Spectrophotometric Trace Determination of Iron in Food, Milk, and Tea Samples using a New Bis-azo Dye as Analytical Reagent
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- Sharma, A.K. & Singh, I. Food Anal. Methods (2009) 2: 221. doi:10.1007/s12161-008-9054-z
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A newly synthesized bis-azo dye, 2,6-bis(1-hydroxy-2-naphthylazo)pyridine (PBN) was used as a sensitive reagent for iron. To determine the metal ion using a spectrophotometer in the concentration range between 0.3 and 2.76 ppm (molar absorptivity of 2.65 × 104 l mol−1 cm−1 at 550 nm). In a phosphate-buffered medium, none of the transition metals, except Fe(II), Co(II), Ni(II), Cu(II), and Hg(II), produced color with the reagent; however, colors produced by Co(II), Ni(II), Cu(II), and Hg(II) could be masked using thiosemicarbazide, therefore, making the reagent highly selective for iron determination. The reagent was applied for the estimation of iron levels in milk, food grains, and tea samples and the results were compared with the iron levels found in those samples using AAS.
KeywordsChromogenicIron DeterminationSpectrophotometry2,6-bis(1-hydroxy-2-naphthylazo)pyridine (PBN)
Iron is the most abundant transition metal in the living system and serves more biological roles than any other metal. Although iron is required for a number of vital functions, the main role of iron is to carry oxygen to the tissues where it is needed. Iron is also essential for the proper functioning of numerous enzymes involved in DNA synthesis, energy metabolism, and protection against microbes and free radicals (Bothwell et al. 1979). The total iron content in an adult body is approximately 4 g, i.e.,70 mmol, of which about two-thirds is in hemoglobin. Iron stores, mainly spleen, liver, and bone marrow, contain about one-quarter of the body’s iron; the remainder is in myoglobin and other hemoproteins. Only 0.1% of the total body iron is in plasma where almost all is bound to a transport protein—transferrin. Iron deficiency affects about 30% of the world population and is one of the main deficiency disorders in Europe (Schuemann and Weiss 2002). Recent surveys in Ireland (IUNA 2001), the Netherlands (Gezondheidsraad 2002), and the UK (UK Office for National Statistics 2003) suggest that inadequate intake of iron is widespread among women. Iron is widely distributed in foodstuffs; normally about 1 mg of iron is absorbed from food every day (Cook et al. 1979). Plant sources are cereals, pulses, and vegetables. Vegetarian diets have an intermediate iron bioavailability and are the commonest cause of iron deficiency in the developing countries (Cook et al. 1979). The low bioavailability of iron in plant food is owing to the presence of phylates and oxalates which interfere with iron absorption. The iron content in milk is low in all mammalian species. The average iron content of human milk is less than 1.0 μg/ml (Picciano et al. 1976). Hence, the foods fortified with iron make a valuable contribution to iron intake, which is inadequate in certain groups of the population, including children and adolescents (Serra-Majem 2001; Sichert-Hellert et al. 2000). But high doses of supplemental iron have been associated with gastrointestinal side effects, especially when taken on an empty stomach. This risk was used by the Institute of Medicine’s Food and Nutrition Board to establish a Tolerable Upper Intake Level of 45 mg/day for iron (Institute of Medicine, Food and Nutrition Board 2001). Thus, adequate quantities of iron are essential for normal physiological processes, but excess intake poses a threat to human health. Therefore, this metal requires special attention in the food selection.
Comparison of the present method with the other spectrophotometric methods for the determination of Iron(II)
Molar absorptivity (l mol−1 cm−1)
2,6 bis(1-hydroxy-2-naphthylazo) pyridine (PBN)
2.65 × 104
2-Diethylamino-4-hydroxy-5-nitroso-6-aminopyrimidine (EHNA) (Schuemann and Weiss 2002)
3.0 × 104
Tsuchiya and Iwanami 1990
Diformylhydrazine (DFH) (IUNA 2001)
0.32 × 104
Nagabhushana et al. 2002
(1,2-dihydroxy-3,4-diketo-cyclobutene) (squaric acid) (Gezondheidsraad 2002)
3.95 × 103
Stalikas et al. 2003
2-Carboethoxy-1, 3-indandione sodium salt (CEIDNa) (UK Office of National Statistics 2003)
0.65 × 104
3-(2-pyridyl)-5,6-bis(4-phenyl sulfonic acid)-1,2,4-triazine (TBA) (Cook et al. 1979)
2.8 × 104
Akl et al. 2006
Materials and Methods
A Beckman spectrophotometer (PC based) with 10-mm matched glass cells was used for recording the spectra. An Elico pH-meter (model L1 614) was used for making pH adjustments. For comparison purposes, an atomic absorption spectrophotometer (AAS) ECIL model 4129 (PC based) was used to analyze the same samples for iron.
