PA CHEMISTRY

Joseph Pelaelo

EXPERIMENT 2 18000267

SPECTROTROPHOTOMETRIC DETERMINATION OF IRON (II) WITH 1, 10-

PHENANTHROLINEIN
SPECTROPHOTOMETRIC DETERMINATION OF IRON (II)
WITH 1, 10-PHENANTHROLINEIN
OBJECTIVE
-To be able to apply the theoretical background of UV/visible spectroscopy in qualitative and
quantitative analysis of a given analyte.
-to be able to measure the absorbance and determine the maximum absorbance wavelength of
a Fe (II)-1, 10-phenanthrolinein complex.
-To be able to apply Beer’s law to construct calibration curve for quantitative analysis.
INTRODUCTION
UV-Vis Spectrometry. Ultraviolet-visible molecular absorption spectroscopy, commonly referred to
as UV-Vis spectrometry, is an important analytical method for identifying and quantifying a large
variety of chemical species. In UV-Vis spectrometry, a sample is exposed to a spectrum of light
ranging from approximately 200-800nm in wavelength and the instrument records the molecular
absorption at each wavelength versus a blank. The instrument used in this experiment is a Perkin-
Elmer Lambda 40 UV/Vis spectrometer. This instrument utilizes two lamps, one halogen and one
deuterium, and a monochromator to scan through the working UV-vis spectrum of wavelengths. The
light beam is split by a beam splitter and reflects through a series of mirrors before passing through a
sample and a solvent blank simultaneously. The absorbance is then recorded by two separate
detectors and the sample absorption less the blank absorption is plotted versus wavelength. This
type of electronic spectroscopy, with the application of radiation in the visible to near visible
spectrum, is commonly referred to as spectrophotometry. Absorption and Charge Transfer.
Absorption of light occurs when an electron is briefly excited to a higher energy state by a photon. In
aqueous transition metal complexes, this excitation takes the form of a charge-transfer absorption.

In charge transfer complexes, an electron is transferred from an electron donor group to an orbital
with mostly electron acceptor character when radiation is absorbed. In most inorganic complexes,
the transition metal ion acts as the electron acceptor, receiving an electron from the complexed
ligand. However, in the Fe(II) complex studied in this experiment, an electron is transferred in an
opposite fashion from the metal to the 1,10-phenanthroline ligand when radiation is absorbed.
Beer’s Law. In order to make determinations from molecular absorption spectroscopic data, Beer’s
law, an equation relating absorption to sample concentration, must be used. Beer’s law states that
Abs = ε c l. The reaction of Fe(II) with 1,10-phenanthroline to form the [Fe(phen)3] 2+ complex was
first used in 1931 by Walden, et al. as a titration indicator because of the strong red coloration of the
complex. The strong coloration of this complex allows for the determination of the concentration of
[Fe(phen)3] 2+ and subsequently, Fe(II) by absorption spectrophotometry. This method was first
developed and performed by Fortune and Walden in 1938 [4]. In this experiment, the standards and
unknowns are prepared in a solution of MilliQ water containing sodium acetate, hydroxylamine
hydrochloride, and 1,10- phenanthroline. In order to accurately determine the concentration of
aqueous iron, all of the iron in the sample must be in the Fe(II) form in order for the [Fe(phen)3] 2+
complex. The hydroxylamine hydrochloride is added to the solution to lower the pH and act as a
reducing agent to prevent the oxidation of Fe(II) to Fe(III). The sodium acetate is added to the
solution to act as a pH buffer to maintain the pH in a range where all of the aqueous iron will reside
in the Fe2+ oxidation state. The intensity of color has been shown not to change with proton
concentration within the pH range of 2.0-9.0 indicating that this is the acceptable experimental pH
range. 1,10-phenanthroline (C12H8N2) is a bidentate ligand, commonly abbreviated “phen”, which is
able to bind to transition metals through each of the ligand’s two nitrogen atoms. Three equivalents
of phen form a six coordinate complex with iron in the 2+ oxidation state. The Iron(II) complex is
stabilized by the 1,10-phenanthroline ligand because it is a π-acceptor. Applications of
Spectrophotometry for Iron Determinations. In the field of chemical oceanography, the presence of
areas of high nutrient concentrations such as nitrate and phosphate and lower than expected
chlorophyll levels are widely accepted to exist. A prominent high nutrient, low chlorophyll (HNLC)
region exists in the equatorial Pacific Ocean. The low chlorophyll levels indicate reduced biodiversity
and low abundance of microorganisms compared to expected values in areas of similar nutrient
concentrations. For years, the reason for this scarcity was unknown. In the early 1980’s, following a
series of reproducible low iron concentration measurements in the equatorial Pacific, it was
hypothesized that limitation of iron as a micronutrient could be to blame. Iron is an important
micronutrient for phytoplankton photosynthesis. Iron limitation has been shown to impair
photochemical energy conversion by decreasing the quantum efficiency of the photochemistry in
Photosystem II (PSII) which contains two iron atoms. The iron limitation hypothesis was tested
though mesoscale iron-seeding experiments in which large amounts of free iron were added to an
HNLC region of the Pacific Ocean. Following seeding of the water with iron, samples were taken over
a time series and determinations of nutrient, chlorophyll, and iron concentrations were made. A
large phytoplankton bloom was observed following the seeding showing a spike in chlorophyll
concentrations accompanied by a rapid decrease in macronutrients and a slow decrease of iron
concentrations. This pattern proved the hypothesis that iron is in fact the limiting factor in HNLC
regions. Without a practical method for measuring aqueous iron concentrations, this hypothesis
could not have been formed and the explanation of HNLC regions could still remain a mystery.
Spectrophotometry is used by public utility companies to measure heavy metal concentrations in
ground and drinking water. Although excess iron in drinking water does not pose a significant human
health hazard, the Environmental Protection Agency (EPA) suggests that public water utilities
monitor and maintain iron levels <0.3 mg/L for aesthetic and taste purposes.

