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Spectrophotometric
determination of Iron(III)
94
Section 1: Introduction
The word ferric is derived from the Latin word “Ferrus” for iron. Ferric refers
to iron containing materials or compounds. In chemistry, iron with an oxidation
number of +3, also denoted iron (III) or Fe+3
which is usually the most stable from of
iron in air.
Iron is an abundant element with a Clarke number of 4.70, the fourth largest
among the elements [1], and it is an essential component of almost every organism in
the biosphere. It occurs in a variety of rocks and soil minerals of oxidation states 2
and 3; but it is only a trace element in biological system. Iron plays a central role in
the biosphere. It is essential component or cofactor of numerous metabolic reactions
and living cells including, both plants and animals. It is involved in oxygen transport
and electron transfer and in enzymes including hydroxylayed peroxidases and
dismutases [2]. Iron deficiency anemia is one of the world’s most common nutritional
deficiency diseases. Evidence has been presented that at low levels iron is an essential
element in the diet whereas at higher concentrations it is toxic [3]. Excess of iron in
body causes “haemochromatosis”. The toxicity of iron and in particular iron overload
has are used considerable interest in recent year [4].
The average adult human body contains 4-6 grams of iron. In human beings,
the majority of iron is found in the blood as a pigment called hemoglobin. The
function of this is to transport oxygen from lungs to various tissues in the body where
it is used to produce energy. One of the byproduct of this metabolism, carbon
dioxide, is thus transported back to the lungs by hemoglobin. Both the oxygen and
carbon dioxide molecules bind to the iron ion present in hemoglobin during transport.
Humans obtain the iron necessary for the formation of hemoglobin from their diet in
foods such as meat and leafy, green vegetables etc.
95
The bioavailability of iron is of great interest because all known forms of life
require iron and ordinary iron (III) compounds are insoluble in an aerobic
environment. The low bioavailability of iron affects all forms of life. The impact of
increasing the bioavailability of iron was famously demonstrated by an experiment,
where a large area of the ocean surface was sprayed with iron (III) salts. After several
days, the phytoplankton within the treated area bloomed to such an extent that the
effect was visible from outer space. This fertilizing process has been proposed as a
means to mitigate the carbon dioxide content of the atmosphere [5].
Ferric iron is a d5 center, means that the metal has fine ‘valence electrons” in
the 3d orbital shell. The magnetism of ferric compounds is mainly determined by
these five electrons, and their behavior depends on the number and type of ligands
attached to iron, as described by ligand field theory usually ferric ions are surrounded
by the ligands arranged in octahedron. Sometimes three and sometimes as many as
seven ligands are observed.
Due to its importance in the contest of clinical diagnosis, intoxication,
environmental pollution monitoring etc., a number of sensitive analytical methods are
available for the determination of iron. There are certain reagents applied for
determination of iron by solvent extraction method [6-8]. Flame and graphite furnace
atomic absorption spectroscopy (AAS) [9-13] are the most commonly used techniques
for iron determination. But these methods are disadvantageous in terms of cost and
instruments used in routine analysis. AAS is often lacking in sensitivity and affected
by many conditions of samples such as salinity. Extraction methods [14-17] are
highly sensitive but generally lack in simplicity. Spectrophotometry is essentially a
trace analytical technique and is one of the most powerful tools in chemical analysis.
A wide variety of reagents have been proposed for the spectrophotometric
96
determination of iron (III) [18-28]. Among them, 1-10 phenanthroline is considered
as most selective reagent for the iron determination [29]. But this method suffers from
interference of foreign ions, stability, simplicity and range of determination. Among
existing methods for the spectrophotometric determination of Fe(III), some are
extractive methods, some have low sensitivity and some have interference with other
metal ions.
Though large number of spectrophotometric methods are reported for the
determination of Fe(III), still there is a demand for simple, highly sensitive and
selective methods for its determination is complex material. Hence, the present
method explores 2-hydroxy3-methoxy benzaldehyde thiosemicarbazone
(HMBATSC) as a analytical reagent used for the spectrophotometric determination of
Fe(III). The developed method can be employed for efficient determination of iron at
microgram level. The proposed method is sensitive, rapid and free from limitations.
The following section comprises the results obtained in the present investigations. In
this thesis, spectrophotometric method for the determination of Fe(III) was developed
by measuring the absorbance of greenish yellow colored complex solution (pH 6.0) of
[Fe(III)-HMBATSC] at 385 nm.
