**5. A simple flow injection spectrophotometric determination method for iron (III) based on O-acetylsalicylhydroxamic acid complexation (Reproduced with permission from the paper of Andac Muberra et al., 2009. Copyright of Institute of Chemistry, Slovak Academy of Sciences)**

1,10-phenanthroline and salicylic acid are the most reported chelating agents applied for the determination of iron(III) and total iron after oxidation to iron(III) (Tesfaldet et al., 2004; Udnan et al., 2004). A number of other chelating agents that have been reported for the spectrophotometric and/or flow-injection spectrophotometric determination of iron(III) and total iron include 2-thiobarbituric acid (Morelli, 1983), norfloxacin (Pojanagaron et al., 2002) tiron (van Staden & Kluever, 2002) DMF (Asan et al., 2003), tetracycline (Sultan et al., 1992) and chlortetracycline (Wirat, 2008). Flow-injection spectrophotometric methods based on the above chelating agents are either not selective, or a masking agent has to be used. However, highly selective, simple and economical methods for routine determination of iron(III) in different sample matrices are still required. In the present study, a simple and rapid flowinjection spectrophotometric method for the determination of iron (III) and total iron is proposed. The method is based on the reaction between iron (III) and *O*acetylsalicylhydroxamic acid (AcSHA) in a 2 % methanol solution resulting in an intense violet complex with strong absorption at 475 nm. The reagent itself is sparingly soluble in water and did not absorb in the visible region of the spectrum, therefore, it might be well suited for flow-injection analysis of iron(III) and total iron. An addition of copper sulphate (1*×*10*−*4 mol L*−*1) into the reagent carrier solution resulted in baseline absorbance, and possible interfering ions were eliminated without a significant decrease in the sensitivity of the method. The method was successfully applied in the determination of iron (III) and total iron in water and ore samples. The method was verified by analysing a certified reference material Zn/Al/Cu 43XZ3F and also by the AAS method.

#### **5.1 Experimental**

438 Macro to Nano Spectroscopy

also given for comparison. The results obtained with the standard addition and the calibration curve methods, and the AAS measurements were in good agreement with each

 Found3 Found4 Found3 Found4 AAS *Ec* (%) Seaport (Sea water) 52.16 (0.12) 52.92 (0.21) 67.25 (0.10) 67.52 (0.12) 66.98 (0.05) 0.60 Atakum(River water) 25.41 (0.10) 26.01 (0.19) 37.41 (0.14) 38.01 (0.17) 38.15 (0.07) 1.16 Kurtun river 32.84 (0.24) 33.57 (0.28) 48.14 (0.19) 48.57 (0.27) 49.12 (0.09) 1.55 Spring water (1) 10.95 (0.15) 11.25 (0.27) 16.75 (0.32) 16.20 (0.28) 16.62 (0.18) 0.88 Spring water (2) 12.65 (0.09) 13.18 (0.12) 21.83 (0.08) 21.32 (0.24) 21.75 (0.14) 0.81 Spring water (3) 38.17 (0.11) 38.12 (0.19) 52.54(0.04) 52.73 (0.16) 52.95 (0.12) 0.60

2. Values in parantheses are the relative standard deviations for *n* =5 with confidence level of 95 %.

Accuracy of the proposed method was also tested by analyzing a certified metal alloy solution (MBH Zn/Al/Cu 43XZ3F). Three replicates of the solution using the sampling volume of 20 μL were analyzed. The certified and the obtained values were 0.085 % and (0.084 *±* 0.006) % of iron, respectively. An excellent agreement between the found and the certified values was obtained for the certified metal alloy solution. The obtained results show that the proposed method can be applied to the determination of iron(III) and total

**5. A simple flow injection spectrophotometric determination method for iron (III) based on O-acetylsalicylhydroxamic acid complexation (Reproduced with** 