REAGENTS

Sodium Acetate (CH3CO2Na) 1.2M, Hydroxylamine hydrochloride (NH2OH•HCl) 0.29M, 1,10-

Phenanthroline (C12H8N2•H20) 0.005M, H2SO4 2M, . Ammonium iron(II) sulfate [Mohr’s Salt,
(NH4)2Fe2+(SO4)2•6H2O] 0.0004M.

PR0CEDURE

Six standards were prepared by pipetting 0, 1, 2, 3, 4, 5ml of the standard iron solution into a series
of six 50ml volumetric flasks. The following solutions were transferred into each volumetric flask
using a pipet;

a) 1ml of the hydroxylamine solution

b) 5ml of the sodium acetate solution
c) 5ml of the 1, 10-phenanthrolinein solution

Each flask was filled with deionised water to the mark and the solutions were mixed thoroughly and
were allowed to stand for 10mins. The solutions were mixed again before measuring absorbance.
Deionised water was used as the blank to determine any possible absorbance from the matrix. The
cuvette were rinsed with each solution three times, filled with the solution and placed in the holder.
The absorbance was then measured. The measurement was repeated with fresh aliquot if the
solution.
MESURING THE UNKNOWN WATER SAMPLES

Two challenge samples were prepared, the one that ended up near mid-range on the calibration
curve and one near the lower end. To test for method recovery and accuracy. 25ml aliquots of cold
and hot tap water were pipetted into 50ml volumetric flasks, and two unknown samples. The
absorbance of the solutions were measured.

RESULTS

Table 1. Absorption data for standard solutions of Fe(II)

VOLUME OF CONCENTRATION ABSORBANCE OF WAVELENGTH OF

IRON SOLUTION CONCENTRATION OF IRON IN IRON RECORDED MAXIMUM
ADDED (mL) OF IRON IN SOLUTION (ppm) (abs) ABSORBANCE(nm)
SOLUTION(M)
0 0.00 0.00 -0.005 600.00
0.5 5.0 X 10-5 1960.0 0.116 507.16
1.0 1.0 X 10-5 3921.4 0.125 507.26
1.5 1.5 X 10-5 5882.1 0.174 507.29
2.0 2.0 X 10-5 7842.8 0.233 507.40
2.5 2.5 X 10-5 9803.5 0.322 507.27

