Research Article

THE ANALYTICAL APPLICATION OF CLOUD POINT EXTRACTION: A SIMPLE SPECTROPHOTOMETRIC METHOD FORSELENIUM DETERMINATION IN KANGKONG

(IPOMOEA REPTANS P.)

YASINDA OKTARIZA, OKKY DWICHANDRA PUTRA*, MUHAMAD INSANU, AMIR MUSADAD MIFTAH

School of Pharmacy, Bandung Institute of Technology,Jalan Ganesa No 10, Bandung 40132, Indonesia. Email: dwichandraputra@fa.itb.ac.id

Received: 31 July 2013, Revised and Accepted: 01 Sep 2013

ABSTRACT

Objective: The aim of the study was to develop a simple spectrophotometric method to quantify Selenium in kangkong (Ipomoea reptans P.).

Method: The method was based on complex formation between Selenium and dithizone with cloud point extraction using Triton X-100 to simplify the technique.

Result: Optimum condition was established with using of 45 µmol L-1for dihizone, 0.4 M for HCl, 0.4% w/v for Triton X-100, and 15 minutes for incubation time. Under optimum condition, the response was linear between 10 and 110 ppb with correlation coefficient 0.998 and in all cases relative standard deviation was less than 5%. Limit of detection and limit of quantification was found 6.634 and 20.104 ppb respectively. The method was successfully applied to determination of Selenium with recovery 92.833 %.

Conclusion: A simple spectrophotometric method has been developed using dithizone as chromogenic agent and cloud point extraction to simplify the technique. The analytical performance full filled the requirement for validation method.

Keywords: Selenium, Dithizone, Kangkong and Spectrophometric.

INTRODUCTION

Selenium (Se) has been recognized as one of the nutrients for plant, animal and human [1]. In human, Se has also known as antioxidant in diet with mechanism of preventing oxidative stress [2]. In other hands, essential elements such as Se can become toxic at high concentration. Because the range between essential and toxic concentration is narrow, Se can be considered as the toxic compound which is needed for living organisms [3-4]. Se was found in the environment and biological materials. In environment, Se is naturally distributed in soil, water and air in very low concentration (≤ 1 µgg-1) [5-6]. Meanwhile, the broad applications of technologies such as electronic, glass, pharmaceutical, and chemical industries increased the prevalence of Se as a pollutant. A plant which grows in exposed environment can accumulate Se in leaves, roots and blades [7-9].

The high concentration in environment has a role of causing Se accumulation and thereby enter to food chain. Edible vegetable such as kangkong (Ipomea reptans P.) may contain Se which came from contaminated water and soil. Kangkong which is from family convolvulaceae is important vegetable in Indonesia, Malaysia, Thailand and some regions of China where it is intensively grown and eaten[10]. Consumption of kangkong may increase risk toxicity of Se to human. Therefore, it is necessary to quantify Se in kangkong for risk analysis.

The most widely analytical method for determining Se are instrumental neutron activation analysis (INAA), atomic absorption spectrometry (AAS), atomic emission spectrometry (AES), atomic fluorescence spectroscopy (AFS), inductive couple plasma-mass spectrometry (ICP-MS), high performance liquid chromatography (HPLC), voltammetry, and spectrophotometric [11-20]. Among those methods, spectrophotometric is preferable and popular because of its simplicity. Complex formation between metal ions and ligands can be utilized for development of analytical method [21-22]. One of the popular of spectrophotometric determinations of Se is based complex formation between Se and dithizone (H2DZ) to form Se-dithizonate (Se(HDZ)4) complex. It is soluble in organic solvent whereas Se and H2DZ are soluble in aqueous solvent [19-20]. The application of liquid-liquid extraction (LLE) was developed before to measure this complex. Nevertheless, some shortcomings of this method were emulsion formation, the using of toxic organic solvent,

large volume of sample and solvent made LLE time consuming, expensive and environmentally unfriendly [23].

