Simultaneous Ultraviolet-visible (uv–vis) Spectrophotometric Quantitative Determination of Pb, Hg, Cd, as and Ni Ions in Aqueous Solutions Using Cyanidin as a Chromogenic Reagent

The use of cyanidin (3, 3 1 4 1 5, 7 – pentahydroxyflavylium chloride) extracted from a tropical plant Hibiscus sabradiffa L. as a chelating reagent for simultaneous spectrophotometric determinations of ions of Pb, Hg, Cd, As and Ni in mixed aqueous solution is reported. The purified extract of the dried calyces of the Roselle plant was characterized and the results compared with literature values. Complexation of the metals ions with cyanidin and scanning through 200-700 nm in a UV –VIS spectrophotometer gave the wavelength of maximum absorption (λ max) of these metal complexes to be complexes respectively indicating appreciable bathochromic shifts compared to pure cyanidin absorption at 283.2 nm. The effect of pH on the determinations was studied and a pH value of 5 was found to be the optimal. Calibration curve plots of the complexes showed linearity between concentration of 0.1 to 5.0 ppm. This method offer cheap, simple, rapid, sensitive, and eco-friendly technique for simultaneous determination of trace heavy metals in mixed aqueous solutions and have potentials for environmental and biological samples. INTRODUCTION Determination of trace metals is of interest because while some are essential nutrients some others are toxic. Metals like zinc, manganese, copper, chromium, iron and cobalt are essential trace elements for humans, animals and plants; but become toxic if the homeostatic mechanisms maintaining their physiological limit are disrupted. On the other hand, lead, cadmium, nickel, arsenic and mercury etc are toxic even at low levels. The need to estimate the levels of these metals in materials or samples have increased tremendously after reports on different roles they play in human health and disease. Numerous analytical techniques exist in literature for use in trace heavy metals assay in mixed solutions. Among the methods, atomic absorption spectrophotometry (AAS) is well recognized because of its attractive features such as sensitivity, reliability, versatility, accuracy and precision (Okoye, 2005; Khamms et al., 2009). However, the use of AAS is limited by the need for high technical skills, huge capital and maintenance cost (Strong and Martin, 1990). Furthermore determinations of metals like arsenic and mercury pose health risk to the analyst using AAS. Some other methods employed in simultaneous determination of metals include polarography, voltammetry, inductively coupled plasma-mass spectroscopy (ICP–MS), inductively coupled plasma-atomic emission spectroscopy (ICP–AES), liquid chromatography (LC) etc (Rouhollahi et al., 2007). Like AAS, these techniques require very costly equipment and reagents that are not easily available in …


INTRODUCTION
Determination of trace metals is of interest because while some are essential nutrients some others are toxic.Metals like zinc, manganese, copper, chromium, iron and cobalt are essential trace elements for humans, animals and plants; but become toxic if the homeostatic mechanisms maintaining their physiological limit are disrupted.On the other hand, lead, cadmium, nickel, arsenic and mercury etc are toxic even at low levels.The need to estimate the levels of these metals in materials or samples have increased tremendously after reports on different roles they play in human health and disease.Numerous analytical techniques exist in literature for use in trace heavy metals assay in mixed solutions.Among the methods, atomic absorption spectrophotometry (AAS) is well recognized because of its attractive features such as sensitivity, reliability, versatility, accuracy and precision (Okoye, 2005;Khamms et al., 2009).However, the use of AAS is limited by the need for high technical skills, huge capital and maintenance cost (Strong and Martin, 1990).Furthermore determinations of metals like arsenic and mercury pose health risk to the analyst using AAS.
Some other methods employed in simultaneous determination of metals include polarography, voltammetry, inductively coupled plasma-mass spectroscopy (ICP-MS), inductively coupled plasmaatomic emission spectroscopy (ICP-AES), liquid chromatography (LC) etc (Rouhollahi et al., 2007).Like AAS, these techniques require very costly equipment and reagents that are not easily available in poor nations of the world coupled with very specialized skills needed.Also, most of the reagents are not eco friendly.The use of UV-VIS spectrophotometry in determination of heavy metals in samples is becoming popular in many laboratories because it provides for easy, simple and rapid determination in low to high concentrations at cheap cost (Soomro et al., 2008).Simultaneous determination of metal ions using UV-VIS methods was reported to be difficult without separation due to overlap absorption spectra (Nai-Liang et al., 2005).The problems associated with spectroscopic determination of these trace heavy metals are complicated by the nature of the photochromic or chromogenic ligands which are mainly synthetic and are toxic in the environment.Some of the reported simultaneous determination of metals using UV spectrophotometry were carried out using toxic and expensive reagent (Rouhollahi et al., 2007;Kachbi, 2010;Naguraja et al., 2009).Other works in literature use specific ligands for specific metals at even different pH values for multi-elements determinations.
This study reports the simultaneous determination of some trace heavy metals by UV-VIS spectrophotometry using cyaniding as a chelating reagent.We relied on the shifts of maximum absorption wavelength (λ max ) of cyanidin after complexation with the metals.The objective of the work was to develop a rapid, sensitive, specific and simple method for simultaneous determination of metals in environmental and industrial samples.

