**4. Spectrophotometry and chromatography as tools for iodine assessment in miscellaneous matrices**

It is known that the choice of the proper analytical method depends on the intended application, the number of samples, the cost of analysis and the technical capability.

A Review of Spectrophotometric and Chromatographic Methods

catalyse this reaction in the same manner as I2, while Mn and MnO4

or tolerate IO3– that are found in natural waters (Heckwan, 1979).

Br-. Among substances reducing Ce4+ they listed NO2-, SCN-

CuSO4).

and Sample Preparation Procedures for Determination of Iodine in Miscellaneous Matrices 381

 I2 + As3+ →2I- + As5+ (3) Iodine has a catalytic effect upon the course of reaction (1), i.e., the more iodine is present in the preparation to be analyzed, the more rapidly proceeds the reaction (1). The speed of reaction is proportional to the iodine concentration. In this manner it is possible to determine iodine even in the nanogram range. Sandell and Kolthoff found that Os and Ru

classed by them as oxidising As3+. These authors also pointed out that certain substances, such as F-, form compounds with Ce4+ giving a stable complex. Ag+, CN-, and Hg+ react with I- as well. The effect of various concentrations of NaCl, NaF, KH2PO4, ZnSO4, KCl, MgSO4, KBr and of CuSO4 on the described reaction was studied by Stolc (Stolc, 1961). According to the author the substances studied may be grouped into two categories, i.e. reaction inhibiting (NaF, KH2PO4, ZnSO4, KCl ) and reaction stimulating agents (NaCl, MgSO4, KBr,

The method is achieved in the following manner: a measured amount of an arsenous oxide (As2O3) solution in concentrated H2SO4 is combined with the test solution. This mixture is then adjusted to its reaction temperature, usually between 20 and 60 degree C. Cerium (IV) sulfate in sulfuric acid is then added, after which the solution is able to react for a limited time at the set temperature. The reaction time ranges from 10 to 40 minutes, and subsequently the content of the test solution of cerium (IV) ions is photometrically determined. The lower the determined concentration of cerium (IV) ions, the faster the reaction, thus a larger amount of catalyzing agent, i.e., iodine. By these means it is possible to directly and quantitatively measure the iodine concentration of the test solution, though execution of such processes is complicated and demands extensive measuring times (Sandell & Kolthoff, 1934, 1937). The above-described method was modified in various ways, for example by replacing H2SO4 with HNO3 (used for acidifying the reaction mixture). It was found that the catalytic activity of iodine in HNO3 solution is 20 times that in H2SO4 and is also far less sensitive towards accompanying ions, making the system far more useful for the determination of traces of iodine (Knapp & Spitzy, 1969). The reaction mixture's change in composition multiplies the sensitivity of the reaction by twenty. Consequently, test solutions of an iodine content that, according to the conventional catalytic reaction method utilizing sulfuric acid, required a reaction time of approximately 20 minutes in order to display a notable decrease in cerium (IV) ion concentration need only 1 minute to produce the same result. These results were achieved while operating at the same reaction temperature. Rodriguez and Pardue (Rodriguez & Pardue, 1969) studied the effect of H2SO4, HClO4, Ag(I), Hg(II), Cl- and temperature on the aforemetioned kinetic reaction. Their studies utilized the catalytic action of iodide on the decomposition of the FeSCN2+complex ion. This indicator reaction is characterized by an induction period, the length of which depends on the reagent concentration, pH and temperature. The mentioned method was adopted as a standard method for iodide determination in natural and waste waters as well as in food and biological samples. However, high inter-laboratory relative standard deviations have frequently been reported for this method. Some authors have suggested that this might be partly attributed to the limitations of the method to quantitatively detect