97
Section 2: Zero order spectrophotometric determination of Fe(III)
The reaction between Fe(III) and 2-hydroxy-3-methoxy benzaldehyde
thiosemicarbazone (HMBATSC) in the pH range 2.0 – 7.0 was resulted in a greenish
yellow colored water soluble complex. The color formation was instantaneous, and
found to be maximum and constant in the pH range 5.5 to 6.5.
a) Absorption Spectra
The absorption spectra of greenish yellow colored [Fe(III)–HMBATSC]
complex and yellow colored HMBATSC solutions were measured in a wavelength
region of 320–600 nm using the general procedure 3.a, and corresponding absorption
spectra were presented in Fig. 5.2.1. The Fe(III) metal complex shows a absorption
maximum at 385 nm where the reagent has considerably low absorbance. Therefore,
Fe(III) was determined by measuring the absorbance at 385 nm using reagent blank as
reference solution.
b) Effect of pH
The greenish yellow color formation between Fe(III) and HMBATSC was pH
dependent and occurs only in acidic buffer medium. Hence, the optimum pH range in
which maximum and constant color intensity was determined by measuring the
absorbance of Fe(III) complex solution at different pH values employing the
procedure given in 3.b. The results presented in the form of a plot in Fig. 5.2.2 which
reveals that maximum sensitivity can be obtained in the pH range 5.5 to 6.5.
Therefore, pH 6.0 was chosen as the optimum pH to get maximum sensitivity for the
determination of Fe(III).
98
Fig. 5.2.1. Absorption Spectra of
(a) [Fe (III) – HMBATSC] Vs HMBATSC blank
(b) HMBATSC Vs buffer blank
[Fe (III)] = 5×10-4
M; HMBATSC = 1×10-3
M
pH = 6.0
Fig. 5.2.2. Effect of pH on absorbance of [Fe (III) – HMBATSC]
[Fe(III)] = 2.5 × 10-4 M; [HMBATSC] = 1 × 10-3 M
Wavelength = 385 nm
99
c) Effect of reagent concentration
The absorbance data presented in Table 5.2.1 for solutions containing different
molar ratios of metal to reagent concentrations at pH 6.0 has been confirmed that a
10-fold excess of reagent compared to the metal ion concentration was necessary to
get maximum and constant coloration.
Table 5.2.1. Reagent effect
[Fe(III)] = 5 × 10-4
M
[HMBATSC] = 5 × 10-3
M
pH = 6.0
Wavelength = 385 nm
[Fe(III)] : [HMBATSC] Absorbance
1 : 1 0.345
1 : 2.5 0.382
1 : 5 0.451
1 : 7.5 0.488
1 : 10 0.514
1 : 12.5 0.511
1 : 15 0.507
d) Calibration and Precision
The absorbance values of experimental solutions containing variable amounts
of Fe(III) and fixed amounts of HMBATSC and buffer pH 6.0 measured at 385 nm as
described in 3.d were plotted against the amount of Fe(III) and presented in Fig. 5.2.3.
A straight line obtained as shown in the Fig. 5.2.3, indicated that Beer’s law was
obeyed over the range of 0.2795 – 5.3105 µg mL-1
of Fe(III). The straight line
corresponds to the equation A385 = 0.1781C - 0.0264 with a correlation coefficient of
0.9998. The molar absorptivity of the proposed method calculated from the slope of
100
the calibration graph was 1.024 x 104 l mol
-1 cm
-1 at 385 nm. The Sandell’s sensitivity
was calculated to be 0.0054 µg cm-2
.
Fig. 5.2.3 Calibration plot [HMBATSC] = 5 × 10
-3 M
Wavelength = 385 nm pH = 6.0
e) Effect of foreign ions
The selectivity of a spectrophotometric method can be determined from the
tolerance limits of other associated ions with the analyte ion evaluated using the
procedure described in 3.e. The tolerance limits of various diverse ions in the present
method are placed in Table 5.2.2. The amount of diverse ion which causes a change in
the absorbance value by +2% was taken as its tolerance limit.
