A New Spectrophotometric Method for Determination of Iron in Herbs, Spices and Beans with 2,6-Diacetylprydine Dioxime and 2-Acetylpyridine Monoxime

The reaction of iron (II) with 2, 6-diacetylprydin e dioxime (H2dapd) and 2-acetylprydine monoxime (Hapm) in 10% and 60% v/v ethanol-water so lution at pH 2.5 and 7.5, respectively, were studied using direct and first derivative spectroph otometry. Fe (III) is reduced by H 2dapd and gives iron (II) complex. A simple, rapid, selective and s en itive method for the determination of Fe (II), F e (III) and a mixture of them with the H 2dapd reagent in acid water-ethanol medium after the solution attained to stand periods of 30 sec, 4 hr and 4 hr, respectively, are proposed. Hapm also reacts with Fe (II) to form [Fe(Hapm) 3] 2+ complex with less selectivity and sensitivity than H2dapm reagent. Calibration graph with [Fe(H 2dapd)2] 2+ is linear over the range 0.28 μg mL̄ 1 with an apparent molar absorptivity of 8.481 ×10 L mol̄ cm̄ 1 at λm 428 nm. Linear dynamic ranges are 0.01-11.0 and 0. 0711.0 μg mL̄ 1 iron (II) as [Fe(H2dapd)2] complex for direct and derivative modes, respectiv ly. The analytical sensitivity is 4.44 ×10 μg mL̄ 1 for direct and 9.15 ×10 μg mL̄ 1 for derivative spectrophotometry with [Fe(H 2dapd)2] 2+ complex. First derivative method enhances the sens itivity of the Fe(H2dapd)2] 2+ more than direct one with two folds for Ni , Co, Cu and Cr. A linear equation was derived from iron (II) determining as Fe (H 2dapd)2] 2+ in the presence of synthetic solution containing Ni , Co, Cu and Cr. The use of H2dapd reagent for the determination of total iron spectrophotometerically in foodstuffs, herbs, spice s (rocks) after wet ashing (wet digestion) in the absence of reducing agent is compared with that emp loying AAS.


INTRODUCTION
Recently, there has been a growing concern about the role of iron (II) in biochemical and environmental systems, regarding man, animals and plants. Iron (II) lead to many diseases such as giddiness, diarrhea and cardiac collapse-conditions found amongst people, to damage of the gastrointestinal tract and liver [1] . These diseases can lead to a problem in iron absorption and in turn iron toxicity.
Because of the ever-increasing interest in the determination of different oxidation state of iron in outdoor samples, there is a need for the development of more sensitive and selective analytical methods. The common availability and low cost of instrumentation, the simplicity of procedures, the accuracy and selectivity of the technique make spectrophotometric method advantage for the determination of iron. Flow Injection Analysis (FIA), intensively a developed over the past 20 years, offers several valuable advantages for speciation of iron [2] , where individual signals were obtained for Fe (II) and Fe (III). However, the determination of iron with the FIA is expensive and with complicated experimental conditions that makes it not an alternative for routine analysis.
The iron reaction with DCDT is unusual due to the high acidity of the medium. In addition, results showed that molar absorptivity, Beer's low and detection limit were 8.9×10 3 L mol¯1 cm¯1, 0.7-5.0 µg mL¯1 and 0.05 µg mL¯1 respectively. Results also showed that Co 2+ , Ni 2+ , Cr 3+ and Cu 2+ foreign ions in synthetic iron (II) solutions could be tolerated to 1.0, 5.0, 25.0 and 50.0 folds, respectively. Whereas after extracting iron into amyl alcohol the tolerance limit of Co 2+ and Cr 3+ were 5.0×10 -3 and 1.5×10 -2 µg mL¯1, respectively.
Considering the relative stabilities and specific character of the axioms (=N-OH) complex of iron (II), 2,6-diacetylprydine dioxin (H 2 dapd) and 2acetylpyridine monoxide (Hapm) had much attention for the spectrophotometric kinetic studies [11][12] . Previous studies indicated that oximate anions were characterized with strong α-nucleophilies [13] . Finally, H 2 dapd possesses structural features in common with DCDT organic reagents. Based on the preliminary studies, H 2 dapd and Hapm oxen are chosen as analytical reagents for iron (II) determination.
Recently, spices and herbs have gained an important role in agronomy production and pharmacy because of their increased needs as a raw material for flavorings beverage, food metabolites and medicinal components. A Vicia faba bean is considered to be one the most popular food in Egypt. Generally, direct determination of iron in herb, spice and bean samples is difficult due to high amounts of protein, amino acids, alkaloids, cellulose, starch, volatile and fixed oil and pigments in the extracts [14] , which caused a serious interference. Therefore, the iron determination is a requisite routine plant control analysis. Procedures usually used wet ashing in the preparation of the solutions for analysis [15] . Most organic matter in the extracts was destroyed by this process.
In this work, the analytical properties of H 2 dapd or Hapm with iron (II) in acid or faintly alkaline medium are examined Spectro photo metrically. Comparison between direct and first derivative spectral methods has been established. The ability to measure trace amounts of Fe (III) and a mixture of Fe (II) and Fe (III) with H 2 dapd is also exploited with the aim of H 2 dapd that could reduce the Fe (III) to Fe (II). This work is considered as an attempt to develop a new, rapid and sensitive method to determine two different oxidation states of iron individually and in a binary mixture. H 2 dapd organic reagent is applied for the determination of total iron as Fe (II) -complex in foodstuffs, herbs, spices and rocks.

