**3.1.1 Reagents, chemicals, equipment**

All reagents used were of analytical reagent grade and the solutions were prepared with double distilled and deionized water. The reagent diphenylamine-4- sulfonic acid sodium salt (DPA-4-SA) was provided by Merck. The chemical formula of the DPA-4-SA is shown in Fig. 4. Standard iron(III) (1 mg mL*−*1) and iron(II) (5 mg mL*−*1) solutions were prepared by dissolving FeCl3 6H2O and FeCl2 4H2O in 0.05 M nitric acid and standardized by titration with EDTA. The stock solution of DPA-4-SA (1*×*10*−*2 M) was prepared by dissolving the diphenylamine-4-sulfonic acid sodium salt in deionized water. All stock solutions were stored in polyethylene containers. All polyethylene containers and glassware used for aqueous solutions containing metallic cations were cleaned with (1+1) nitric acid while the rest were cleaned with 3 % Decon 90, all were rinsed with deionized water before use. The working standard solutions were prepared by appropriate dilution immediately before use. Interference studies were carried out using chloride or nitrate salts of the metal cations, and sodium or potassium salts of anions. All solutions were degassed before use using a sonicator (LC 30). A certified metal alloy sample consisting of 0.085 % Fe (Zn/Al/Cu 43XZ3F) was provided by MBH Analytical Ltd. (UK).

The pH measurements were carried out using a Jenway 3040 Model digital pH-meter consisting of a contained glass pH electrode. UV-Visible spectra of the DPA-4-SA reagent and metal-DPA-4-SA complexes were taken with a Unicam spectrophotometer (GBC Cintra 20, Australia). A peristaltic pump (ISMATEC; IPC, Switzerland) 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 injection loop. Absorbance of the colored complex formed in the flow system was measured using a UV-visible spectrophotometer equipped with a flowthrough micro cell (Spectra SYSTEM UV 3000 HR, Thermo Separation Products, USA), and connected to a computer (IPX Spectra SYSTEM SN 4000) incorporated with a PC 1000 software program. The reaction coil was made of PTFE tubing (1 m, 0.5 mm, i.d.). A UNICAM 929 model (Shimadzu AA-68006) flame atomic absorption spectrophotometer with 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, airacetylene flame (fuel gas flowrate 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 manifold of the flow-injection system was similar to that proposed in our previous study (Asan et al., 2003). The peristaltic pump was used for propelling the reagent carrier solution at a flow-rate of 1 mL min*−*1. Samples were injected into the reagent carrier solution, soon load the reaction coil. The reaction zone containing the complex was moving towards the flow-through spectrophotometric detector cell in which the presence of iron(III)-DPA-4- SA complex was selectively monitored, and the absorbance of the complex at 410 nm was continuously recorded.

### **3.1.2 Preparation of water samples and certified metal alloy solution**

Sea and river water samples collected in Nalgene plastics were acidified by adding 1 mL of nitric acid (0.1 M) per 100 mL of sample solution after filtration over a 0.45 μm Millipore

Flow-Injection Spectrophotometric Analysis of Iron (II), Iron (III) and Total Iron 431

absorption for the Fe(III)-DPA-4-SA complex. Iron(III) reacts with DPA-4-SA in the pH range of 2.0–6.0 forming a complex with absorption maxima at 410 nm and molar absorptivity of 1.60*×*104 L mol*−*1 cm*−*1. The Fe(II)-DPA-4-SA complex presents only a slight absorption at this wavelength. Seventeen different metals do hardly react with DPA-4-SA in aqueous medium to from complexes. This can be an important advantage when developing a simplified FIA method for iron(III). Therefore, the specific absorbance maximum of the Fe(III)-DPA-4-SA complex at this wavelength can be applied in the selective determination

The optimum experimental conditions were determined using a standard iron(III) solution. The concentration of DPA-4-SA, pH, and the flow-rate were the main variables influencing the intensity of the signal in the FIA system. Optimization of the FIA system was therefore performed by changing these variables one by one while applying 10 μg L*−*1 and 90 μg L*−*1 of iron(III) standard solutions in order to obtain the highest signal and better reproducibility at

Influence of the DPA-4-SA concentration in the carrier solution on the peak height was examined by changing the DPA-4-SA concentration in the range of 1*×*10*−*2 M to 5*×*10*−*4 M in an acetate buffer solution (pH = 5.5), at the flow rate of 1 mL min*−*1. Maximum peak heights were found using 1*×*10*−*3 M of the DPA-4-SA solution for both, 10 μg L*−*1 and 90 μg L*−*1, iron(III) levels. Therefore, 1*×*10*−*3 M of DPA-4-SA was chosen as the color-developing