, MnO4 were

, Fe2+, while BrO3-

Currently, there are multiple distinctive analytical methods for determining concentrations of iodine species. The methods vary in principle, reliability, accuracy, precision, availability, detection limit, sample throughput, time and reagent consumption, ease of performance and cost of analysis. These factors all play a role in the choice of the most suitable method but ultimately the purpose of the analysis determines the method, e.g., whether the analysis is routine or if an analysis of a reference material is necessary. For any given purpose, one of the first factors taken into account is whether the method's detection limit is adequately low. Several methods of iodine determination have been proposed, including catalytic methods (with LOD=0.1 μg/ l) (Kamavisdar & Patel, 2002), chromatography in various modes (eg., IC with LOD = 0.1-0.8 μg/l (Hu et al., 1999; Bichsel & Von-Gunten, 1999), (chromatographic methods are especially useful for iodine speciation when coupled with ICP-MS or elecrochemical detection), GC-EC: gas chromatography with electron capture detection (0.11μg/l) (Maros et al., 1989), GC–MS: gas chromatography–mass spectrometry(0.010 μg/l) (Das et al., 2004), FAAS: flame atomic absorption spectrometry (2.75 μg/l) (Yebra & Bollaín, 2010), NAA (0.1-0.2 μg/l) (Hou et al., 1999), ETAAS: electrothermal atomic absorption spectrometry (1.2 -3.7 μg/l) (Bermejo-Barrera et al., 1999), inductively coupled plasma mass spectrometry ICP-MS (1.0-9.0 μg/l) (Fernandez-Sanchez & Szpunar 1999), ICP-AES (40.0- 470.0 μg/l) (Anderson & Markowski, 2000), inductively coupled plasma optical emission spectrometry (ICP- OES) (2 µg/l) (Naozuka et al., 2003), ion selective electrodes (1.96 μg/l) (Kandhro et al., 2009), X-rayfluorescence (XRF) (180 μg/L) (Varga, 2007), VG-ICP-OES: vapour generation inductively coupled plasma optical emission spektrometry (20 μg/l) (Niedobová at al., 2005). The iodine content can also be measured by the use of titrimetric methods usually combined with potentiometric measurements. They are also used for verifying other methods (Gottardi, 1998). The titrimetric method is mainly used for samples without complex matrices (i.e. water or salt). Generally such methods involve acidification of the sample solution and adding an excess of KI solution to determine the liberated iodine by titration with sodium thiosulphate. Despite numerous advantages of the abovementioned methods, very few of them are widely used due to very high costs of instrumentation, software, and maintenance. Spectrophotometric and chromatographic methods are used very frequently for the analysis of iodine and its various chemical forms. Chosen examples of applications of iodine determinations are presented below.

#### **4.1 Spectrophotometric methods**

#### **4.1.1 Water samples**

Spectrophotometric analysis continues to be one of the most widely used analytical techniques available. Kinetic spectrophotometric methods, which are based on the reaction, found by Sandell and Kolthoff (1934) set the foundation for the development of different methods for the determination of iodine in environmental samples (mostly water). The said reaction proceeds according to the following equation (1):

$$2\text{ Ce}^{4\*} + \text{As}^{3\*} \rightarrow 2\text{ Ce}^{3\*} + \text{As}^{5\*} \tag{1}$$

By adding an arsenious acid (H3AsO3) solution and an ammonium cerium sulfate ((NH4)2Ce(SO4)3) solution as reagents to I- in a specimen, yellow Ce4+ is reduced to produce colorless Ce3+ ((2) and (3)).

$$2\text{Ce}^{4\*} + 2\text{I}^{\cdot} \text{-} 2\text{Ce}^{3\*} + \text{I}\_2 \tag{2}$$

