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Chem126 Lab Instrumental Analysis

EXPERIMENT 1
Spectrophotometric Determination of Fe2+ Ions using 1,
10-Phenanthroline
(External Calibration Method)
Vanessa Olga J. Dagondon and Ken M. Menez
Department of Chemistry, College of Arts and Sciences,
University of the Philippines Visayas, Miag-ao Iloilo

ABSTRACT
The determination of the Fe 2+ concentration in a branded iron
supplement tablet was developed using the external calibration
method. This method uses a series of standardized solutions to
obtain data through the UV VIS spectrophotometer to construct a
graphs that relate the wavelength to absorbance and, ultimately, to
the concentration of a solution. The standard used was a hydrated
derivative of ferrous sulphate, which is called ferrous ammonium
sulphate hexahydrate. Because of ferrous ions typically exist as
having a sulphate base in compounds; it was assumed that the iron
tablet contained mainly a high percentage of this ferrous sulphate.
This experiment also tackled to explain the principle as to how a
solution absorbs light, and how this could be related to the
concentration of ions in the solution. This experiment had also
provided first-hand experience to operating a UV VIS
spectrophotometer and practice to calculating necessary data
through the least squares method. This least squares method was
considered important at the event that an automatic calculating
device was not on hand. The principles followed by each property of
absorbance follow the Beers Law, which is reintroduced and
expanded in definition and use in this experiment. Lastly, since this
was a class experiment, the Fe 2+ concentration obtained by each
group was correlated and compared with each other in the whole
class. This was performed to verify the reliability of the method for
determining the said Fe2+ concentration.

INTRODUCTION
Spectrophotometric methods are based on the interaction of a
sample and of the light passing through it. These methods measure the
amount of light absorbed by the sample and thereby identifying the
amount or concentration of a part of the sample or of the sample which
interacts with it.
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Chem126 Lab Instrumental Analysis

The principle governing spectrophotometry is the Beer-Lamberts


Law or simply Beers law. According to this law, the absorbance of light is
directly proportional to the concentration and the pathlength or the
thickness of the sample.
A=bc
[1]
Equation [1] gives the mathematical relation of Beers law where A
is the proportionally constant or the molar
is the absorbance,
absorptivity, c is the concentration, and b is the pathlength of the
absorbing sample or species.
Spectrophotometer is the instrument used to measure the
absorbance of the sample at different concentrations. The basic
components of a spectrophotometer include the light sources, a
monochromator, sample holder, light detector and a read-out meter. An
analytical instrument such as a spectrophotometer must undergo
calibration so as its measured signal (for a spectrophotometer,
absorbance) will be free from determinate errors. Calibration is done
against a standard. Standardization is defined as the process of
determining the measured signal and the amount of analyte. A method is
standardized if the proportionality constant of the signal and amount of
analyte is known.
Many approaches in standardization are being used and improved to
lessen possible determinate errors that might occur in analytical
measurements. One of these standardization methods is the multi-point
external standard calibration method. This standardization method uses
two or more standards of known analyte prepared separately from the
samples containing the analyte. This is accomplished by constructing a
calibration curve. A calibration curve is the plot of the signal against the
concentration of the standards prepared. If the calibration curve is linear,
the slope of the line is the proportionality constant.
This experiment aims to use the multi-point external standard
calibration method in the spectrophotometric determination of Fe 2+ ions in
Ferrous sulfate tablets using 1, 10- Phenanthroline.
METHODOLOGY
A standard ferrous solution was formulated to be approximately 10
parts per million (milligrams per liter) by using a weighed amount of
standardized ferrous ammonium sulfate hexahydrate, or Fe (NH4)2 (SO4)2
6H2O. The solid was dissolved part-by-part before transferred into a one
liter volumetric flask, and dissolved with concentrated sulfuric acid before
being diluted to mark.

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Chem126 Lab Instrumental Analysis

