A Simple Spectrophotometric Method for the Trace Determination of Iron in Some Real, Environmental, Biological, Food, Pharmaceutical and Soil Samples Using 2-aminophenol

A very simple, ultra-sensitive and highly selective non-extractive spectrophotometric method is presented for the rapid determination of iron (III) at trace levels using ortho aminophenol or 2aminophenol (OAP) as a new spectrophotometric reagent (max = 402 nm) in slightly acidic (0.00050.0015 M H2SO4) aqueous solution. The reaction is instantaneous and absorbance remains stable for over 24 h. The average molar absorption coefficient and Sandell’s sensitivity were found to be 6.65×105 L mol cm and 5 ng cm, respectively. Linear calibration graphs were obtained for 0.016 mg L -1 of iron with a correlation co-efficient value 0.9998 for Fe-OAP complex. The stoichiometric Original Research Article Ahmed et al.; ACSj, 7(4): 236-253, 2015; Article no.ACSj.2015.075 237 composition of the chelate is 1:3 (Fe: OAP). The detection limit and quantification of limit of the reaction system were found 1 μg L -1 and 10 μg L -1 , respectively. Large excesses of over 50 cations, anions and complexing agents (e.g. tartrate, oxalate, citrate, phosphate, thio-urea, SCN) do not interfere in the determination. The developed method was successfully used in the determination of iron in several standard reference materials (alloys and steels) as well as in some environmental waters (portable and polluted), biological samples (human blood and urine), food samples (bean, meat, banana, tomato, egg etc.), soil samples, pharmaceutical samples (tablets, capsules etc.), some solution containing both iron (II) and iron (III) and complex synthetic mixtures. The results of the proposed method for biological and food samples were comparable with AAS and were found to be in excellent agreement. The method has high precision and accuracy (s = ±0.01 for 0.5 mg L).


INTRODUCTION
Iron plays a dual role in human biochemistry as in trace amounts; it is an essential nutrient, while large amounts are toxic and carcinogenic [1]. The essentiality and toxicity of iron depend on its oxidation states or the forms in which it was supplied. Iron is the most abundant transition metal in the living system and serves more biological roles than any other metal. Although iron is required for a number of vital functions, the main role of iron is to carry oxygen to the tissues where it is needed. Iron is also essential for the proper functioning of numerous enzymes involved in DNA synthesis, energy metabolism and protection against microbes and free radicals [2]. Iron salts are widely used in industrial materials [3], paint products, fertilizers, feeds and disinfectants [4]. They are important building components in biological systems [5]. The total iron content in an adult body is approximately 4 g, i.e.70 m mol, of which about two thirds is in hemoglobin. Iron stores, mainly spleen, liver, and bone marrow, contain about one-quarter of the body's iron; the remainder is in myoglobin and other hemoproteins. Only 0.1% of the total body iron is in plasma where almost all is bound to a transport protein-transferrin. Iron deficiency affects about 30% of the world population and is one of the main deficiency disorders in Europe [6]. Iron deficiency is characterized by anaemia, stunted growth, fatigue lowered resistance to infection, anorexia and death [7]. Iron is involved in oxygen transport from the lungs to tissues by hemoglobin and in oxygen storage in myoglobin; divalent Fe is a cofactor in heme enzyme such as catalase and cytochrome and in nonheme enzymes such as aldolase and tryptophan oxygenase [8]. On the other hand, excess amount of iron can result in toxicity and even death [9]. Toxicology considerations are important in terms of iron deficiency (anemia) and accidental acute exposure and chronic iron overload due to idiopathic hemochromatosis or as a consequence of excess dietary iron or frequent blood transfusions. In human poisonings, symptoms of iron intoxication include vomiting, cirrhosis of liver, hemochromatosis, diarrhea, lethargy, coma, irritability, seizures and abnormal pain [10]. All these findings cause great concern regarding public health, demanding accurate determination of this metal ion at trace and ultra-trace levels. Spectrophotometry is essentially a trace analysis technique and is one of the most powerful tools in chemical analysis. Ortho aminophenol or 2aminophenol (OAP) has not previously been used for the spectrophotometric determination of iron. This paper reports on its use in a very sensitive, highly selective spectrophotometric method for the trace determination of iron. The method possesses distinct advantages over existing methods [11][12][13][14][15][16][17][18][19] with respect to sensitivity, selectivity, range of determination, simplicity, speed, pH / acidity range, thermal stability, accuracy, precision and ease of operation. A comparison between existing methods 11-19 and the present method are shown in Table 1. The method is based on the reaction of nearly non-absorbent OAP in slightly acidic (0.0005-0.0015 M H 2 SO 4 ) aqueous solution with iron (III) to produce a highly absorbent orange chelate product, followed by a direct measurement of the absorbance in an aqueous solution. With suitable masking, the reaction can be made highly selective and the reagent blank solution show negligible absorbance.

