Solid Phase Extraction and Spectrophotometric Determination of Mercury in Tobacco and Tobacco Additives with 5-( p-Aminobenzylidene )-Thiothiorhodanine

Foi desenvolvido um método altamente sensível, seletivo e rápido para a determinação de mercúrio, a partir da reação rápida de mercúrio(II) com 5-(p-aminobenzilideno)-tiorodanina (ABTR) e posterior extração em fase sólida do quelato colorido, utilizando discos C 18 . Em pH 3,5 e na presença do emulsificante-OP, ABTR reage com mercúrio(II) para formar um quelato vermelho na razão molar 1:2 (mercúrio:ABTR). O quelato foi enriquecido pela extração em fase sólida com discos C 18 e o quelato retido, eluído com dimethyl formamida (DMF). Um fator de enriquecimento na ordem de 50 foi obtido. Em DMF, a absortividade molar do quelato é 1,21×10 L mol cm a 545 nm, e a lei de Beer é obedecida no intervalo 0,01~3 μg mL na solução medida. O desvio padrão relativo para onze replicatas a 0,01 μg mL é 1,7%. Este método foi aplicado para a determinação de mercúrio em tabaco e aditivos de tabaco. Bom coeficiente de preconcentração foi encontrado, comparando-se o método proposto com outros similares.


Introduction
Mercury is a toxic heavy metal.Thus, trace mercury determination in tobacco and tobacco additives is a very important issue.The Quality Standards of Tobacco in Chinese Tobacco Company says that the concentration of mercury should not exceed 0.2 μg g -1 in tobacco and tobacco additives. 1][4][5][6][7][8] However, the spectrophotometric method has still the advantage of its simplicity and accessibility, not needing expensive or complicated equipments.0][11][12][13][14][15][16][17][18][19] Each chromogenic system has its advantages and disadvantages with respect to sensitivity, selectivity and analysis speed.
Nevertheless, for the routine spectrophotometric determination of mercury trace, a preconcentration step is usually required.1][22][23][24] In the present work, a new chromogenic reagent, 5-(paminobenzylidene)-thiothiorhodanine (ABTR) was synthesized, and its reaction with mercuric ions and solid phase extraction of the formed chelate with C 18 disks were studied.Based on this, a highly sensitive, selective and rapid method for the determination of total mercury in tobacco and tobacco additives was developed.

Reagents
The ABTR was synthesized by the following procedure: 40 mL of acetic acid was added to 1.5 g of thiorhodanine and 1.2 g of p-aminobenzaldehyde, and the mixture was gently heated until total dissolution.The solution was refluxed for about 1.5 h, and 1 mL of concentrated sulfuric acid was added dropwise.After the solution has turned red, the reflux was stopped and the sample was poured into 200 mL of distilled water.After the precipitation of a red solid took place, a small amount of aqueous ammonia was added.The solid was separated by filtration.They were recrystallized twice from absolute alcohol and the yield was 36% (mp 282 ~ 285 °C).The structure (Figure 1 All of the solutions were prepared with ultra-pure water obtained from a Milli-Q50 SP Reagent Water System (Millipore Corporation, USA).High purity dimethyl formamide (DMF) (Fisher Corporation, USA) was used.A 3.0×10 -4 mol L -1 ABTR solution was prepared by dissolving ABTR in ethanol.A stock standard solution of mercury (1.0 mg mL -1 ) was obtained from the Chinese Standard Material Center, and a working solution (0.5 μg mL -1 ) was prepared by adequate dilutions of this solution.Sodium acetateacetic acid buffer solution ( 0.5 mol L -1 , pH 3.5, containing 0.2 mol L -1 of pyrophosphoric acid) was prepared by dissolving the 40.8 g sodium acetate, 180 mL acetic acid and and 44.2 g of sodium pyrophosphate in water and diluted to 1 L. Emulsifier-OP solution (2.0%, v/v) was prepared by dissolving the emulsifier-OP in water.All chemical used were of analytical grade unless otherwise stated.

General procedure
To a proper volume (up to 40 mL) of standard or sample solution containing no more than 3.0 μg of Hg(II), 5 mL of pH 3.5 buffer solution, 3.0 mL of 3.0×10 -4 mol L -1 ABTR solution and 2.0 mL of 2.0% Emulsifier-OP solution were added.The mixture was made up to 50 mL and well mixed.After 10 min, the solution was passed through the C 18 disks at a flow rate of 50 mL min -1 .The elution was then performed at a flow rate of 5 mL min -1 in the reverse direction with 1.0 mL of DMF, and the final volume accurately made up to 1.0 mL in a 1.0 mL calibrated flask by adding microamounts of DMF with a 200 μL syringes.The absorbance of this solution was measured at 545 nm in a 1 cm microcell (0.5 mL) against a reagent blank prepared in a similar way.

Sample preparation
The samples (0.50 g) were accurately weighted into the PTFE high-pressure microwave acid-digestion vessels, and 3.0 mL of concentrated nitric acid plus 5.0 mL of 30% hydrogen peroxide were added.The vessels were sealed tightly and then positioned in the carousel of the microwave oven.The system was operated at full power for 8.0 min.The digest was evaporated to near dryness.The residue was dissolved with 5 mL 5% (m/v) nitric acid, and quantitatively transferred to a 50 mL volumetric flask for further analysis.