1,10-phenanthroline and salicylic acid are the most reported chelating agents applied for the determination of iron(III) and total iron after oxidation to iron(III) (Tesfaldet et al., 2004; Udnan et al., 2004). A number of other chelating agents that have been reported for the spectrophotometric and/or flow-injection spectrophotometric determination of iron(III) and total iron include 2-thiobarbituric acid (Morelli, 1983), norfloxacin (Pojanagaron et al., 2002) tiron (van Staden & Kluever, 2002) DMF (Asan et al., 2003), tetracycline (Sultan et al., 1992) and chlortetracycline (Wirat, 2008). Flow-injection spectrophotometric methods based on the above chelating agents are either not selective, or a masking agent has to be used. However, highly selective, simple and economical methods for routine determination of iron(III) in different sample matrices are still required. In the present study, a simple and rapid flowinjection spectrophotometric method for the determination of iron (III) and total iron is proposed. The method is based on the reaction between iron (III) and *O*acetylsalicylhydroxamic acid (AcSHA) in a 2 % methanol solution resulting in an intense violet complex with strong absorption at 475 nm. The reagent itself is sparingly soluble in

**permission from the paper of Andac Muberra et al., 2009. Copyright of** 

Sample Fe(III)2 (μg L-1) Total iron 2 (μg L-1)

1. Samples were collected at Samsun, Turkey.

Table 7. Determination of total iron in water samples1

iron content in water samples without a pre-concentration process.

**Institute of Chemistry, Slovak Academy of Sciences)** 

3. Calibration curve method. 4. Standard addition method.

other.

All chemicals used were of analytical reagent grade, and solutions were prepared from double deionised water. Standard iron(II) and iron(III) stock solutions were prepared by dissolving 278.02 mg of iron(II) and 489.96 mg of iron(III) sulphate (Merck; Darmstadt, Germany) in 100 mL of 0.01 mol L*−*1 hydrochloric acid to give 0.01 mol L*−*1 stock solution of iron(II) and iron(III). Iron(II) and iron(III) working standard solutions were prepared daily by suitable dilution of the stock solutions with double deionised water. Standard reference material consisting of 0.085 % Fe (Zn/Al/Cu 43XZ3F) was provided from MBH Analytical Ltd. (UK). Hydrogen peroxide solution of 30 vol. % was obtained from Merck. AcSHA was synthesised according to the procedure described previously (Asan et al., 2003). A stock solution of AcSHA (0.01 mol L*−*1) was prepared by dissolving 0.095 g of AcSHA in 100 mL of aqueous methanol (2 vol. %). For the spectrophotometric study, AcSHA complex solutions of various metals were prepared by mixing 1 mL of 1×10−4 mol L−1standard solution of each metal in double deionised water with the suitable volume of 1×10−4 mol L−1 AcSHA stock solution. Reagent carrier solution was composed of AcSHA in a 2 % methanol solution and 1×10−4 mol L−1 CuSO4 in 0.001 mol L−1 HCl 98 % (pH 2.85). UV-VIS spectra of metal-AcSHA complexes were taken with a Unicam spectrophotometer (GBC Cintra 20, Australia). A Jenway 3040 Model digital pH-meter was used for the pH measurements. In the FIA system, a peristaltic pump (ISMATEC; IPC, Switzerland) 0.50 mm i.d. PTFE tubing was used to propel the samples and reagent solutions. Samples were injected into the carrier stream by a 7125 model stainless steel high pressure Rheodyne injection valve provided with a 20 μL loop. Absorbance of the coloured complex formed was measured with a UV-VIS spectrophotometer equipped with a flowthrough micro cell (Spectra SYSTEM UV 3000 HR,Thermo Separation Products, USA), and connected to a computer incorporated with a PC1000 software programme. A UNICAM 929 model (Shimadzu AA-68006) flame atomic absorption spectrophotometer with a deuterium-lamp background correction was used for the determination of iron in reference to the FIA method. The measuring conditions were as follows: UNICAM hollow cathode lamp, 10 cm 1-slot burner, air-acetylene flame (fuel gas flow-rate 1.50 L min−1), 0.2 nm spectral bandwidth, and 7 mm burner height. The wavelength and the lamp current of iron were 248 nm and 5 mA, respectively.

The FIA system used, similar to that proposed in our previous works (Asan et al., 2003), is quite simple. The sample solution was introduced into the reagent carrier solution by the Rhodyne injection valve. A water-soluble complex (*λ*max = 475 nm) was then formed on the passage of the reagent carrier solution in the mixing coil. As a mixing coil, PTFE tubing (50 cm long) was attached before the flow-through detection cell. The absorbance of the coloured complex was selectively monitored in the cell at 475 nm. The transient signal was recorded as a peak, the height of which was proportional to the iron(III) concentration in the sample, and it was used in all measurements. Five replicate injections per sample were made.

