EXPT.

1 SPECTROPHOTOMETRIC
DETERMINATION OF Fe2+ IONS USING
1, 10-PHENANTHROLINE
Structure
1.1 Introduction
Objectives
1.2 Principle
1.3 Requirements
1.4 Solutions Provided
1.5 Procedure
1.6 Observations and Calculations
1.7 Result
1.8 Precautions

1.1 INTRODUCTION
UV-VIS spectrophotometry is probably the most useful tool available for quantitative
determinations in diverse areas. It is due to its versatility, accuracy and sensitivity. You
have read about UV-VIS spectrophotometry in Unit 2 of the MCH-003 course. You
have learnt that the UV-VIS spectrum results from the interaction of electromagnetic
radiation in the UV-VIS region with molecules, ions or complexes. You would recall
that the absorption of radiation in the UV-VIS region by the absorbing species is
governed by the Beer-Lambert’s law which relates it to the thickness of the absorbing
medium and the concentration of the absorbing species. This law forms the basis of a
number of methods for the determination of micro and semimicro quantities of analyte.

UV-VIS spectrophotometry can be used for direct determination of a large number of

organic, inorganic and biochemical species accurately at fairly low concentrations; viz.,
10 −4 to 10 −5 M or even lower. A significant feature of UV-VIS spectrophotometry is
that it can also be used for the quantitative determination of analyte which do not
absorb in the UV-VIS region. It is achieved by making them react with a reagent that
gives a product which strongly absorbs in the region. In the first experiment of this
course you would perform a determination based on this very aspect of UV-VIS
spectrophotometry. Perhaps you will like to read Section 2.3 and subsection 2.6.3 of
Unit 2 of MCH - 003 course again to understand the principle behind the UV-VIS
spectrophotometry better.

Objectives
After studying and performing this experiment you should be able to:
• explain the principle underlying spectrophotometric determination of an element
like iron at low concentrations,
• make absorbance measurements on a spectrophotometer and draw the UV-VIS
spectrum of a substance,
• determine the wavelength of maximum absorption and compute the
corresponding molar absorption coefficient,
• draw a calibration curve using standard solutions of the analyte and determine
the concentration of iron in an unknown sample using the calibration plot, and
• adapt the method for determination of iron in real samples such as alloys.

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Spectroscopic Methods
Lab 1.2 PRINCIPLE
As mentioned in the introduction, UV-visible absorption spectrophotometry provides a
convenient method of determination of concentration of any substance which can be
treated to form a coloured solution in which the colour intensity is proportional to the
concentration of the substance. This experiment is based on the determination involving
the formation of a complex species that absorbs in the visible region. The general
procedure usually involves the following basic steps.
• Treatment of the properly prepared sample with a reagent to form a coloured
solution,
• Controlling factors influencing absorption by the coloured species,
• Measurement of absorbance of the coloured solution at the appropriate
wavelength,
• Preparation of an absorbance-concentration plot (calibration plot) by
measurements of the absorbance of the standard solutions of known
concentrations, and
• Estimation of concentration in the unknown sample corresponding to the
absorbance measured by using the calibration plot.
In the determination of iron (II) in aqueous solutions, a tricyclic nitrogen heterocyclic
compound,1, 10-phenanthroline (C12H8N2, ortho-phenanthroline or o-Phen) is used as
the ligand that reacts with metals such as iron, nickel, ruthenium, and silver to form
strongly coloured complexes. With ferrous ions (Fe2+), it reacts in a ratio of 1:3 to form
an orange red coloured complex [(C12H8N2)3Fe]2+ in aqueous medium as per the
following equation.
Fe2+ + 3 Phen Fe(Phen)32+
The ligand is a weak base that reacts to form phenanthrolinium ion, phenH+, in acidic
medium. Accordingly, the complex formation may be represented as follows,
Fe2+ + 3PhenH+ Fe (Phen) 32+ + 3H+
The molar absorption coefficient ( ε ) of the ferrous complex, [(C12H8N2)3Fe]2+ so
obtained is 11,100 dm3 mol-1 cm-1 at the wavelength of maximum absorbance, λmax
= 508 nm. The large value is indicative of strong absorption by the complex and forms
the basis of the quantitative determination of iron (II). The absorbance of the coloured
complex is measured at 508 nm using a spectrophotometer or with the help of a filter
photometer using a blue-green filter.

The colour intensity is not affected by change of pH over the range 2-9 and is also
stable for a long time. However, a pH of about 4.5 is ordinarily recommended to
prevent precipitation of iron salts. Further, the cations like Ag+, Bi3+, Cu2+, Ni2+, Co2+
and anions such as perchlorate, cyanide, molybedate and tungstate interfere
significantly in this determination, therefore, these must be absent in the analyte
solution.

