**4.1 Experimental**

432 Macro to Nano Spectroscopy

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)

Over 1000 Co(II), Cr(III), Al(III), Cu(II), Cd(II), Ni(II), Pb(II), Sn(II),

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

 Found3 Found4 Found3 Found4 AAS Seaport (Sea water) 45.16 (0.06) 45.92 (0.21) 53.46 (0.19) 53.78 (0.27) 54.93(0.24) Industry (Sea water) 56.28 (0.18) 56.11 (0.14) 76.45 (0.27) 76.13 (0.15) 78.19(0.16) Atakum (River water) 21.45 (0.05) 21.18 (0.12) 32.69 (0.08) 31.85 (0.24) 34.47(0.36) Mert (River water) 38.17 (0.11) 38.12 (0.19) 1.18 (0.04) 41.27 (0.16) 43.76(0.32)

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

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

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

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).

> Total iron2 (μg L-1)

CN-, NO3-, NO2-, SO42-, CO32-, Cl-, Br-

Mn(II), Zn(II), K(I), Na(I), Ag(I), Ca(II), Mg(II), Ba(II), Hg(II),

, PO43-, NH4+

Total iron2 (μg L-1)

gave a positive interference.

Tolerance limit (mg L-1) Foreign ion

Over 2 Fe(II)

Sample Fe(III)2

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

1. Samples were collected at Samsun, Turkey.

(μg L-1)

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 prepared with doubly distilled water.

Flow-Injection Spectrophotometric Analysis of Iron (II), Iron (III) and Total Iron 435

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

Fig. 6 shows the fluorescence emission spectra of 5*×*10*−*5 mol L*−*1 salicylic acid in a buffer solution at pH 8.5 before and after the reaction with 1*×*10*−*5 mol L*−*1 iron(II) and iron(III), respectively, in batch experiments. As can be seen, the intensity of salicylic acid fluorescence decreased significantly in the presence of iron(III). From these spectra, the emission wavelength chosen for the FIA measurement was 409 nm, using 299 nm for the fluorescence

Fig. 6. Emission spectrum of 5*×*10*−*5 M salicylic acid in batch experiment (in the absence and presence of 1*×*10*−*5 M Fe(III) and 1*×*10*−*5 M Fe(II) ions): a) salicylic acid, b) salicylic acid +

Optimization of the flow system was performed to establish the best FIA variables. A fixed standard Fe (III) solution, 10 μg L*−*1 was injected into the flow system for the determination of optimum experimental conditions. The main variables influencing the intensity of the signal were: flow-rate, pH, and the concentration of salicylic acid. Therefore, optimization of

The effect of salicylic acid in the carrier solution on the peak height was examined by changing the amount of salicylic acid in the range of 5*×*10*−*7–5*×*10*−*5 mol L*−*1 in buffer solution at pH 8.5, at the flow rate of 1.0 mL min*−*1. Peak heights were found maximum using a 2*×*10*−*6 mol L*−*1 salicylic acid solution for 10 μg L*−*1 iron(III) levels. Therefore, 2*×*10*<sup>−</sup>*<sup>6</sup>

The effect of flow-rate on the peak height of iron(III) was examined by varying the flow-rate from 0.5 mL min*−*1 to 1.5 mL min*−*1. Peak heights decreased at flow-rates above 1.2 mL min*−*1

mol L*−*1 salicylic acid was chosen as the fluorescence reagent in the carrier solution.

the FIA system was carried out by changing these variables one by one.

determination of total iron.

**4.2 Results and discussion** 

Fe(II), c) salicylic acid + Fe(III).

**4.2.1 Optimization of FI manifold** 

excitation.

Buffer solution, 0.1 mol L*−*1 NH4+ /NH3 at pH: 8.5, was used to produce analytical signal in the FIA system. Salicylic acid was provided from Merck (Darmstadt, Germany). Standard salicylic acid solutions were prepared daily by dissolving the appropriate amount of salicylic acid in an ethanol:water mixture (30 : 70). The reagent carrier solution was composed of 2*×*10*−*6 mol L*−*1 salicylic acid and 0.1 mol L*−*1 NH4 + /NH3 buffer solution (90:10) at pH 8.5.

Fluorescence measurements for the batch experiments were performed with an SPF-500 model spectrofluorometer (American Instrument Co, Jessup, USA) using 1 cm quartz cells. Instrument excitation and emission slits were fixed at 10 nm. The light source was a 150 W Xenon lamp (American Instrument Co, Jessup, USA). Excitation and emission wavelengths were set at 299 nm and 409 nm, respectively. An eight-channel ISMATEC IPC peristaltic pump (Z¨urich, Switzerland), 0.75 mm i.d. PFTE tubing, was used to propel the samples and reagent solutions. Samples were injected into the carrier stream by a Rheodyne injection valve provided with a 20 μL loop. A Varian 2070 spectrofluorometer (Tokyo, Japan) using a 15 μL flow cell was used for the on-line measurements of analytical signals. Instrument excitation and emission slits were set at 20 nm. The light source was an ozoneless 75 W Xenon lamp (Tokyo, Japan). A strip chart recorder was attached to the instrument. Cationexchange resin, sodium form of A650 W (100–200 mesh), was provided by the BioRad Labs (Hercules, CA, USA). The cation-exchange resin minicolumn (6 cm long, 2 mm i.d) was prepared in our laboratory.

pH measurements were carried out using a Jenway digital pH-meter model 3040 (Essex, England). An ATI UNICAM 929 model AAS (Cambridge, UK) 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 of 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 flow injection manifold was similar to that proposed in our previous study (Isildak et al., 1999). Peristaltic pump was used to transport the reagent carrier solution through the system. The sample was injected using an injection loop (20 μL). The reagent carrier solution and the sample were allowed to mix in the flow stream and in the mini-column. The decrease in the fluorescence intensity of the salicylic acid as a function of Fe(III) concentration was measured in the flow cell using 299 nm for excitation and 409 nm for emission. Water samples were obtained from different places of the river, sea and thermal spring in Samsun, Turkey. They were filtered through a 0.45 μm Millipore Filter (Millford, MA, USA). Water samples were split into two portions: one part was directly injected into the FIA system for the determination of iron(III). Before the analysis of the other part, 1 mL of H2O2 (10 mass %) was added to a 9 mL sample solution for complete oxidation of iron(II) to iron(III). Then, 20μL of this solution were injected into the system for the determination of total iron, 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 + 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 deionized water, 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.