Flow-Injection Spectrophotometric Analysis of Iron (II), Iron (III) and Total Iron 441

As can be seen from Fig. 8, only AcSHA reacted efficiently with iron to form iron-(AcSHA)*n*  complexes with the absorbance maxima at 475 nm. At this wavelength, AcSHA itself has no absorption while Ac- SHA complexes of copper(II), nickel(II), cobalt(II), and zinc(II), among all metal ions with the anions tested, show a negligible absorption. The FIA setup shown in Fig. 9. was used in order to develop an FIA method based on the above phenomenon.

Fig. 9. Flow diagram of the flow-injection analysis system used for the determination of iron (III) and total iron, R; reagent carrier solution (1x10-4 M AcSHA, 1x10-4 M CuSO4, pH: 2.85), P, Peristaltic pump, S; Rheodyne sample injection valve, MC; mixing coil (50 cm long, 0.5 mm i.d), D; spectrophotometric detector (*max* = 475 nm), W; waste, C; computer, P; printer.

Various variables closely related to iron determination were examined using a simple flowinjection analysis system with a fixed iron(III) concentration of 5 μg L*−*1. The AcSHA concentration was varied from 1*×*10*−*5 mol L*−*1 to 1*×*10*−*2 mol L*−*1. The peak height was found to increase with the AcSHA concentration increasing up to 1*×*10*−*4 mol L*−*1, no noticeable increase was found at higher concentrations. Therefore, 1*×*10*−*4 mol L*−*1 AcSHA was used as the colour developing component of the carrier solution. With the concentration of AcSHA fixed at 1*×*10*−*4 mol L*−*1, pH of the carrier solution was varied from 1.5 to 5.5. The interference effect of iron(II) was found to increase with pH increasing up to 3.5 and to remain constant at higher pH. Also, the peak heights were found to increase with pH increasing up to 3.0, to remain constant up to 4.0, and to decrease slightly above this value. pH of the reagent carrier was, however, adjusted to 2.85 to obtain the maximum peak height and minimum iron(II) interference in the analysis. To obtain a reasonable background of absorption and a smooth baseline, CuSO4 was added into the carrier solution. The CuSO4 concentration was varied from 1*×*10*−*5 mol L*−*1 to 1*×*10*−*2 mol L*−*1. When the concentration of CuSO4 was 1*×*10*−*4 mol L*−*1, the baseline was stable and the interference effects of nickel(II), cobalt(II), and zinc(II) were found minimum. Over the CuSO4 concentration of 1*×*10*−*4 mol

In order to proceed with the final system design, the effects of sample volume, mixing coil length and flow-rate were studied at the optimal pH (2.85), and fixed concentrations of AcSHA (1*×*10*−*4 mol L*−*1) and CuSO4 (1*×*10*−*4 mol L*−*1). The sample volume was varied from 5–50 μL. The peak height was decreased by decreasing the sample size, and the peaks were broadened with the increasing sample size due to the sample zone dispersion. The sample injection volume of 20 μL was selected as a compromise between the sensitivity and sample throughput rate. The mixing coil (MC) was examined using PTFE tubing (0.5 mm i.d.) of

**5.2.2 Optimisation of chemical variables and FIA manifold** 

L*−*1, the sensitivity of the method decreased.

Sea and river water samples collected in Nalgene plastics were acidified by adding 1 mL of nitric acid (0.1 mol L*−*1) per 100 mL of sample solution after filtration over a 0.45 μm Millipore Filter (Millford, MA). After the filtration, water samples were injected directly into the FIA system for the determination of iron(III).