Synthesis of bis-azo dye, 2,6 bis(1-hydroxy-2-naphthylazo)pyridine (PBN)
A 5 × 10−4 M reagent solution was prepared by dissolving 0.2092 g of PBN in 1 l ethanol.
A 0.01 M stock solution of iron(II) was prepared by dissolving calculated amount of ferrous ammonium sulfate in acidulated doubly distilled water, standardized with EDTA, and further diluted as required for working standards.
The phosphate buffer (Thomas and Chamberlin 1980) (pH 6.0) was prepared by diluting the solution containing 250 ml of 0.2 M potassium dihydrogen phosphate and 28.2 ml of 0.2 M sodium hydroxide to 1 l with distilled water.
All the reagents used were of analytical grade.
One hundred milliliters of milk (procured from local dairies) was added drop wise to a heated crucible to evaporate it without frothing. After the moisture has been removed, it was heated strongly to 450–500 °C for ∼1 h. Utmost care was taken to avoid the loss by sputtering. The white ash obtained was dissolved in minimum volume of diluted nitric acid and volume was made up to 25 ml.
Five grams of food grains (dried for ∼24 h at 70 °C in an oven) was wet ashed with nitric and perchloric acids. Also, 5 ml of hydrochloric acid (1 + 9) was added to the ash and evaporated to dryness. This step was repeated. The dry residue was dissolved in water and filtered into a 25-ml standard flask; one or two drops of conc. HCl were added and made up to 25 ml.
Two grams of the material was dry ashed at 450 °C and then digested with 2 ml of 3:1 mixture of nitric and perchloric acids. The sample was heated gently almost to dryness, repeated again with 2 ml of acid mixture, and diluted finally to 25 ml with water. The sample was set aside overnight and filtered to remove impurities.
Iron(II) in a solution
To an aliquot containing iron(II) ions between 7.5 and 69 μg, add 5 ml of 1.0 × 10−3 M PBN solution, 2 ml of 0.1 N thiosemicarbzide, and 2 ml of phosphate buffer solution (pH 6.0) and make up the volume to 25 ml, maintaining 50% (v/v) ethanol concentration in the final solution. Record the absorbance at 550 nm against a reagent blank prepared under similar conditions.
Iron(II) in processed samples
Take 1 ml of the processed sample and analyze it for iron(II) ions following the recommended procedure. Dilute the processed sample, if necessary.
Results and Discussion
The reagent and its color reaction
As is evident from the literature, an azo dye of α-naphthol is obtained if 1,2-naphthoquinone is reacted with an aromatic hydrazine (Anderson and Nicless 1967, 1968; Kamel and Amin 1964). Thus, a bis-azo dye was obtained by reacting 2 mol of 1,2-naphthoquinone with 1 mol of 2,6-dihydrazinopyridine. Its infra-red spectrum confirmed that the compound obtained had an enol-form (absence of the νC=O (1,670 cm−1 for quinione)) and appearance of a new strong frequency νOH at 3,200–3,500 cm−1. The dye showed a light orange color up to pH 9.0. It was observed that only one complex was formed absorbing maximum at 572 nm at all pH levels; however, a maximum absorbance was exhibited in the pH range 5.2–7.2.
