Experiment No: 02

Experiment Name:
Spectrophotometric determination of iron
Date of Performance- 13.05.18

Introduction:

Spectrophotometry is a method to measure how much a chemical substance absorbs light by

measuring the intensity of light as a beam of light passes through sample solution. The basic
principle is that each compound absorbs or transmits light over a certain range of wavelength.
This measurement can also be used to measure the amount of a known chemical substance.
Spectrophotometry is one of the most useful methods of quantitative analysis in various fields
such as chemistry, physics, biochemistry, material and chemical engineering and clinical
applications.

Theory
When light (monochromatic or heterogeneous) falls upon a homogeneous medium a portion of
the incident light is reflected, a portion is absorbed and the remainder is transmitted so that,
Io = I a + I t + I r

Here,
Io= Intensity of the incident light
It= Intensity transmitted
Ia= Intensity absorbed
Ir= Intensity reflected
For air-glass interface, which occur when using glass cells, about 4% of the incident light is
reflected. Ir is usually eliminated by the use of a control, such as a comparison cell, hence

Io = I a + I t

The Beer-Lambert Law

The Beer-Lambert law relates the attenuation of light to the properties of the material through
which the light is traveling. This page takes a brief look at the Beer-Lambert Law and explains
the use of the terms absorbance and molar absorptivity relating to UV-visible absorption
spectrometry.
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The Absorbance of a Solution
For each wavelength of light passing through the spectrometer, the intensity of the light passing
through the reference cell is measured. This is usually referred to as Io

Figure 4.1 - Light absorbed by sample in a cuvetter

The intensity of the light passing through the sample cell is also measured for that wavelength -
given the symbol, I. If I is less than Io, then the sample has absorbed some of the light (neglecting
reflection of light off the cuvetter surface). A simple bit of math is then done in the computer to
convert this into something called the absorbance of the sample - given the symbol, A. The
absorbance of a transition depends on two external assumptions.
The absorbance is directly proportional to the concentration (c) of the solution of the sample used
in the experiment.
The absorbance is directly proportional to the length of the light path (l), which is equal to the
width of the cuvette.
Assumption one relates the absorbance to concentration and can be expressed as
A∝c
The absorbance (A) is defined via the incident intensity Io and transmitted intensity I by
Io
A=log10 ( )
I
Assumption two can be expressed as
A∝l
Combining Equations
A∝cl
This proportionality can be converted into an equality by including a proportionality constant (ϵ).
A=ϵcl
This formula is the common form of the Beer-Lambert Law, although it can be also written in
terms of intensities:

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 Io
A= log10 ( )=ϵlc
I

The constant ϵ is called molar absorptivity or molar extinction coefficient and is a measure of the
probability of the electronic transition. On most of the diagrams you will come across, the
absorbance ranges from 0 to 1, but it can go higher than that. An absorbance of 0 at some
wavelength means that no light of that particular wavelength has been absorbed. The intensities
of the sample and reference beam are both the same, so the ratio Io/I is 1 and the log10 of 1 is zero.

In the above equation if ϵ and l are kept constant then absorbance and concentration become
proportional to each other. If a graph is plotted with different absorbance at different
concentrations it will give a straight line. If we can determine the absorbance of a solution of
unknown concentration, from the graph, we can easily find out the corresponding concentration.
In this way spectrophotometry can be used for determining unknown concentrations of a solution.

Figure 4.2 - Typical Absorbance vs. Concentration Graph.

Chemistry of the Process

Since spectrophotometric estimation involves physical property only, so no reaction takes place.
Only addition of KSCN forms a complex with Fe3+ which is given below

Fe 3+ + 6(SCN)- = [Fe (SCN)6]3-

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Experimental Setup
Chemicals
Iron (iii) (Fe3+ )solution
Cyanide (SCN-) solution
Nitric Acid (HNO3)
Distilled water

Apparatus
Pipette
Beaker
Round bottom flask
Spectrophotometer

Experimental Procedure
Procedure mentioned below is maintained during conducting this experiment –
1. Fe3+ solution of concentration 1 to 5 ppm are prepared and then 20ml 10% KSCN and 3ml
6(N) HNO3 acid is added in each of the prepared ferric solution
2. Solution of concentration of 4 ppm is taken as standard solution
3. Absorbency data are collected against wavelengths ranging from 400 to 520 nm provided
that each wavelength is adjusted to 100% transmittance for solvent i.e. distilled water
4. Setting the spectrophotometer at the maximum wavelength, absorbance against each
concentration is evaluated
5. Then absorbance versus concentration curve is constructed
6. From the plot, which is a straight line, knowing absorbance of the unknown solution,
concentration of that solution is evaluated
7. In order to evaluate maximum wavelength, absorbency versus wavelength curve is plotted

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Observed Data

Table 2.1- Observed data for Concentration of Fe3+ and absorbance at 474 nm

Sample Concentration(ppm) Absorbance

1 0 0
2 1.0 0.178
3 2.0 0.295
4 3.0 0.417
5 4.0 0.526
6 5.0 0.647
X Unknown 0.361

Graphical Representation

Absorbance Vs. Concentration

0.7
f(x) = 0.13 x
0.6

0.5
Absorbance(A)

0.4

0.3

0.2

0.1

0
0 1 2 3 4 5 6

Concentration(ppm)

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 Figure 4.3- Graphical presentation of Absorbance vs. Concentration

4.8 Calculation

From Graph,

The plotted data follows the equation:

y = 0.1338 x

Where, y = absorbance and x = concentration

For unknown, the obtained absorbance = 0.361

Putting this value in equation,

0.361
Concentration of unknown solution = = 2.70 ppm
0.1338

Real value = 2.5 ppm

Result

Concentration of the unknown sample was determined to be 3.82 ppm from the calibration curve.

Discussion

The prediction of Beer Lambert’s law is verified in this experiment. By plotting absorbance vs.
concentration a straight line should be obtained which the linear relation between absorbance and
concentration is true.

The calibration curve of absorbance versus concentration (at constant wavelength) should pass
through the origin since at the zero concentration (Infinite dilution) absorbance must be zero. But
in this experiment, least square fitting of the curve yields a straight line while passed straightly
above the origin. The following reasons may be responsible for this –

 Some of the solutions were more than 100 mL in volume. This included in the error found.

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 Same pippete was used to measure Fe3+solution and acid. This definitely issued some error
in the results.

 In complete conversion of ferrous ion (Fe2+) to ferric ion (Fe3+) may occurred.
 Impurities present in the sample solution (solution of 3 ppm concentration) which may
absorb light at the absorption wavelength.
 Impurities present in the distilled water used in preparing sample solution.

In the spectrum of Fe [(SCN)6]-3 the highest peak indicates strong (maximum absorption) while
the small peak indicates weak absorption. Maximum absorption occurs at a particular wavelength
but not at any wavelength. The sample compound (Fe 3+ solution) doesn’t absorb radiation
appreciably in the provided wavelength regions. It is necessary to form an absorbing substance by
reacting the sample with other reagents. That is way KSCN is added to Fe 3+ solution. This forms
a complex with Fe3+ solution.

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