MATERIALS AND METHODS
A Jasco V-530E Spectrophotometer with 1.0 cm quartz cells, a Buck Scientific Model 210 VGP Atomic Absorption Spectrophotometer (AAS), Shimadzu FTIR-8101 Spectrophotometer, Perkin-Elmer 240C Elemental Analyzer and an Orion pH electrode fitted with a combined glass electrode were used. Milliporewater purification system was used.

Preparation of H 2 dapd and Hapm Reagents:
The H 2 dapd or Hapm reagents were prepared from 2,6diacetylpyridine or 2-diacetylpyridine via the modified procedure [16] . Briefly, into a 250 mL flask 4.1 g of 2,6diacetylpyridine or 20 mmol of 2-diacetylpyridine were mixed with 4.6 or 2.5 g of hydroxylamine hydrochloride and 4.8 or 2.4 g of barium carbonate, respectively, in 100 mL ethanol. The warmed mixture was stirred on a heating-stirrer and then refluxed for 5 hr. After filtration, the filtrate was left to stand overnight. The H 2 dapd and Hapm oxime compound was re-crystallized from ethanol and dried.

Chemicals and Reagents:
All entire reagents and solvents used were from BDH chemical grade except where indicated. 2,6-diacetylpyridine and 2-diacetylpyridine were obtained from Aldrich. HCl and HNO 3 were ultrapur acids (Merck). A stock solution of 1.0 (10 -2 M H 2 dapd and Hapm reagents were prepared by dissolving reagent in ethanol. Stock solutions containing 1.0×10 -3 M iron(II) and iron(III) (Merck) were prepared by dissolving an accurately weighed amounts of ferrous and ferric ammonium sulphate in 0.05 M H 2 SO 4 and standardized titrimetrically with a standard K 2 Cr 2 O 7 solution (daily) and gravimetrically by weighing as F 2 O 3 , respectively. Working solutions were prepared by appropriate dilution with ultrapure water. Air must be removed from all solutions by purging with free oxygen-nitrogen prior to the experiments and all additions of reagents must be carried out. All glassware was cleaned as the procedure described elsewhere [17] .
Calibration Plot: Under the optimum conditions, a linear calibration graph was obtained up to 11.0 and 16.0 µg mL¯1 of iron (II) with H 2 dapd and Hapm reagents, respectively. A plot showed that the optimum range was 0.28 to 11.0 (0.56 to 16.0) µg mL¯1 iron (II) with H 2 dapd (Hapm) reagents using direct and derivative modes. The concentration of iron was calculated by the regression equations (Table 1).