Equipment and reagents
Salts of Pb, Cd, Hg, Ni and oxide of Arsenic were purchased from Riedel -de Haen, Germany in analytical grade.Jenway (6405model) Spectrophotometer and Jenway (3015-model) pH meter were used for absorbance and pH measurement respectively.The chemicals used to prepare buffer solutions were also of analytical grade and deionized water was used.All glass wares were first washed with detergents and cupiouslyrinsed with deionized water.

Cyanidin extraction
Cyanidin was extracted from calyces of Roselle plant (Hibiscus sabdariffa L.) according to the method of Ukwueze et al. (2009).A 500 g of dry calyces were ground to powder and macerated in 2.5 L methanol: HCl mixture (85:15% v/v) for 72 h and filtered.The filtrate was concentrated to 500 and 100 ml of conc.HCl was added and Okoye et al. 99 content was refluxed for 2 h.The solution was then put in a beaker and cooled in a refrigerator until crystals settled out.The crystals were filtered out under suction and re-crystallized from hot methanol, air dried and weighed.

Preparation of buffer solutions
Buffer solutions of pH 1 to 8 were prepared in accordance with methods described earlier using KCl, HCl, KHC 8 H 4 O 4, KH 2 PO 4 in varying concentrations and mixings (Lange, 1973;Meities, 1963).

Determination of λ max of cyanidin
5% cyanidin solution was prepared by dissolving 5 g of the purified crystals in methanol containing 0.01% conc.HCl and made up to 100 cm 3 in a standard flask. 1 cm 3 of this solution was diluted to 10 cm 3 and its λ max was determined by scanning from 200-700 nm using Jenway (6405 model) spectrophotometer in a 1 cm 3 cuvette.

Preparation of stock and working solutions of metals
Stock solutions (1000 ppm) of Pb, Cd, Hg, Ni, and As were prepared from lead nitrate, cadmium, mercury and nickel chlorides and arsenic oxide.The solutions were serially diluted to the required working standards.2.5 cm 3 of each stock solution was diluted in a 250 cm 3 standard flask to give 10 ppm solution.

Determination of wavelength of maximum absorption (λ max ) of metalcyanidin complexes
5 cm 3 metal solutions (10 ppm) were diluted with 5 cm 3 of the 5% cyanidin solution and the wavelength of absorption was scanned from 200 to 700 nm in a 1 cm 3 cuvette using the spectrophotometer.

Determination of optimum pH (pH opt ) of metal-cyanidin complexes
Each of the 8 beakers containing 5 cm 3 of 5% cyanidin in methanol were added 5 cm 3 of standard solutions of one of the studied metals.50 cm 3 of the solution in each beaker was adjusted to a given pH from 1 to 8 using the buffer solutions prepared.The absorbance of each solution was read at the λ max of the analyte metal at the adjusted pH.

Simultaneous determination of various metalcyanidin complexes at pH (opt)
Working standard solutions from 1.0 -9.0 ppm of Pb(II); Cd(II); Hg(II), As(III) and Ni(II) solutions were prepared from the 10 ppm solution of each metal by diluting appropriately.These were used to plot a calibration graph for each metal-cyanidin complex by determining the absorbance at λ max of 5 cm 3 of standard solution diluted by 5 cm 3 of 5% cyanidin solution in a 1 cm 3 cuvette at pH (opt) determined.5 cm 3 of 1.0 ppm mixed standard solution was diluted with 5 cm 3 of 5% cyanidin chloride solution adjusted to pH opt and the absorbance read at the λmax of each metalcyanidin complex.Similar procedures were repeated for 2-9 ppm mixed metal solutions.

