Liu Hongcheng, Er Zhu, Li Qiwan, Hu Qiufen ^#, Yin Jiayuan ^#
(Supervision & Testing Center For Farm Products Quality, Ministry Of Agriculture,   Kunming, 650223; ^# Department Of Chemistry, Yunnan University, Kunming 650091, China )

1. INTRODUCTION
The RP-HPLC technique with pre-column derivatization has been proved to be a favorable and reliable technique for the separation and determination of trace amount of metal ions. Many kinds of reagents have been examined as pre-column derivatization regents, and several review articles have appeared on this approach ^{[1- 8]}. Among the various kinds of reagents, porphyrin ligands are useful because of its high molar absorptivity and high stability. The molar absorptivity of metal-porphyrin chelates above 10⁵ is often found. Porphyrins can form very stable 1:1 chelates with many metal ions and these chelates are very difficult to decomposing during the HPLC separation ^[1-3]. Due to these facts, porphyrin reagents have received more and more attentions and are widely applied to the simultaneous determination of metal ions ^[9-12].
    Determination of trace lead, cadmium, mercury, nickel, cobalt, silver and tin in water and food is very important because of their biological significance ^[13]. In this paper, a ZORBAX Stable Bound microcolumn (2.0×50 mm, 1.8 m m) with pH range 1.5-12 was used for the separation of Hg-T₄-APP, Cd-T₄-APP, Pb-T₄-APP, Ni-T₄-APP, Co-T₄-APP, Ag-T₄-APP and Sn-T₄-APP chelates with pH 10 mobile phase. The seven chelates were separated completely within 2 min. Based on this; a rapid, sensitive and selective method for the simultaneous determination of seven transition metal ions in water and food was developed.

2. EXPERIMENTAL PROCEDURES
2.1 Apparatus
The HPLC system consisted of a Waters 2690 Alliance separation model and a 996 photodiode array detector (Waters Corporation, USA). The pH values were determined with a Beckman F-200 pH meter. The absorbance measurement was measured with a Shimidzu UV-2401 spectrophotometer. The atomic absorption spectrometry analysis was made with a Shimidzu AA-6701F atomic absorption spectrophotometer.
    The separation column used was a ZORBAX Stable Bound microcolumn (2.0´ 50 mm, 1.8 m m) (Agilent Corporation, USA). The cartridge used is Zorbax C₁₈ solid phase extraction cartridge (1 cc/50mg，30 m m) (Agilent Corporation, USA). The extraction was performed on Waters Solid Phase Extraction (SPE) Device (which can prepare twenty samples simultaneously).
2.2 Chemicals
All of the solutions were prepared with ultra-pure water, which was obtained from a Milli-Q50 SP Reagent Water System (Millipore Corporation, USA). Pb(II), Cd(II), Hg(II), Ni(II), Co(II), Ag(I) and Sn(IV) standard solution: 1.0 mg/ml (Obtained from Chinese Standards Center), a working solution of 0.2 mg/ml was prepared by diluting this standard solution. HPLC grade methanol and THF (Fisher Corporation, USA). Pyrrolidine-acetic acid buffer solution: 0.5 mol/L, pH=10. TritonX-100 solution, 2.0% (v/v). T₄-APP was synthesized by our laboratory as described in the literature [14], and was dissolving with THF to make a 1.5´ 10^-4 mol/L of solution. The glass and Teflon wares used were soaked in 5% of nitric acid for a long time, and then thoroughly wash with pure water.
2.3 Standard procedure
An appropriate volume of standard or the sample was transferred into a 50 ml of volumetric flask. To which, 5.0 ml of T₄-APP THF solution, 2 ml of Triton X-100 solution and 5 ml of 0.5 mol/L pH=10 pyrrolidine-acetic acid buffer solution were added. The solution was diluted to a volume of 50 ml with water and mixed well. The mixture was heated in boiling water bath for 15 min. After cooled, the solution was passed through the C₁₈ cartridge at a flow rate of 10 ml/min. After the enrichment had finished, the retained chelates were eluted from the cartridge with 1.0 ml of THF at a flow rate of 5 ml/min in an opposite direction. The solution was filtered with filters of 0.45 m m and adjusted to the volume of 1.0 ml. 2.0 ml of sample was injected for HPLC analysis. A tridimensional was recorded from 350~ 600 nm with photodiode array detector and the chromatogram of 435 nm is shown in Fig.1.

Fig.1 Chromatogram of standard sample and real sample: 1) Real sample, 2) Standard sample
The Injection volume is 2.0 ml. Detection wavelength is 435 nm. Other conditions as in standard procedure.