Apparatus
A Shimadzu (Kyoto, Japan) (Model-1800) double-beam UV-VIS spectrophotometer and a Jenway (England, UK) (Model-3010) pH meter with combined electrodes were used for the measurements of absorbance and pH, respectively. A Thermo Fisher company (Model-iCE-3000) Atomic Absorption Spectrophotometer equipped with a microcomputer controlled nitrous oxide-acetylene flame was used for comparison of the results. Infrared spectrum was recorded with FTIR Spectrophotometer, Shimadju (Model-IR Prestige 21, Detector-DTGS KBr) in the range 7500-350 cm -1 .

Reagents and Solutions
All chemicals used were of analytical-reagent grade or the highest purity available. Deionised water was used throughout the study. Triply distilled ethanol (from lime), which is non absorbent under ultraviolet radiation, was also used. High purity water was obtained by passing tap water through cellulose absorbent and to mixed-bed ion exchange columns, followed by distillation in a corning AG-11 unit. The A1 level in the high-purity water was found to be below the spectrophotometric detection limit (3s of the blank) of 1 µg L -1 . Glass vessels were cleaned by soaking in acidified solutions of KMnO 4 or K 2 Cr 2 O 7 , followed by washing with concentrated HNO 3 and were rinsed several times with highpurity deionized water. Stock solutions and environmental water samples (1000 mL each) were kept in polypropylene bottle containing 1 mL of concentrated HNO 3 . More rigorous contamination control was applied when the iron levels in specimens were low.

2-Aminophenol (OAP) Solution
The reagent solution was prepared by dissolving the requisite amount of 2-aminophenol (BDH chemicals, proanalysis grade, 99% pure), in a known volume of distilled de-ionized water. More dilute solutions of the reagent were prepared as and when required.

Iron (II) standard solution (1.7910 -2 M)
A 100 mL amount of stock solution (1 mg mL -1 ) of divalent iron was prepared by dissolving 497 mg of ferrous sulfate (FeSO 4 .7H 2 O) purifiedgrade (Merck pro analysis grade) in deionized water. More dilute standard solutions were prepared by appropriate dilution of aliquots from the stock solution with deionized water as and when required. Concentrations were checked using the standard potassium dichromate solution [20].
Aliquots of this solution were standardized with potassium dichromate solution [20]. More dilute standard solutions were prepared by appropriate dilution of aliquots from this stock solution with de-ionized water as and when required.

Potassium permanganate solution
A 1% potassium permanganate (Merck) solution was prepared by dissolving in de-ionized water. Aliquots of this solution were standardized with oxalic acid.

Potassium dichromate solution
A 100 mL amount of stock solution (0.1 N) was prepared by dissolving 500 mg of finely powdered K 2 Cr 2 O 7 (Merck) in 100 mL de-ionized water.

Tartrate solution
A 100 mL stock solution of tartrate (0.01% w/v) was prepared by dissolving 10 mg of ACS-grade (99%) potassium sodium tartrate tetra hydrate in (100 mL) de-ionized water.

Aqueous ammonia solution
A 100 mL solution of aqueous ammonia was prepared by diluting 10 mL concentrated NH 4 OH (28-30%, ACS grade) to 100 mL with de-ionized water. The solution was stored in a polypropylene bottle.