Recommended Procedures for Direct and First
0.9997 3.99×10 -7 A=Absorbance; D= dA/dλ=Vertical Amplitude from Zeroth Line; r =Correlation Coefficient; *n =10 Measurements; ** n = 10 measurements  Transfer both solutions to a 10.0 ml flask and make up to the mark with ultrapure water. In procedures (I) and (II), the absorption (A) and amplitude signals (dA/dλ) against a reagent blank were recorded. Then the concentration of ferrous ion as Fe (II) -and Fe (II) -Hapm complexes were measured at 428 (523) or 524 (555) NM with direct (first derivative) mode, (Table 1).  Table 1).

Determination of Total Iron in Herb, Spice, Bean and Rock Samples:
The recommended procedure (I) was applied to determine iron in four herbs and spices (kakade, anise, cumin and black pepper), one Vicia faba bean (testa, cptyledons and testa+ cptyledons) and six rock samples. Four herbs and spice or six rock samples were purchased from a supermarket in Sohag area are collected from the red sea area, Upper Egypt. To prepare dry weight of herb, spice and bean, the samples washed with water to avoid any contamination on the surface [15] . After drying, the samples were ground with titanium blade (food blender) to make the homogeneous and sieved to separate particle size fraction (<150 µm). Whereas rock samples were ground with ceramic mortar and sieved to separate particle size fraction (<150 µm). A certain amount of ground sample (herb, spice and bean) was heated at 500 o C for 2 hr in a muffle furnace. A 1.0 g of resultant or rock sample was mixed with 10 mL concentrated HCl and HNO 3 (1:1). Evaporate tell appearance of white residue, then dissolve in 10 mL water and evaporated again. After cooling, 50.0 mL of water was added and the solution was filtered into a 100 mL flask, brought to volume. Aliquots of 1.0-3.0 or 5.0 ml from an herb, spice and bean or rock solutions were used in spectrophotometric determination of iron by procedure (I). The absorbance of iron as [Fe(H 2 dapd) 2 ] 2+ complex was measured at 428 (523) nm using direct (first derivative) mode after standing 4 hr. The iron concentration was calculated using regression equations (Table 1).

RESULTS AND DISCUSSION
Effect of pH: The influence of pH on the formation of Fe(H 2 dapd) 2 ] 2+ ([Fe(Hapm) 3 ] 2+ ) complex in 10% (60%) v/v water-ethanol mixture was studied in the pH range from 0.5 to 12.0, Fig. 1 (a, b). Obviously, observance is constant and maximal at pH 2.0-3.0 (7.0-10.0) for Fe (II) -H 2 dapd (Fe (II) -Hapm) complex. So, pH 2.5 or 7.5 were selected for the iron (II) determination with H 2 dapd or Hapm reagents. Figure 2 (a) shows the influence of varying period times (30.0 sec to 12 hr) on the stability of 1.0x10 -3 M H 2 dapd at pH 2.5. Obviously, the absorbance at λ m 297 nm attained constant value from 30 sec to 4.0 hr with relative error less than 1.8%. On further increasing period times in 12 hr, the absorbance decreases slowly with relative error range 2.5-11.2%. After three days, the absorbance decreases rapidly with relative error 33%. The same result is also found in λ m 258 nm, suggesting that H 2 dapd reagent is stable within 4.0 hr. For this fact, it is advisable to prepare this reagent daily.