The effect of flow-rate on the peak height of 10 μg L*−*1 and 90 μg L*−*1 iron(III) was examined by varying flow-rates from 0.2 mL min*−*1 to 2.0 mL min*−*1. Peak heights decreased at flowrates above 1.2 mL min*−*1 and below 0.7 mL min*−*1. Flow-rates below 0.7 mL min*−*1 peaks also broadened. In the flow-rates range of 0.7–1.2 mL min*−*1, there were slight differences in the peak heights. However, taking into consideration the stability of the pump, peak shape, and sampling time, the flow-rate of the reagent carrier solution was adjusted to 1 mL min*−*1. This

pH of the carrier solution consisting of 1*×*10*−*3 M of DPA-4-SA was adjusted by adding simple acids and bases into the buffer to obtain the pH range of 3.30– 6.10. The peak shape and height were found maximum at pH 5.5. Therefore, 1*×*10*−*2 M of the acetate buffer

Reaction coil was used for the interaction of iron(III) and DPA-4-SA in the flow-injection system. The effect of the reaction coil (RC) length was examined by changing the coil length from 10 cm to 150 cm. The peak height decreased with the increase of length due to fast kinetics of the color forming reaction. The 10 cm length reaction coil was chosen since it

The calibration graph for the determination of iron(III) was maintained under the optimized conditions as described above. A good linear relationship was observed for iron(III) ranging from 5 μg L*−*1 to 200 μg L*−*1. The calibration curve equation was *A* = 0*.*4018*C* + 2*.*0196; *r*2 = 0*.*9958; *n* = 6, where A represents the absorbance measured as peak height and C the iron concentration in μg L*−*1. The confidence limits of the intercept and the slope were calculated at the 95 % confidence level. The same calibration graph can be used for the determination

of iron(III) in the flow-injection system.

different concentration levels.

component of the carrier solution.

provided a sampling frequency of 60 h*−*1.

solution at pH 5.5 was used throughout the study.

produced the best peak height together with a good reproducibility.

of total iron. The detection limit estimated (S/N = 3) was 1 μg L−1 of iron(III).

Filter (Millford, MA). After the filtration and pre-treatment, water samples were injected directly into the FIA system for the determination of iron(III).

Fig. 5. Absorption spectra for DPA-4-SA and metal-DPA-4- SA complexes: (○) 5 *×* 10*−*5 M DPA-4-SA and 5*×* 10*−*5 M of each Co(II), Cu(II), Cr(III), Al(III), Cd(II), Ni(II), Mn(II), Ba(II), Ca(II), Ag(I), K(I), Na(I), Hg(II), Zn(II), and Mg(II); (●) 5 *×* 10*−*5 M of Fe(II)–DPA-4-SA; and (▲) 5 *×* 10*−*5 M of Fe(III)–DPA-4-SA.

Oxidizing iron(II) to iron(III) was used to determine the total iron amount. Hydrogen peroxide was chosen as the oxidizing agent for the determination of total iron. A concentration of 0.25 mol L*−*1 of H2O2 ensured total oxidation of iron(II) to iron(III) (Pons et al., 2005). Before the determination, 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. Analyses were performed with the least possible delay.

A 0.10 g sample of the certified metal alloy (Zn/Al/Cu 43XZ3F) was dissolved in 12 mL of concentrated HCl + 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 were diluted to 100 mL with deionized water. The solution was filtered and transferred quantitatively to a 1000 mL volumetric flask and filled up to the volume with deionized water. The volume of 10 mL of this solution was treated with H2O2 (10 mass %) for iron(II) oxidation. After the oxidation step, the solution was diluted 100 fold, and then, 20 μL of this solution were used for the determination of total iron.

#### **3.2 Results and discussion**

According to the spectrophotometric studies, iron (II) and iron(III) react with DPA-4-SA in aqueous medium to form complexes. As shown in Fig. 5, the absorption spectra corresponding to solutions of 5*×*10*−*5 M of each metal complex in water demonstrate strong

Filter (Millford, MA). After the filtration and pre-treatment, water samples were injected

Fig. 5. Absorption spectra for DPA-4-SA and metal-DPA-4- SA complexes: (○) 5 *×* 10*−*5 M DPA-4-SA and 5*×* 10*−*5 M of each Co(II), Cu(II), Cr(III), Al(III), Cd(II), Ni(II), Mn(II), Ba(II), Ca(II), Ag(I), K(I), Na(I), Hg(II), Zn(II), and Mg(II); (●) 5 *×* 10*−*5 M of Fe(II)–DPA-4-SA; and

Oxidizing iron(II) to iron(III) was used to determine the total iron amount. Hydrogen peroxide was chosen as the oxidizing agent for the determination of total iron. A concentration of 0.25 mol L*−*1 of H2O2 ensured total oxidation of iron(II) to iron(III) (Pons et al., 2005). Before the determination, 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. Analyses were performed