As we are attempting to determine the concentration of iron (II) ions in the analyte
sample, it must be free from any iron (III) ions which may be present due to the partial
oxidation of the ferrous ions. This is achieved by adding a reducing agent before the
coloured complex is formed. Ferric ion (Fe3+) is reduced to ferrous state (Fe2+) by
hydroxylamine before complexation as per the following equation.
4Fe3+ + 2NH2OH Fe2+ + N2O + 4 H+ + H2O
Under these conditions the complex obeys Beer-Lambert’s law in the range of the
concentrations being determined (~0.5‒2.0 ppm). The range for the validity of Beer-

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Lambert’s law can be determined by plotting a calibration curve by measuring the
absorbance values for a series of standard solutions of the complex being determined at
a fixed wavelength of 508 nm. The curve so obtained is then used for the determination
of concentration of unknown solutions from a measurement of its absorbance at the
same wavelength.

This method can be modified for the determination of a mixture of iron (II) and iron
(III) in a given solution. The principle of the method is given in the box below.

Simultaneous determination of iron (II) and iron (III) in a solution

The spectrophotometric determination of Fe2+ and Fe3+ ions simultaneously in a

given solution is based on the fact that Ferric ions (Fe3+) form a similar complex
with the ligand orthophenanthroline as the ferrous ions (Fe2+) but the complex is
yellow in colour. Both, the ferrous and ferric complexes have identical absorption
at 396 nm and are additive. Therefore, the measurement of absorption at 396 nm
would give the amount of total iron present. For this the solution is made slightly
acidic with dilute sulphuric acid, treated with 1,10-phenanthroline and buffered
with potassium hydrogen phthalate at a pH of 3.9.

The absorption at 508 nm on the other hand corresponds to ferrous complex only.
Therefore, the absorbance at 508 nm gives the amount of ferrous ions. Thus, the
absorbance at 396 nm gives the total iron content while at 508 nm gives iron (II).
The subtraction of the latter from the former gives the ferric iron present in the
solution.

1.3 REQUIREMENTS
Apparatus Chemicals
Spectrophotometer/ Filter photometer 1 Ferrous ammonium sulphate hexahydrate
Matched cuvette 2 1, 10-Phenanthroline
Volumetric flasks (1 dm3) 1 Hydroxylamine hydrochloride
Volumetric flasks (50 cm3) 12 Sulphuric acid
Graduated pipette, 10 cm3 1 Acetic acid
Pipettes (5, 10 cm3) 1 Sodium acetate
Weighing bottle 1

1.4 SOLUTIONS PROVIDED

i) Standard ferrous solution (10 ppm (10 mg/ dm3)); prepared as follows.
• Weigh 0.0702 g of analytical grade ferrous ammonium sulphate
hexahydrate [Fe(NH4)2(SO4)2.6H2O].
• Quantitatively transfer the weighed sample to a volumetric flask of 1 dm3
capacity and add sufficient distilled water to dissolve it.
• Add 2.5 cm3 of concentrated sulphuric acid and make up the solution to the
mark.
ii) 1, 10-phenanthroline; prepared by dissolving 100 mg of the reagent in 100 cm3
of distilled water. The reagent can be stored in a bottle.

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Spectroscopic Methods iii) Hydroxylamine hydrochloride; prepared by dissolving 10 g of hydroxylamine
Lab hydrochloride in 100 cm3 of distilled water.
iv) Sodium acetate (0.1M); prepared by dissolving 10 g of sodium acetate in
100 cm3 of water.
v) Acetic acid (0.1M); prepared by diluting about 6 cm3 of glacial acetic acid to
100 cm3
vi) Acetic acid-sodium acetate buffer (pH = 4.5); prepared by mixing 65 cm3 of
0.1 M acetic acid and 35 cm3 of 0.1 M sodium acetate in a 100 cm3 flask.
(Prepare the buffer whenever required.)

1.5 PROCEDURE
1. Pipette out 1, 2, 3, 5, 10, 15 and 20 cm3 of the standard ferrous ion solution into a
series of 100 cm3 standard flasks labelled from 1 to 7.
2. In another flask, labelled ‘Sample’, take 10 cm3 of the unknown sample.
3. To another 100 cm3 standard flask, labelled ‘Blank’, add about 20 cm3 of
distilled water to prepare the blank solution.
4. To each of the above flasks (standards, sample and blank) add 1 cm3 of
hydroxylamine hydrochloride and 5 cm3 of 1, 10-phenanthroline.
5. Buffer each solution by adding 8 cm3 of acetic acid / sodium acetate buffer.
6. Allow at least 15 minutes after the addition of the reagents for full colour
development (The colour once developed is stable for hours).
7. Dilute each solution exactly to 100 cm3 mark with distilled water and mix well.
8. The standard solutions so obtained correspond to 0.1, 0.2, 0.3, 0.5, 1.0, 1.5 and
2.0 ppm respectively. You may label the flasks accordingly.
9. Record the absorption spectrum for the 2.0 ppm standard solution against the
reagent blank in the range of 400 – 700 nm.
10. If the instrument is of manual type, measure the absorption value after every
10 nm over the spectral range and record the readings in Observation Table 1.1.
Draw the spectrum by plotting the absorbance as a function of the wavelength in
the graph provided in Fig.1.1.
11. Select the wavelength which gives maximum absorbance ( λmax ) and record the
same under observations. The reported value is 508 nm.
12. Calculate the molar absorption coefficient ( ε ) of the complex from the molar
concentration and path length of the cuvette. Use the relation A = ε cb.
13. Measure the absorbance for all the standard solutions at the wavelength of
maximum absorption and record the readings in the column no. 4 of the
Observation Table 1.2.
14. Measure and record the absorbance for the ‘Sample’ also in the same way.
15. Make a plot of absorbance at Y-axis (column 4) vs. concentration of the standard
solutions at X-axis (column 3) to get the calibration curve. (The linear region of
the curve obeys Beer- Lambert’s law and is used for the estimation of unknown
samples.)
16. Determine the concentration of the given sample solution with the help of the
calibration curve.
17. Calculate the ferrous ions present in the unknown sample solution by accounting
for the dilution factor and report the value.