Oxidation-reduction Character of H 2 dapd:
The presence of ascorbic acid does not affect the Fe (II) -H 2 dapd complex, whereas changes occur in the presence of H 2 O 2 . This observation revealed that H 2 dapd reagent behaves as a reducing agent. To assess the ability and power of H 2 dapd as a reducing agent, a study was carried out with four standard solutions: Two solutions contain 2.8 and 5.6 µg mL¯1 Fe (II) with 18.3 µg mL¯1 H 2 dapd ; Fig 2 (d, e), While other two solutions contain 2.5 and 5.6 µg mL¯1 Fe(III) with 38.6 µg mL¯1 H 2 dapd ; Fig 2 (b,d), following the optimum condition of spectrophotometric procedure (I). The ratio between iron: reagent is about 1:10. The absorbance of Fe(II) or Fe(III) complexes at λ m 428 nm after allowed periods range 30 sec -12.0 hr were measured. Figure 2 (b,d) shows that the observance of Fe (III) system increases gradually with increasing time from 30 sec to 4.0 hr. With increasing time from 4 to 6.5 hr, the observance remains constant. At still increasing time, the absorbance decreases slowly. Figure 2(c,e) shows the rapid formation of the Fe(II)-complex indicating that Fe(II)complex is formed and it's stable for at least 8 hr but with increasing time to 12 hr, absorbance decreases slowly.   The results are illustrated in Fig. 3. Obviously, the absorbance values in Fe (III) system increase gradually with increasing time of reaction. In addition, the absorbance value of Fe (III) system will be fixed at time ranges from 4 to 6 hr. Results also show that the recovery of ferric reduction to ferrous ion with H 2 dapd are 73.6, 85.6, 91.6, 96.2, 99.1, 99.5 and 99.7 % at 30 Sec, 0.5, 1.0, 2.0, 3.5, 4.0 and 6.0 hr, respectively, assuming the standard value of absorbance for ferrous at 30 Sec. On the other hand, observance of iron in the ferrous system (curve h) at 30 Sec is analogous with absorbance in iron mixture at 4.0 and 6.0 hr (curves f and g). These observations reveal that H 2 dapd reagent reduced ferric ion completely to ferrous within period at least 4 hr without shift in λ m (428 nm). Also, ferrous ion in a binary mixture of ferric and ferrous ions could not be measured at 30 Sec, i.e. The reaction is instantaneous, resulting incomplete reduction process of ferric system.

Spectral Characteristics:
The iron (II) reacts rapidly within a minute with H 2 dapd and Hapm reagents forming. Stable red and orange complexes at pH 2.5 and 7.5, respectively. Figure 4 shows the direct and first derivative spectra of Fe (II) -H 2 dapd (curves a, á) and Fe-(II) -Hapm (curves b, b )  complex indirect spectra was explained by a pronounced reduction in the ligand field caused by the greater stress requirements of the two legends molecules. Two absorption bands were assigned to the 5 A 1 and 5 B 1 states derived from the splitting of 5 E spectroscopic term in the tetragonal ligand [18] . Maximum amplitude in the first derivative mode for di-(mono) complex appeared in 412, 439, 481 and 523 (480 and 555) NM.

Composition of the Complexes:
The continuous variations and molar ratio methods were applied to ascertain the stoichiometric composition of Fe (II) -H 2 dapd or Fe (II) -Hapm complex at pH 2.5 or 7.5. A 1:2 and 1:3 of the Fe (II) -H 2 dapd and Fe (II) -Hapm complexes were indicated, respectively. The ratios are in agreement with that found by analysis of solid complexes with the CHN elemental analyzer and IR.