3. RESULT AND DISCUSSION
3.1. The selection of porphyrin Reagent
In this paper, seven porphyrin reagent, tetra-(4-bromophenyl)-porphyrin (T₄-BPP), tetra-(4-chlorophenyl)- porphyrin (T₄-CPP), tetra-(4-methoxyphenyl)-porphyrin (T₄-MOPP), tetra-(4-methylphenyl)-porphyrin (T₄-MPP), tetra-(4-sulfophenyl)-porphyrin (T₄-SPP), tetra-(4-hydroxyphenyl)-porpyrin (T₄-HPP) and tetra-(4-aminophenyl)-porpyrin (T₄-APP) were studied as pre-column derivatization regents for Co(II), Ni(II), Sn(IV), Hg(II), Pb(II), Ag(I) and Cd(II) ions. The experiment show that all of the seven reagents can form colored chelates with Co(II), Ni(II), Sn(IV), Hg(II), Pb(II), Ag(I) and Cd(II). The T₄-MPP, T₄-CPP, T₄-MOPP, T₄-BPP and their metal-chelates have a very poor solubility in water. To get a stable solution, it is required adding large proportion of organic solvents to improve the solubility, which is inconvenient. The T₄-SPP and T₄-HPP can form soluble chelates with metal ions. But in alkaline medium, the chelates have a poor retention on reversed-phase column because the sulfonic group or the hydroxy group on porphyrin reagents can ionize. So T₄-SPP and T₄-HPP are unsuitable to use as pre-column derivatization reagents in this experiment. T₄-APP can form soluble chelates in aqueous medium when Triton X-100 is existed, and the chelates have good retention on reverse-phase column. So T₄-APP was selected as pre-column derivatization regents in this experiment.
3.1 Precolumn derivation
According to the literature ^[14-16], the optimal pH is 8.2 ~ 11.8 for the reaction of Co(II), Ni(II), Sn(IV), Hg(II), Pb(II), Ag(I) and Cd(II) with T₄-APP, so a 0.5 mol/L of pH=10 pyrrolidine-acetic acid buffer solution was recommended to control pH. It was found that 1.0 ml of 1.5×10^-4 mol/L T₄-APP THF solution was sufficient to complex 3.0 mg of Ni(II), Sn(IV), Co(II), Hg(II), Pb(II), Ag(I) and Cd(II). But in real samples, the foreign ions, such as Mg²⁺, Pd²⁺, Fe³⁺, Mn²⁺, Zn²⁺, Bi³⁺, Ba²⁺, Cu²⁺ and the like, can complex with T₄-APP to consume reagents. So more T₄-APP was needed. In this experiment, A 5.0 ml of 1.5´ 10^-4 moL/L T₄-APP solution was recommended in this experiment.
    The reaction of Co(II), Ni(II), Sn(IV), Hg(II), Pb(II), Ag(I) and Cd(II) with T₄-APP was slow at room temperature. Heating can accelerate the reaction. The reaction was complete for heating in boiling water bath for 10 min and the chelate can keep stable for 5 h after cooling, so heating 15 min was selected.
3.2 Solid phase Extraction
Both of 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 10 ml/min when enrichment and 5 ml/min when elution.
    Some experiments were carried out in order to investigate the retention of metal-T₄-APP chelates on the cartridge. It was found that the Sn-T₄-APP, Ni-T₄-APP, Co-T₄-APP, Hg-T₄-APP, Pb-T₄-APP, Ag-T₄-APP and Cd-T₄-APP chelates could be retained on the cartridge quantitatively when they pass the cartridge as aqueous solution. The capacity of the cartridge for metal-T₄-APP chelates was 32 mg in a 50 ml of solution. In this experiment, the cartridge has adequate capacity to enrich the metal-T₄-APP chelates.
    In order to choose a proper eluant for the retained T₄-APP and its metal-chelates, various organic solvents were studied. It was found that the THF, isopentyl alcohol, acetonitrile, acetone, ethanol and methanol could elute the metal-T₄-APP chelates from cartridge quantitatively. The effect of the various eluants for the retained metal-T₄-APP chelates was in the following squence: THF > isopentyl alcohol > acetonitrile > acetone > ethanol > methanol. So THF was selected. The metal-T₄-APP chelates have a good stability in a weak alkaline medium. A pyrrolidine-acetic acid buffer salt of 0.05 mol/L (pH=10) in THF could increase the stability of the metal-T₄-APP chelate during the elution. So THF (containing 0.05 mol/L pyrrolidine-acetic acid buffer salt (pH=10)) was selected as eluant. Experiment showed that it was easier to elute the retained T₄-APP and its metal-chelate on cartridge in reverse direction than in forward direction, so it was necessary to upturned the cartridge when elution. 1.0 ml of the THF eluant was sufficient for eluted the metal-T₄-APP chelate from cartridge quantitatively at a flow rate of 5 ml/min.
3.3 Spectrophotometric properties
From tridimensional chromatogram recorded by photodiode array detector, the absorption spectrum of metal-T₄-APP chelates was obtained. The maximum absorption wavelengths of Ni-T₄-APP, Sn-T₄-APP, Co-T₄-APP, Hg-T₄-APP, Pb-T₄-APP, Ag-T₄-APP and Cd-T₄-APP are 428 nm, 432 nm, 432 nm, 451 nm, 466 nm, 428 nm and 438 nm. To get maximum sensitivity, each metal-T₄-APP chelates were monitored at its maximum absorption wavelength.
3.4 Chromatographic Separation
The Co-T₄-APP, Sn-T₄-APP, Ni-T₄-APP, Hg-T₄-APP, Pb-T₄-APP, Ag-T₄-APP and Cd-T₄-APP chelates were stable in weak alkaline medium. The pH of mobile phase within 8.5-11.8 can avoid the chelates decomposing and get a good peak shape. So methanol-tetrahydrofuran (95:5, v/v, containing 0.05 mol/L pyrrolidine-acetic acid buffer salt, pH=10.0) was selected as mobile phase. Because the routine silica bonds reserved phase chromatographic column was not stable in pH 10, a ZORBAX Stable Bound microcolumn (2.0´ 50 mm, 1.8 m m) was selected as analytical column in this experiment. ZORBAX Stable Bound column have a good stability in pH 1.5-12.
3.5 Calibration graphs
Under optimum conditions, regression equations of metal-T₄-APP chelates were established based on the standard samples injected and its peak areas. Limits of detection are calculated by the ratio of signal to noise (S/N=3). The results were shown in Table-1. The reproducibility of this method was also examined for 10 mg/L of Ni(II), Sn(IV), Co(II), Pb(II), Cd(II), Ag(I) and Hg(II). The relative standard deviations (n=10) were shown in Table 1 too.