Amount of Fe(III) (µg mL–1)
101
Table 5.2.2. Tolerance limit of foreign ions
Amount of Fe(III) = 2.236 µg mL-1
Diverse ion Tolerance limit
(µg mL-1
) Diverse ion
Tolerance limit
(µg mL-1
)
Thiosulphate 1220 Te(IV) 1050
Thiourea 1060 U(VI) 1020
Thiocyanate 890 Na(I) 880
Fluoride 810 K(I) 790
Sulphate 780 Ti(IV) 700
Phosphate 740 Zn (II) 650
Iodide 720 W(VI) 555
Bromide 600 Y(III) 510
Ascorbate 560 Ce(IV) 340
Nitrate 520 Cd(II) 280
Bromate 440 Ag(I) 150
Tartrate 400 Mn(II) 120
Acetate 380 Ru(III) 100
Chloride 365 Mo(VI) 90
Formate 350 Ga(III) 90
Oxalate 280 Pd(II) 75
Citrate 210 Cr(VI) 45
In(III) 40
V(V) 30
Ni(II) 25
Co(II) 20
Cu(II) 10, 110a
Fe(II) 8, 105a
V(IV) 10, 100b
Au(III) 10, 120c
In the presence of (a) 1220 µg of thiosulphate; (b) 810 µg of fluoride; (c) 720 µg of iodide.
102
The clear investigation on interference studies (Table 5.2.2) indicates that all
the anions did not interfere even when present in more than 200-fold excess. Majority
of cations did not interfere when they were present in more than 50-fold excess.
Cr(VI) and In(III) were tolerable up to 40-fold excess. V(V), Ni(II) and Co(II)
interfered when present in between 20-30-fold excess. The metal ions, Cu(II), Fe(II),
V(IV) and Au(III) are interfered under 10-fold excess. However, Cu(II) and Fe(II), in
presence 1220 µg of thiosulphate, V(IV) in presence of 810 µg of fluoride and Au(III)
in presence of 720 µg of iodide were tolerable up to 100-fold excess.
f) Composition and stability constant
The stoichiometry of the greenish yellow colored [Fe(III)-HMBATSC]
complex solution was determined by Job’s continuous variation method (Fig. 5.2.4)
and molar ratio method (Fig. 5.2.5). Both the methods gave a 1:2 (metal : ligand)
stoichimetry. The stability constant of the complex was calculated from the Job’s
curve and obtained as 3.75 × 109.
103
Fig. 5.2.4. Jobs Curve
[Fe(III)] = [HMBATSC] = 5×10-3
M
Wavelength = 385 nm; pH = 6.0
Fig. 5.2.5. Molar ratio plot
[Fe(III)] = [HMBATSC] = 5×10-3 M
Wavelength = 385 nm; pH = 6.0
104
g) Applications
The proposed method was applied for the determination of Fe(III) in steel
alloys and environmental water samples.
(i) Determination of Fe(III) in Stainless steel and Chromium-nickel type steel:
A known amount of the samples (~1.0 g) were placed in a 100 ml beaker,
added 10 ml of 20% (v/v) sulfuric acid and carefully covered with a watch glass until
the brisk reaction subsided. The solution was heated and simmered gently after
addition 5 ml of 14 M HNO3 until all carbides were decomposed. Then, 2 ml solution
of H2SO4 (1:1) was added and the mixture was evaporated carefully until the dense
white fumes dried off the oxides of nitrogen and then cooled at room temperature.
After appropriate dilution with water, the contents of the beaker were warmed to
dissolve the soluble salts. The solution was then cooled, neutralized with NH4OH
solution and filtered through a Whatman 41 filter paper into a calibrated flask of
known volume. The residue was washed with a small volume of hot 1% H2SO4
followed by water and the volume was made up to the mark with water. The
analytical results are shown in Table 5.2.3. The amount of Fe(III) in these sample
solutions could be determined from a predetermined calibration plot.
(ii) Determination of Fe(III) in environmental water samples
The water samples collected in a clean 1 liter beakers from different place of
Anantapur and Kurnool districts (Andhra Pradesh, India) were filtered through 0.45
µm pore size membrane filters immediately after sampling. The water samples were
slowly evaporated to about 25 ml. 5 ml of H2O2 was added and evaporated to dryness
[30]. It was then dissolved in 2 mL of water and filtered to remove insoluble
substance. The filtrate was collected in 100 mL volumetric flask quantitatively and
105
diluted to the mark with distilled water. The filtered water samples were analyzed
using the proposed to determine iron(III) using zero order method. A known amount
of Fe(III) was added to the water samples and the recovery was evaluated as an
average of five determinations. The results were presented in Table 5.2.4 and indicate
the recoveries were in the acceptable range.
