Food Analytical Methods

, Volume 2, Issue 3, pp 221–225

Spectrophotometric Trace Determination of Iron in Food, Milk, and Tea Samples using a New Bis-azo Dye as Analytical Reagent

Authors

    • Department of ChemistryG. J. University of Science & Technology
  • Ishwar Singh
    • Department of ChemistryM. D. University
Article

DOI: 10.1007/s12161-008-9054-z

Cite this article as:
Sharma, A.K. & Singh, I. Food Anal. Methods (2009) 2: 221. doi:10.1007/s12161-008-9054-z

Abstract

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.

Keywords

ChromogenicIron DeterminationSpectrophotometry2,6-bis(1-hydroxy-2-naphthylazo)pyridine (PBN)

Introduction

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.

Heterocyclic azo dyes have been used as chromogens in spectrophotometric determination of metal ions. In our laboratory, the chromogens such as 2-(4,6-dimethyl-2-pyrimidylazo)-1-naphthol-4-sulfonic acid, 2-(4,6-diamino-4-pyrimidylazo)-phenol, 2,6-bis(7-hydroxyacenaphthyl-8-azo)pyridine, and 2,6-bis(1-hydroxy-2-naphthylazo)pyridine have been synthesized and used (Singh 2000; Singh and Yadav 2000; Singh et al. 2003, 2006). This paper describes the application of a newly synthesized bis-azo dye, 2,6-bis(1-hydroxy-2-naphthylazo)pyridine (PBN), using the Anderson and Nickless (1967, 1968) method that showed a good sensitivity and high selectivity for iron(II) ions. Keeping in view the biological importance of iron, this reagent was utilized to determine iron levels in cereals, legume grains, and milk and tea samples commonly consumed by Indian vegetarians. This developed method is proven to be sensitive when compared to some other reported methods (Table 1).
Table 1

Comparison of the present method with the other spectrophotometric methods for the determination of Iron(II)

Reagent

pH

λmax (nm)

Molar absorptivity (l mol−1 cm−1)

Reference

2,6 bis(1-hydroxy-2-naphthylazo) pyridine (PBN)

6.0

550

2.65 × 104

Present method

2-Diethylamino-4-hydroxy-5-nitroso-6-aminopyrimidine (EHNA) (Schuemann and Weiss 2002)

10.3

660

3.0 × 104

Tsuchiya and Iwanami 1990

Diformylhydrazine (DFH) (IUNA 2001)

7.3–9.3

470

0.32 × 104

Nagabhushana et al. 2002

(1,2-dihydroxy-3,4-diketo-cyclobutene) (squaric acid) (Gezondheidsraad 2002)

2.7

515

3.95 × 103

Stalikas et al. 2003

2-Carboethoxy-1, 3-indandione sodium salt (CEIDNa) (UK Office of National Statistics 2003)

3–6

490

0.65 × 104

Ghazy 1995

3-(2-pyridyl)-5,6-bis(4-phenyl sulfonic acid)-1,2,4-triazine (TBA) (Cook et al. 1979)

5.0

2.8 × 104

Akl et al. 2006

Materials and Methods

Instruments

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.

Reagents

Synthesis of bis-azo dye, 2,6 bis(1-hydroxy-2-naphthylazo)pyridine (PBN)

The reagent as a bis-azo dye was synthesized by the method described in the literature (Anderson and Nickless 1968). The purity of the compound was checked with thin layer chromatography and subjected to elemental analysis [theoretical (%) C = 71.599, H = 4.09, N = 16.706; found (%) C = 71.65, H = 4.10, N = 16.55]. The infra-red spectrum of the compound showed the complete absence of the νC=O (1,670 cm−1 for naphthoquinione) and νC≡N (1,630–1,680 cm−1 for hydrazones) frequencies with the appearance of a new strong frequency νOH (phenolic) at 3,200–3,500 cm−1, confirming thereby the enol-form of the compound rather than the keto-form.
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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.

Processing of Samples (Thomas and Chamberlin 1980; Sandel and Onishi 1978)

Milk samples

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.

Foodstuffs

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.

Tea leaves

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.

Recommended Procedure

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).