Currently, there are multiple distinctive analytical methods for determining concentrations of iodine species. The methods vary in principle, reliability, accuracy, precision, availability, detection limit, sample throughput, time and reagent consumption, ease of performance and cost of analysis. These factors all play a role in the choice of the most suitable method but ultimately the purpose of the analysis determines the method, e.g., whether the analysis is routine or if an analysis of a reference material is necessary. For any given purpose, one of the first factors taken into account is whether the method's detection limit is adequately low. Several methods of iodine determination have been proposed, including catalytic methods (with LOD=0.1 μg/ l) (Kamavisdar & Patel, 2002), chromatography in various modes (eg., IC with LOD = 0.1-0.8 μg/l (Hu et al., 1999; Bichsel & Von-Gunten, 1999), (chromatographic methods are especially useful for iodine speciation when coupled with ICP-MS or elecrochemical detection), GC-EC: gas chromatography with electron capture detection (0.11μg/l) (Maros et al., 1989), GC–MS: gas chromatography–mass spectrometry(0.010 μg/l) (Das et al., 2004), FAAS: flame atomic absorption spectrometry (2.75 μg/l) (Yebra & Bollaín, 2010), NAA (0.1-0.2 μg/l) (Hou et al., 1999), ETAAS: electrothermal atomic absorption spectrometry (1.2 -3.7 μg/l) (Bermejo-Barrera et al., 1999), inductively coupled plasma mass spectrometry ICP-MS (1.0-9.0 μg/l) (Fernandez-Sanchez & Szpunar 1999), ICP-AES (40.0- 470.0 μg/l) (Anderson & Markowski, 2000), inductively coupled plasma optical emission spectrometry (ICP- OES) (2 µg/l) (Naozuka et al., 2003), ion selective electrodes (1.96 μg/l) (Kandhro et al., 2009), X-rayfluorescence (XRF) (180 μg/L) (Varga, 2007), VG-ICP-OES: vapour generation inductively coupled plasma optical emission spektrometry (20 μg/l) (Niedobová at al., 2005). The iodine content can also be measured by the use of titrimetric methods usually combined with potentiometric measurements. They are also used for verifying other methods (Gottardi, 1998). The titrimetric method is mainly used for samples without complex matrices (i.e. water or salt). Generally such methods involve acidification of the sample solution and adding an excess of KI solution to determine the liberated iodine by titration with sodium thiosulphate. Despite numerous advantages of the abovementioned methods, very few of them are widely used due to very high costs of instrumentation, software, and maintenance. Spectrophotometric and chromatographic methods are used very frequently for the analysis of iodine and its various chemical forms.

Chosen examples of applications of iodine determinations are presented below.

Spectrophotometric analysis continues to be one of the most widely used analytical techniques available. Kinetic spectrophotometric methods, which are based on the reaction, found by Sandell and Kolthoff (1934) set the foundation for the development of different methods for the determination of iodine in environmental samples (mostly water). The said

 2 Ce4+ +As3+→2 Ce3+ + As5+ (1) By adding an arsenious acid (H3AsO3) solution and an ammonium cerium sulfate ((NH4)2Ce(SO4)3) solution as reagents to I- in a specimen, yellow Ce4+ is reduced to produce

2Ce4+ + 2I- →2Ce3+ + I2 (2)

**4.1 Spectrophotometric methods** 

reaction proceeds according to the following equation (1):

**4.1.1 Water samples** 

colorless Ce3+ ((2) and (3)).

$$\rm I\_2 + As^{3\*} \rightarrow 2I^{\cdot \cdot} + As^{5\*} \tag{3}$$

Iodine has a catalytic effect upon the course of reaction (1), i.e., the more iodine is present in the preparation to be analyzed, the more rapidly proceeds the reaction (1). The speed of reaction is proportional to the iodine concentration. In this manner it is possible to determine iodine even in the nanogram range. Sandell and Kolthoff found that Os and Ru catalyse this reaction in the same manner as I2, while Mn and MnO4- do so in the presence of Br-. Among substances reducing Ce4+ they listed NO2-, SCN- , Fe2+, while BrO3-, MnO4 were classed by them as oxidising As3+. These authors also pointed out that certain substances, such as F-, form compounds with Ce4+ giving a stable complex. Ag+, CN-, and Hg+ react with I- as well. The effect of various concentrations of NaCl, NaF, KH2PO4, ZnSO4, KCl, MgSO4, KBr and of CuSO4 on the described reaction was studied by Stolc (Stolc, 1961). According to the author the substances studied may be grouped into two categories, i.e. reaction inhibiting (NaF, KH2PO4, ZnSO4, KCl ) and reaction stimulating agents (NaCl, MgSO4, KBr, CuSO4).

