Solid phase extraction and spectrophotometric determination of cobalt in environmental samples

Weng Zhehui, Jia Zhiruo, Dong Xuechang^# , Miao Mingming, Hu Qiufen^#
(Yunnan Academy of Tobacco Science, Kunming, Yunnan 650106; ^#School of Chemistry and Bioscience, Yunnan University of Nationality, Kunming, Yunnan 650031 )

1 INTRODUCTION
Cobalt is an element both important for industry and biological systems. In rapidly expanding the analytical fields such as environmental, biological and material monitoring of trace metals, there is an increasing need to develop simple, sensitive and selective analytical techniques that do not use expensive or complicated test equipment. Many sensitive instruments, such as spectrofluorimetry, X-ray fluorescence spectrometry, neutron activation analysis, atomic absorption spectrometry, chemiluminescence, and the like have widely been applied to the determination of cobalt.^[1-7] But the spectrophotometric method still has the advantages for which is simple and do not need expensive or complicated test equipment. For this reason, a wide variety of spectrophotometric methods for the determination of cobalt have been developed. The main chromogenic reagents are pyridylazo reagents, thiazolylazo reagents, benzothiazolylazo reagents, 8-aminoquinoline derivatives, porphyrins, nitroso dyes and the similar.^[8-24]
In the previous works, some 2-quinolylazo-phenol reagents were reported for the determination of metal ions.^[25-30] This kind of reagent has a higher sensitivity than pyridylazo reagents due to its larger conjugated system. However, the 2-quinolylazo-phenol reagent has also a disadvantage of its poor selectivity because both the oxygen atoms and nitrogen atoms donate elections to the metal ions. To select a more sensitive and selective reagent, in this work, a compound 2-(2-quinolinylazo)-5- dimethylaminoaniline (QADMAA) was synthesized, and its color reaction with cobalt was studied. This reagent has higher selectivity than that of 2-quinolylazo-phenol reagents because of it has only donating elections to metal ions.
The routine spectrophotometric methods are often not sensitive enough to determine low concentration of cobalt ion in environmental samples, as the cobalt concentration existing only at mg/L level. Consequently, a preconcentration step is usually required. Solid phase extraction is an attractive technique because of its notable advantages.^[31-36] In this paper, based on the color reaction of QADMAA with cobalt and combining with the solid phase extraction of the colored chelate with C18 disks, a highly sensitive, selective and rapid method for the determination of cobalt in environmental samples was developed.

2 EXPERIMENTAL
2.1 Synthesis of QADMAA
2-Aminoquinoline (6.9 g) was dissolved in 500 mL anhydrous ethanol. To which, sodamide (2.0 g) was added and the mixture was refluxed in a boiling water bath for 5 h, followed by the addition of isoamyl nitrite (7.4 mL). The solution was refluxed for 30 min with boiling water bath. The solution was cooled and placed over night under 0°C. The diazo salt was obtained by filtering this solution with an isolation yield of 95%. The diazo salt was dissolved in 200 mL anhydrous ethanol, followed by the addition of m-dimethylaminoaniline (5.7g; 0.042 mol). The carbon dioxide was ventilated into the solution with stirring until the pH reaches to about 8.0. The solution was placed for two days, and evaporated to dryness. The residue was re-crystallized with 30% ethanol. QADMAA was obtained with 36% yield. The structure of QADMAA was verified by elemental analysis, IR (Fig.1), ¹HNMR (Fig.2), and MS (Fig.3). Elemental analysis: C₁₇H₁₇N₅ found (calculated) C 69.82 (70.08), N 23.83 (24.04), H 6.04 (5.88). All these show that the QADMAA has the structure in Fig.4.