Total iron was determined by oxidising iron(II) to iron(III). Hydrogen peroxide was chosen as the oxidising agent for the determination of total iron. A 0.25 mol L*−*1 H2O2 concentration ensured total oxidation of iron(II) into iron(III) (Pons, et al., 2005). Before the determination of total iron, H2O2 (10 mass %) was added to the water sample solution for complete oxidation of iron(II) to iron(III). Then, 20 μL of this solution were injected into the system, as in the procedure described above. A 0.10 g sample of the certified metal alloy (Zn/Al/Cu 43XZ3F) was dissolved in 12 mL of concentrated HCl and HNO3 (3 : 1) in a 100 mL beaker. The mixture was heated on a hot plate nearly to dryness; 5 mL of HNO3 were added to complete the dissolution and the solution was diluted to 100 mL with deionised water. The solution was filtered and transferred quantitatively to a 1000 mL volumetric flask and filled up to volume with deionised water. 9 mL of this solution were treated with 1 mL of H2O2 (10 mass %) for iron(II) oxidation. After the oxidation step, 20 μL of this solution were used in the determination of total iron. Metal ore samples (0.10 g) were powdered (*≥* 500 mesh) and prepared as in the procedure described above. All analyses were performed with the least possible delay.

#### **5.2 Results and discussion**

#### **5.2.1 Spectrophotometric studies of AcSHA-metal complexes**

Metal ions react with AcSHA in aqueous media in the range of pH 2.0–10.0 forming coloured complexes with different stoichiometry. These complexes are fairly soluble in aqueous media (O'Brien et al., 1997). Their absorption spectra corresponding to solutions of 5 *×* 10*−*5 mol L*−*1 metal complexes measured against a reagent blank are shown in Fig. 8.

Fig. 8. Absorption spectras of 5x10-5 M AcSHA and M-(AcSHA)*<sup>n</sup>* complexes. a) Fe(III)- (AcSHA)n; b) Fe(II)-(AcSHA)n; c) Cu-(AcSHA)n; d) M-(AcSHA)n; (M: Ni, Co, Zn, Pb); e) AcSHA only.

Sea and river water samples collected in Nalgene plastics were acidified by adding 1 mL of nitric acid (0.1 mol L*−*1) per 100 mL of sample solution after filtration over a 0.45 μm Millipore Filter (Millford, MA). After the filtration, water samples were injected directly into

Total iron was determined by oxidising iron(II) to iron(III). Hydrogen peroxide was chosen as the oxidising agent for the determination of total iron. A 0.25 mol L*−*1 H2O2 concentration ensured total oxidation of iron(II) into iron(III) (Pons, et al., 2005). Before the determination of total iron, H2O2 (10 mass %) was added to the water sample solution for complete oxidation of iron(II) to iron(III). Then, 20 μL of this solution were injected into the system, as in the procedure described above. A 0.10 g sample of the certified metal alloy (Zn/Al/Cu 43XZ3F) was dissolved in 12 mL of concentrated HCl and HNO3 (3 : 1) in a 100 mL beaker. The mixture was heated on a hot plate nearly to dryness; 5 mL of HNO3 were added to complete the dissolution and the solution was diluted to 100 mL with deionised water. The solution was filtered and transferred quantitatively to a 1000 mL volumetric flask and filled up to volume with deionised water. 9 mL of this solution were treated with 1 mL of H2O2 (10 mass %) for iron(II) oxidation. After the oxidation step, 20 μL of this solution were used in the determination of total iron. Metal ore samples (0.10 g) were powdered (*≥* 500 mesh) and prepared as in the procedure described above. All analyses were performed with the least

Metal ions react with AcSHA in aqueous media in the range of pH 2.0–10.0 forming coloured complexes with different stoichiometry. These complexes are fairly soluble in aqueous media (O'Brien et al., 1997). Their absorption spectra corresponding to solutions of 5 *×* 10*−*5 mol L*−*1 metal complexes measured against a reagent blank are shown in Fig. 8.

Fig. 8. Absorption spectras of 5x10-5 M AcSHA and M-(AcSHA)*<sup>n</sup>* complexes. a) Fe(III)- (AcSHA)n; b) Fe(II)-(AcSHA)n; c) Cu-(AcSHA)n; d) M-(AcSHA)n; (M: Ni, Co, Zn, Pb); e)

the FIA system for the determination of iron(III).

possible delay.

AcSHA only.

**5.2 Results and discussion** 

**5.2.1 Spectrophotometric studies of AcSHA-metal complexes** 

As can be seen from Fig. 8, only AcSHA reacted efficiently with iron to form iron-(AcSHA)*n*  complexes with the absorbance maxima at 475 nm. At this wavelength, AcSHA itself has no absorption while Ac- SHA complexes of copper(II), nickel(II), cobalt(II), and zinc(II), among all metal ions with the anions tested, show a negligible absorption. The FIA setup shown in Fig. 9. was used in order to develop an FIA method based on the above phenomenon.