The method is achieved in the following manner: a measured amount of an arsenous oxide (As2O3) solution in concentrated H2SO4 is combined with the test solution. This mixture is then adjusted to its reaction temperature, usually between 20 and 60 degree C. Cerium (IV) sulfate in sulfuric acid is then added, after which the solution is able to react for a limited time at the set temperature. The reaction time ranges from 10 to 40 minutes, and subsequently the content of the test solution of cerium (IV) ions is photometrically determined. The lower the determined concentration of cerium (IV) ions, the faster the reaction, thus a larger amount of catalyzing agent, i.e., iodine. By these means it is possible to directly and quantitatively measure the iodine concentration of the test solution, though execution of such processes is complicated and demands extensive measuring times (Sandell & Kolthoff, 1934, 1937). The above-described method was modified in various ways, for example by replacing H2SO4 with HNO3 (used for acidifying the reaction mixture). It was found that the catalytic activity of iodine in HNO3 solution is 20 times that in H2SO4 and is also far less sensitive towards accompanying ions, making the system far more useful for the determination of traces of iodine (Knapp & Spitzy, 1969). The reaction mixture's change in composition multiplies the sensitivity of the reaction by twenty. Consequently, test solutions of an iodine content that, according to the conventional catalytic reaction method utilizing sulfuric acid, required a reaction time of approximately 20 minutes in order to display a notable decrease in cerium (IV) ion concentration need only 1 minute to produce the same result. These results were achieved while operating at the same reaction temperature. Rodriguez and Pardue (Rodriguez & Pardue, 1969) studied the effect of H2SO4, HClO4, Ag(I), Hg(II), Cl- and temperature on the aforemetioned kinetic reaction. Their studies utilized the catalytic action of iodide on the decomposition of the FeSCN2+complex ion. This indicator reaction is characterized by an induction period, the length of which depends on the reagent concentration, pH and temperature. The mentioned method was adopted as a standard method for iodide determination in natural and waste waters as well as in food and biological samples. However, high inter-laboratory relative standard deviations have frequently been reported for this method. Some authors have suggested that this might be partly attributed to the limitations of the method to quantitatively detect or tolerate IO3– that are found in natural waters (Heckwan, 1979).

A Review of Spectrophotometric and Chromatographic Methods

addition of the method was 96.2–99.2% (Zhai et al., 2010).

& Zarei 2001; Ensafi & Dehaghi, 2000).

and iodine in pharmaceutical preparations.

**4.1.3 Foodstuffs** 

& Zaitsev, 2004).

IO3-

estimated to be around 2 µg/kg.

and Sample Preparation Procedures for Determination of Iodine in Miscellaneous Matrices 383

(Lepidium sativum) was determined by an experiment in which different amounts of iodine were added to the potted plants. The iodine fertiliser used was natural caliche. The results show a very close correlation between the iodine supply and iodine concentration in the

A semi-automated method for determination of the total iodine in milk was described by Aumont (Aumont, 1982). The method involved destruction of organic matter by alkaline incineration and automated spectrophometric determination of iodide based on the Sandell and Kolthoff's reaction. The recoveries of the added iodide before calcination were between 90.05 +/- 7.36% and 97.14 +/- 4.56% (mean +/- S.D.). The coefficient of variation ranged from 2.15 to 7.21% depending on the iodine content in the milk. The limit of detection was