Fig. 1 The infrared spectrum of QADMAA

Fig. 2 The ¹H nuclear magnetic resonance spectrum of QADMAA

Fig. 3 The mass spectrum of QADMAA

Fig. 4 The structure of QADMAA

2.2 Experimental Apparatus
A UV-160A spectrophotometer (Shimidzu, Japanese) equipped with a 1 cm microcells (0.5 ml) was used for all absorbance measurements. The pH values were determined with a Beckman F-200 pH meter. The extraction was performed on Waters Solid Phase Extraction (SPE) device (which can prepare twenty samples simultaneously), and Zorbax C₁₈ membrane disks [47 mm (diameter)× 0.5 mm (thickness), 8 mm, 50 mg] (Agilent Technologies, USA) was used.
2.3 Reagents
All of the solutions were prepared with ultra-pure water obtained from a Milli-Q50 SP Reagent Water System (Millipore Corporation, USA). Isopentyl alcohol (Fisher Corporation, USA) was used. QADMAA solution (5×10^-4 mol/l) was prepared by dissolving QADMAA with 95% of ethanol. A stock standard solution of cobalt (1.0 mg/mL) was obtained from Chinese Standard Center, and a work solution of 1.0 m g/mL was prepared by diluting this solution. The buffer solution of 0.5 mol.L^-1 acetic acid-sodium acetate (containing 5% of Zn-EGTA and 5% of NaF) was prepared by dissolving 30 g acetic acid, 50 g sodium fluoride and 50 g glycoletherdiamine tetraacetic acid zinc salt (Zn-EGTA) in 600 mL of water, then adjusted pH to 5.5 with sodium hydroxide solution and diluted to volume of 1000 mL with water. Cetyl trimethylammonium bromide (CTMAB) solution: 1.0%, dissolving with 10% of ethanol. All chemical used were of analytical grade unless otherwise stated.
2.4 General procedure
To a standard or sample solution containing no more than 2.4 m g of Co(II) in a 200 mL of calibrated flask, 10 mL of 0.5 mol/L acetic acid-sodium acetate buffer solution (containing 5% of Zn-EGTA and 5% of NaF) with pH 5.5, 6 mL of 5×10^-4 mol/L QADMAA solution and 5.0 mL of 1.0 % CTMAB solution were added. The mixture was diluted to the volume of 200 mL and mixed well. After 10 min, the solution was passed through the C₁₈ disks at a flow rate of 50 mL/min. After the enrichment had finished, the retained chelates were eluted from the disks with 4 mL of isopentyl alcohol at a flow rate of 5 mL/min. The absorbance of the eluant was measured in a 1 cm cell at 616 nm against a reagent blank prepared in a similar way without cobalt.

3 RESULTS AND DISCUSSION
3.1 Absorption Spectra
The absorption spectra of QADMAA and its Co(II) complex in isopentyl alcohol medium are shown in Fig.5. The absorption peaks of QADMAA and its complex are located at 462 nm and 616 nm.

Fig.5 Absorption spectra of QADMAA and its Co(II) complex: (1) QADMAA-CTMAB blank against water; (2) QADMAA-Co(II)-CTMAB complex against reagent blank		Fig.6 Effect of pH on the formation of Co(II) complex, Co(II) concentration was 5.0×10-6 mol.l-1, other conditions as general procedure.

3.2 Effect of Acidity
Results showed that the optimal pH (Fig.6) for the reaction of Co with QADMAA is 4.2- 6.8. A pH 5.5 acetic acid-sodium acetate buffer solution was recommended to control pH. The use of 6-15 mL of buffer solution per 25 mL was found to give a maximum and constant absorbance. So 10 mL of buffer solution was recommended. In this work, the routine ions Ni(II), Cu(II), Zn(II) and Fe(III) react with QADMAA to cause a serious positive interference. According to the literatures, ^[37,38] for the determination of cobalt with pyridylazo reagent, the interference of Fe(III) can be masked with NaF, and the interference Ni(II), Cu(II), Zn(II) can be masked with Zn-EGTA. So the experiments of containing an appropriate amount of Zn-EGTA and NaF in buffer solution to mask Ni(II), Cu(II), Zn(II) and Fe(III) ions were studied. The results showed containing 4- 6% of Zn-EGTA and 3- 10 % of NaF in buffer solution can greatly enhance the tolerance limits of Ni(II), Cu(II), Zn(II), Fe(III) and do not affect the sensitivity of this method. So containing of 5% Zn-EGTA and 5% NaF in the buffer solution were recommended to mask Ni(II), Cu(II), Zn(II) and Fe(III) ions.
3.3 Effect of surfactants
The Co-QADMAA complex has a poor solubility in water solution. It needs to add a suitable amount of surfactant to enhance the solubility of the complex. Experiments showed that all the anionic surfactants, nonionic surfactants and cationic surfactants have good effect to enhance the solubility. In addition to enhance the solubility, in the nonionic surfactants and cationic surfactants medium, the sensitivity of the Co-QADMAA chelates was increased markedly too. The effect of the nonionic surfactants and cationic surfactants for improving the sensitivity is shown in Table 1. The results showed that CTMAB was the best additive and the use of 3.0- 8.0 mL of 10% CTMAB solution gave a constant and maximum absorbance. Accordingly, 5.0 ml CTMAB solution was recommended.

