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DATE PERFORMED: AUGUST 29, 2008

EXPERIMENT NO. 9: SPECTROPHOTOMETRIC DETERMINATION


OF IRON IN AQUEOUS SOLUTIONS AS A COMPLEX OF 1,10PHENANTHROLINE
J.G.S. REAMILLO
DEPARTMENT OF CHEMICAL ENGINEERING, COLLEGE OF ENGINEERING
UNIVERSITY OF THE PHILIPPINES, DILIMAN, QUEZON CITY, PHILIPPINES
RECEIVED SEPTEMBER 8, 2008

ABSTRACT
The experiment aims to determine the concentration of an unknown Fe(II) solution
by using a spectrophotometer and applying the Beer-Lamberts Law. Iron(II) was reacted
with 1,10-phenanthroline to produce the complex tris(1,10-phenanthroline)iron(II). Five
complex standard solutions were used to obtain a calibration curve y = 0.1946 (x) 0.0501,
relative to a reagent blank, with a correlation factor of R 2 = 0.9954. Using this curve, the
concentration of the unknown Fe(II) stock solution was determined and was found to have
an average of 3.71 ppm or 6.64E-05 moles/liter with a standard deviation of 0.030.
However when pooled together with the other groups data, the standard deviation
increases to 0.180, which might be due to the lower concentration values of group 4s data
set. Q-test showed that the said data set was not an outlier and was still included in the
calculations. Errors might have been caused by the inconsistencies in the cuvette handling
as well as by the limitations of Beers law, like ion interactions, which were not taken into
account.

INTRODUCTION
Spectrophotometry is one of the most
common
instrumental
techniques
in
quantitative
analysis,
wherein
the
concentration of a solution is determined by
measuring the radiant energy absorbed by a
chemical system containing the analyte or
its derivative. It is possible to determine the
analyte concentration with appreciable
accuracy and precision, especially if its
derivative is an intensely colored complex
while the reactants are in the form of
solutions which are colorless or of lesser
color
intensity,
by
using
a
spectrophotometer. (Christian, 2004)
Every molecular analyte is capable of
absorbing
certain
characteristic
wavelengths of electromagnetic radiation
depending on its functional groups. In this
process, the energy of the radiation is
transferred temporarily to the molecule,

matic radiation is illustrated by the BeerLamberts Law: A = abc, where A is the


absorbance, a is the wavelength-dependent
absorptivity coefficient, b is the path length,
and c is the analyte concentration. The BeerLamberts law could be employed to setup a
calibration curve through a series of
standard solutions, which can then be used
to determine the unknown concentration.
(Skoog, 2004)
The procedure is useful especially in
the quantitative determination of trace
metals, like Fe(II). Iron(II) readily reacts
with 1,10-phenanthroline (C12H8N2) to form
an intensely-colored red-orange complex,
tris(1,10-phenanthroline)iron(II), as given by
the reaction:
Fe2+(aq) + 3C12H8N2
Fe(C12H8N2)3

2+

(aq)

resulting to a decrease in the radiation


intensity. Similarly, when visible light passes
through a colored solution, a fraction of it is
absorbed by the solution; the unabsorbed
light is then transmitted and can be
quantitatively measured (Skoog et al, 1994.
A spectrophotometer makes use of
this concept in order to determine the
absorbance of a certain solution by
measuring the transmitted light relative to a
blank solution used in the calibration.
The linear relationship between
absorbance and analyte concentration as
well as path length for monochro-

The absorbance readings for the


colored complex can then be used to
quantify the iron(II) concentration through
the pre-obtained calibration curve.
This experiment aims to determine
the iron(II) concentration in aqueous
solution
samples
through
the
spectrophotometric analysis of its colored
complex
tris(1,10-phenanthroline)iron(II)
and demonstrate the principles involved in
the spectrophotometric method of analysis.
The experiment also aims to demonstrate
the pertinent calculations in calibration and
analysis, illustrate the Beer-Lamberts Law,
as well as account for the possible
deviations from and limitations of the
aforementioned law.