A 0.10 g sample of the certified metal alloy (Zn/Al/Cu 43XZ3F) was dissolved in 12 mL of concentrated HCl + 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 were diluted to 100 mL with deionized water. The solution was filtered and transferred quantitatively to a 1000 mL volumetric flask and filled up to the volume with deionized water. The volume of 10 mL of this solution was treated with H2O2 (10 mass %) for iron(II) oxidation. After the oxidation step, the solution was diluted 100 fold, and then, 20 μL of this solution were used

According to the spectrophotometric studies, iron (II) and iron(III) react with DPA-4-SA in aqueous medium to form complexes. As shown in Fig. 5, the absorption spectra corresponding to solutions of 5*×*10*−*5 M of each metal complex in water demonstrate strong

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

(▲) 5 *×* 10*−*5 M of Fe(III)–DPA-4-SA.

with the least possible delay.

for the determination of total iron.

**3.2 Results and discussion** 

absorption for the Fe(III)-DPA-4-SA complex. Iron(III) reacts with DPA-4-SA in the pH range of 2.0–6.0 forming a complex with absorption maxima at 410 nm and molar absorptivity of 1.60*×*104 L mol*−*1 cm*−*1. The Fe(II)-DPA-4-SA complex presents only a slight absorption at this wavelength. Seventeen different metals do hardly react with DPA-4-SA in aqueous medium to from complexes. This can be an important advantage when developing a simplified FIA method for iron(III). Therefore, the specific absorbance maximum of the Fe(III)-DPA-4-SA complex at this wavelength can be applied in the selective determination of iron(III) in the flow-injection system.

The optimum experimental conditions were determined using a standard iron(III) solution. The concentration of DPA-4-SA, pH, and the flow-rate were the main variables influencing the intensity of the signal in the FIA system. Optimization of the FIA system was therefore performed by changing these variables one by one while applying 10 μg L*−*1 and 90 μg L*−*1 of iron(III) standard solutions in order to obtain the highest signal and better reproducibility at different concentration levels.

Influence of the DPA-4-SA concentration in the carrier solution on the peak height was examined by changing the DPA-4-SA concentration in the range of 1*×*10*−*2 M to 5*×*10*−*4 M in an acetate buffer solution (pH = 5.5), at the flow rate of 1 mL min*−*1. Maximum peak heights were found using 1*×*10*−*3 M of the DPA-4-SA solution for both, 10 μg L*−*1 and 90 μg L*−*1, iron(III) levels. Therefore, 1*×*10*−*3 M of DPA-4-SA was chosen as the color-developing component of the carrier solution.

The effect of flow-rate on the peak height of 10 μg L*−*1 and 90 μg L*−*1 iron(III) was examined by varying flow-rates from 0.2 mL min*−*1 to 2.0 mL min*−*1. Peak heights decreased at flowrates above 1.2 mL min*−*1 and below 0.7 mL min*−*1. Flow-rates below 0.7 mL min*−*1 peaks also broadened. In the flow-rates range of 0.7–1.2 mL min*−*1, there were slight differences in the peak heights. However, taking into consideration the stability of the pump, peak shape, and sampling time, the flow-rate of the reagent carrier solution was adjusted to 1 mL min*−*1. This provided a sampling frequency of 60 h*−*1.

pH of the carrier solution consisting of 1*×*10*−*3 M of DPA-4-SA was adjusted by adding simple acids and bases into the buffer to obtain the pH range of 3.30– 6.10. The peak shape and height were found maximum at pH 5.5. Therefore, 1*×*10*−*2 M of the acetate buffer solution at pH 5.5 was used throughout the study.

Reaction coil was used for the interaction of iron(III) and DPA-4-SA in the flow-injection system. The effect of the reaction coil (RC) length was examined by changing the coil length from 10 cm to 150 cm. The peak height decreased with the increase of length due to fast kinetics of the color forming reaction. The 10 cm length reaction coil was chosen since it produced the best peak height together with a good reproducibility.

The calibration graph for the determination of iron(III) was maintained under the optimized conditions as described above. A good linear relationship was observed for iron(III) ranging from 5 μg L*−*1 to 200 μg L*−*1. The calibration curve equation was *A* = 0*.*4018*C* + 2*.*0196; *r*2 = 0*.*9958; *n* = 6, where A represents the absorbance measured as peak height and C the iron concentration in μg L*−*1. The confidence limits of the intercept and the slope were calculated at the 95 % confidence level. The same calibration graph can be used for the determination of total iron. The detection limit estimated (S/N = 3) was 1 μg L−1 of iron(III).