A solution of 1, 10 phenanthroline was formulated from dissolving


a known weight of its solid form into a small beaker. This solution was
transferred into a plastic reagent bottle for storage.
Next, a solution of hydroxylamine hydrochloride was prepared
similarly to the phenanthroline solution, but was transferred into a brown
glass reagent bottle.
A buffer solution was needed in the experiment to allow a
permanent color change. A solution of 0.1 molar sodium acetate and 0.1
molar acetic acid were prepared, and a specified ratio was used to mix the
two solutions and form a buffer solution which was placed in a small
beaker.
A solution for the sample of ferrous sulfate, which naturally
appeared as ferrous ammonium sulfate or Fe (NH 4)2 (SO4)2, was prepared
by using a 325 milligram RiteMed iron tablet. This tablet was dissolved in
water and 6 molar hydrochloric acid. Additional heating was required to
dissolve the tablet completely. The solution was taken care as to not turn
brittle solid as this will make the sample invalid. However, due to
malpractice and inexperience with the experiment, the beaker containing
the sample solution was heated too high and produced an invalid product.
The group had to utilize a correctly prepared sample from another group.
Seven solutions of different volumes from the standard ferrous
solution were prepared and labeled. Another solution was taken from the
unknown sample solution, and a blank solution was prepared from distilled
water.
Each flask was added with a measured amount of each prepared
solution of hydroxylamine hydrochloride, 1, 10 phenanthroline, and the
acetic acid sodium acetate buffer solution. The solutions were allowed to
settle form a permanent color change.
The solution with the highest ppm concentration was used as the
basis for the maximum wavelength of all other solutions. The
spectrophotometer was used to measure this wavelength and the varying
absorbances obtained from each different solution contained in its
cuvette.
The molar absorption coefficient, denoted by , was determined
using the relation of A =

bc. The absorbance data taken from the non-

blank solutions were tabulated and graphed against the corresponding


concentrations, with absorbance as the y-value, to form the calibration
curve. This required for the sample concentration to be solved before
graphing.
The standard deviation was solved by using the x value taken from
the samples calibration curve and the calculated mean of the whole class.
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Chem126 Lab Instrumental Analysis

Then, the weight of iron present in the tablet was calculated using the
concentration of the sample multiplied by a number of dilution factors.
RESULTS

AND

DISCUSSION

The maximum wavelength was determined from the absorption


spectrum in Figure 1. The absorption spectrum was made by plotting the
absorbance against the wavelength. The maximum wavelength, or the
max, was determined to be 510 nanometers. The standard solution with
the highest concentration was chosen as it can absorb the most light out
of all the solutions. The wavelength component of the highest absorbance
is considered the maximum wavelength. As concentration is directly
proportional to absorbance, this maximum wavelength was used as the
basis for all the other solutions with lower concentrations.
0.45
0.4
0.35
0.3
0.25

Absorbane

0.2
0.15
0.1
0.05
0
400
-0.05

450

500

550

Wavelength (nm)

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600

650

Chem126 Lab Instrumental Analysis

Fig. 1. Absorption Spectrum


0.45
0.4
0.35
0.3
0.25
Absorbance

0.2
0.15
0.1
0.05
0
0

0.5

1.5

Concentration (ppm)

Fig. 2. Calibration Curve from the Standard Ferrous Ion Solution

Beers Law states that the absorbance transmitted by a substance is


directly proportional to its concentration in solution and path length of the
cuvette. This relationship is shown mathematically in Equation 1. Using
this relationship the proportionality constant or molar absorptivity,
denoted by , was calculated to be 0.1980 L/mg cm. The path length

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2.5

Chem126 Lab Instrumental Analysis

for the cuvette used has a constant value of 1.00 centimeter, which
effectively cancels its value from the Beers Law equation.
Fig. 2 shows the plot of the absorbance against the concentration of
the prepared standard ferrous solutions. This calibration curve describes
the linear relationship between the absorbance and concentration. The
slope of the line is the proportionality constant between the absorbance
and concentration of the standard solutions. The resulting slope is equal
to the molar absorptivity calculated using Beers Law. This also denotes
how the absorbance of a solution is directly proportional to its
concentration as a suggested in Equation 1. It was observed that as the
concentration of the analyte increases in each of the standard solution,
the color intensity also increases. This indicated a direct proportionality
between the two components.
The standardization technique used in the experiment was a multipoint external standard calibration method. It is widely used to analyze a
series of samples using a single calibration curve. In this technique the
samples matrix and that of the standard solutions matrix is assumed to
be the same and that would not affect the value of the proportionality
constant. However, this is not always the case and proportional
determinate error is introduced into the analysis. This is a limitation in this
kind of technique.
The equation of the line, obtained by least squares method, in the
calibration curve contained the value for the concentration of Fe 2+ in the
sample solution. This equation is in the form y = mx + b, where y
corresponds to the absorbance of the sample solution, m is the slope or
proportionality constant, b is the y intercept gained from the linear
regression process, and x is the concentration of the sample. The
concentration of the sample solution was then multiplied to a series of
dilution factors to obtain 61.7624 mg Fe2+.
The obtained concentration of ferrous ion in the sample is analyzed
statistically. Originally, triplicates of the sample should have been
analyzed however due to time and budget constraints only a single
sample was analyzed. The standard deviation from the results of the
calibration curve (sc) was calculated to measure the reliability of the result
of the external standard calibration. The standard deviation in the
regression line (sr), the relative standard deviation (RSD) was also
calculated. To measure the linearity of the calibration curve, R 2 was
calculated. Table 1 summarizes the statistics done in the groups sample.
The class standard deviation of the obtained concentration of the
ferrous ion in the sample was calculated. Also, the relative standard
deviation and coefficient of variance were determined to measure the
precision of measurements. Using Grubbs test on the concentrations of

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Chem126 Lab Instrumental Analysis

the five class sample solutions, there were no outliers detected in the
class data. Table 2 summarizes the statistics done on the class data.