The iodide-catalyzed reaction between As(III) and Ce(IV) stopped by the addition of diphenylamine-4-sulfonic acid was used for the development of a sensitive kinetic procedure for determining iodides with a detection limit of 2 ng/mL. The developed procedure was suitable for the determination of the total iodine in foodstuffs (Trokhimenko

Another modification of the catalytic kinetic spectrophotometric method has been established for the determination of iodine using the principle that potassium periodate oxidize rhodamine B (RhB) to discolor and I− has a catalytic effect on the reaction. The absorbance difference (ΔA) is linearly related with the concentration of iodine in the range of 0 – 2.6 µg/mL and fits the equation ΔA = 0.1578 C(C: μg/mL) + 0.0052, with a regression coefficient of 0.9965. The detection limit of the method is 7.10 ng/mL. The method was used to determine iodine in kelp, potato, tap water, and rain water samples. The relative standard deviation of 13 replicate determinations was 1.81–2.10%. The recovery of the standard

Some researchers reported that the spectrophotometric methods for the determination of

Balasubramanian and Nagaraja (Balasubramanian & Nagaraja, 2008) described a sensitive spectrophotometric method for the determination of multiple iodine species such as I-, I2, IO3- and IO4-. The method involved oxidation of iodide to ICl2- in the presence of iodate and chloride in an acidic medium. The formed ICl2- bleaches the dye methyl red. The decrease in the intensity of the colour of the dye is measured at 520 nm. Beer's law is obeyed in the concentration range 0-3.5 μg of iodide in an overall volume of 10 ml. The relative standard deviation was 3.6% (n=10) at 2 μg of iodide. The developed method can be applied to the samples containing iodine, iodate and periodate by prereduction to iodide using Zn/H(+) or NH2NH2/H(+). The effect of interfering ions on the determination was pointed out. The described method was successfully applied to determine iodide and iodate in salt samples

Silva *et al. (*Silva et al., 1998) outlined a new method for the determination of iodate in table salt. KIO3, after being converted to I3 by reacting with iodide in the presence of phosphoric

are based on its reaction with the excess I- to liberate I2 which forms tri iodide (Afkhami

cress which increased to more than 30 mg/kg dry matter (Jopke et al., 1996).

An alternative flow injection spectrophotometric method for the determination of I- in the ground and surface water was reported by Kamavisdar and Patel (Kamavisdar & Patel, 2002). The method was based on the catalytic destruction of the colour of the Fe(III)–SCN−– CP+–nBPy quarternary complex. The detection limit of the method was reported to be 0.1 ng ml−1 of iodide. Another redox reaction between chloramine-T and N,N' tetramethyldiaminodiphenylmethane (Feigl's Catalytic Reaction) was applied for the determination of traces of iodine in drinking water (Jungreis & Gedalia, 1960).

An alternative to the Sandell-Kolthoff method was developed by Gurkan et al., (Gurkan et al., 2004). Iodides were determined in waters by inhibition kinetic spectrophotometric method based on the inhibitory effect of I- on the Pd(II)-catalyzed reduction of Co(III)-EDTA by the hypophosphite ion in a weak acid medium. The main advantage of this method was related to the pretreatment step of the analysis which would be omitted (a time-consuming alkaline ashing preparative procedure is necessary in order to apply the standard method). The sensitivity of the method allowed determinations in the range of 2-35 ng/ml of I*−* (LOD=1.2 ng/ml). Koh et al. (Koh et al., 1988) separated I- from other chemical species by its oxidation and subsequent extraction into carbon tetrachloride. The proposed spectrophotometric method was based on the extraction of the back-extracted iodide into 1,2-dichloroethane as an ion pair with methylene blue. The authors applied that method to determine various amounts of iodide in natural water samples (at the 10–6 mol l–1 level). Spectrophotometric determination of the total dissolved sulfide in natural waters allowed also simultaneous determination of other UV-absorbing ions, including I- (Guenther et al., 2001).