Selection of Wavelength:
In order to select the best analytical signal for Fe(II)-H 2 dapd (Fe(II)-Hapm) complex, calibration graphs by direct or derivative modes were plotted at λ m 428 and 502 (524) nm or 412, 439, 481 and 523 (480 and 555), respectively. Linear equations (y= bx+a) of A or D signal against Fe (II) concentration for both complexes were listed in Table 1. Molar absorptivity in direct and derivative modes of Fe(II)-H 2 dapd (Fe(II)-Hapm) complexes were 8.481×10 3 (6.905x10 3 ) L mol¯1 cm¯1 at 428 and 524 nm and 217.6 (134) L mol¯1 cm¯1 at 523 (555) nm, respectively, suggesting that H 2 dapd ligand is the best one for determining iron(II). Miller [19] has indicated that if the r closes 1 or -1, the gentle calibration graph is obtained. Table 1 shows a slight deviation from ideal linear. This deviation is attributed to random experimental errors, which make difficult to select the best wavelength or pulse amplitude. Accordingly, ŷ and y-ŷ residuals are introduced. Y-ŷ represents the difference between the experimental y values and fitted ŷ values. By comparing ŷ-residual in direct and derivative modes for both irons-complexes, results show that low value in ŷ-residual appeared at λ m 428 and D at 523 nm for [Fe(H 2 dapd) 2 ] 2+ complex. This is confirmed graphically, on plotting individual yŷ-residuals vs. y signals (Fig. 5). Clearly, Fig. 5 (a, á) shows that the residuals should thus take if the unweighted regression line is a good model for the experimental spectra. There is a clear sequence of the positive residual, followed by sequence negative ones followed by a second positive sequence. The three numbers of runs give a significant data. While Fig. 5 (b,  b ) shows that, the regression line is inappropriate. Because the sign (+) or (-) of the residual is in random. Therefore, results indicate that H 2 dapd reagent is considered the best reagent for iron(II) determination at 428 and 523 nm with direct and first derivative, respectively, while iron(II) measurement with Hapm reagent at 524 (555) nm with direct (first derivative) is inappropriate.

Sensitivity, Detection Limits, Dynamic Range, Accuracy and Precision:
Analytical sensitivity (S A ) [20] , detection limit (y L ) and dynamic range [20] 3 ] 2+ ) was measured at λ m 428 and 523 NM is using direct and D at 524 and 555 using first derivative, respectively. The mean value and standard deviation for a procedure I or II were 2.85 (0.018 or 5.55 (0.021 using direct and 2.81 (0.021 or 5.72 (0.018 µg mL¯1 iron (II) using first derivative, while relative errors were 0.71 and 0.35 % for direct and 5.0 and 2.8% for the first derivative, respectively. Thus, Fe (H 2 dapd) 2 ] 2+ complex has the advantages of reasonable selectivity, good precision and low detection limit than Fe (Hapm) 3 ] 2+ complex.

Effect of Foreign Ions:
The effect of 24 foreign ions on the determination of 2.8 or 5.6 µg mL¯1 iron (II) with H 2 dapd or Hapm reagent were examined individually by measuring the absorbance and vertical distance under the recommended procedures I and II. Metal ions were added as Na + or K + while the anions was added as NO 3 ions. The tolerance limit was taken as the amount that caused 2.5% error. The results are given in Table 3. Tolerance limit of iron (II) as Fe (H 2 dapd) 2 ] 2+ in presence of Cu 2+ , Cr 3+ , Ni 2+ and Co 2+ , with direct and derivative modes is higher than that obtained with [Fe (Hapm) 3 ] 2+ complex. Derivative spectra improved the tolerance limit of iron (II) fold twice than the direct spectra. Figure 6 shows the influence of 0.152 (0.044) mg mL¯1 Cu 2+ , 0.155 (0.052) mg mL¯1 Cr 3+ , 0.017 (0.007) mg mL¯1 Ni 2+ and 0.017 (0.009) mg mL¯1 Co 2+ , an iron -(II) as [Fe (H 2 dapd) 2 ] 2+ ([Fe (Hapm) 3 ] 2+ ). Obviously, the amplitude signal in the first derivative (D) of iron (II) as [Fe (H 2 dapd) 2 ] 2+ complex was not affected with no significant difference in the presence of Cu 2+ , Cr 3+ , Ni 2+ and Co 2+ ions (Fig. 6á), while indirect mode, the observance of iron (II) -complex affected seriously with them ions (Fig. 6a), suggesting that Cu 2+ , Cr 3+ , Ni 2+ and Co 2+ , interfering ions are completely masked with derivative mode. In contrast, direct and derivative spectra of iron (II) as [Fe (Hapm) 3 ] 2+ could not prevent the deviation in A and D, respectively, as shown in (Fig. 6 b, b ). Thus, the first derivative mode is used successfully for the determination of iron (II) with the H2dapd reagent in presence Cu 2+ , Cr 3+ Ni 2+ and Co 2+ , ions. Although H 2 dapd reagent improved tolerance limits two folds with first derivative spectra, values of Ni 2+ and Co 2+ ions are still low (Table 3). This is possibly attributed to the formation of Ni 2+ dimmer complexes [Ni (H 2 dapd) 2 ] 2+ and [Ni (dapd) 2 ] 2+ and quite oxidation of Co 2+ to Co 3+ in presence of H 2 dapd ligand [21,12] . The absorbance of iron mixture was recorded under the optimum condition of procedure I (0.1M NaNO 3 , pH = 2.5 and 10% ethanol) after standing 4 hr at λ m 428 nm.    There is a good agreement between true and found concentration of iron. The recovery of measurements was found to be higher than 99.3%. Thus, H 2 dapd reagent can be recommended for the determination of total iron in a mixture containing ferrous and ferric mixture as [Fe(H 2 dapd) 2 ] +2 complex after an allowed period of 4 hr for complete reduction Fe(III) to Fe(II).

