**2. A very sensitive flow-injection spectrophotometric determination method for iron(II) and total iron using 2', 3, 4', 5, 7-pentahydroxyflavone**

Spectrophotometric detection based on the measurement of the absorbance at a characteristic wavelength of complex formed between a chelating agent and iron has been mainly applied (Kass M. and Ivaska A. 2002; Saitoh K., et al. 1998; Weeks D.A. and Bruland K.W. 2002; Giokas D.L., et al. 2002; Themelis D.G., et al. 2001; Bagheri H., et al. 2000; Molina-Diaz A., et al. 1998; Teshima N., et al. 1996; Tesfaldet Z.O., et al. 200); Udnan Y., et al. 2004; Morelli B., et al. 1983; Pojanagaroon T., et al. 2002; van Staden J.F. and Kluever L.G. 1998). 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 B., et al. 1983), norfloxacin (Pojanagaroon T., et al. 2002), tiron (Mulaudzi L.V., et al. 2002; Van Staden J.F. and Kluever L.G. 1998), tetracycline (Ahmed M.J. and Roy U.K. 2009) and chlortetracycline (Sultan S.M. and Suliman F. 1992). Flow-injection spectrophotometric methods based on above chelating agents are not either selective, or a masking agent should be used (Wirat R., 2008). However, highly selective, simple and economical methods are still required for the routine determination of iron (II) in different sample matrices. An ultrasensitive and highly selective, rapid flow-injection spectrophotometric method for the determination of iron (II) and total iron has been proposed. The method was based on the reaction between iron (II) and 2', 3, 4', 5, 7-pentahydroxyflavone (Morin) in slightly acidic solution (pH:4.50) with a strong absorption at 415 nm. The chemical structure of Morin is shown Fig. 1. The reagent itself is sparingly soluble in water and does not absorb in the visible region of the spectrum, therefore, might be well suited for flow-injection analysis of iron (II) and total iron. The method has been successfully applied to the determination of iron (II) and total iron in water samples and ore samples.

Fig. 1. The chemical structure of 2', 3', 4', 5', 7-pentahydroxyflavone (Morin)

#### **2.1 Experimental**

#### **2.1.1 Reagent and standards**

All chemicals used were of analytical reagent grade or the highest purity available. Doubly distilled deionized water was used throughout the study. Glass vessels were cleaned by soaking in acidified solutions of KMnO4 or K2Cr2O7 followed by washing with concentrated HNO3, and were rinsed several times with high-purity deionized water. Stock solutions and environmental water samples (1000 mL each) were kept in polypropylene bottles containing 1 mL of concentrated hydrochloric acid. Standard iron (II) and iron (III) stock solutions were prepared by solving 278.02 mg of iron (II) and 489.96 mg of iron (III) sulphate (Merck) in 0.01 M 100 mL hydrochloric acid to give 0.01 M stock solution of iron (II) and iron (III). Iron (II) and iron (III) working standard solutions were prepared daily by suitable dilution of stock solutions with double deionized water. Standard reference material consisting of 0.085 % Fe (Zn/Al/Cu 43XZ3F) was provided from MBH Analytical Ltd. (UK). Hydrogen peroxide solution 30 % (v/v) used was from Merck.

A stock solution of Morin (5x10-3M) was prepared by dissolving requisite amount of Morin (BDH Chemicals) in 100 mL of ethanol:water (4:96 v/v) because of it's low solubility in water only. For spectrophotometric study, morin complex solutions of various metals were prepared by mixing 1 mL of 1x10-4 M standard solution of each metal in double deionised water with a suitable volume of 1x10-4 M Morin solution. 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. All solutions were degassed before use using a sonicator (LC 30). Reagent carrier solution was composed of Morin in 0.1 M HAc/Ac buffer (pH:4.50) solution consisting of metanol 4 %.