Modern trends in analytical chemistry are toward simplification and miniaturization of sample preparation. Cloud point extraction (CPE) is the development of conventional LLE where the micelles mediated extraction is generated to miniaturize and simplify extraction process [24]. CPE was firstly developed to extract organic soluble metal complex which is similar to Se-dithizonate complex [25]. The cloud point is the temperature which aqueous solution of surfactant (nonionic and zwitterionic) become turbid [25-26]. The technique is based on the property of surfactant in aqueous solution to form micelles and to separate into surfactant-rich phase of small volume and diluted aqueous phase when put at cloud point condition [27-28]. Triton X-100 was used in CPE for analyzing Se in tablet and shampoo by complex formation with H2DZ at 45oC for cloud point temperature [29]. Meanwhile, our preliminary study showed that heating affected stability of complex Se-dithizonate in plant matrices and decreased absorption value of complex mixture.

Because the application of LLE has several weaknesses, CPE was used to overcome these problems. The aim of this research wasto develop an alternative method for quantifying Se in kangkong (I. reptans P.) with H2DZ as chromogenic agent.

MATERIALS AND METHODS

Materials

Amanda seeds of I. reptansP. and soil werepurchased fromToko Tani Sugih, Bandung, Indonesia. Selenium metal was obtained from May and Baker. Triton X-100 was supplied by Promega. Na2SeO3, HNO3(65%), dithizone, NaOH, ascorbic acid, HCl(37%), methanol, phenol, H2SO4 (95-97%), H2O2(30%) were obtained from Merck. All reagents were analytical grade.Deionized water was from IKA Pharmaceutical and it was used for preparing all solutions.

Instruments and Apparatus

A Beckman Coulter DU 720 UV-Visible spectrophotometer with 1.00 cm quartz cells was used for all absorbance measurements. A Hettich centrifuge EBA 20 was used for separation surfactant-rich phase and aqueous phase. Measurments of pH were made with a Mettler Toledo Seven Easy pH meter. Hotplate IKA RH Basic KT/C

International Journal of Pharmacy and Pharmaceutical Sciences

ISSN- 0975-1491 Vol 5, Issue 4, 2013

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with thermostat was used.All glass apparatus which is common in laboratory was also used in this experiment.

Preparation of solution

The stock solution of Se (IV) (1000 ppm) was prepared by dissolving 100 mg of Se metal in hot concentrated nitric acid and the volume was adjusted to 100 mL in volumetric flask with deionized water. The stock solution of Se was diluted with deionized water to obtain various concentrations of 10, 30, 50, 70, 90, 110 ppbof selenium (IV). The stock solution of H2DZ (10-3 molL-1) was prepared by dissolving 12.8 mg in 5 mL NaOH 0.1 N (containing 50 mg of ascorbic acid as stabilizing agent) by heating in water bath while the volume was adjusted to 50 mL using deionized water. The stock solution of Triton X-100 and phenol (4 % w/v) was prepared by dissolving 4 g Triton X-100 and phenol separately in water and the volume was adjusted to 100 mL with deionized water.

Procedure

Each level concentration of Se(IV) was made as an aliquot of 10 mL solution containing H2DZ (45 µmol L-1), HCl (0.4 M), and Triton X-100 (0.4% w/v). All reagents were optimized to know which concentration that gave a maximum absorbance. The solutions were kept for 15 minutes until the color development was completed. Cloud point was induced by adding phenol (0.8% w/v) to the sample solution. Centrifugation was used at 3500 rpm for 15 minutes.The surfactant-rich phase was added up to 5 mL by adding methanol and measured at 417 and 592 nm, against methanol as blank solution. The same procedure was applied for preparation of H2DZ solution (without Se) for correcting the absorbance between complex and dithizon which were overlapping.

Application toKangkong (I. reptans P.)