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1.6 OBSERVATIONS AND CALCULATIONS
A. Recording the visible spectrum of the iron-phen complex

Observation Table 1.1: Absorbance of the iron-phen complex obtained from

2ppm standard solution of iron (II) as a function of the wavelength
Wavelength Absorbance Wavelength Absorbance Wavelength Absorbance
(nm) (nm) (nm)
410 510 610
420 520 620
430 530 630
440 540 640
450 550 650
460 560 660
470 570 670
480 580 680
490 590 690
500 600 700

B. Obtaining the spectrum of the complex: Graph between wavelength and

absorbance for the standard solution (from Observation Table 1.1).
Absorbance

400 500 600 700 800

Wavelength (nm)
Fig. 1.1: Visible spectrum of the Iron-phen complex
As per the spectrum obtained above, the wavelength of maximum absorption,
λmax is found to be: ……….. nm.

C. Calculation of molar absorption coefficient

The absorbance at the λmax = A = ..........
Path length of the sample (cuvette cell length) = b = ....... cm
Concentration of the standard solution = c = 2.0 ppm = 2.0 mg / dm3
2 × 10 -3 g
=
605.85 g mol dm 3
= 3.3 × 10-6 mol dm-3

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Spectroscopic Methods Using the relation, A = ε cb
Lab
A
The value of molar absorption coefficient = ε =
cb
.............
= −6
3.3 × 10 mol dm −3 × ........cm

= .................... cm‒1 mol‒1 dm3

D. Collecting absorbance data for the calibration curve

Observation Table 1.2: Absorbance values of the phen complexes of

standard and sample solutions of ferrous ions

Column
1 2 3 4
S. No. Volume of the Conc. of Fe2+ ions Absorbance at λmax
standard / sample (ppm)
solution (cm3)
1 1 0.1
2 2 0.2
3 3 0.3
4 5 0.5
5 10 1.0
6 15 1.5
7 20 2.0
Sample 10 ?

E. Plotting the calibration curve: Graph between the absorbance and the
concentration of ferrous ion in the standard solution
Absorbance

0 0.5 1.0 1.5 2.0

Concentration of the standard ferrous solution (ppm)
Fig. 1.2: Calibration plot of absorption vs. Concentration

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F. Determining the concentration of the ferrous ions in the sample from
calibration curve
Plot the value of absorbance of the sample solution on the calibration curve
obtained in Fig.1.2 and determine the corresponding concentration value (in
ppm).
The concentration of ferrous ions in the given sample =
(Value obtained from the calibration curve × 10) ppm

= ...........× 10 ppm = ..........ppm

[The factor of ten comes from the fact that we took 10 cm3 of the sample solution
and finally diluted it to 100 cm3.]

1.7 RESULT
i) The wavelength of maximum absorption for the iron-phen complex has been
found to be = ............nm.
(Ref. step C in the observations and calculations.)
ii) The molar absorption coefficient of the complex is found to be
= .......... cm‒1 mol‒1 dm3.
(Ref. step D in the observations and calculations.)
iii) The concentration of ferrous ions in the given sample solution =........... ppm.
(Ref. step F in the observations and calculations.)

1.8 PRECAUTIONS

• The standard solution of iron must be prepared fresh.

• Do not handle the lower portion of a cuvette through which the light beam passes.
• Rinse the cuvette with the solution being analysed before taking a measurement.
• Wipe off any liquid drops or smudges on the lower half of the cuvette with the
help of a lens paper. (You may use a soft tissue paper but make sure that no fibers
are left on the cuvette in the optical path.)
• Don’t use a test tube brush to clean the cuvettes after the experiment is over.
• The instrument must be recalibrated each time the wavelength setting is changed.

Instructions for the Counsellor

• The total volume of standard/unknown solution can be 25, or 50 cm3 since the
sample holder (cuvette) hold about 3 -5 cm3 volume. Compute the concentration
accordingly.
• You can choose any suitable concentration range for preparing the calibration
curve, provided the calibration plot is linear and obeys Beer‒ Lambert’s law.
• Dilution at different stages should be recorded properly for computing the final
value.

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