Table 5.2.3. Determination of Iron(III) in Steel alloys
Alloy sample Composition (%) Amount of Fe(III) (%) Relative
error
(%) Taken Found*
1 Cr (11-13), Ni (10), C (0.1-
0.4), Fe (77) 77 76.2 98.96
2 Cr (16), Mn (14), Ni (1), Fe
(69) 69 69.8 101.16
3
Mn (0.81), Cr (0.66), Mo
(0.58), Ni (2.55), Cu(0.088),
Sn (0.011), Fe (95.0)
95 94.1 99.05
* Average of five determinations.
Table 5.2.4. Determination of Fe(III) in environmental water samples
Sample Amount of Au(III) ( µg mL
-1)
Recovery (%) Added Found
*
Ground water 0.50 0.51 102
Rain water 1.00 1.03 103
Drain water 1.50 1.54 102.5
Lake water 2.00 1.98 99
* Average of five determinations.
106
Section 3: Derivative spectrophotometric determination of Fe(III)
In the proposed zero order method, serious interference from certain ions was
observed in the determination of Fe(III). To overcome these interferences and to
improve the selectivity of the method, an attempt has been made to develop some
derivative spectrophotometric methods for the determination of Fe(III).
a) Derivative Spectra
The second and third order derivative spectra were recorded for known
aliquots of experimental solutions containing variable amounts of Fe(III) at pH 6.0 in
the wavelength region 320-600 nm and presented in Fig. 5.3.1 and 5.3.3, respectively.
In second order spectra a trough at 400 nm and an intensified crust at 425 nm, and in
the third order spectra a crust at 385 nm a trough at 395 nm and another crust at 405
nm were noticed and the curve showed zero amplitude at 389 nm and 402 nm.
b) Determination of Fe(III)
By adopting the general procedure as described in 3.d. linear plots were
constructed between the amount of iron(III) and measured derivative amplitudes at
400 nm and 425 nm for 2nd
order (Fig. 5.3.2), and at 395 and 405 nm for 3rd
order
(Fig. 5.3.4). The linear plots show that, the calibration plots are perfectly linear in the
concentration range, λ400 = 0.140 – 5.088 and λ425 = 0.140 – 5.470 µg mL-1
at 400 nm
(2st order) and λ395 = 0.210 – 5.46 and λ405 = 0.210 – 5.67 µg mL
-1 at 405 nm for third
order derivative method. The analytical results of the present investigations are
compared and presented in the Table 5.3.2.
107
Fig. 5.3.1. Second derivative spectra of
[Fe (III) – HMBATSC] Vs reagent blank [Fe(III)] (µg mL-1) = (a) 0.2357; (b) 0.4714; (c) 0.7071; (d) 0.9428
0 1 2 3 4 5 60.0
0.5
1.0
1.5
2.0
2.5
λλλλ425
= 0.4088C + 0.007
λλλλ400
= 0.4600C + 0.030
Am
pli
tud
e
Amount of Fe(III) (µg mL-1)
Fig. 5.3.2. Beer’s Law plot of Second order derivative data
[HMBATSC] = 5 × 10-3
M; pH = 6.0
108
Fig. 5.3.3. Third order spectra of [Fe (III) – HMBATSC] Vs Reagent Blank
[Fe(III)] (µg mL-1
) = (a) 0.2357; (b) 0.4714; (c) 0.7071; (d) 0.9428
0 1 2 3 4 5 60.00
0.04
0.08
0.12
0.16
λλλλ405
= 0.0190C - 0.002
λλλλ395
= 0.0260C - 0.004
Am
plit
ude
Amount of Fe(III) (µg mL-1
)
Fig. 5.3.4. Beer’s Law plot of third derivative data [HMBATSC] = 5 × 10-3 M; pH = 6.0
109
c) Effect of diverse ions
The effect of various cations and anions on the derivative amplitude was
studied by following the general procedure in chapter 3.2.e. It was noticed that all the
ions that did not interfere in the zero order determinations of Fe(III) (cf. Table 5.2.2)
also did not interfere in both second and third order derivative methods. In the zero
order, the metal ions Cu(II), V(IV) and Au(III) were interfered in 10-fold excess, but
in all the derivative methods they were tolerable up to 25 fold excess. Further, Fe(II)
was interfered 8-fold excess in zero order, also intereferes in the derivative methods.