The ethanolic solution of PBN gave very deep color reactions with a number of metal ions at different pH levels: deep blue to violet color with zinc(II), cadmium(II), mercury(II), copper(II), silver(I), cobalt(II), nickel(II), and manganese(II) in neutral to alkaline media; violet color with iron(II), vanadium(V), and thallium(I) in alkaline media; green color with palladium(II) at neutral to alkaline media and a pink color with chromium(III) in alkaline media. In all these color reactions, the ethanol content was kept above 50% otherwise precipitates appeared in most of the cases.
Preliminary studies on color reactions of PBN with metal ions also showed that in a phosphate-buffered medium only copper(II), iron(II), cobalt(II), nickel(II), and mercury(II) gave colored complexes while complexation avoided with other metals. Further investigations revealed that the colors produced by copper(II), cobalt(II), nickel(II), and mercury(II) are masked by thiosemicarbazide thus making the present method highly selective for iron. With this view, detailed spectrophotometric studies were made on PBN as a reagent for iron(II).
Precision and accuracy of the method
Fe(II) added (ppm)
Fe(II) found (ppm)
In the determination of iron(II) at a 1.12 μg ml−1 level, chloride, bromide, iodide, nitrate, nitrite, acetate, thiosulfate, sulfide, thiosemicarbazide(TSC), phosphate, borate, Ca(II), Sr(II), Ba(II), Nb(V), Ta(V), Al(III), and lanthanides did not interfere at all. Chromium(III) 100 fold, cobalt(II) 3 fold, nickel(II) 3 fold, copper(II) 6 fold, mercury(II) 20 fold, silver(I) 10 fold, and palladium(II) 30 fold are masked by TSC. Complexation of manganese(II) 5 fold, zinc(II) 6 fold, cadmium(II) 10 fold, lead(II) 40 fold, and thallium(II) 200 fold was prevented by phosphate buffer. However, EDTA, fluoride, oxalate, citrate, tartrate, and cynide interfered seriously.
Iron(II) in milk, foodstuffs, and tea samples
Contents of Iron in various foodstuffs
No. of analyses
Sample ashed (ml or g)
Fe found in whole sample using PBN (μg)
Fe found in whole sample using AAS
Range of Fe levels (mg/100 g or mg/100 ml)
(a) Milk samples
49.3, 47.8, 46.3
48.9, 48.0, 46.4
58.2, 62.7, 61.2
59.1, 62.6, 61.0
50.5, 48.5, 42.7
50.4, 48.4, 42.5
(b) Food samples
Phaseolus aureus (mung)
125.5, 123.9, 128.5
125.7, 124.0, 128.5
Cicer arietinum (gram)
42.7, 44.38, 43.82
42.72, 44.37, 43.80
Oryza sativa (bran rice)
194.2, 200.2, 201.6, 198.6
194.2, 200.6, 200.5, 199.0
Pennisetum typhoidem (bajra)
124.5, 132.9, 129.9
124.7, 132.9, 130.2
Zea mays (maize)
91.12, 89.62, 94.10, 95.5
91.10, 89.80, 93.70, 95.0
Lens culinaris (masur)
285.8, 289.8, 286.8
285.3, 288.5, 286.0
Triticum aestivum (wheat flour)
122.5, 123.98, 119.5
121.6, 123.9, 119.9
28.6, 29.2, 26.9
27.6, 29.0, 27.0
(c) Tea and coffee samples
144.9, 140.4, 143.4
144.6, 140.8, 143.9
128.5, 132.9, 135.9
128.8, 133.0, 136.0
80.6, 77.68, 82.17
80.70, 77.75, 82.28
65.73, 58.26, 64.24
65.70, 59.0, 64.85
59.75, 55.26, 58.26
59.70, 55.46, 58.0
A new reagent for the determination of trace amounts of iron in different samples is proposed. Ethanolic solution of 2,6 bis(1-hydroxy-2-naphthylazo)pyridine (PBN) formed a violet colored water-soluble complex with dilute solution of Fe(II) ion at pH 6. The colored complex has high molar absorptivity and is made a basis of the spectrophotometric determination of the metal ion. The potentiality of the reagent was further explored successfully by analyzing iron in various foodstuffs consumed by the local gentry of the area. The proposed method is very simple, highly selective, reproducible, and relatively inexpensive. The AAS studies reveal that PBN can successfully be used determine iron(II) ions in diverse samples.