Determination of Total Iron in Outdoor Samples
Using H 2 dapd Reagent: The proposed procedure I was applied to determine iron in herb, spice, bean (rock) after wet ashing (wet digestion) of the samples. The results are listed in Table 5. The method based on the reduction of Fe(III)-complex to F(II)-complex with H 2 dapd reagent after standing for 4 hr. The A or D signals were measured at 428 or 523 nm, respectively against the blank solution. The results obtained were compared with that measured by AAS. There are agreements between the results obtained by direct and derivative modes and AAS. No significant differences are observed (F=1. 048 in direct mode-AAS and F=1. 032 in derivative mode-AAS). The relative error was found to be less than 5%. Thus, H 2 dapd reagent can be used successfully to determine total iron as [Fe (H 2 dapd) 2 ] 2+ complex in the majority of wet ashing herb, spice and bean or wet digested rock samples without reducing agent. A comparison between present (average of three values) and published results [22] ; that investigated the amount of iron in kakade (10.62 vs. 6.57 mg g¯1) and black pepper (8.32 vs. 3.9 mg g¯1) is higher whereas anise (3.95 vs.7.67 mg g¯1) and cumin (7.75 vs.8.28 mg g¯1) is lower than in the published results. Higher concentration may be possibly attributed to contaminated samples, which comes from the irrigation with contaminated water and addition of fertilizer and/or herbicides to soil.

CONCLUSION
One of the most important aspects of the present work is the simple, rapid method for spectrophotometric determination of ferrous and ferric individually or a mixture of ferrous and ferric as [Fe (H 2 dapd) 2 ] 2+ complex in acidic water-ethanol medium (pH 2.5). H 2 dapd reagent has the ability to react instantaneously with ferrous whereas ferric within a period of 4.0 hr. The sensitivity and selectivity of the method were improved with first derivative. Comparing the present procedure with the DCDT reagent before or after extraction into amyl alcohol concerning detection and tolerance limits for Cu 2+ , Cr 3+ , Ni 2+ and Co 2+ indicated that H 2 dapd is sensitive and selective (except for Cu 2+ ). On comparing one method with existing flow injection analysis methods, indicates that the use is readily available, relatively inexpensive apparatus and simplicity of the experimental conditions make the proposed method a good alternative for routine analysis. Recommended method is economical since it is cheaper than AAS and does not need any gas maintenance. The method has been successfully used for determining total iron directly in digesting foodstuffs, herbs, spices and rock solution as [Fe (H 2 dapd) 2 ] 2+ in the absence of reducing agents.