DATA AND RESULTS


GROUP I
A. Absorption Spectrum
Wavelength at maximum absorption (max):
509.80 nm
B. Calibration Curve
Concentration of working std Fe(II)
solution: 25 ppm
Table 1.1 Absorbance Readings from
Calibration
Volume of
Concentration Absorban
Working
of Standard
ce
Standard
Fe(II),
Solution, ml
ppm
1.00
0.5
0.114
2.00
1.0
0.215
3.00
1.5
0.320
4.00
2.0
0.427
5.00
2.5
0.526
C. Sample Analysis
Volume of stock sample solution:
100 ml
Volume of aliquot from stock solution:
10 ml
Volume of diluted unknown solution:
50 ml
Table 1.2 Absorbance Readings from
Samples
Unknown Sample
Absorbance
Number
1
0.184
2
0.184
3
0.183
D. Tabulated Results
Linear equation of the calibration curve:
y = 0.2072 (x)

GROUP II
A. Absorption Spectrum
Wavelength at maximum absorption (max):
509.80 nm
B. Calibration Curve
Concentration of working std Fe(II)
solution: 25 ppm
Table 2.1 Absorbance Readings from
Calibration
Volume of
Concentration
Absorba
Working
of Standard
nce
Standard
Fe(II),
Solution, ml
ppm
1.00
0.5
0.112
2.00
1.0
0.215
3.00
1.5
0.313
4.00
2.0
0.430
5.00
2.5
0.518
C. Sample Analysis
Volume of stock sample solution:
100 ml
Volume of aliquot from stock solution:
10 ml
Volume of diluted unknown solution:
50 ml
Table 2.2 Absorbance Readings from
Samples
Unknown Sample
Absorbance
Number
1
0.188
2
0.204
3
0.190
D. Tabulated Results
Linear equation of the calibration curve:
y = 0.2054 (x)

0.0096
Correlation Factor (R2):
R2 = 0.9999
Table 1.3 Calculated Concentration of
Samples
Unknow Concentrati
Concentration of
n
on of Fe(II),
Fe(II) in Stock
Sample
ppm
Solution
#
mg/L
moles/
(ppm)
L (M)
1
7.54E0.842
4.21
05
2
7.54E0.842
4.21
05
4
7.49E0.837
4.18
05
Average
7.52E4.20
05

0.0095
Correlation Factor (R2):
R2 = 0.9987
Table 2.3 Calculated Concentration of
Samples
Unknow Concentrati
Concentration of
n
on of Fe(II),
Fe(II) in Stock
Sample
ppm
Solution
#
mg/L
moles/
(ppm)
L (M)
1
7.78E0.869
4.35
05
2
8.48E0.947
4.73
05
4
7.87E0.879
4.39
05
Average
8.04E4.49
05

Figure 1. Group I Calibration Curve:


Graph
of
the
Absorbance
vs
Concentration of the complex tris(1,10phenanthroline)iron(II) at max = 509.80
nm
GROUP III
A. Absorption Spectrum
Wavelength at maximum absorption (max):
510.00 nm
B. Calibration Curve
Concentration of working std Fe(II)
solution: 25 ppm
Table 3.1 Absorbance Readings from
Calibration
Volume of
Concentration Absorban
Working
of Standard
ce
Standard
Fe(II),
Solution, ml
ppm
1.00
0.5
0.116
2.00
1.0
0.219
3.00
1.5
0.323
4.00
2.0
0.426
5.00
2.5
0.521
C. Sample Analysis
Volume of stock sample solution:

Figure 2. Group II Calibration Curve:


Graph
of
the
Absorbance
vs
Concentration of the complex tris(1,10phenanthroline)iron(II) at max = 509.80
nm
GROUP IV
A. Absorption Spectrum
Wavelength at maximum absorption (max):
509.40 nm
B. Calibration Curve
Concentration of working std Fe(II)
solution: 25 ppm
Table 4.1 Absorbance Readings from
Calibration
Volume of
Concentration
Absorba
Working
of Standard
nce
Standard
Fe(II),
Solution, ml
ppm
1.00
0.5
0.135
2.00
1.0
0.253
3.00
1.5
0.354
4.00
2.0
0.440
5.00
2.5
0.528
C. Sample Analysis
Volume of stock sample solution:

100 ml
Volume of aliquot from stock solution:
10 ml
Volume of diluted unknown solution:
50 ml
Table 3.2 Absorbance Readings from
Samples
Unknown Sample
Absorbance
Number
1
0.190
2
0.192
3
0.191
D. Tabulated Results
Linear equation of the calibration curve:
y = 0.2034 (x)
0.0159
Correlation Factor (R2):
R2 = 0.9997
Table 3.3 Calculated Concentration of
Samples
Unknow Concentrati
Concentration of
n
on of Fe(II),
Fe(II) in Stock
Sample
ppm
Solution
#
mg/L
moles/
(ppm)
L (M)
1
7.66E0.856
4.28
05
2
7.75E0.866
4.33
05
4
7.71E0.861
4.30
05
Average
7.71E4.30
05

100 ml
Volume of aliquot from stock solution:
10 ml
Volume of diluted unknown solution:
50 ml
Table 4.2 Absorbance Readings from
Samples
Unknown Sample
Absorbance
Number
1
0.195
2
0.193
3
0.195
D. Tabulated Results
Linear equation of the calibration curve:
y = 0.1946 (x)
0.0501
Correlation Factor (R2):
R2 = 0.9954
Table 4.3 Calculated Concentration of
Samples
Unknow Concentrati
Concentration of
n
on of Fe(II),
Fe(II) in Stock
Sample
ppm
Solution
#
mg/L
moles/
(ppm)
L (M)
1
6.67E0.745
3.72
05
2
6.57E0.734
3.67
05
4
6.67E0.745
3.72
05
Average
6.64E3.71
05

Figure 3. Group III Calibration Curve:


Graph
of
the
Absorbance
vs
Concentration of the complex tris(1,10phenanthroline)iron(II) at max = 510.00
nm

Figure 4. Group IV Calibration Curve:


Graph
of
the
Absorbance
vs
Concentration of the complex tris(1,10phenanthroline)iron(II) at max = 509.40
nm

DISCUSSION

to
measurements
obtained
monochromatic radiation since its

from
linear

In order for a species to be viable for


spectrophotometric determination, it must
exhibit radiation absorption. However in the
absence of such characteristics, a radiation
absorbing
derivative
might
be
used.
(Christian, 2004) Aqueous iron(II) reacts
readily with 1,10-phenanthroline to form a
relatively
stable
red-orange
tris(1,10phenanthroline)iron(II)
complex.
The
corresponding product-favored reaction is
given by the chemical equation:
Fe2+(aq) + 3C12H8N2
2+
Fe(C12H8N2)3 (aq)
The color intensity of the red-orange
complex is relatively high even in dilute
solutions,
making
it
suitable
for
spectrophotometric analysis (Twigg, 1972).
However,
in
the
spectrophotometric
determination of the concentration of iron(II),
mere
absorbance
readings
would
be
inconclusive without the application of the
Beer-Lamberts Law.
According to the Beer-Lamberts Law,
for
a
monochromatic
radiation,
the
absorbance of a solution is directly
proportional to the concentration of the
absorbing species and the path length of the
absorbing medium with some proportionality
constant a, called the wavelength-dependent
absorptivity coefficient. The law is best
illustrated by the equation:
A = abc,
where A is the absorbance, a, the
wavelength-dependent
absorptivity
coefficient; b, the path length, and c is the
analyte concentration. Although transmitted
light is the one being directly measured by
the spectrophotometers detector, Beers law
is expressed in terms of absorbance in order
to preserve the linear relationship between
the light-derived quantity and the analyte
concentration, which will prove to be very
useful in the setting-up of the calibration
curve.
Although
the
Beer-Lambert
relationship of absorbance and concentration
is very useful in determining the unknown
concentration
of
solutions
given
the
absorbance readings of a spectrophotometer,
it has, in itself, a couple of limitations (Skoog
et al, 2004). The Beer-Lamberts law holds
true only for concentrations lower than 0.01
M. At concentrations higher than 0.01 M, the
average distances between ions or molecules
are lessened such that each particle affects