CONCLUSION
The method used to standardize the sample was the external
standard calibration method.
The concentration of Fe2+ ion in the sample of 325 milligrams of
ferrous sulfate was calculated to be 61.7624 mg/tablet. The s c was equal
to 0.02235, representing reliability for the external standard calibration
method to produce data points with precision. The mean class data for the
concentration of the sample solution had an average of 61.7722
mg/tablet. The standard deviation for the whole class was calculated to be
1.9666. This suggests that there is a slight variation in the results relative
to the mean of the class data.
LITERATURE CITED
Harvey, D. May 2002. External Standards or Standard Additions? Selecting
and Validating a Method of Standardization. Journal of Chemical
Education Vol. 79: 613-615.
Harvey, D. Modern Analytical Chemistry. United State of America: The
McGraw-Hill Companies, Inc.; 2000 [cited 2016 February]. Available
from: http://elibrary.bsu.az/

Skoog D. A., West D. M., Holler F. J., Crouch S. R. 2014. Fundamentals of


Analytical Chemistry Ninth Edition. Canada: Nelson Education, Ltd.
1026p.

APPENDICES

x
5.6616
Sxx

x
7.8090
Syy

TABLES
y
1.1590
Sxy

3.2299
sr

0.1267
Xunknown

0.6394
sc

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y2
0.3186
Slope (m)

0.1980
RSD

xy
1.5769
Y
intercept
(b)
0.005448
R2

Chem126 Lab Instrumental Analysis

0.004041
61.7624
0.02235
0.0003618
Table 1. External Standard Calibration Method Group Data

xsample

ssample

sample

0.9994

RSDsample

308.8608
61.7722
1.9666
0.0318
Gmax
Gmin
Gexp
CV
1.0147
1.4148
1.5712
3.1836
Table 2. External Standard Calibration Method Class Data
CALCULATIONS
x= x i=0.1011+0.2022+0.3033+ 0.5055+ 1.011+1.5165+2.0220=5.6616
x 2= xi2 =0.0102+ 0.0408+0.0920+0.2555+1.0221+2.2998+ 4.0885=7.8090
y=y i=0.029+0.042+0.068+ 0.103+0.202+0.311+0.404=1.1590
2

y =y i = 0.000841 + 0.001764 + 0.004624 + 0.010609 + 0.040804


+ 0.096721 + 0.163216 = 0.318579
xy= 0.002932 + 0.008492 + 0.020624 + 0.05207 +

0.471632 + 0.816888 = 1.576857


S xx = x 2

( x )2
( 5.6616 )2
=7.8090
=3.2299
n
7

( y )2
( 1.1590 )2
S yy = y
=0.318579
=0.1267
n
7
2

( xy )2
[(5.6616)(1.1590)]
S xy =xy
=1.5769
=0.6394
n
7

m=

S xy 0.6394
=
=0.1980
S xx 3.2299

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0.204222 +

Chem126 Lab Instrumental Analysis

b=

y
x 1.1590
5.6616
m
=
( 0.1980 )
=0.005448
n
n
7
7

( )

y b
m

sr =

0.250.005448
=61.7624
0.1980
x unknown=

2
S yy m2 S xx
0.1267( 0.1980 ) (3.2299 )
=
=0.004041
n2
72

( m

S xx |=

RSD=

x unknown

R =1

0.25

1.1590
7

0.004041 1 1
+ +
=0.02235
0.1980 1 7 ( 0.19802 ) ( 3.2299 )

1 1
+ +
M N
sr

m
s c =

sc

y
n

0.02235
=0.0003618
61.7624

[ y i( b +m x i ) ]
S yy

=0.9994

x sample =58.9899+63.7677+63.5354 +61.0000+61.5678=308.8608

x sample =

x sample 308.8608
=
=61.7722
n
5

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Chem126 Lab Instrumental Analysis

( x ix )
( 58.989961.7722 )2+ ( 63.767761.7722 )2 + ( 63.535461.7722 )2+ ( 6161.7722 )2+ ( 6
s sample =
=
n
5
1.9666

RSD sample =

ssample 1.9666
=
=0.0318
x sample 61.7722

Gmax =

x max x sample 63.767761.7722


=
=1.0147
s sample
1.9666

Gmin =

x sample x min 61.772261.0000


=
=1.4148
s sample
1.9666

CV =RSD sample 100 =0.0318 100 =3.180

Gexp=

( )(
n1
n

) ( )(

CV 2
51
=
2
n2+CV
5

3.1802
=1.5712
52+3.1802

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