Fig. 9. Flow diagram of the flow-injection analysis system used for the determination of iron (III) and total iron, R; reagent carrier solution (1x10-4 M AcSHA, 1x10-4 M CuSO4, pH: 2.85), P, Peristaltic pump, S; Rheodyne sample injection valve, MC; mixing coil (50 cm long, 0.5 mm i.d), D; spectrophotometric detector (*max* = 475 nm), W; waste, C; computer, P; printer.

#### **5.2.2 Optimisation of chemical variables and FIA manifold**

Various variables closely related to iron determination were examined using a simple flowinjection analysis system with a fixed iron(III) concentration of 5 μg L*−*1. The AcSHA concentration was varied from 1*×*10*−*5 mol L*−*1 to 1*×*10*−*2 mol L*−*1. The peak height was found to increase with the AcSHA concentration increasing up to 1*×*10*−*4 mol L*−*1, no noticeable increase was found at higher concentrations. Therefore, 1*×*10*−*4 mol L*−*1 AcSHA was used as the colour developing component of the carrier solution. With the concentration of AcSHA fixed at 1*×*10*−*4 mol L*−*1, pH of the carrier solution was varied from 1.5 to 5.5. The interference effect of iron(II) was found to increase with pH increasing up to 3.5 and to remain constant at higher pH. Also, the peak heights were found to increase with pH increasing up to 3.0, to remain constant up to 4.0, and to decrease slightly above this value. pH of the reagent carrier was, however, adjusted to 2.85 to obtain the maximum peak height and minimum iron(II) interference in the analysis. To obtain a reasonable background of absorption and a smooth baseline, CuSO4 was added into the carrier solution. The CuSO4 concentration was varied from 1*×*10*−*5 mol L*−*1 to 1*×*10*−*2 mol L*−*1. When the concentration of CuSO4 was 1*×*10*−*4 mol L*−*1, the baseline was stable and the interference effects of nickel(II), cobalt(II), and zinc(II) were found minimum. Over the CuSO4 concentration of 1*×*10*−*4 mol L*−*1, the sensitivity of the method decreased.

In order to proceed with the final system design, the effects of sample volume, mixing coil length and flow-rate were studied at the optimal pH (2.85), and fixed concentrations of AcSHA (1*×*10*−*4 mol L*−*1) and CuSO4 (1*×*10*−*4 mol L*−*1). The sample volume was varied from 5–50 μL. The peak height was decreased by decreasing the sample size, and the peaks were broadened with the increasing sample size due to the sample zone dispersion. The sample injection volume of 20 μL was selected as a compromise between the sensitivity and sample throughput rate. The mixing coil (MC) was examined using PTFE tubing (0.5 mm i.d.) of

Flow-Injection Spectrophotometric Analysis of Iron (II), Iron (III) and Total Iron 443

measurements taken in water samples are also given for comparison (Table 10). The analytical value of total iron in water is in good agreement with that obtained by the AAS

(1) Values in parenthesis are the relative standard deviations for n=5 with confidence level of 95 %.

 Found(3) Found(4) Found(3) Found(4) AAS Kurtun river 38.33(0.24) 38.55(0.12) 42.33(0.02) 42.91(0.18) 43.65(0.17) Seaport 78.84(0.32) 78.65(0.24) 95.13(0.12) 95.75(0.06) 97.12(0.12) Baruthane sea water 47.51(0.18) 47.62(0.14) 57.24(0.04) 57.65(0.15) 58.97(0.24)

(2) Values in parenthesis are the relative standard deviations for n=5 with confidence level of 95 %.

The results obtained show that the proposed method can be applied in the determination of

A number of highly sensitive, selective and rapid flow-injection spectrophotometric and spectrofluorimetric analysis methods for the determination of iron (II), iron (III) and total iron in a wide concentration range, without employing any further treatment, have been described. The methods were based on the reactions of iron (II) and iron (III) with different complexing agents in different carrier solutions in FIA. In addition to the simplicity and low reagent consumption of the methods, the complexing agents used are commercially available and may not have a risk of serious toxicity, thus enhancing the potential applicability of the methods for iron analysis in real samples. Several parameters affecting to the determination of iron (II) and iron (III) were examined. The methods developed have been successfully applied to the determination of iron (II), iron (III) and total iron different types of water samples including river, sea, industry and spring water samples. The

Ahmed M. J. and Roy U. K. (2009) A simple spectrophotometric method for the

determination of iron(II) aqueous solutions. Turkish journal of chemistry. 33, 709-

Table 10. Determination of iron (III) and total iron in river and sea water samples

iron(III) and total iron content in water samples without a preconcentration process.

methods were also verified by applying certified reference materials.