equation does not account for polychromatic


light. Gratings or filters are required to set
the radiation to a band of wavelengths near
the target wavelength where the law would
be applicable (Skoog et al, 2004).
In the experiment, the iron(II) was not
treated with 1,10-phenanthroline alone; a
couple of reagents were added before and
after to maintain the necessary conditions for
an optimum reaction system. For the
calibration setup, six different concentrations
of iron were prepared, starting with the
reagent blank, the one with all the reagents
except the species to be analyzed and is also
known as the one with 0 ppm concentration.
The next five solutions, namely, 0.5, 1.0, 1.5,
2.0, and 2.5 ppm, were made in increments of
0.5 ppm with respect to the reagent black.
Evenly spaced increments were used in order
to get convenient X values in the calibration
curve as well as to enhance the precision of
the equation for the linear regression to be
used. The six setups were then treated with
hydroxylamine hydrochloride in order to
reduce all the Fe(III) in the solution into the
desired analyte, Fe(II), and keep them in the
Fe(II) state. The chemical reaction is given by
the following equation:
4Fe3+ + 2NH2OHHCl 4 Fe2+ + N2O +
4H+ + H2O
The solution, which originally has only
Fe(II) in it, might have traces of Fe(III) since
Fe(II) has a tendency to be oxidized in the
presence of water and acidic media.
Conversion of virtually all Fe(III) into Fe(II) is
necessary
to
facilitate
good
complex
formation.
The next step in the setup preparation
is the addition of excess 1,10-phenanthroline.
The Fe(II) was the limiting reagent for all the
standard solutions; only small fractions of the
25 ppm working standard was used, as
compared with the 0.20% w/v concentration
of the 1,10-phenanthroline for the standards.
The forward reaction was assumed to have
proceeded completely, leaving no Fe(II) ion
behind.
Consequently,
the
tris(1,10phenanthroline)iron(II)
complex
concentration was considered equal to the
concentration of the limiting reagent, Fe(II).
Most reactions of the same aforementioned
reaction
systems
with
approximately
equimolar reactants do not go into

the charge distribution, and consequently, the


absorption of nearby particles, resulting to
deviations from its linear nature (Skoog et al,
2004). Molar absorptivity of the analyte is
also altered when particles and electrolytes
interact electrostatically, leading to further
departures from the linear nature of the
Beers law. This occurs when the solution has
a relatively high concentration of interfering
species, especially electrolytes, since Beers
Law does not account for species undergoing
dissociation, association, and reaction with
the solvent (Skoog et al, 2004).
Another limitation of Beers law is that
it applies only
of the hydroxide of iron, which could possibly
lead to deviations from the actual Fe(II)
concentrations.
It is therefore necessary to add each
reagent according to the sequence mentioned
above to ensure the complete reaction of
iron(II) with 1,10-phenanthroline, to minimize
the possibility of interfering ions like iron(III)
and hydroxides, and to obtain optimum
complex absorbance readings.
After all solutions have been prepared,
the next step was to calibrate the
spectrophotometer
according
to
the
conditions
necessary
for
the
Fe(II)
determination.
Since the complex tris(1,10phenanthroline)iron(II) is the absorbing
species, the spectrophotometer was set to a
wavelength range of 350 to 600 nma range
where the red orange complex can exhibit
absorption of green light.
Interestingly, the tris(1,10phenanthroline)iron(II) complex is colored
red not because the complex absorbs red
radiation, but because it absorbs its
complementary color, green. The green
radiation is the one that experiences
maximum absorbance change with respect to
concentration, while the red radiation is
almost
unaltered,
resulting
to
its
transmission. The transmission of red light
and the absorption of green light is the one
that
gives
the
tris(1,10phenanthroline)iron(II)
complex
its
distinctive color.