Table 1 The effect of surfactants on Co-QADMAA chromogenic system

3.4 Effect of QADMAA concentration
For up to 2.4 mg of Co(II), the use of about 5 -10 mL of 5×10^-4 mol/L of QADMAA solution has been found to be sufficient for a complete reaction. Accordingly, 6 mL of QADMAA solution was added in all further measurement.
3.5 Stability of the chromogenic system
After mixing the components, the absorbance reaches its maximum within 6 min at room temperature and remains stable for 6 h in aqueous solution. The chelates are stable at least 20 h when it was extracted into the isopentyl alcohol medium.
3.6 Solid phase extraction
Both the enrichment and the elution were carried out on a Waters SPE device (which can prepare twenty samples simultaneously). The flow rate was set to 50 mL/min during enrichment and 5 mL/min while elution.
    Some experiments were carried out in order to investigate the retention of QADMAA and its Co(II) chelate on the disks. It was found that the QADMAA and its Ni(II) chelate was retained on the disks quantitatively when they pass the disks as aqueous solution. The capacity of the disks for QADMAA was 31 mg and for its Co(II)-chelate was 22 mg in a 200 mL of solution. In this experiment, the disks has adequate capacity to enrich the Co(II)-QADMAA chelate and the excessive QADMAA.
    In order to choose a proper eluant for the retained QADMAA and its Co(II) chelate. Various organic solvents were studied. The effect of various organic solvents was in the following sequence: isopentyl alcohol > acetone > acetonitrile > ethanol > methanol. So isopentyl alcohol was selected as eluant. Experiment show that it was easier to elute the retained QAMAA and its Co(II) chelate in reverse direction than in forward direction, so it is necessary to upturned disks when elution. 4.0 mL of isopentyl alcohol was sufficient to elute the QADMAA and its Co(II) chelate from disks at a flow rate of 5 mL/min. The volume of 4.0 mL eluant was selected.
3.7 Calibration Curve and Sensitivity
The calibration curve show that Beer's law is obeyed in the concentration range of 0.01~ 0.6 m g Co(II) per mL, The linear regression equation obtained was: A = 2.28 C (mg/mL) + 0.0115, (r=0.9991). The molar absorptivity was calculated to be 1.32×10⁵L.mol^-1.cm^-1 at 596 nm. The relative standard deviation at a concentration level of 0.2 mg Co(II) per mL (11 repeat determination) was 1.35 %.
3.8 Composition of the complex
The composition of the complex was determined by continuous variation and molar ratio method. Both showed that the molar ratio of Co(II) to QADMAA is 1:2.
3.9 Interference
The selectivity of the proposed method investigated by the determination of 2.0 mg/200ml of Co(II) in the presence of various ions within a relative error of ± 5% are given in Table 2.

Table 2 Tolerance limits in the determination of 5 mg of Co with QADMAA (relative error ± 5% )

3.10 Application
The proposed method has been successfully applied to the determination of cobalt in biological sample and water.
    For biological samples, 0.20 g of sample was weigh accurately and put into the Teflon high-pressure microwave acid-digestion bomb (Fei Yue Analytical Instrument Factory, Shanghai, China). 2.5 mL of concentrated nitric acid and 2.5 mL of 30% hydrogen peroxide were added. The bombs were sealed tightly and then positioned in the carousel of the microwave oven (Model WL 5001, 1000 W, Fei Yue Analytical Instrument Factory, Shanghai, China). The system was operated at full power for 6.0 min. The digest was evaporated to near dryness. The residue was dissolved with 1% of hydrochloric acid, and the cobalt contents were analyzed according to general procedure. The results are shown in Table 3.
    For water sample, the samples were acidified with hydrochloric acid and filtrated by 0.45 mm filter. The cobalt contents were analyzed according to general procedure. The results were shown in Table 4, together with the results of a recovery test. A standard method using ICP-MS has also been used as reference method. The results are also shown in Table 4.

Table 3 Determination of cobalt in the certified standard biological samples

Table 4 Determination of Cobalt in the water sample

Table 5 Comparison of reagents for spectrophotometric determination of cobalt

4 CONCLUSION
A comparison of the possibilities of the QADMAA with those of other methods for the determination of cobalt was given in Table 5. In this work, a high sensitive and selective reagent for cobalt was synthesized. Experiments show that the molar absorptivity of the Co(II)-QADMAA-CTMAB chelate reaches 1.32×10⁵ L.mol^-1.cm^-1 in the measured solution. Most foreign ions do not interfered with the determination in the presence of sodium thiosulfate and sodium fluoride medium. By solid phase extraction with C₁₈ disks, the enrichment factor of 50 was achieved. The detection limit reaches 0.02 mg/L in the original samples, and low concentration of cobalt in samples can be determined with good results. The consuming of organic solvents in this method is much lower than those consumed in liquid-liquid extraction method. By using Waters SPE device, twenty samples can be prepared simultaneously. This method is rapid for simultaneously preparing large amount of samples.

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固相萃取分光光度法测定环境样品中的钴
翁哲慧, 贾智若, 董学畅^# , 缪明明 胡秋芬^#
(云南烟草科学研究院, 昆明, 云南 650106; ^#云南民族大学化学与生物技术学院, 昆明, 云南 650031 )
摘要  基于钴（II）和2-(2-喹啉偶氮)-5-二甲氨基苯氨(QADMAA)的快速反应以及C₁₈固相萃取盘对Co(II)-QADMAA螯合物的固相萃取，建立一种灵敏度高、选择性好、快速的钴测定方法。在pH=5.0的缓冲液和十六烷基三甲铵溴化胺（CTMAB）介质中，QADMAA和钴形成络合比为2:1的紫红色络合物。将此络合物用C₁₈固相萃取盘进行固相萃取，用少量的异戊醇可洗脱，富集倍数为50倍。在异戊醇介质中，络合物在616 nm处的摩尔吸光系数为1.32×10⁵ L.mol^-1.cm^-1。钴含量在0.01- 0.6 mg/ml范围内符合比尔定律。对0.01 mg/ml的样品11次重复测定，相对标准偏差为1.35%。样品中的检测限达到0.02 mg/L。将此方法用于环境样品中钴的测定，结果令人满意。
关键词  2-(2-喹啉偶氮)-5-二甲氨基苯氨，钴，固相萃取光度法