OBJECTIVE

The goal of this experiment is to use a spectrophotometric method to determine the concentration
of an unknown iron solution. This experiment also teaches you about this method and how it
works.
Theory
When light strikes a homogeneous medium, some of the incident light is reflected, some is
absorbed, and the rest is transmitted. The light intensities are denoted as Io for incident light, Ia
for absorbed light, It for transmitted light, and Ir for reflected light.
I o=I a + I t + I r

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 = Ia + It
Lambert is frequently credited with investigating the change in light absorption with a medium
thickness. Lambert's law and Beer's law are the two separate laws that govern absorption. They
are known as the Beer-Lambert law when combined.
Beer-Lambert Law
According to Beer-Lambert law, when monochromatic light passes through a transparent
medium, the intensity of the emitted light decreases exponentially as the concentration and
thickness of the medium increase arithmetically.

−ϵbc
I =I o e

I
¿ , ln =ϵbc= A
Io

Here, A = absorbance, ε = molar extinction coefficient, b = thickness of the medium, c =

concentration

The Beer-Lambert law states that the absorbance is directly proportional to the path length b of
the sample and its concentration

A=ϵlc

where, l = path length, c is the molar concentration (in moles.dm-3), and l, the path length, is
measured in centimeters.
The molar extinction coefficient is constant for a given solute and varies with light wavelength.
An absorption spectrum is a plot against the wavelength. Such spectra, as illustrated above, could
be in the ultraviolet or visible ranges of the electromagnetic spectrum.

If and l are held constant in the equation I absorbance and concentration become proportional to
each other. A straight line is produced when a graph with different absorbances at different
concentrations is plotted. If we can determine the absorbance of an unknown concentration
solution from the graph, we can easily determine the corresponding concentration.
Spectrophotometry can thus be used to determine unknown concentrations of a solution.
Chemistry in the process
Since spectrophotometric estimation involves physical property only, so no reaction takes place.
Only the addition of KSCN forms a complex with Fe3+ which is given below –

Fe 3+ + n (SCN)- = Fe (SCN)n 3- (blood red color)

Advantages

Absorption spectroscopy is one of the most useful quantitative analysis methods available in
chemistry. Important photometric method characteristics include: (a) broad applicability; (b)
high sensitivity; (c) moderate to high selectivity; (d) accuracy; and (e) convenience.
Spectrophotometers can provide a narrow bandwidth of radiation and also handle absorption
spectra in the ultraviolet region, making the spectrophotometric method superior to the
spectrometer or photometer.Spectrophotometric estimation can be a simple way to determine
minute amounts of substances. It can detect 10-4 to 10-6 mol/liter in any solution, for example.

Limitations

Some issues with spectrophotometric estimation arise when analyzing very concentrated or very
dilute solutions, i.e., solutions with transmittances outside the range of 0.2 to 0.65. Whereas the
transmittance scale in the ordinary method is set to zero (to represent infinite concentration),
precession is less than that obtained with volumetric or gravimetric techniques. The main
limitation of spectrophotometric estimation is this.

Wavelength selection

The Beer-Lambert Law states that absorbance is proportional to concentration at each

wavelength. In theory, we could use any wavelength to estimate concentration quantitatively.
However, the magnitude of the absorbency is critical, particularly when attempting to detect very
small amounts of material. The distance between curves 1 and 2 in the spectra above reaches a
maximum at 525 nm, and at this wavelength, the change in absorbance is greatest for a given
change in concentration. That is, at this wavelength, the measurement of concentration as a
function of concentration is most sensitive. As a result, in most cases, the wavelength of
maximum absorbance for a given sample is chosen and used in absorbance measurements.

APPARATUS:
1. Pipette
2. Beaker
3. Round bottom flask
4. Spectrophotometer
CHEMICALS:
1. Fe3+ solution
2. SCN- solution
3. Nitric acid (HNO3)

4. Distilled water

PROCEDURE:

1. Fe3+ solutions of different concentrations were prepared adding 1 ml HNO3, 10 mL

NH4SCN and make up distilled water.

2. The Spectrophotometer was calibrated by a reference solution.

3. The Fe3+ solutions were taken one by one to produce calibration curves using a

spectrophotometer for determining the maximum absorbance and max of the solution.
4. Then maximum absorbance versus concentration curve was constructed.

5. From the plot, which is a straight line, knowing the absorbance of the unknown solution

from the calibration curve, the concentration of the unknown solution was evaluated.

Data Collection and Calculation

The wavelength of the incident light λ=480 nm

Concentration, c
Observation Absorbance, A
(ppm)
1 0 0
2 1 0.108
3 2 0.208
4 3 0.326
5 4 0.405
6 5 0.512
7 Unknown 0.366
Graphical Representation
The calibration curve -

Absorbance vs Concentration of the Sample Solutions

0.6

0.5
f(x) = 0.101971428571429 x + 0.00490476190476191
R² = 0.998218146914064
0.4
Absorbance, A

0.3

0.2

0.1

0
0 1 2 3 4 5 6
Concentration, C (ppm)

Calculation

The Absorbance vs Concentration graph in Figure 3 is plotted and it follows a linear equation.
From the best fitted curve, it is found-
 y ( A )=0.102 x ( c )+ 0.0049………………….(1)

For unknown concentration of sample Fe3+ solution, Absorbance is found, A = 0.366.

(0.366−0.0049)
If we put y= A=0.366 then the concentration is found, x = c =
0.102

=3.54 ppm

DISCUSSION

This experiment validates Beer Lambert's law's prediction. A straight line should be obtained by
plotting the absorbance vs concentration graph, indicating that the linear relationship between
absorbance and concentration is true.

The calibration curve of absorbance versus concentration (at constant wavelength) should pass
through the origin because absorbance must be zero at zero concentration (infinite dilution).
However, in this experiment, the curve's least square fitting yields a straight line when passed
straight above the origin. This could be due to the following factors:

1. There may have been an incomplete conversion of ferrous ion (Fe2+) to ferric ion (Fe3+).

2. Impurities in the sample solution that may have absorbed light at the wavelength of absorption

3. Impurities in the distilled water used to make the sample solution.

The highest peak in the spectrum of Fe [SCN)6]3- indicates strong (maximum absorption), while
the lowest peak indicates weak absorption. Maximum absorption happens at a specific
wavelength but not at any other wavelength. The sample compound (Fe3+ solution) does not
absorb radiation significantly in the wavelength regions specified. By reacting the sample with
other reagents, an absorbing substance is formed. This is why KSCN is included in the Fe3+
solution.