Other solutions
Solutions of a large number of inorganic ions and complexing agents were prepared from their analytical grade or equivalent grade water-soluble salts (or the oxides and carbonates in hydrochloric acid).

General Procedure
A volume of 0.1-1.0 mL of a neutral aqueous solution containing 0.1-60 g of iron (III) in a 10-mL calibrated flask was mixed with a 1:220-1:800 fold molar excess of the 2-aminophenol (OAP) reagent solution (preferably 1 mL of 4.510 -3 M) followed by the addition of 0.5-1.5 mL (preferably 1 mL) of 0.01 M sulphuric acid. After one minute the mixture was diluted to the volume with deionized water. The absorbance was measured at 402 nm against a corresponding reagent blank. The iron content in an unknown sample was extrapolated from a concurrently prepared calibration graph.

Environmental samples
Water samples were collected in polyethylene bottles from different places of Bangladesh. After collection, conc. HNO 3 (1 mL per Liter) was added as preservative.

Blood and urine
Blood and urine samples were collected in polythene bottles from effected persons of Chittagong Medical College Hospital, Bangladesh. Immediately after collection they were stored in ice and later, in the laboratory, at -20°C.

Soil samples
Soil samples were collected from different location of Bangladesh. Samples were dried in air and homogenized with a mortar.

Food samples
Food samples (banana, meat, tomato, bean, egg, arum and lentil) were collected from local market of Chittagong. After collection the samples were stored in refrigerator for preservation. Samples (arum, lentil, bean) were used as dry condition and homogenized with mortar.

Pharmaceutical samples
Pharmaceutical samples (tablets and capsules) of different companies were collected from local pharmacy of Chittagong, Bangladesh. Samples were homogenized with a mortar.

Determination of iron in alloys and steels (certified reference materials)
A 0.1 g amount of a brass or alloy or steel sample containing 1.56-34.26% of iron was weighed accurately and placed in a 50 mL Erlenmeyer flask following a method recommended by Parker et al. [21]. To it, 10 mL of concentrated HNO 3 , 1 mL of concentrated H 2 SO 4 and 1-2 mL of 1% KMnO 4 were added to oxidize Fe (II) to Fe (III), excess of KMnO 4 was removed by addition of 1-2 mL freshly prepared 2.5% sodium azide solution and carefully covering the flask with a watch glass until the brisk reaction subsided. The solution was heated to drive off excess azide solution and simmered gently after the addition of 5 mL of concentrated HNO 3 until all carbides were decomposed. The solution was carefully evaporated to dense white fumes to drive off the oxides of nitrogen and then cooled to room temperature (25±5)ºC. After suitable dilution with de-ionized water, the contents of the Erlenmeyer flask were warmed to dissolve the soluble salts. The solution was then cooled and neutralized with a dilute NH 4 OH solution. The resulting solution was filtered, if necessary, through a Whatman No. 40 filter paper into a 25-mL calibrated flask. The residue was washed with a small volume of hot (1+99) H 2 SO 4 , followed by water and the volume was made up to the mark with de-ionized water.
A suitable aliquot (1-2 mL) of the above solution was taken into a 10-mL calibrated flask and the iron content was determined as described under the general procedure, using tartrate and 1,5diphenylcarbazide as masking agents. Based on five replicate analyses, average iron concentration determined by spectrophotometric method was in close agreement with the certified values ( Table 5). The results are shown in Table 5.