Samples(1) Iron (III)(2) (g L-1) Total iron(2) (g L-1)

Sample Total Fe(1) (%) Certified Fe (%)

Table 9. Total iron content of iron alloys and standard reference material

Alloy (1) 8.23(0.24) 8.58 Alloy (2) 16.15(0.17) 16.62 Std Zn/Al/Cu 43XZ3 F 0.083(0.022) 0.085

(1) Samples were collected at Samsun, Turkey.

(3) Calibration curve method. (4) Standard addition method.

**6. Conclusions** 

**7. References** 

726

method.

different lengths ranging between 10 cm and 150 cm. The peak height increased with the increasing mixing coil length from 10–50 cm, decreased at lower concentrations and broadened at higher concentrations and longer coil lengths. The mixing coil length of 50 cm was chosen since it resulted in the best peak height and good reproducibility.

The flow-rate was varied from 0.2 mL min*−*1 to 2 mL min*−*1. The peak height decreased with the increasing flow-rate, probably due to the extent of the reaction decrease. The flow-rate of 0.8 mL min*−*1 was selected as a compromise between the sample throughput rate and sensitivity. A linear calibration graph for 4–150 μg L*−*1 iron(III), with the regression coefficient of 0.9914, was obtained under optimum conditions. The relative standard deviation for the determination of 5 μg L*−*1 iron(III) was 0.85 % (10 replicate injections), RSD of the data was below 3 %. The limit of detection (blank signal plus three times the standard deviation of the blank) was 0.5 μg L*−*1. The sample throughput of the proposed method was almost 60 h*−*1.


Table 8. Effect of foreign ions on the determination of 5 μg L-1 of iron (III) in solution

The interference effects of many cations and anions on the determination of 5 μg L*−*1 iron(III) were examined. The results summarised in Table 8 represent tolerable concentrations of each diverse ion taken as the highest concentration causing an error of 3 %. Most of the ions examined did not interfere with the determination of iron(III). The major interference was caused by iron(II) at the amount of 100 μg L*−*1. It is known that zinc and cobalt are the main interference metal ions in the determination of iron (Ensafi et al., 2004). In this study, the interference of these ions was completely eliminated by an addition of copper sulphate (1*×*10*−*4 mol L*−*1) to the reagent carrier solution. Background absorbance of copper(II) maintained in the reagent carrier solution eliminated possible interfering ions and improved the determination of iron(III). It is apparent from Table 1 that the proposed method tolerates all interfering species tested in satisfactory amounts, and it is therefore adequately selective for the determination of iron(III) and total iron.

#### **5.2.3 Applications**

The FIA method was applied in the determination of iron(III) and total iron in water and ore samples. In order to evaluate the accuracy of the proposed method, the determination of total iron in a standard reference material (Zn/Al/Cu 43XZ3F) and in a metal alloy sample was carried out. The analytical results obtained by the proposed method are in good agreement with the certified values as shown in Table 9.

For the application of the proposed FIA method to water samples; river and sea water samples collected from different sources were analysed using both the calibration curve and the standard addition methods. The values obtained from the calibration curve and the standard addition methods are in good agreement as shown in Table 10. Atomic absorption measurements taken in water samples are also given for comparison (Table 10). The analytical value of total iron in water is in good agreement with that obtained by the AAS method.


(1) Values in parenthesis are the relative standard deviations for n=5 with confidence level of 95 %.



(1) Samples were collected at Samsun, Turkey.

(2) Values in parenthesis are the relative standard deviations for n=5 with confidence level of 95 %.

(3) Calibration curve method.

(4) Standard addition method.

Table 10. Determination of iron (III) and total iron in river and sea water samples

The results obtained show that the proposed method can be applied in the determination of iron(III) and total iron content in water samples without a preconcentration process.