Thisexperiment used 4 different concentrations of added Se (0,1,2, and 4 ppm). In the pot eight seeds of kangkong was cultivated. Se was applied into the pot containing 2.5 kg soil as Na2SeO3 after dissolving in deinoized water. Nitrogen, phospor, and potassium as the three major fertilizer elements were used for growth development of the plants.The plants were harvested after 50 days and they were sliced until finely pounded. The destruction of sample was carried out by adding 5 mL concentrated HNO3 into 1 g of sliced plant. Sample was heated at 175oC for 40 minutes and the temperature was decreased into 150oC for 90 minutes. After the sample was cooled down in room temperature, 1 mL of concentrated HNO3 and 5 mL concentrated H2SO4 were added and then heated at 175oC for 60 minutes. After it was cooled, 1-2 mL of concentrated H2O2 was addeddropwisely and then it was heated at 140oC for 10 minutes. After the sample was cooled down, 8 mL of HCl was added and the volume was adjusted to 25 mL in volumetric flask with deionized water.

RESULT AND DISCUSSION

Optimization of Analytical Parameters

Se (IV) reacted with H2DZ to form hydrophobic complex Se-dithizonate (Se(HDZ)4) which were soluble in organic solvent or micelles of surfactant. The solubility of complex in micelles caused separation from aqueous phase after centrifugation. Our preliminary study showed that temperature affected the stability of complex formation, it was indicated by the decreasing of absorbance. Thus, in our experiments, heating was not used. Fig 1 showed the absorption spectra of H2DZ and Se(HDZ)4 in surfactant-rich phase against metanol as blank solution.The maximum absorption of complex was 417 nm and maximum absorptions of dithizone were 423 and 592 nm. Since the spectra were overlapping, the absorbance should be corrected to overcome the problem based on following equation:

𝐴𝑐𝑜𝑟𝑟 = 𝐴417 − 𝐴417𝐴592

𝐿

× 𝐴592

Where A417 and A592were the absorbance in 417 and 592 nm. (A417/A592)L is the absorbance of H2DZ in 417 and 592 nm with assumption the complex did not absorb at 592 nm. This equation is

used in optimization, validation and application of analytical method.

Fig. 1: The absorption spectra of (a) Se(HDZ)4 complex and (b) H2DZ in methanol (conditions of measurement:0.4 M HCl , 0.4 %

w/v Triton , 45 µmol L-1 H2DZ, and 110 ppm Se(IV)).

Different experimental parameters were extensively studied in order to get optimum condition of complex formation. Concentration of H2DZ was the first parameter which was optimized. As shown in Fig 2, the absorbance was increased following H2DZ concentration. Then, after optimum concentration, the absorbance was decreased. The phenomenon indicated that the complete formation complex was achieved and 45 µmol L-1H2DZ was selected for further step.

The second parameter was Triton X-100 concentration. It was necessary to be optimized because it would determine the number of complex that was trapped in surfactant-rich phase. Triton X-100 is non-ionic surfactant and popular in CPE due to the easy modification of cloud point. The origin cloud point of Triton X-100 is 63.7oC. If 0.8 % w/v phenol solution was added, cloud point was changed into 25oC[29]. Different concentrations of Triton X-100 were used to observe their effect to the absorbance. As shown in Fig 3, low concentration of Triton X-100 resulted low absorbance. It might indicate that at low concentration, the number of the micelles were not adequate to trap hydrophobic complex of Se(HDZ)4. After peak point, the addition of Triton decreased the absorbance. This phenomenon caused by H2DZ which was slightly soluble in organic phase that was also trapped in the micelle. Then, 0.4 % w/v was selected as the optimized concentration optimum of Triton X-100 and was used for further analysis.

Fig. 2: Effects of H2DZ concentration on corrected absorption (condition: 150 ppb Se(IV), 0.6 % w/v Triton X-100, 0.35M HCl,

and 15 minutes equilibration time).

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

350 450 550 650 750

Ab

sorb

an

ce

Wavelength (nm)

a

b

0.000

0.200

0.400

0.600

0.800

30 35 40 45 50 55

Aco

rr

Concentration of H2DZ (μmol L-1)

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Fig. 3: Effects of Triton X-100 concentration on corrected absorption (condition: 150 ppb Se(IV), 45 µmol L-1, 0.35 M HCl,

and 15 minutes equilibration time).