Flow-Injection Spectrophotometric Analysis of Iron (II), Iron (III) and Total Iron 433

the certified values has been obtained for the certified metal alloy solution. The results obtained show that the proposed method can be applied in the determination of iron(III)

**4. Flow injection spectrofluorimetric determination of iron (III) in water using salicylic acid (Reproduced with permission from the paper of Asan Adem et al., 2010. Copyright of Institute of Chemistry, Slovak Academy of Sciences)**  In general terms, sensitivity of the spectrofluorimetric method is much higher than that of the spectrophotometric method. However, fluorescence reagents and methods suitable for the determination of iron are scarce and they suffer from serious interference of some metal cations such as aluminium, copper, and tin or they require a matrix separation step. Also, the reagents used for the determination of iron have a risk of toxicity (Tamm & Kalb, 1993; Yan et al., 1992; Cha et al., 1996; Ragos et al., 1998). Therefore, it is still important to develop simple and economical procedures that could be directly applied to real samples without

In literature (Cha et al., 1998), salicylic acid has been used as a fluorescence reagent for the spectrofluorimetric determination of iron(III) in batch conditions. Experimentally it was found to be a very sensitive emission reagent for the spectrofluorimetric determination of iron(III) in the absence of iron (II). A very strong emission peak of salicylic acid in aqueous solution, which decreased linearly with the addition of iron(III), occurred at 409 nm with excitation at 299 nm. Also, salicylic acid is a commercially available reagent and it does not

A simple and fast flow injection fluorescence quenching method for the determination of low levels of iron(III) in water has been developed. For this purpose, a preconcentration minicolumn consisting of cation-exchange resin was coupled to the FIA system. The use of mini-column in the system provided an improvement in sensitivity and the developed FIA method was successfully applied to the on-line determination of low levels of iron in real samples without the pre-concentration process. Fluorimetric determination was based on the measurement of the quenching effect of iron on salicylic acid fluorescence. An emission peak of salicylic acid in aqueous solution occurs at 409 nm with excitation at 299 nm. The effect of interferences from various metals and anions commonly present in water was also studied. The method was successfully applied to the determination of low levels of iron in

Analytical reagent grade chemicals were employed for the preparation of the standard, and the solutions were prepared using double distilled water. Standard iron(III) and iron (II) stock solutions (5*×*10*−*3 mol L*−*1 Fe(III) and Fe(II)) were prepared by dissolving FeNH4(SO4)2 *·*  12H2O and Fe(NH4)2(SO4)2 *·*6H2O in water and were standardized by titration with EDTA. Iron(II) and iron(III) working standard solutions were prepared by appropriate dilution of the stock solutions with water immediately before use. Hydrogen peroxide solution, 30 mass %, was purchased from Merck (Darmstadt, Germany). Standard solutions of other metal ions (all of them from Merck (Darmstadt, Germany)) at different concentrations were

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

the matrix separation step and with minimized reagent consumption.

real samples (river, sea, and spring waters).

prepared with doubly distilled water.

**4.1 Experimental** 

have a risk of serious toxicity when compared to the reagents used previously.

The limit of quantification(LOQ) was calculated as recommended (Currie, 1995); based on a ten fold of the standard deviation of 10 consecutive injections of the blank, the value of 1.65 μg L*−*1 was obtained. The reproducibility of the method calculated as the relative standard deviation (RSD) of peak heights obtained from 5 injections of 10 μg L*−*1 iron(III) was 3.5 %.

Possible interferences in the determination of iron(III) were examined under the optimum experimental conditions. The effect of potential interfering ions on the determination of iron was investigated at the 5 % interference level. To carry out this study, 20 μL of a 20 μg L*<sup>−</sup>*<sup>1</sup> iron(III) standard were injected. Table 4 summarizes the tolerance limits of the interfering ions. Most of the ions examined did not interfere with the iron(III) determination up to at least a 50000 fold excesses. The only interfering ion was iron(II), even 2 mg L*−*1 of iron(II) gave a positive interference.


Table 4. Effect of foreign ions on the determination of 20 μg L-1 of iron(III) in solution

The proposed method was applied in the determination of total iron in river and seawater samples. Iron(III) and total iron were determined according to the FIA procedure as described in the experimental section. The results obtained by both, standard addition and calibration curve, methods were in good agreement with each other. Atomic absorption measurements taken in water samples 1 and 2 are also given for comparison (Table 5).


1. Samples were collected at Samsun, Turkey.

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

3. Calibration curve method.

4. Standard addition method.

Table 5. Analytical results of iron(III) and total iron in natural water samples1

The analytical value of total iron in water is in good agreement with that obtained by the AAS method. The accuracy of the proposed method was tested by the analysis of 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 has been obtained for the certified metal alloy solution. 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.