The maximum and constant absorbance was found in pH range of 5.0 to 7.0 and 2 ml of phosphate buffer solution of pH 6.0 was suitable for this purpose. Various physico-chemical constants of the complex formed are as follows: λmax 550 nm, Beer’s law validity 0.0–2.85 ppm, optimum concentration range 0.3 to 2.76 ppm, and molar extinction coefficient (ɛ) of the color developed 2.65 × 104 l mol−1 cm−1. Job’s method of continuous variation was employed to elucidate the composition of the complex (M:L) to be 1:2 (Fig. 1).
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Fig. 1

Composition of Fe(II)–PBN complex by Job’s method

The mean absorbance of a series of solutions containing 1 ml of 5 × 10−4 M iron(II) and excess of PBN in a total volume of 25 ml at pH 6.0 was calculated. The accuracy of the method was checked by preparing a series of solutions containing different amounts of iron(II) (7.5–69.0 μg) and following the recommended procedure. The recoveries of known additions to different samples lay within the range 99.3–100.8%. The standard deviation and relative standard deviation values clearly indicate that the precision and accuracy of the method are good (Table 2).
Table 2

Precision and accuracy of the method

Sr. no.

Fe(II) added (ppm)

Fe(II) found (ppm)

% Recovery

SD

RSD%

1.

14.0

14.0

100.0

0.1581

1.13

2.

21.0

20.90

99.52

0.2413

1.15

3.

28.0

28.0

100.0

0.1516

0.54

4.

35.0

34.90

99.7

0.1204

0.34

5.

42.0

41.70

99.3

0.2915

0.67

6.

56.0

56.25

100.4

0.1746

0.31

7.

63.0

63.50

100.8

0.1673

0.26

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

The procured samples were processed using standard methods (Anderson and Nickless 1967, 1968) and were analyzed for iron following the recommended procedure as stated above. The samples were also analyzed for iron using an atomic absorption spectrophotometer (AAS). The amounts of iron(II) found in various samples using the present method and by AAS are summarized in Table 3.
Table 3

Contents of Iron in various foodstuffs

Sample

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

Cow

3

100

49.3, 47.8, 46.3

48.9, 48.0, 46.4

0.0463–0.0493

Buffalo

3

100

58.2, 62.7, 61.2

59.1, 62.6, 61.0

0.0582–0.0493

Goat

3

100

50.5, 48.5, 42.7

50.4, 48.4, 42.5

0.0427–0.0505

(b) Food samples

Phaseolus aureus (mung)

3

2

125.5, 123.9, 128.5

125.7, 124.0, 128.5

6.195–6.425

Cicer arietinum (gram)

3

5

42.7, 44.38, 43.82

42.72, 44.37, 43.80

0.854–0.887

Oryza sativa (bran rice)

4

2

194.2, 200.2, 201.6, 198.6

194.2, 200.6, 200.5, 199.0

9.710–10.08

Pennisetum typhoidem (bajra)

3

5

124.5, 132.9, 129.9

124.7, 132.9, 130.2

2.490–2.658

Zea mays (maize)

4

4

91.12, 89.62, 94.10, 95.5

91.10, 89.80, 93.70, 95.0

2.240–2.387

Lens culinaris (masur)

3

4

285.8, 289.8, 286.8

285.3, 288.5, 286.0

7.132–7.245

Triticum aestivum (wheat flour)

3

5

122.5, 123.98, 119.5

121.6, 123.9, 119.9

2.390–2.479

Milk powder

3

5

28.6, 29.2, 26.9

27.6, 29.0, 27.0

0.0539–0.0584

(c) Tea and coffee samples

Lipton

3

2

144.9, 140.4, 143.4

144.6, 140.8, 143.9

7.020–7.245

Brooke-Bond

3

2

128.5, 132.9, 135.9

128.8, 133.0, 136.0

6.425–6.795

Taj

3

2

80.6, 77.68, 82.17

80.70, 77.75, 82.28

3.884–4.108

Rich

3

2

65.73, 58.26, 64.24

65.70, 59.0, 64.85

2.913–3.286

Nescafe

3

2

59.75, 55.26, 58.26

59.70, 55.46, 58.0

2.763–2.987

Conclusion

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.

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© Springer Science+Business Media, LLC 2008