Absorption spectra
The absorption spectra of ABTR in water and its Hg(II) chelate in DMF are shown in Figure 2. The absorption peak maxima of ABTR in water and its complex in DMF medium are located at 420 nm and 545 nm, respectively.

Effect of acidity and masking
Results showed that the optimal pH for the complexation of Hg(II) by ABTR is at the range 1.2~4.2.The use of the sodium acetate-acetic acid buffer solution (pH 3.5) is recommended, and the addition of 4~6 mL of this solution to a final volume of 50 mL was found to give a maximum and constant absorbance.A volume of 5 mL of the buffer solution was then recommended.The addition to the buffer solution of 0.15~0.25 mol L -1 pyrophosphoric acid do not affect the sensitivity and can greatly increase the selectivity.For instance, the tolerance limits of foreign ions such as Cu(II), Ag(I), Pd(II) and Pb(II), increased from 0.005, 0.005, 0.002 and 0.002 to 0.5, 0.2, 0.1 and 0.1 mg per 50 mL, respectively if pyrophosphoric acid is present.Thus, the presence of 0.2 mol L -1 of pyrophosphoric acid in the buffer solution is recommended.

Effect of surfactants
The influence of surfactants on the Hg(II)-ABTR chromogenic system was studied.The results (Table 1) showed that in the absence of surfactants, or in the presence both anionic or cationic surfactants, lower molar absorptivity (ε) values were found, whereas in the presence of nonionic surfactants, the chromogenic system ε values increases markedly.Considering the various nonionic surfactants invatigated the absorbance enhancement follows the sequence: Emulsifier-OP >Tween-80 > Tween-20 > Tween-60.Accordingly, the use of 0.5~3 mL of the Emulsifier-OP solution give a constant and maximum absorbance.The use of 2.0 mL of this surfactant was then recommended.

Effect of ABTR concentration
For up to 3.0 μg of Hg(II), the use of about 3 mL of 3.0×10 -4 mol L -1 of ABTR solution has been found to be sufficient for a complete reaction.Accordingly, 3.0 mL of ABTR solution were added in all further measurement.

Stability of the chromogenic system
After mixing the components, the absorbance reaches its maximum within 5 min at room temperature and remains stable for at least 8 h.After extracted into the DMF medium, the chelate was stable for at least 12 h.

Solid phase extraction
Both the enrichment and elution were carried out on a Waters SPE device (able to prepare twenty samples simultaneously).The flow rate was set to 50 mL min -1 during enrichment and 5 mL min -1 during elution.Some experiments were carried out in order to investigate the retention of ABTR and its Hg(II) chelate on the disks.It was found that the ABTR and its Hg(II) chelate could be quantitatively retained on the disks as they pass through the disks in aqueous solution.The capacity of the disks was determined as 30 mg for ABTR and 25 mg for its Hg(II) chelate both in 50 mL solution.This experiment has shown that the disks have adequate capacity to enrich the Hg(II)-ABTR chelate to adequate levels.In order to choose a proper eluent for the retained ABTR and its Hg(II) chelate, various organic solvents were studied.Their effectivences was evaluated and the following sequence was obtained: DMF > acetonitrile > acetone > ethanol > methanol.So DMF was selected as eluent.Experiments have shown that it is easier to elute the retained ABTR and its Hg(II) chelate in the reverse direction in comparison to the forward direction.Thus, it is also recommended to invert the disks before elution.Only 1.0 mL of eluent was sufficient to elute all the ABTR and its Hg(II) chelate retained at the disks at a flow rate of 5 mL min -1 .

Figures and merit
The calibration curve showed that the Beer's law is obeyed in the 0.01 to 3 μg Hg(II) per mL concentration range.The linear regression equation obtained was A =0.581 C (μg mL -1 ) + 0.0126 (r=0.9994).The molar absorptivity was calculated to be 1.21×10 5 L mol -1 cm -1 at 545 nm.The relative standard deviation (n=11) at a 0.01 μg mL -1 concentration level was 1.7%.The detection limit, based on the three times of standard deviation is 0.02 μg L -1 , in the original sample solutions.

Composition of the chelate
The composition of the complex was determined by the continuous variation (Figure 3) and molar ratio (Figure 4) methods.Both showed a 1:2 Hg(II) to ABTR molar ratio.A possible chelate structure is suggested in Figure 5.

Interferences
The selectivity of the proposed method was investigated by analysis of 20 μg L -1 Hg(II) solutions containing various potentially interfering ions.A relative deviation of ±5% was tolerated and the results are given in Table 2.The results showed that the ions commonly present in Hg determinations do not interfere at a preoccupting concentration range.

Application and validation
The proposed method has been successfully applied to the determination of mercury in tobacco and tobacco additives.The digested sample was quantitatively

Table 1 .
The effect of surfactants on Hg(II)-ABTR chromogenic system

Table 4 .
The comparison reagents for the spectrophotometric determination of mercury

Table 3 .
Determination of mercury in real samples