completion; the limiting reagent is not


completely transformed into the product. But
since the [1,10-phenanthroline]>> (was
much greater than that of)[Fe(II)], there was
an excess [1,10-phenanthroline] in the
solution. The Le Chateliers principle ensured
that the excess [1,10-phenanthroline] would
push the forward reaction further, thus
converting all the Fe(II)
into tris(1,10phenanthroline)iron(II) (Whitten et al, 2007).
The last reagent to be added was the
acetate buffer with pH 4. The reaction system
needs to be buffered within the said pH range
to facilitate the full color development of the
complex and neutralize the H+ from the
aforementioned reaction of Fe3+ with
hydroxylamine hydrochloride. Moreover, the
buffer solution would prevent the formation
the errors due to interfering contaminants
from the experiment reagents and vessels,
and
the
absorbance
readings
would
correspond
to
the
tris(1,10phenanthroline)iron(II)) concentration with
minimal loss of accuracy and precision.
The remaining five solutions
absorbances were then obtained one after
another using the spectrophotometer, relative
to the blank solution.
The absorbance readings from the five
solutions were then used to setup the
calibration curve using linear regression. A
calibration curve is needed since the
absorptivity constant is not always a known
quantity, and may vary from one setup to
another. A calibration curve of y = 0.1946
(x) 0.0501 with a correlation factor of R2
=
0.9954 was obtained by plotting the
absorbance against the concentration.
The calibration curve derived from the
experiment was a linear equation of the BeerLamberts law. The y component corresponds
to the absorbance, the slope m to the product
of absorptivity and path length, ab, and the x
component to the concentration c:
y
=
mx
+
b
A
= (ab)c
The calibration curve was then used to
calculate the concentration of the unknown
samples by plugging in their absorbance
value in the calibration curve. Solving for X,

Using the most concentrated Fe(II)


solution,
spectral
scanning
was
then
employed to determine the lambda max and
the baseline of the absorption spectrum. The
lambda max corresponds to the absorbance
wavelength with maximum sensitivity. The
max corresponds to the absorption peak of
the solution wherein the sensitivity (change
in
absorbance
per
unit
change
of
concentration) is highest. Moreover, the
corresponding absorption curve is often flat
at a maximum, which facilitates a good
adherence to the Beer-Lamberts law and less
uncertainty due to precise reproduction
failures in the wavelength setting of the
instrument (Skoog, et al, 1994). On the other
hand, the spectral scanning is not concerned
in determining the absolute value for a
maximum; it pertains to a maximum value
relative to a reference point, the baseline, in
a similar way that absorbance is not an
absolute quantity but is relative to a
reference point known as the blank.
When the spectrophotometer was set
to the max, which, in the four experiment
setups, fell between 509.40 510.00 nm, the
calibration procedure was begun. The first
step was to calibrate the spectrophotometer
according to the blank solution that was
prepared. The prepared blank solution
contained all the reagents and solvents
except for the analyte, which in this case, was
the Fe(II). All necessary steps were also
performed on the blank solution, which will
be used to correct the spectrophotometric
measurements. The calibration with the blank
solution would eliminate the
higher concentrations, fingerprints on the
cuvette during blank solution calibration
could yield to a lower absorbance reading for
the succeeding standards.
Errors can also result from the
inconsistencies of the solution volumes used,
which can be brought about by adhesion of
solution molecules to the containers glass
surfaces. A lot of liquid transfer was
employed in the experiment, which could
yield to compounded error.
Errors in the calibration curve can also
greatly affect the concentration of the
unknown samples. Since absorbance was