Fig. 4: Effects of HCl concentration on corrected absorption (condition: 150 ppb Se(IV), 45 µmol L-1, 0.4 % w/v Triton X-100

, and 15 minutes equilibration time).

Fig. 5: Effects of incubation time on corrected absorption (condition: 150 ppb Se(IV), 45 µmol L-1, 0.4 % w/v Triton X-100

, and 0.4 M HCl).

Acidity plays important role in where the complex formation involve weak acid or base as chelating agent. After peak point, the absorbance decreased and showed plateau response(Fig 4). The plausible reason was the stability of the complex decreasing at higher pHs. Hence, 0.4 M of HCl was selected as the optimum point and used in further analysis.

At this research, incubation time was the only parameter in reaction condition. As shown in Fig 5, the optimum point of incubation time was 15 minutes. If the incubation time was shorter than 15 minutes,

the lower absorbance was present. This was caused by incomplete complex formation. If the incubation time was longer than 15 minutes, lower absorbance was detected. It might be caused by the decreasing stability of complex of Se(HDZ)4.

Analytical Performances

The analytical performance parameters were conducted at optimum conditions. The results were summarized in Table 1, they showed that the response was linear between 10 and 110ppb with correlation coefficient and relative standard deviation (RSD) were0.998 and 3.351 %. The accuracy was determined by spiked method which recovery was92.833 %. Respectively, limit of detection (LOD) and limit of quantification (LOQ) was found to be6.634and 20.104ppb.

Table 1: The analytical parameter of Se determination in kangkong (I. reptans P.) using dithizone as chromogenic agent

with CPE using Triton X-100

Parameter Result Range (ppb) 10-110 λmax of Se(HDZ)4 (nm) 417 Molar absorptivity (L mol-1 cm-1) 8.753 × 103 LOD (ppb) 6.634 LOQ (ppb) 20.104 Recovery (%) 92.833 Linnearity b a Sy/x Vx0 (%) R

0.005 0.254 0.010 3.351 0.998

RSD (%) Intra day Inter day

4.343 3.504

Application to Real Samples

The treatment of kangkong was firstly applied by addition selenium with 4 level concentrations which were 0, 1, 2, and 4 ppm for 18 times. The results of analysis wasshown in Table 2. The result showed that I. reptans P. could accumulate selenium at very high levels (hundred micrograms per gram dry weight) that are probably phytotoxic. The content of Se in plants was decreased at level of 4 ppm added. It might be caused by the competition from other ions which came from soil and fertilizer[30].

Table 2: The results of selenium determination in kangkong (I. reptans P.)(water content 90%) using proposed method

Conc. of added Se (ppm)

Total of added Se(IV) (µg/g soil)

Total Se(IV) conc. in plant (µg/g dry weight)

0 1 2 4

0 0.45 0.90 1.80

58.250 461.263 744.673 371.318

HNO3 and H2SO4 is strong acid that will accelerate digestion process and dissolve the formed ash. The addition of H2O2 had a purpose to complete digestion process which indicated by formation of clear solution. In the other hands, the addition of H2O2 would cause the oxidation of Se (IV) to Se (VI). Because the existence of Se (VI) which was unwanted in complex formation, addition of HCl was necessary to transform Se (VI) to Se (IV).

CONCLUSION

The methods for quantifying Se in kangkong (I. reptans P.) was conducted at optimum conditions which were45 µmol L-

1forH2DZ,0.4 M forHCl,0.4% w/vfor Triton X-100, and 15 minutes for incubation time. The analytical performance of this method has been appropriate with standard requirement for validation method.This method should be applicable to routine analysis for plant materials.

0.000

0.200

0.400

0.600

0.800

0.00 0.50 1.00

Aco

rr

Concentration of Triton X-100 (%

w/v)

0.000

0.200

0.400

0.600

0.800

0.3 0.5 0.7 0.9

Aco

rr

Concentration of HCl (M)

0.000

0.200

0.400

0.600

0.800

5 10 15 20 25 30

Aco

rr

Incubation Time (minutes)

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