3.6 Interference
Under the pre-column derivatization condition, the foreign ions of Mg²⁺, Cu²⁺, Pd²⁺, Rh³⁺, Fe³⁺, Mn²⁺, Zn²⁺, Pt²⁺, Ba²⁺, Bi³⁺ and Ru³⁺, which can reacts with T₄-APP to form color chelates. To examine the selectivity of this method, the interference of foreign ions was investigated. When 5.0 ml of 1.5×10^-4 mol/L T₄-APP was used, with 10 mg/L of Ni(II), Sn(IV), Pb(II), Co(II), Cd(II) and Hg(II) respectively, the tolerance amount with an error of ± 5% was 5000 mg/ml for Fe(III), Mg(II), 1000 mg/ml for Cu(II), Bi(III), Zn(II), Ba(II) and 500 mg/ml for Pd(II), Pt(II), Ru(III), Rh(III). The results show most foreign ions do not interfer with the determination.
3.7 Application to Food samples
0.2-0.30 g of sample was weighed 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 5 ml of 5% of nitric acid and transferred into a 50 ml of calibrated flask quantitatively, and diluted to volume with water. The Co, Ni, Sn, Hg, Pb, Ag and Cd contents were analyzed by using a proper volume of this solution according to general procedure. The results (deducted the reagents blank) were shown in Table 2.

Table2 Determination results of certified standard food samples

3.8 Application to water samples
For the fresh water (tap water, river water and lake water) the water sample was analyzed according to the general procedure. The results (deducted the reagents blank) were shown in Table 3, together with the results of a recovery test by added 0.2mg of Ni, Co, Sn, Ag, Pb, Cd and Hg in water sample and diluted to 50 ml of final solution. For plant effluents, the sample was digested as literature ^[13] and analyzed according to the general procedure. The results (deducted the reagents blank) were shown in Table-3 too, together with results of a recovery test by added 0.2 mg of Ni, Co, Sn, Ag, Pb, Cd and Hg in water sample and diluted to 50 ml of final solution. A standard method using atomic absorption spectrometry (AAS) had also been used as reference method. The results (AAS) were shown in braces.

Table 3 Determination results (mg/L) of the water sample with this method

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