Determination of iron in environmental water samples
Each filtered (with Whatman No. 40) environmental water sample (1000 mL) evaporated nearly to dryness with a mixture of 2 mL concentrated H 2 SO 4 and 10 mL of concentrated HNO 3 in a fume cupboard and 1-2 mL of KMnO 4 , following a method recommended by Greenberg et al. [22] Excess of KMnO 4 was removed by 2.5% freshly prepared azide solution and was heated with 10 mL of deionized water in order to remove excess azide solution and dissolved the salts. The solution was then cooled and neutralized with dilute NH 4 OH solution. The resulting solution was then filtered (if necessary) and quantitatively transferred into a 25-mL calibrated flask and made up to the mark with deionized water. An aliquot (1-2 mL) of this pre-concentrated water sample was pipetted into a 10-mL calibrated flask and the iron content was determined as described under the general procedure, using mixture of tartrate and 1,5diphenylcarbazide as masking agents. The analyses of environmental water samples for iron from various sources are shown in Table 6.
Most spectrophotometric method for the determination of iron in natural and sea-water require pre-concentration of iron [22]. The concentration of iron in natural and sea-water is a few µg L -1 in Japan [16]. The mean concentration of iron found in UK drinking water is less than 1 mgL

Determination of iron in biological samples
Human blood (2-5 mL) and urine (20-30 mL) was collected in polythene bottles from the affected persons. Immediately after collection, they were stored in a salt-ice mixture and later, at the laboratory, were kept at -20ºC. The samples were taken into a 100 mL micro-Kjeldahl flask. Glass bead and 10 mL of concentrated nitric acid were added and the flask was placed on the digester under gentle heating. When the initial brisk reaction was over, the solution was removed and cooled following a method recommended by Stahr [24]. 1 mL volume of concentrated sulfuric acid and 1-2 mL of 1% KMnO 4 were added carefully and excess of KMnO 4 was removed by 2.5% freshly prepared sodium azide solution followed by the addition of 0.5 mL of 70% HClO 4 and heating was continued for at least ½ hr to remove excess azide solution and then cooled. The solution of flask then neutralized with dilute NH 4 OH solution in the presence of 1-2 mL of a 0.01% (w/v) tartrate. The resultant solution was then transferred quantitatively into a 10-mL calibrated flask and made up to the mark with deionized water.
A suitable aliquot (1-2-mL) of the final solution was pipetted into a 10-mL calibrated flask and the iron content was determined as described under the general procedure using tartrate and 1,5-diphenylcarbazide as masking agents. The results of biological analyses by the spectrophotometric method were found to be in excellent agreement with those obtained by AAS. The results are shown in Table 7.
The abnormally high value for the liver cirrhosis patient is probably due to the involvement of high iron concentration with Cu and Zn. Occurrence of such high iron contents are also reported in liver cirrhosis patients from some developed countries [25].

Determination of iron in food samples
An air dried food sample bean (2 gm), chicken (2 gm), banana (2 gm), tomato (5 gm), arum (1 gm), lentil (1 gm), egg (1 g) was taken in a 100-mL micro-Kjeldahl flask. A glass bead and 10 mL of concentrated nitric acid were added and the flask was placed on the digester under gentle heating. When the initial brisk reaction was over, the solution was removed and cooled following a method recommended by Stahr [24]. 1 mL volume of concentrated sulfuric acid and 1-2 mL of 1% KMnO 4 were added carefully and excess of KMnO 4 was removed by 2.5% of freshly prepared sodium azide solution followed by the addition of 0.5 mL of 70% HClO 4 and heating was continued for at least ½ hr to remove excess azide solution and then cooled. The solution of flask then neutralized with dilute NH 4 OH solution. The resultant solution was then transferred quantitatively into a 25-mL calibrated flask and made up to the mark with deionized water.
A suitable aliquot (1-2 mL) of the final solution was pipetted into a 10-mL calibrated flask and the iron content was determined as described under the general procedure using tartrate and 1,5-diphenylcarbazide as masking agents. High value of iron for banana (Musa acuminata) is probably due to the involvement of high iron concentration in the soil. The results are shown in Table 8.

