results

Mole-ratio Method

x=3,

which means 1 mole Iron (II) is to 3 moles of

phenanthroline.

It

can be seen in the graph that the ratio of iron

(II) to phenanthroline is 1:3.

 Flask

A:

nphenanthroline = (0.0007M)(.001L) = 7.0x10 -7

mole ratio = 1.4x10-6/7.0x10-7 = 0.5
Flask

B:

nphenanthroline = (0.0007M)(.003L) = 2.1x10 -6

mole ratio = 1.4x10-6/2.1x10-6 = 1.5
Flask

C:

nphenanthroline = (0.0007M)(.005L) = 3.5x10 -6

mole ratio = 1.4x10-6/3.5x10-6 = 2.5

Flask D:

nphenanthroline = (0.0007M)(.008L) = 5.6x10-6

mole ratio = 1.4x10-6/5.6x10-6 = 4.0

Flask E:

nphenanthroline = (0.0007M)(.012L) = 8.4x10-6

mole ratio = 1.4x10-6/8.4x10-6 = 6.0

Flask F:

nphenanthroline = (0.0007M)(.015L) = 1.05x10-5

mole ratio = 1.4x10-6/1.05x10-5 = 7.5

Results : Slope-ratio
method
can

be applied to systems in which the

complexes have large dissociation
constants and are, therefore, not readily
suited to the continuous variations or
mole-ratio method

 based

upon absorbance measurements

of solutions in which dissociation of the
complex is repressed by a large excess
of one of the reactants; no long
uncertain extrapolations are involved.

Results

Slope-ratio

method: constant Fe(II)

Flask

mL of C12H8N2

[C12H8N2] in M
Absorbance

1.4 x 10-5 2.8 x 10-5 4.2 x 10-5 5.6 x 10-5 7.0 x10-5
0.050

0.099

0.147

0.195

0.253

Results

Slope-ratio method: constant Fe(II)

Constant Amount of Fe (II)

0.3
0.25
0.2
0.15
0.1
0.05

Absorbance

[C12H8N2]

Results
Flask

Slope-ratio method: constant phen

A

mL of Fe (II)

0.5

1.0

1.5

2.0

2.5

[Fe (II)] in M

7.0 x 10-6

1.4 x 10-5

2.1 x 10-5

2.8 x 10-5

3.5 x 10-5

Absorbance

0.053

0.118

0.197

0.259

0.318

Result
s

Slope-ratio method: constant phen

Constant amount of [C12H8N2]

0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
Absorbance

[Fe2+]

Slope-ratio method

Results

Constant Fe:

[MxLy] = CM(1 mol MxLy)

(x mol ironII)
A = bCM = bCM
x
x
y = mx + b
m1 = b
x

Constant phen:

[MxLy] = CL(1 mol MxLy)

(y mol phen)
A = bCL = bCL
y
y
y = mx + b
m = b
y

Using linear regression:

r2 = 0.998693267
A2 = -0.0123
m2 = 9585.714286

Results

Slope-ratio method

Getting the slope ratio of the two graphs

gives the ratio of Fe(II) to phenanthroline :

m fe
m ph

3585.714286

0.374
9585.714286

This means based from the experimental

values the ratio of Fe(II) to phenanthroline
is 0.374:1 or about 1:3.

Discussion

The stoichiometry for the formation of the

complex of iron (II) and the non-absorbing ligand
1,10 phenanthroline was experimentally
determined via three methods: Continuous
variation, slope ratio and mole ratio method.
Theoretically, the said complex ferroin, possesses
a 3:1 phenanthroline-iron ratio.
The iron-phenanthroline complex is a deep
orange-red, highly absorbing substance with an
absorption maximum at 508 nm.

Discussion

N
Fe2+ +

Fe2+

3
N

1,10-phenanthroline
3

Discussion

For the experiment, iron (II) came from the

prepared 0.0007M Fe(NH4)2(SO4)2,6H2O.
hydroxylamine hydrochloride must be added to
counter the rapid oxidation of iron (II) to iron (III)
which may complex with phenanthroline giving a
pale blue solution instead of the desired reddish
orange color.

Discussion

phen)3Fe+2

(phen)3Fe+3 + e-1

red-orange
2Fe+3

pale blue

+ 2NH2OH + 2OH 2Fe+2 + N2 + 4H2O

Discussion

Fe(phen)32+ forms in acidic environment, however

like most metal ion, it competes with the H3O+,
hence would not form in strongly acidic solution,
on the other hand, most metal ions forms
insoluble hydroxides in basic solution. For these
reasons, the iron determination must be carried
out in slightly acidic solution with pH of about 4-6.
This was done via addition of acetate buffer.

Discussion

Possible sources of error:

Personal errors in reagent preparation

Instrument error
Undetected oxidation of iron
Photo decay

Conclusion

With the use of various methods, the ratio of

phenanthroline and iron was determined to be about
3:1,

sample preparation plays the most important factor

in the experiment.

Ignoring all errors, and tedious procedures as well as

computations and graphing, the general concept of
the experiment proved to be simple and accurate.

THE END

PICTURES!!!