ABSORBANCE VS CONCENTRATION OF IRON IN SOLUTION

0.35
0.3
f(x) = 11628.57 x + 0.02
0.25 R² = 0.95
absorbance

0.2
0.15
0.1
0.05
0
0 0 0 0 0 0 0
-0.05
concentration(M)

Figure 1graph of absorbace vs concentration

Table 2. Parameters obtained from Beers law plot

Slope 11629
Pathlength (cm) 1.0
ε 11629

Table 3: Absorption data for solutions containing unknown absorbances of Fe(II)

UNKNOWN SAMPLE No ABSORBANCE WAVELENGTH FOR MAXIMUM

ABORBANCE(nm)
1 0.154 507.28
2 0.210 507.27
Table . concentration of iron in tap water

TAP WATER Concentration (M) Absorbance

Hot 0.28 0.006
cold 0.00 0.0

Table . limit of detection and quantization from excel spreadsheet

7.75939E-
LOD 06
2.35133E-
LOQ 05

SAMPLE CALCULATIONS

1. Concentration of the Fe(II) standard solutions

In Mol/L(M)

C1V1=C2V2

5 X 10-4 M*0.5 mL=50 mL* C2

C2 = (5 X10-4 M*0.5 ml)/ 50 mL=0.000005M

In ppm

Moles=c x v

=0.000005mol/L*0.05L=0.00000025mol

Mass=n x mr

=0.00000025mol*392.14g/mol=0.000098035g (mass of iron)

Ppm = (mass of solute/mass of solution)*106=(0.000098035g/0.05kg) *106=1960

ε=m/b
=10 441 M /cm

1. Concentration of unknown 1
y = 11629x + 0.0155
0.154=11629x+0.0155
X=0.1385 /11629
X = 0.000011909 M
X= 1.19 x 10-5M
2. Conentration of unknown 2
y = 11629x + 0.0155
 0.210=11629x + 0.0155
X=0.1945/11629
X=0.000016725M
X= 1.67 x 10-5 M

3. Molar absortivity of the [Fe(phen)3]2+ complex

Absorbtion= a b c
Absorbtion=average absorbtion= 0.160833333
A=0.161 abs
B= 1cm
C= average concentration= 0.0000125 M
a=A/bc

a=0.161/0.0000125

a=12880M-1 cm-1

DISSCUSION

The standard used in the experiment, FeSO4• (NH4)2SO4•6H2O, though not a primary
standard, is available with a purity greater than 99%, which is adequate.

The iron (II) could not be directly used in the analysis since Fe 2+ is only weakly colored (pale
blue green). It was reacted with 1, 10-phenanthroline prior to analysis to produce an intense red
color. [6] The stoichiometric reaction of Fe2+ with three molecules of the ligand is shown below:

Figure 2: Reaction of Fe (II) with 1, 10-phenanthroline

Hydroxylamine hydrochloride (NH2OH.HCl) is a reducing agent that was added to ensure that all of
the dissolved irons were Fe2+ It reduced any iron (III) present to iron (II). Although there are many
other ways of reducing the Fe 3+ to Fe2+, the hydroxylamine hydrochloride is the best since it does not
interfere in the absorption measurement:
Acetic acid –sodium acetate was added in order control the pH of the solutions since the complex
formed is very sensitive to pH to avoid errors in the analysis due to deviation from the Beer’s LAW.
The maximum wavelength was first measured, 508- 511 nm but the wavelength chosen was 511
though theoretically 509-510 nm would be better.

CONCLUSION

In spectrophotometric methods, like UV-VIS, the sample solution absorbs

electromagnetic radiation from the appropriate source, and the amount absorbed is
related to the concentration of the analyte in the solution. The comparison of the
intensity of the color of solutions of known concentration with the intensity of an
unknown could be a tool in identification of the concentration of the unknown solution.
In the experiment, the Beer’s law was used to quantify the amount of iron and ferrous sulfate in
tap water. Based on the result of experiment, the concentration of the unknown 1 was found to
be 1.19 x 10-5M and unknown 2 was 1.67 x 10 -5 M. Systematic error could occur during the
calibration process (preparation of standard, sampling process). The difference in the
composition of standards and matrix also contributed in the error. For the future experiment, it is
recommended to use a volumetric pipette when transferring any amount of liquid from a
volumetric flask to a volumetric flask.