Cyanidin extract characterization
The extracted cyanidin was found to conform to the characteristics earlier reported by Ukwueze and others (2009).Table 1 shows the R f values of thin layer and paper chromatographs (TLC and PC) obtained in various solvents compared to literature values.Both experimental and literature R f values are very close.The spectral data obtained from the analysis of the extract in comparison with literature values are shown in Table 2.The R f values and λ max's of the extract showed no significant variation from the authentic literature values (Harborne, 1958).

Effects of the metal ions on the absorption wavelength of cyanidin
The result of the investigation of the effects of the metal ions on the λ max of cyanidin is shown in Table 3. From the table, all the metals caused a marked bathochromic shift in cyanidin absorbance wavelength in the UV region.Shifts in VIS region are very small causing an overlap in absorbances when plotted.The simultaneous determination of these metals can be measured with satisfaction in the UV region.The affinity of metal ions for ligands is controlled by size, charge and electronegativity.These metals are highly polarisable, have lower charge density, large ionic size and their d-orbitals are available for πbonding and due to these, they form covalent complexes showing absorption in the UV region.

Selection of pH optimum at λmax of metal ions
The effect of pH on the formation of metal-cyaniding complexes was studied at different pH values in methanol and the result shown in Figure 1.
From the graph, Cdcyanidin complex has pH = 3 as the optimum pH; Pbcyanidin has pH = 4; Hgcyanidin has pH = 5; Ascyanidin has pH = 2 and Nicyanidin has pH = 6.At these various pH values, these metalcyaniding complexes showed reproducible results.However, all the metal complexes showed prominent absorbances at pH = 5.As a midway or compromise between selectivity and sensitivity of the developed method, pH value of 5 was chosen as the optimum pH for the simultaneous determination of these metals complexes with cyanidin in aqueous media.

Simultaneous determination of the metal ions
At the optimal pH value, the UV spectra of the mixed metal ionscyanidin complexes was studied for possible spectral overlaps or interferences.The results are shown in Table 4.
From the table, only Pb(II), Hg(II) and As(III) were absorbed at their original λ max .It was noted that Cd(II) showed no peaks which could be attributed to the closeness of its λ max value to As(III).The absorption   intensity of As(III) was greatly enhanced in the spectra.Ni(II) absorbed at higher wavelength.Such interferences might result from the formation of mixed oxides or from suppression of ionized gaseous metals pressures which occur in the molecular environment adjacent to the chromophore.

Linear detection range of metalcyanidin complexes
Absorbance of the five complexes at various concentrations at various λ max's and optimum pH (pH = 5) were investigated and the result was shown in Figure 2. The calibration curves obtained showed linearity between concentration ranges of 1.0-10.0ppm.At this range, Beer-Lambart law was obeyed.

Conclusion
Complexation of cyanidin simultaneously with Pb, Hg, Cd, As and Ni ions markedly altered the wavelength of absorption of the cyanidin and this phenomenon can be utilized in spectrophotometric determination of these metal ions.The complexes were stable and yielded reproducible results at pH = 5.It was found that all the metal ions determined show linearity at concentration range of 1.0 to 10.0 ppm indicating that at these concentrations, UV spectroscopy can conveniently be used for their simultaneous determination.This offers a simple, rapid, sensitive and cheap method that promotes the spirit of green chemistry in chemical quantitative analysis.It has potentials in evaluation of trace heavy metals in environmental, biological and food samples.

Figure 1 .
Figure 1.pH variation at λ max of absorption by metal-cyanidin complexes.

Figure 2 .
Figure 2. Mixed graph of Abs vs. Conc.Of the metal complexes.

Table 1 .
R f values (X 100) of TLC and PC of cyaniding in various solvents.

Table 2 .
Spectral results of Cyanidin extract compared with literature values.

Table 3 .
Effect of metal ions on wavelength of absorption of cyaniding.

Table 4 .
λmax for simultaneous determination of mixed metalcyanidin complexes.