Determination of iron in soil samples
An air dried homogenized soil sample (100 g) was weighed accurately and placed in a 100-mL micro-Kjeldahl flask. The sample was digested in the presence of a oxidizing agent (1% KMnO 4 ), following the method recommended by Hesse [26]. Excess of KMnO 4 was removed by 2.5% freshly prepared sodium azide solution and heating was continued for at least half an hour to remove excess azide solution and then cooled. The content of the flask was filtered through a Whatman No. 40 filter paper into a 25-mL calibrated flask and neutralized with dilute NH 4 OH solution. Then the solution of the flask was made up to the mark with deionized water.
Suitable aliquots (1-2 mL) were transferred into a 10 mL calibrated flask. The iron content was determined as described under the general procedure using tartrate and 1,5diphenylcarbazide as masking agents. The iron content was then determined by the above procedure and quantified from a calibration graph prepared concurrently. The results are shown in Table 9. An suitable aliquot (1-2 mL) of the final solution was pippetted into a 10 mL calibrated flask and then iron content was determined as described under the general general procedure using tartrate and 1,5-diphenylcarbazide as masking agents. The results of some pharmaceutical analyses are in excellent agreement with the reported value. The analyses of pharmaceutical samples from several pharmaceutical companies for iron are given in Table 10.

Absorption spectra
The absorption spectra of the Fe(III)-OAP system in 0.01 M H 2 SO 4 medium was recorded using the spectrophotometer. The absorption spectra of the Fe(III)-OAP is a symmetric curve with maximum absorbance at 402 nm and the average molar absorption coefficient of 6.65×10 5 L mol -1 cm-1 was shown in Fig. 1. The reagent blank exhibited negligible absorbance despite having wave length in the same region. The reaction mechanism of the present method was as reported earlier [28]. The structure of 2aminophenol are shown in Scheme 1.

Effect of Acidity
Of the various acids (nitric, sulfuric, hydrochloric, phosphoric) studied, sulfuric acid was found to be the best acid for the system. The absorbance was maximum and constant when the 10 mL of solution (1 mg L-1) contained 0.5-1.5 mL of 0.01 M sulfuric acid at room temperature (25±5ºC). Outside this range of acidity, the absorbance decreased (Fig. 2). For all subsequent measurements 1 mL of 0.01 M sulfuric acid was added.

Effect of Time
The reaction was very fast and instantaneous. Constant maximum absorbance was obtained within few seconds after dilution to volume and remained strictly unaltered for over 24 hours. A longer period of time was not studied.

Effect of Temperature
Effect of various temperatures (20-60ºC) on Fe (III)-OAP system was studied. The Fe (III)-OAP system attained maximum and constant absorbance at room temperature (25±5ºC).

Effect of Reagent Concentration
Different molar excess of 2-aminophenol (OAP) were added to a fixed metal ion concentration and absorbance were measured according to the standard procedure. It was observed that at 1 mg L -1 Fe (III) metal, the reagent molar ratios of 1:220-1:800 produced a constant absorbance of the Fe-chelate (Fig. 3). For all the subsequent measurements 1 mL of 4.5×10 -3 M OAP reagent was added.

Effect of Metal Concentration
The well-known equation for spectrophotometric analysis in very dilute solutions derived from Beer's law. The effect of metal concentration was studied over 0.01-100 mg L of iron (III), respectively. Of the four calibration curve, the first three pass through the origin and the fourth (Fig. 4) one shows the deviation from linearity. The selected analytical parameters obtained with the optimization experiments are summarized in Table 1.

Effect of Foreign Ions
The effect of over 50 anions, cations and complexing agents on the determination of only 1 mg L -1 of Fe (III) was studied. The criterion for an interference [30] was an absorbance value varying by more than 5% from the expected value for Fe (III) alone. The results are summarized in Table 3. As can be seen, a large number of ions have no significant effect on the determination of iron. The most serious interference were from V (V) and Cr (VI) ions. In order to eliminate interference of V (V) and Cr (VI) ions, tartrate and 1,5-diphenylcarbazide used as masking agent, respectively. During the interference studies, if a precipitate was formed, it was removed by centrifugation. The amount mentioned is not the tolerance limit but the actual amount studied. However, for those ions whose tolerance limit has been studied, their tolerance ratios are given in Table 3.

Composition of the Absorbent Complex
Job's method [31] of continuous variation method was applied to ascertain the stoichiometric composition of the complex under the optimum conditions (Table 2). A Fe (III)-OAP (1:3) complex was indicated by this method (Fig. 5).