From the interference study it can be observed that the metal ions which interfere in
zero order method are greatly enhanced in the derivative methods indicating the
greater selectivity of derivative methods than the direct method.
d) Application
Based on the results, the proposed second and third order derivative methods
were applied for the determination of Fe(III) in different water samples.
Procedure
A known aliquot of the sample solution was taken in a 10 ml volumetric flask
containing 5 ml of buffer solution (pH 6.0) and 1 ml of the HMBATSC solution (1 x
10-3
M). The contents were made up to the mark with distilled water. The derivative
amplitudes at 425 nm and 405 nm were measured for 2nd
and 3rd
order derivative
methods, respectively. The amounts of Fe(III) present in the sample solution was
determined from the predetermined calibration plots. The results are presented in
Table 5.3.1.
110
Table 5.3.1. Determination of Fe(III) in water samples
Water
Samples
2nd
order derivative
Recovery
(%)
3rd
order derivative
Recovery
(%)
Amount of Fe(III)
(µg mL-1
)
Amount of Fe(III)
(µg mL-1
)
Added Found* Added Found
*
Drinking
water 0.500 0.493 98.60 0.500 0.512 102.4
Natural
water 0.750 0.761 101.47 0.750 0.740 98.67
Bore well
water 1.000 1.028 102.80 1.00 0.989 98.90
Polluted
water 1.500 1.486 99.07 1.250 1.260 100.80
* Average of five determinations
Table 5.3.2. Analytical Characteristics of [Fe(III)–HMBATSC]
Parameter Zero Order Second Derivative Third Derivative
Analytical Wavelength (nm) 385 400 and 425 395 and 405
Molar absorptivity, ε (lmol-1
cm-1
) 1.024 × 104 -- --
Beer’s law range (µg mL-1
) 0.279– 5.31 λ400 = 0.140 – 5.088
λ425 = 0.140 – 5.470
λ395 = 0.210 – 5.46
λ405 = 0.210 – 5.67
Sandell’s sensitivity (µg cm-2
) 0.0054 -- --
Angular coefficient (m) 0.1781 0.2243 0.2594
Y-intercept (b) 0.0264 0.007 & 0.030 0.004 & 0.002
Correlation coefficient (r) 0.9998 0.9997 0.9996
Standard deviation (%) 0.057 0.048 0.039
Complex ratio 1 : 2 -- --
Stability constant 3.75 × 109 -- --
Detection limit (µg mL-1
) 0.022 0.015 0.018
Determination limit (µg mL-1
) 0.066 0.045 0.054
111
Discussion
The developed direct and derivative spectrophotometric methods for the
determination of iron(III) are simple, fast, less cumbersome, sensitive and reasonably
selective. The reaction between the Fe(III) ion and the reagent was quite fast and the
resultant light green colour was stable for more than 24 hours. The pH dependence of
the color formation was also not critical (Fig. 5.2.2). The molar absorptivity (1.024 ×
104
l mol-1
cm-1
), the detection limit (0.022 µg mL-1
) and determination limit (0.066
µg mL-1
) indicate the sensitivity of the proposed direct method. The relative standard
deviation (0.57%) and correlation coefficient (0.9998) show the precision and
linearity of the calibration plot of the present method respectively. The zero order
method was successfully applied for the analysis of alloy-steels and water samples.
The results indicate the acceptability of the proposed method.
The derivative method developed was found to be more sensitive and at some
wavelengths more selective than the direct method (Table 5.3.3). The determination
and detection limits as well as the RSD values were calculated for the derivative
method. The derivative method was applied for the determination of iron (III) in
environmental water samples with good acceptability.
The acid dissociation constants 1KP and
2KP of HMBATSC were found to be
4.0 and 7.0 which correspond to the dissociation of hydroxyl proton and SH proton
respectively. At pH 6.0 where the analytical studies are carried out, the hydroxyl
proton undergoes dissociation to give a mono anionic ligand. The composition of the
resultant Fe(III)-HMBATSC complex was determined by Job’s method and molar
ratio method and obtained as 1:2 complex [(Fe(III): HMBATSC)].
112
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