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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