Precision and Accuracy
The precision of the present method was evaluated by determining different concentrations of iron (each analyzed at least five times). The relative standard deviation (n = 5) was 0-2% for 0.1-60 μg of iron (III) in 10-mL, indicating that this method is highly precise and reproducible. The detection limit (3s of the blank) and Sandell's sensitivity (concentration for 0.001 absorbance unit) for iron (III) were found to be 1 µg mL -1 and 5 ng cm -2 , respectively. The method was tested by analyzing several synthetic mixtures containing iron (III) and diverse ions ( Table 4). The results for total iron were in good agreement with certified values ( Table 5). The reliability of our Fe-chelate procedure was tested by recovery studies. The average percentage recovery obtained for addition of iron (III) spike to some environmental water samples was quantitative as shown in Table 6. The results of biological analyses by the spectrophotometric method were in excellent agreement with those obtained by AAS (Table 7).

APPLICATIONS
The proposed method was successfully applied to the determination of iron (III) in a series of synthetic mixtures of various compositions (Table 4) and also in a number of real samples e.g. several Certified Reference Materials (CRMs) ( Table 5). The method was also extended to the determination of iron in a number of environmental, biological, soil, food and pharmaceutical samples. In view of the unknown composition of environmental water samples, the same equivalent portions of each such sample were analyzed for iron content; the recoveries in both the "spiked" (added to the samples before the mineralization or dissolution) and the "unspiked" samples are in good agreement ( Table 6). The results of biological analyses by spectrophotometric method were found to be in excellent agreement with those obtained by AAS (Table 7). The results of food samples by the spectrophotometric method are shown in Table 8. The results of soil analyses by the spectrophotometric method are shown in Table 9. The results of pharmaceutical samples by the spectrophotometric method are shown in Table 10. The results of speciation of iron (II) and iron (III) in mixtures are shown in Table 11.

Determination of Iron in Synthetic Mixtures
Several synthetic mixtures of varying compositions containing iron and diverse ions of known concentrations were determined by the present method using tartrate and 1,5diphenylcarbazide as masking agent [32,33]. The results were found to be highly reproducible. The results are shown in Table 4. Accurate recoveries were achieved in all solutions.        An equal aliquot of the above iron (III+II) mixture was taken into a 25-mL beaker. 1 mL of 0.01% (w/v) 1,10-phenanthroline was added to mask iron (II) and neutralize with dilute NH 4 OH. Then, the content of the beaker was transferred into a 10-mL calibrated flask and its iron (III) content was determined according to the general procedure. The iron concentration was calculated in g L -1 or mg L -1

Determination of Iron (Ii) and Iron (Iii) Speciation in Mixtures
with the aid of a calibration graph. This gives a measure of iron originally present as iron (III) in the mixture. The value of the iron (II) concentration was calculated by subtracting the concentration of iron (III) from the corresponding total iron concentration. The results were found to be highly reproducible. The occurrence of such reproducible results is also reported for different oxidation states of iron [31]. The results of a set of determination are given in Table 11.
The present method was compared with a reported method [27] statistically. It was found that present method is much superior that of the reported method. The results are shown in Table 12.

CONCLUSIONS
In this paper, a new, simple, sensitive, selective and inexpensive method with the Fe (III)-OAP complex was developed successfully for the determination of iron in some real, environmental, biological, soil, food and pharmaceutical samples for continuous monitoring to establish the trace levels of iron in difficult sample matrices. It offers also a very efficient procedure for speciation analysis.
Although many sophisticated techniques such as pulse polarography, HPLC, AAS, ICP-OES and ICP-MS are available for the determination of iron at trace levels in numerous complex materials, factors such as the low cost of the instrument, easy handling, lack of requirement for consumables and almost no maintenance have caused spectrophtometry to remain a popular technique, particularly in laboratories of developing countries with limited budgets. The sensitivity in terms of molar absorptivity and precision in terms of relative standard deviation of the present method are very reliable for the determination of iron in real samples down to ng g -1 levels in aqueous medium at room temperature (25±5ºC).