the concentration of the three solutions from


one unknown sample was found to be 0.745,
0.734, and 0.745 ppm, respectively. However,
these concentrations are for the diluted
solutions. In order to determine the
concentration of the stock solution, the
obtained concentration must be multiplied by
the dilution factor, which in this case, is 5. To
convert this corrected ppm concentration of
the stock solution into molarity, the
concentrations must be divided by 1000 and
the molar mass of Fe(II), which is 55.845
g/mol. The reported ppm values for the Fe(II)
unknown stock solution were 3.72, 3.67, and
3.72, with an average of 3.71 ppm. The
molarity counterparts however were 6.67E05, 6.57E-05, 6.67E-05 M respectively, with a
mean of 6.64E-05 M. The relative standard
deviation for the said set of data was only
about 8.01 ppt with standard deviation
0.030 (see attached calculation sheet), which
means a low scattering of the data obtained.
However, when pooled together with the data
off all four groups, the pooled standard
deviation rises to 0.108, probably because
group 4s concentrations are lower than that
obtained by all the other groups. However, Qtest determined that the mean of group 4s
data cannot be considered as an outlier.
Possible sources of error might be the
presence of fingerprints on the surface of the
cuvette
during
the
cuvette
handling.
Although fingerprints in the standard
solutions will yield higher absorbance
readings and consequently

in water samples, iron determination in blood


samples, and analysis of metal and mineral
content in food and medicines.
REFERENCES
[1]Skoog, D.A., West, D.M., and F.J. Holler.
Analytical Chemistry: An Introduction.
6th ed. Saunders College Publishing.
USA. 1994.
[2]Skoog, D.A, West, D.M., Holler, F.J., and S.
R. Crouch. Fundamentals of Analytical
Chemistry. 8th ed. Thomson Brooks
Cole, Thomson Learning, Inc. USA.
2004
[3]Twigg, M. V. Aquation of Tris(1,10-

derived from transmittance, a value based on


the logarithmic relationship of light intensity,
slight discrepancies can take its toll on the
calibration curve, which can lead to possible
loss of accuracy and precision.
CONCLUSION AND
RECOMMENDATION(S)
The concentration of an unknown
Fe(II) sample can be determined through
spectrophotometry. In the experiment, Fe(II)
was reacted with 1,10-phenanthroline to
produce an absorbing complex tris(1,10phenanthroline).
The different complex
standard solutions were then used to come
up with a calibration curve relative to the
reagent blank which was similar to the linear
relationship of absorbance and concentration
as given by the Beer Lamberts Law: A = abc.
The calibration curve obtained, y =
0.1946 (x) 0.0501 with a correlation factor
of R2 = 0.9954, was used to calculate the
concentration of the unknown sample based
on its absorbance. Calculations showed that
the unknown Fe(II) stock solution has an
average concentration of 3.71 ppm, or 6.64E05 (M) moles per liter. The standard deviation
obtained for the group 4 setup was roughly
0.030 but when pooled together with all the
other groups, increased to 0.108.

phenanthroline)iron(II)
in
Acid
Solution.
Journal
of
Chemical
Education. Volume 49, Number 5, May
1972
[4] Demirhan, N. and Elmali F. T.
Spectrophotometric Determination of
Iron(II) with 5-Nitro-6-amino-1,10phenanthroline. Turk J Chem. 2003
[5]Whitten, K.W., Davis, R.E., Peck, M.L.,
Stanley, G.G. Chemistry. 8th ed.
Thomson Brooks/Cole, CA, U.S.A.
2007. pp. 671-672
[6]Christian, G.D. Analytical Chemistry. 6th
ed. John Wiley and Sons, Inc. U.S.A.
2004
[7]Harris, D.C. Quantitative Chemical
Analysis. 7th ed. W.H. Freeman and
Company, New York, U.S.A.
APPENDIX
A. WORKING EQUATIONS
M1V1 = M2V2
y = mx + b ( Calibration Curve, A =
bc)
See attached data sheet
B. SAMPLE CALCULATIONS
See attached data sheet and excel
sheet

More solutions for the standard


samples would greatly increase the accuracy
of the calibration curve. Multiple identical
cuvettes could improve the absorbance
readings since repetition of the cuvette is
prone to scratch and moisture build-up.
APPLICATIONS
Spectrophotometry and the BeerLamberts law are useful concepts in the
determination of the concentration of colored
solutions, especially complexes. They provide
more accurate results than visual and manual
titration and is relatively easy to employ once
the solutions are prepared.
Spectrophotometry finds a lot of
applications in industries and medicine, like
the analysis of trace metal ions